Hybrid antibody

Abstract

To provide hybrid antibodies targeted for use in the treatment of cancer.SOLUTION: Antibodies have the ability to bind to Fc ε and Fc γ receptors, which can be achieved, for example, by grafting heavy chain constant domain sequences (e.g., CH2 and CH3 domains) from IgG onto IgE.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention resides in the design of synthetic (non-naturally occurring) hybrid antibodies, particularly hybrid IgE antibodies, along with their therapeutic uses. [Background technology]

[0002] Immunoglobulin E (IgE) is a class of antibody (or immunoglobulin (Ig) "isotype") found only in mammals. IgE is synthesized by plasma cells. Like all antibody classes, IgE monomers consist of two larger identical heavy chains (ε chains) and two identical light chains (common to all antibody classes), with the ε chains containing four Ig-like constant domains (Cε1 to Cε4; see Figure 1).

[0003] The different antibody classes are distinguished by the properties of their heavy chains; those of the IgE class are larger and more heavily glycosylated than the heavy chains of the more common IgG class. Each antibody chain is composed of a series of tandemly arranged immunoglobulin domains. The N-terminal domains (one on the light chain and one on the heavy chain) contain regions of highly variable sequence (variable domains) that allow binding to a wide range of antigens. The remaining domains consist of the highly conserved so-called constant (Fc) domains.

[0004] The main function of IgE is immunity against parasites such as helminths. IgE also plays an essential role in type I hypersensitivity, which manifests as various allergic diseases such as allergic asthma, most types of sinusitis, allergic rhinitis, food allergies, and specific types of chronic urticaria and atopic dermatitis. IgE also plays a crucial role in responding to allergens such as anaphylactic drugs, bee stings, and antigen preparations used in desensitization immunotherapy.

[0005] IgE is typically the least abundant isotype, with IgE levels in normal ("non-atopic") individuals representing only 0.05% of the Ig concentration, compared with 75% for 10 mg / ml of IgG, the isotype responsible for most classical adaptive immune responses and capable of eliciting the most potent inflammatory reactions.

[0006] IgG is the main type of antibody found in blood and extracellular fluids, enabling it to control infection of bodily tissues. IgG protects the body from infection by binding to many types of pathogens, such as viruses, bacteria, and fungi. IgG antibodies are large molecules with a molecular weight of approximately 150 kDa, made up of four peptide chains. Each molecule contains two identical gamma heavy chains of approximately 50 kDa and two identical light chains of approximately 25 kDa, resulting in a tetrameric quaternary structure. The two heavy chains are linked to each other and to the light chains by disulfide bonds (see Figure 1). The resulting tetramer has two identical halves that together form a Y-like shape. Each end of the fork contains an identical antigen-binding site.

[0007] Structural differences confer different biological activities among antibody classes due to factors that bind to multiple effector cells and different constant domains of each antibody class. The gamma chain of IgG binds to FcγRI (CD64), FcγRIIa, FcγRIIb, and FcγRIIIa (CD 16) and FcγRIIIb. The Epsilon chain binds to the high affinity receptor FcεRI and the lower affinity receptor FcεRII. The differential expression of these various receptors on various immune effector cells is The presence of IgG and IgE determines the type of immune response that can be generated.

[0008] Receptor molecules that interact with IgG are known to do so within the second constant domain (CH2 domain) of the gamma heavy chain. For example, the receptor on natural killer (NK) cells (FcγRIIIa), which interacts with IgG to enable guidance and activation of these cells for cell / pathogen killing, has a CH2 domain / lower hinge region. In contrast, it is the CH3 domain of IgE that is involved in binding interactions with IgE receptors (FcεRI and FcεRII) on effector cells (mast cells, basophils, monocytes, macrophages, eosinophils; Scott C. et al (2012) Immunobiology 217: 1067-1079). IgE does not interact with FcγRIIIa, whereas IgG does interact with FcεRI and FcεRII. As a result, the two antibody classes recruit distinct populations of effector cells and factors.

[0009] Although IgE is mostly known for its harmful role in allergy, several studies have long been directed toward the natural tumor surveillance function of this antibody isotype (Jensen-Jarolim E. et al (2008) Allergy 63: 1255-1266; Jensen-Jarolim E., Pawelec G. (2012) Cancer Immunol. Immunother. 61: 1355-1357). Pioneering studies using IgG and IgE antibodies of the same epitope specificity tested side-by-side revealed the greater potential of IgE in terms of cytotoxicity (Gould HJ et al (1999) Eur. J. Immunol. 29: 3527-3537).

[0010] IgE has evolved to kill tissue-dwelling multicellular parasites and endows it with several key attributes that make it ideal for use in the treatment of solid tumors, the majority of which are tissue-resident. The epsilon constant region of IgE has an exceptionally high affinity for its cognate receptor (FcεRI) on the surface of immune effector cells, including macrophages, monocytes, basophils, and eosinophils (Ka for FcεRI approximately 10 10 / M, and Ka for CD23 trimeric complex is approximately 10 8 ~10 9 / M;Gould HJ, Sutton BJ (2008) Nat. Rev. Immunol. 8: 205-217). This interaction is up to 10,000 times greater than the affinity that the gamma chain of IgG has for its cognate receptor, which results in the majority of IgE molecules being permanently attached to the surface of immune effector cells (Fridman WH (1991) FASEB J. 5: 2684-2690). The latter are therefore primed and capable of producing IgE. IgE can rapidly destroy cells expressing antigens recognized by IgE. As a result, IgE can penetrate tissues more effectively than IgG and stimulate significantly greater levels of both antibody-dependent cell-mediated phagocytosis (ADCP) and antibody-dependent cell-mediated cytotoxicity (ADCC), the two main mechanisms by which immune effector cells can kill tumor cells. Due to its rapid binding to Fcε receptors on cells, IgE is rapidly cleared from the circulation and has a significantly longer tissue half-life than IgG (2 weeks vs. 2-3 days), which is advantageous from a side effect perspective due to the compound's shorter duration in the bloodstream and also supports its role in killing solid tumors.

[0011] Furthermore, potential IgE immunotherapies should be effectively distributed to tumor tissues, for example, because IgE antibodies bound to Fcε receptors on mast cells can penetrate malignant tumors using these cells as a shuttle system, and this transport would be very efficient because mast cells are tissue-resident immune cells (St John AL, Abraham SN (2013) J. Immunol. 190: 4458-4463).

[0012] Other potential advantages include the high sensitivity of IgE effector cells to activation by antigen, and the speed and magnitude of the response, which can be seen most impressively during allergic and anaphylactic reactions, which typically begin within minutes of allergen exposure. At the same time, this is also the biggest concern with the use of IgE-based immunotherapy for cancer: intravenously applied recombinant IgE always carries the risk of anaphylactic reactions, and therefore careful selection of target epitopes is of utmost importance in this regard.

[0013] A challenge for current immunotherapy using IgG antibodies is that not all human Fcγ receptors are immunostimulatory: one of them, FcγRIIb, is inhibitory (Nimmerjahn F., Ravetch JV (2006) Immunity 24: 19-28). Therefore, IgG-based immunotherapy is difficult. The tumoricidal effect of immunotherapy also depends on the net ratio of binding to activating and inhibitory receptors. As shown for IgG4, a subclass that exhibits relatively high binding affinity to FcγRIIb (Bruhns P. et al (2009) Blood 113: 3716-3725), this antibody Despite being tumor-associated antigen-specific, IgG4 antibodies are unable to induce immune cell-mediated tumor cell killing in vitro. Furthermore, it has been demonstrated that IgG4 antibodies significantly impair the killing potential of IgG1 antibodies of the same specificity in vitro and in vivo (Karagiannis P. et al (2013) J. Clin. Invest. 123: 1457-1474). Strategies to overcome this limitation involves post-translational glycosylation modifications of the heavy chain of the IgG constant region, as these sugar residues have been identified as highly relevant for distinct binding affinities to various Fc receptors (Schroeder HW Jr, Cavacini L. (2010) J. Allergy Clin. Immunol. 125: S41-52). On the other hand, there are no inhibitory receptors for IgE (Karagiannis et al., 2001). SN et al (2012) Cancer Immunol. Immunother. 61: 1547-1564), so again, this isotype may contribute to overcoming current challenges in cancer immunotherapy. [Prior art documents] [Non-patent literature]

[0014] [Non-Patent Document 1] Scott C. et al (2012) Immunobiology 217: 1067-1079 [Non-patent document 2] Jensen-Jarolim E. et al (2008) Allergy 63: 1255-1266 [Non-patent document 3] Jensen-Jarolim E., Pawelec G. (2012) Cancer Immunol. Immunother. 61: 1355-1357 [Non-patent document 4] Gould HJ et al (1999) Eur. J. Immunol. 29: 3527-3537 [Non-patent document 5] Gould HJ, Sutton BJ (2008) Nat. Rev. Immunol. 8: 205-217 [Non-patent document 6] Fridman WH (1991) FASEB J. 5: 2684-2690 [Non-Patent Document 7] St John AL, Abraham SN (2013) J. Immunol. 190: 4458-4463 [Non-patent document 8] Nimmerjahn F., Ravetch JV (2006) Immunity 24: 19-28 [Non-Patent Document 9] Bruhns P. et al (2009) Blood 113: 3716-3725 [Non-Patent Document 10] Karagiannis P. et al (2013) J. Clin. Invest. 123: 1457-1474 [Non-Patent Document 11] Schroeder HW Jr, Cavacini L. (2010) J. Allergy Clin. Immunol. 125: S41-52 [Non-Patent Document 12] Karagiannis SN et al (2012) Cancer Immunol. Immunother. 61: 1547-1564 Summary of the Invention [Problem to be solved by the invention]

[0015] Thus, there is a need for antibodies that have improved properties compared to both the IgE and IgG isotypes and that are useful, for example, in the treatment of cancer. [Means for solving the problem]

[0016] Despite the advantages of IgE over IgG in the solid tumor setting, IgG possesses certain functions that IgE lacks, such as NK cell activation. Thus, by exploiting the high degree of structural similarity between immunoglobulin domains, the present invention provides, in one embodiment, Thus, IgE / IgG hybrid antibodies are provided that possess the combined functionality of IgG and IgE isotypes.

[0017] In one embodiment, the invention provides hybrid antibodies that bind to Fcε and Fcγ receptors. In this context, "binds" typically refers to binding of the hybrid antibody via one or more of its constant domains; i.e., "binds" does not refer to the specificity of the hybrid antibody binding to a target antigen via its variable domains.

[0018] The term hybrid, as used herein, refers to an antibody whose structure is derived from more than one class of antibody. In the present invention, it is typically the hybrid Fc region that provides the antibody with the ability to bind to cell surface receptors of the immune system that are associated with antibodies of various classes. Typically, hybrid antibodies are capable of binding to and activating both Fcε and Fcγ receptors, thereby transducing receptor signaling and effector function in cells of the immune system where these receptors are expressed.

[0019] In one embodiment, an antibody of the invention comprises one or more heavy chain constant domains derived from an IgE antibody (e.g., derived from an epsilon heavy chain). For example, the antibody may comprise one or more domains selected from Cε1, Cε2, Cε3, and Cε4. Preferably, the antibody comprises at least the Cε3 domain, more preferably at least the Cε2, Cε3, and Cε4 domains.

[0020] In one embodiment, the hybrid antibody comprises tetrameric IgE and at least one binding site for one or more Fcγ receptors. The one or more Fcγ receptor binding sites may be attached to the C-terminus of IgE. The tetrameric IgE may comprise a Fab region and an Fc region, with the Fc domain comprising at least the Cε2, Cε3, and Cε4 domains.

[0021] The fragment crystallisable / constant region (Fc region) of an antibody interacts with cell surface Fc receptors and certain proteins of the complement system. This is the tail region, which allows the antibody to activate the immune system.

[0022] The Fcγ receptor binding site or sequence can be provided by one or more constant domains from IgG. Structural regions on IgE can be identified that exhibit homology with regions on IgG to which FcγRs bind. Once such regions are identified, amino acid substitutions can then be made to allow for the transfer of IgG functionality to an IgE background.

[0023] Attachment of one or more constant domains can be by any suitable attachment, linkage, grafting, fixation, or fusion. For example, the construct can include all or part of a hinge region derived from an IgG. It will be understood that all or part of a constant domain sequence, as well as variants thereof, can be used.

[0024] Thus, in one embodiment, a hybrid antibody of the invention comprises one or more heavy chain constant domains derived from an IgG antibody (e.g., derived from a gamma heavy chain). For example, the antibody may comprise one or more domains selected from Cγ1, Cγ2, and Cγ3. Preferably, the antibody comprises at least the Cγ2 domain, more preferably at least the Cγ2 and Cγ3 domains.

[0025] In one embodiment of the invention, the antibody comprises an Fc region comprising the CH2, CH3, and CH4 domains derived from IgE (i.e., Cε2, Cε3, and Cε4 domains) and the CH2 domain or a variant thereof derived from IgG (i.e., Cγ2 domain). The antibody may further comprise a CH3 domain or a variant thereof (i.e., a Cγ3 domain) derived from an IgG, and / or all or a portion of a hinge region derived from an IgG.

[0026] In some embodiments, the antibody may comprise a wild-type IgG hinge region, for example, as shown in SEQ ID NO: 9:

[0027] TIFF2026016524000001.tif12145

[0028] In some embodiments, the antibody may comprise a modified IgG hinge region. For example, a potential free cysteine ​​residue in the IgG hinge region may be replaced with another amino acid residue, e.g., to improve the stability of the hybrid antibody. In one embodiment, the cysteine ​​residue present in the hinge region at position 220 of the IgG heavy chain sequence (numbering based on the EU numbering scheme, with reference to the IgG portion of the hybrid antibody) may be substituted with an alternative amino acid residue (e.g., serine). Thus, the hybrid antibody may comprise, for example, a Cys220Ser amino acid substitution in the heavy chain IgG hinge region. Position 220 in the IgG heavy chain sequence referred to above corresponds to position 5 in SEQ ID NO: 9, i.e., the hybrid antibody may comprise a variant of SEQ ID NO: 9 lacking the C residue at position 5 (i.e., the antibody comprises a hinge region comprising a variant of SEQ ID NO: 9 with a substitution at position 5).

[0029] Thus, in one embodiment, the antibody comprises a modified IgG hinge region as shown in SEQ ID NO: 174:

[0030] TIFF2026016524000002.tif12141

[0031] One or more IgG constant domains may contain one or more amino acid substitutions or post-translational modifications to promote Fc receptor-mediated activity, for example, the CH2 domain may contain glycosylation at its Asn297 position to support Fc receptor-mediated activity.

[0032] In certain embodiments, the IgG-derived sequences, domains, and regions are derived from an IgG1 antibody. The antibody domains described herein can be derived from any species, preferably a mammalian species, and more preferably human.

[0033] In one embodiment, the hybrid antibody binds to FcγRIIIa. In one embodiment, the antibody binds to FcεRI. Preferably, the hybrid antibody binds to both FcγRIIIa and FcεRI.

[0034] In some embodiments, the hybrid antibody may bind to neonatal Fc receptors (FcRn) in addition to the Fcγ receptors typically described above. In alternative embodiments, the hybrid antibody may not bind to FcRn, i.e., the antibody lacks FcRn binding ability. For example, the hybrid antibody may comprise one or more modified heavy chain constant domains derived from an IgG antibody, e.g., whereby FcRn binding of the modified antibody is reduced or eliminated (compared to a native IgG antibody). In one embodiment, the ability of the IgG portion of the hybrid antibody to bind FcRn is eliminated by amino acid substitution at specific residues known to be involved in FcRn binding. Such residues include Ile253, His310, and His435 in the IgG heavy chain sequence (numbering is as follows for the hybrid antibody). (The numbers are based on the EU numbering scheme, with reference to the IgG portion of the hybrid antibody sequence. Thus, the hybrid antibody may comprise an IgG portion with one or more amino acid substitutions at positions 253, 310, or 435 in the IgG heavy chain sequence. For example, the IgG portion of the hybrid antibody may comprise one or more of the following mutations: Ile253Ala, His310Ala, and His435Ala. The sequence of the wild-type IgG CH2 domain is shown in SEQ ID NO: 10:

[0035] TIFF2026016524000003.tif27167

[0036] The sequence of the wild-type IgG CH3 domain is shown in SEQ ID NO: 11:

[0037] TIFF2026016524000004.tif26167

[0038] Positions 253, 310, or 435 in the IgG heavy chain sequence correspond to positions 23 and 80 in SEQ ID NO: 10, and position 95 in SEQ ID NO: 11, respectively. Thus, a hybrid antibody may comprise a variant of SEQ ID NO: 10 (i.e., a modified IgG CH2 domain) comprising one or more amino acid substitutions at positions 23 and / or 80 (e.g., Ile23Ala and / or His80Ala). Alternatively, a hybrid antibody may comprise a variant of SEQ ID NO: 11 (i.e., a modified IgG CH3 domain) comprising an amino acid substitution at position 95 (e.g., His95Ala). Preferably, the hybrid antibody comprises the modified IgG CH2 domain and modified IgG CH3 domain described herein.

[0039] Thus, in one embodiment, the hybrid antibody comprises a modified IgG CH2 domain and / or a modified IgG CH3 domain (i.e., a modified Cγ2 and / or Cγ3 domain) as set forth in SEQ ID NO: 175 and / or SEQ ID NO: 176:

[0040] TIFF2026016524000005.tif49170

[0041] It will be appreciated that other receptor binding sites, and desirable functions specific to IgG in the context of tumor targeting, can also be grafted onto or into the IgE molecule to alter its functionality.

[0042] The hybrid antibody may further comprise a variable domain sequence that determines specific binding to one or more target antigens. Such variable domain sequences may be derived from any immunoglobulin isotype (e.g., IgA, IgD, IgE, IgG, or IgM). In one embodiment, the variable domain sequence may be derived from IgE. In another embodiment, the variable domain sequence may be derived from IgG, e.g., IgG1. Alternatively, the variable domain may comprise sequences derived from two or more different isotypes; for example, the variable domain may comprise a partial sequence derived from IgE and a partial sequence derived from IgG1. In one embodiment, the hybrid antibody comprises one or more complementarity-determining regions (CDRs) derived from an immunoglobulin isotype other than IgE (e.g., IgA, IgD, IgG, or IgM, e.g., IgG1), and one or more framework regions and / or constant domains derived from an immunoglobulin of isotype IgE.

[0043] The variable domains or portions thereof (e.g., complementarity determining regions (CDRs) or framework regions) may be derived from the same or different mammalian species as the constant domains present in the hybrid antibody. Thus, the hybrid antibody may be a chimeric, humanized, or human antibody.

[0044] Typically, the variable domains of the antibody bind to one or more target antigens useful in the treatment of cancer, for example, a cancer antigen (i.e., an antigen that is selectively expressed on or overexpressed on cancer cells), or an antigen that inhibits or suppresses immune-mediated tumor cell killing. The sequence of one such variable domain sequence (i.e., of trastuzumab (Herceptin) IgE, which binds to the cancer antigen HER2 / neu) is shown in SEQ ID NO: 1.

[0045] In some embodiments, the antibody may comprise an IgE amino acid sequence set forth in any one or more of SEQ ID NOs: 1-5, or a variant or fragment thereof. For example, a hybrid antibody may comprise an amino acid sequence having at least 85%, 90%, 95%, or 99% sequence identity to any one or more of the sequences of SEQ ID NOs: 1-5. Preferably, the antibody comprises at least SEQ ID NOs: 3, 4, and 5, or a variant thereof, i.e., the antibody comprises an amino acid sequence having at least 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5, respectively.

[0046] In another embodiment, the hybrid antibody comprises an IgG CH2 amino acid sequence having at least 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 10 or SEQ ID NO: 175. In another embodiment, the antibody further comprises an IgG CH3 amino acid sequence having at least 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 11 or SEQ ID NO: 176. In another embodiment, the antibody further comprises an IgG hinge amino acid sequence having at least 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 9 or SEQ ID NO: 174.

[0047] In certain embodiments, the antibody comprises i) an amino acid sequence (e.g., derived from IgE) that has at least 85%, 90%, 95%, or 99% sequence identity to any one or more of the sequences of SEQ ID NOs: 1-5, preferably an amino acid sequence that has at least 85%, 90%, 95%, or 99% sequence identity to each of SEQ ID NOs: 3, 4, and 5 (more preferably, at least SEQ ID NO: 4); and ii) an amino acid sequence (e.g., derived from IgG1) that has at least 85%, 90%, 95%, or 99% sequence identity to SEQ ID NOs: 9, 10, 11, 174, 175, and / or 176 (more preferably, at least SEQ ID NOs: 10 and 11, or at least SEQ ID NOs: 175 and 176).

[0048] The IgG-derived amino acid sequence is preferably attached to the C-terminus of the IgE-derived amino acid sequence, either directly or using a suitable linker sequence. For example, the sequence of SEQ ID NO: 5 may be adjacent to the sequence of SEQ ID NO: 9, 10, 11, 174, 175, or 176, preferably SEQ ID NO: 9 or SEQ ID NO: 174. Thus, in some embodiments, the hybrid antibody may comprise at least the Cε4 domain and at least the IgG hinge region and Cγ2 domain (including a modified IgG hinge and / or Cγ2 domain), preferably at least the Cε4 domain and at least the IgG hinge region and the Cγ2 and Cγ3 domains. Thus, the antibody may comprise an amino acid sequence having at least 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 23 or SEQ ID NO: 24.

[0049] In preferred embodiments, the antibody comprises an amino acid sequence (e.g., heavy chain) having at least 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO:25 or SEQ ID NO:26, most preferably SEQ ID NO:26, e.g., over at least 50, 100, 200, 300, 500, or 700 amino acid residues of SEQ ID NO:25 or SEQ ID NO:26, or over the entire length of SEQ ID NO:25 or SEQ ID NO:26.

[0050] In further embodiments, the antibody comprises an amino acid sequence (e.g., heavy chain) having at least 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 163, 164, 165, or 166, most preferably SEQ ID NO: 164 or 166, e.g., across at least 50, 100, 200, 300, 500, or 700 amino acid residues of any of SEQ ID NOs: 163-166, or across the entire length of any of SEQ ID NOs: 163-166. In these embodiments, the antibody may further comprise a light chain amino acid sequence having at least 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 167, e.g., across at least 50, 100, 200, 300, 500, or 700 amino acid residues of SEQ ID NO: 167, or across the entire length of SEQ ID NO: 167.

[0051] In further embodiments, the antibody comprises a (e.g., heavy chain) amino acid sequence having at least 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 169, 170, 171, or 172, most preferably SEQ ID NO: 170 or 172, e.g., across at least 50, 100, 200, 300, 500, or 700 amino acid residues of any of SEQ ID NOs: 169-172, or across the entire length of any of SEQ ID NOs: 169-172. In these embodiments, the antibody may further comprise a light chain amino acid sequence having at least 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 173, e.g., across at least 50, 100, 200, 300, 500, or 700 amino acid residues of SEQ ID NO: 173, or across the entire length of SEQ ID NO: 173.

[0052] Also described herein are antibodies comprising at least a CH3 domain or fragment thereof (i.e., a Cε3 domain) derived from IgE and one or more loop sequences derived from an IgG CH2 domain (i.e., a Cγ2 domain). Such antibodies may comprise a Cε3 domain in which one or more loop sequences (e.g., as set forth in SEQ ID NOS: 6-8) are replaced by one or more FcγR-binding loops derived from the Cγ2 domain (e.g., as set forth in SEQ ID NOS: 12-14). The replaced loop sequences in the IgE Cε3 domain may exhibit structural homology to the FcγR-binding loops in the IgG Cγ2 domain. Such antibodies may comprise an amino acid sequence (e.g., encoding a hybrid Cε3 / Cγ2 domain) having at least 85%, 90%, 95%, or 99% sequence identity to any one or more of the sequences set forth in SEQ ID NOS: 15-22.

[0053] In another aspect, the present invention provides a method for treating cancer, e.g., melanoma, Merkel cell carcinoma, non-small cell lung cancer (squamous and non-squamous), renal cell carcinoma, bladder cancer, head and neck squamous cell carcinoma, mesothelioma, virally induced cancers (such as cervical cancer and nasopharyngeal cancer), soft tissue cancer, and the like. and hybrid antibodies as defined above for use in treating or preventing benign or malignant tumors, such as sarcomas, hematological malignancies, e.g., Hodgkin's and non-Hodgkin's disease, and diffuse large B-cell lymphomas (e.g., melanoma, Merkel cell carcinoma, non-small cell lung cancer (squamous and non-squamous), renal cell carcinoma, bladder cancer, head and neck squamous cell carcinoma, and mesothelioma, or virally induced cancers, such as cervical cancer and nasopharyngeal carcinoma, and soft tissue sarcomas).

[0054] In other words, the invention encompasses the use of the hybrid antibodies described above in the manufacture of a medicament for administration to a human or animal to treat, prevent, or delay cancer, e.g., benign or malignant tumors such as melanoma, Merkel cell carcinoma, non-small cell lung cancer (squamous and non-squamous), renal cell carcinoma, bladder cancer, head and neck squamous cell carcinoma, mesothelioma, virally induced cancers (such as cervical cancer and nasopharyngeal carcinoma), soft tissue sarcoma, hematological malignancies such as Hodgkin's and non-Hodgkin's disease, and diffuse large B-cell lymphoma (e.g., melanoma, Merkel cell carcinoma, non-small cell lung cancer (squamous and non-squamous), renal cell carcinoma, bladder cancer, head and neck squamous cell carcinoma, and mesothelioma, or virally induced cancers (such as cervical cancer and nasopharyngeal carcinoma), and soft tissue sarcoma).

[0055] Expressed still further, the present invention encompasses a method of preventing, treating, and / or delaying cancer (e.g., benign or malignant tumors) in a mammal afflicted therewith, the method comprising administering to the mammal a therapeutically effective amount of a hybrid antibody described above. It will be understood that the hybrid antibodies of the present invention may be administered in the form of a pharmaceutically acceptable composition or formulation.

[0056] In yet another aspect, the invention resides in a composition comprising the hybrid antibody described above and a pharmaceutically acceptable excipient, diluent, or carrier. The composition may further comprise a therapeutic agent, such as another antibody or fragment thereof, an aptamer, or a small molecule. The composition may be in a sterile aqueous solution.

[0057] In yet a further aspect, there is provided a (recombinant) nucleic acid encoding all or part of the heavy chain of a hybrid antibody, wherein the heavy chain comprises an amino acid sequence having at least 85%, 90%, 95%, or 99% sequence identity to (i) any one or more of SEQ ID NOs: 3-5, and (ii) SEQ ID NO: 10 and / or SEQ ID NO: 11, or SEQ ID NO: 175 and / or SEQ ID NO: 176. In one embodiment, the nucleic acid encodes an amino acid sequence having at least 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, or SEQ ID NO: 26, preferably SEQ ID NO: 24 or SEQ ID NO: 26, or any one of SEQ ID NOs: 163-166 or SEQ ID NOs: 169-172.

[0058] Also provided is a vector comprising a nucleic acid as defined above, optionally wherein the vector is a CHO vector (ie, an expression vector suitable for expression of a hybrid antibody in Chinese hamster ovary cells).

[0059] In a further aspect, there is provided a host cell comprising a recombinant nucleic acid encoding the hybrid antibody described above, or a vector described herein, wherein the encoding nucleic acid is operably linked to a promoter suitable for expression in a mammalian cell.

[0060] Also provided herein is a method for producing the hybrid antibodies described above, comprising culturing a host cell described herein under conditions for expression of the antibody, and recovering the antibody or fragment thereof from the host cell culture.

[0061] The hybrid antibodies described herein are highly stable, e.g., they typically exhibit high thermal stability in denaturation studies. Preferably, the hybrid antibodies are at least as thermostable as the corresponding IgE antibody (e.g., the IgE antibody from which the hybrid antibody comprises one or more domains). More preferably, the hybrid antibodies exhibit improved stability compared to the IgE antibody. [Brief explanation of the drawings]

[0062] [Figure 1] 1 is a schematic depiction of IgE and IgG antibodies. [Figure 2-1] ~ [Figure 2-2] A. Schematic of a hybrid antibody containing IgG1 hinge, CH2, and CH3 domains (i.e., hinge, Cγ2, and Cγ3 domains) fused to a whole IgE molecule via its C-terminal Cε4 domain. B. SEC-HPLC chromatogram of the purified hybrid antibody. C. SDS-polyacrylamide gel electrophoresis of the purified hybrid antibody under native and denaturing conditions, showing the size of the whole antibody or its single chains against protein markers (in kDa). [Figure 3] FIG. 1 is a schematic diagram of a single-cycle kinetic analysis of purified IgE-IgG hinge-CH2-CH3 fusion protein binding to CD64 (FcγRI). [Figure 4] 1A-1C are assay results showing antibody binding to CD64 (FcγRI). Binding of the IgE-IgG hinge-CH2-CH3 fusion protein to CD64 is similar to that of wild-type IgG1. [Figure 5] 1 is a ribbon diagram illustrating the crystal structure of IgG1 Fc in complex with soluble FcγRIII (shown in green). [Figure 6] Overlaid ball-and-stick images of IgE CH3 (top) and CH4 (bottom) in green, and IgG CH2 (top) and CH3 (bottom) in blue. [Figure 7]Schematics of domain-grafted IgE molecules prepared according to embodiments of the present invention. The red domain is the IgG CH domain, the blue domain is the IgE C domain, the yellow domain is the VH domain, and the green domain is the light chain V and C domains. A. The IgG CH2 domain is fused to the C-terminus of IgE. B. The IgG CH2-CH3 domains are fused to the C-terminus of IgE. [Figure 8] FIG. 1 is a schematic diagram of an alternative single-cycle kinetic analysis of purified hybrid antibodies binding to CD64 (FcγRI) or CD16A (FcγRIIIa). [Figure 9] Figure 1 shows the results of an assay demonstrating the binding of hybrid antibodies to CD64 (FcγRI). Only hybrid antibodies containing the IgG CH2 or IgG CH2-CH3 domains can bind to CD64, but the off-rate for fusions containing only IgG CH2 is faster than for fusions containing IgG CH2-CH3. [Figure 10] 1 shows the results of an assay demonstrating the binding of hybrid antibodies to FcγRIIIA (CD16A), where only hybrid antibodies containing IgG CH2-CH3 domains can bind to CD16A. [Figure 11] FIG. 1 is a schematic diagram of a multiple-cycle kinetic binding analysis of purified wild-type IgE, Herceptin (trastuzumab IgG), and IgE-IgG hinge-CH2-CH3 fusion binding to FcεRIα. [Figure 12] 1 shows the results of an assay demonstrating hybrid antibody binding to FcεRIα. Wild-type IgE and IgE-IgG hinge-CH2-CH3 fusion bind similarly to FcεRIα, whereas Herceptin does not bind to FcεRIα. [Figure 13] 1 is a schematic diagram of a vector expressing IGEG. [Figure 14] Schematic of the Biacore assay used to assess binding of trastuzumab IGEG variants to human Her2 antigen by single-cycle kinetic analysis. [Figure 15-1] ~ [Figure 15-2] FIG. 1 shows 1:1 binding of human HER2:trastuzumab IGEG variants. [Figure 16] 1 is a schematic of the Biacore assay used to assess antibody binding to Fc gamma receptors. [Figure 17-1] ~ [Figure 17-8] Figure 1 shows HMW-MAA IGEG (CH) variant binding to human Fc receptors. (a) 1:1 binding of human FcgRI:CSP4 IGEG variant. (b) 1:1 binding of human FceRIa:HMW-MAA IGEG variant. (c) Binding of human FcγRIIIA176Val:HMW-MAA IGEG variant - raw sensorgram. (d) Steady-state binding of human FcγRIIIA176Val:HMW-MAA IGEG variant - analyzed data. In this figure, "CH" refers to anti-HMW-MAA (i.e., CSPG4) antibody, and variant names are as otherwise described in Example 6. [Figure 18] 1 is a schematic of the Biacore assay used to assess antibody binding to FcRn. [Figure 19-1] ~ [Figure 19-8] Figure 1 shows HMW-MAA (CH) IGEG variant binding to human FcRn. (a) FcRn pH 6.0: Binding of HMW-MAA IGEG variant—raw sensorgram. (b) FcRn pH 6.0: Steady-state binding of HMW-MAA IGEG variant—analyzed data. (c) FcRn pH 7.4: Binding of HMW-MAA IGEG variant—raw sensorgram. (d) FcRn pH 7.4: Steady-state binding of HMW-MAA IGEG variant—analyzed data. In this figure, "CH" refers to anti-HMW-MAA (i.e., CSPG4) antibody, and variant names are as otherwise described in Example 6. [Figure 20-1] ~ [Figure 20-2]Figure 1 shows in vivo stability analysis of HMW-MAA (Hu CH) I GEG variants. (a) Fluorescence thermal melting curve overlay. (b) SLS 473 stability profile curve overlay. In this figure, "CH" refers to the anti-HMW-MAA (i.e., CSPG4) antibody, and variant names are as otherwise described in Example 6. [Figure 21] Figure 1 shows binding of anti-HMW-MAA (HuCH) IgE antibody to A375 cells. (a) Detection with anti-IgG secondary antibody. (b) Detection with anti-IgE secondary antibody. In this figure, "CH" refers to anti-HMW-MAA (i.e., CSPG4) antibody, and variant names are as otherwise described in Example 6. [Figure 22] FIG. 1 shows R1, R2, R3 gating of data acquired from an Attune® NxT Acoustic Focusing Cytometer. [Figure 23-1] ~ [Figure 23-2]

[0023] Figures 1A-1C show the effects of trastuzumab IgG, Herceptin IgG, trastuzumab-IGEG (labeled CH2CH3), trastuzumab-IGEG-C220S (labeled CH2CH3C220S), and isotype IgG antibodies on antibody-dependent cell-mediated phagocytosis (ADCP) and antibody-dependent cell-mediated cytotoxicity (ADCC). (a) Effect of antibodies on ADCP and ADCC at various concentrations (120-7.5 nM). (b) Graph showing the effect of antibodies on ADCP and ADCC. DETAILED DESCRIPTION OF THE INVENTION

[0063] As used herein, the singular forms "a," "an," and "the" include both singular and plural referents unless the context clearly dictates otherwise.

[0064] As used herein, "comprising", "comprises", and The term "consisting of" does not include "including," "includes," or is synonymous with "containing" and "contains" and is inclusive or open-ended and does not exclude additional, unrecited members, elements, or method steps. The term also encompasses "consisting of" and "consisting essentially of."

[0065] While the term "one or more," such as one or more members of a group of members, speaks for itself with further illustration, the term specifically encompasses reference to any one of the members, or any two or more of the members, such as any >3, >4, >5, >6, or >7, etc. of members, and up to all members.

[0066] As used herein, the term "antibody" is used in its broadest sense and generally refers to an immunological binding agent. The term "antibody" not only includes antibodies produced by methods involving immunization, but also includes any polypeptide, such as a recombinantly expressed polypeptide, that is produced to include at least one complementarity-determining region (CDR) that can specifically bind to an epitope on a target antigen. Thus, the term applies to such molecules, regardless of whether they are produced in vitro or in vivo.

[0067] The antibody may be a polyclonal antibody, such as an antiserum or immunoglobulin purified therefrom (e.g., affinity purified). The antibody may be a monoclonal antibody or a mixture of monoclonal antibodies. Monoclonal antibodies may target a particular antigen or a particular epitope within an antigen with greater selectivity and reproducibility. By way of example and not limitation, monoclonal antibodies are those described by Kohler et al. 1975 (Nature 256: 495). Monoclonal antibodies may be made by the hybridoma method first described in U.S. Pat. No. 4,816,567, or may be made by recombinant DNA methods (e.g., as described in U.S. Pat. No. 4,816,567). Monoclonal antibodies may also be produced from phage antibody libraries using the techniques described, for example, by Clackson et al. 1991 (Nature 352: 624-628) and Marks et al. 1991 (J Mol Biol 222: 581-597). It can also be isolated from the

[0068] The term antibody includes antibodies originating from or comprising one or more moieties derived from any animal species, preferably vertebrate species, including, for example, birds and mammals. Without limitation, the antibody may be derived from chicken, turkey, goose, duck, guinea fowl, quail, or pheasant. Also without limitation, the antibody may be derived from human, murine (e.g., mouse, rat, etc.), donkey, rabbit, goat, sheep, guinea pig, camel (e.g., Camelus bactrianus and Camelus dromaderius), llama, or other animal. (e.g., alpacas (Lama paccos), llamas (Lama glama), or vicunas (Lama vicugna)), or horses.

[0069] Those skilled in the art will understand that an antibody can contain one or more amino acid deletions, additions, and / or substitutions (e.g., conservative substitutions) so long as such changes preserve its binding for the respective antigen. An antibody can also contain one or more natural or artificial modifications (e.g., glycosylation, etc.) of its constituent amino acid residues.

[0070] Methods for producing polyclonal and monoclonal antibodies and fragments thereof are well known in the art, as are methods for producing recombinant antibodies or fragments thereof (see, e.g., Harlow and Lane, "Antibodies: A Laboratory Manual", Cold Spring Harbour Laboratory, New York, 1988; Harlow and Lane, "Using Antibodies: A Laboratory Manual", Cold Spring Harbour Laboratory, New York, 1999, ISBN 0879695447; "Monoclonal Antibodies: A Manual of Techniques", by Zola, ed., CRC Press 1987, ISBN 0849364760; "Monoclonal Antibodies: A Practical Approach", by Dean & Shepherd, eds., Oxford University Press 2000, ISBN 0199637229; Methods in Molecular Biology, vol. 248: "Antibody Engineering: Methods and Protocols", Lo, ed., Humana Press 20 04, ISBN 1588290921).

[0071] Thus, methods for immunizing animals, e.g., non-human animals such as laboratory animals or livestock, using any one or more (isolated) markers, peptides, polypeptides, or proteins taught herein, and fragments thereof, optionally attached to a presentation carrier (i.e., using them as immunizing antigens), are also disclosed. Immunization and preparation of antibody reagents from immune serum are well known per se and are described in the documents mentioned elsewhere herein. The animal to be immunized may include any animal species, preferably warm-blooded species, more preferably vertebrate species, including, for example, birds, fish, and mammals. Without limitation, the antibody may be from chicken, turkey, goose, duck, guinea fowl, shark, quail, or pheasant. Also without limitation, the antibody may be from human, murine (e.g., mouse, rat, etc.), donkey, rabbit, goat, sheep, guinea pig, shark, camel, llama, or horse. The term "presenting carrier" or "carrier" generally refers to an immunogenic molecule that, when conjugated to a second molecule, increases the immune response to the latter, usually through the provision of additional T cell epitopes. Presentation carriers can be (poly)peptide or non-peptide structures, such as glycans, polyethylene glycols, peptidomimetics, synthetic polymers, among others. Exemplary, non-limiting carriers include human hepatitis B virus core protein, multiple C3d domains, tetanus toxin fragment C, or yeast Ty particles.

[0072] The invention described herein resides in IgE antibodies with altered heavy chain (Fc) portions, resulting in hybrid IgE molecules. Structural regions on IgE that exhibit homology with regions on IgG that bind FcγRIIIa were identified. Once such regions were identified, amino acid substitutions were made that allowed for the transfer of IgG functionality to an IgE background. In particular, the IgG CH2 domain and IgG CH2-CH3 regions were fused to the C-terminus of IgE to confer gamma functionality to IgE.

[0073] The hybrid antibodies described herein are typically capable of binding to Fcε receptors, such as FcεRI and / or FcεRII receptors. Preferably, the antibodies are capable of at least binding to FcεRI (i.e., the high affinity Fcε receptor) or at least binding to FcεRII (CD23, the low affinity Fcε receptor).

[0074] Typically, to trigger IgE-mediated effector functions, antibodies can also activate Fcε receptors expressed, for example, on cells of the immune system. For example, an antibody can bind to FcγRI and potentially activate mast cells, basophils, monocytes / macrophages, and / or eosinophils.

[0075] The sites on IgE involved in these receptor interactions have been mapped to peptide sequences on the Cε chain and are clearly distinguishable. The FcεRI site is located in the cleft created by residues between Gln301 and Arg376 and includes the junction between the Cε2 and Cε3 domains (Helm, B. et al. (1988) Nature 331, 180183). The FcεRII binding site is located at Val It is located within Cε3 around residue 370 (Vercelli, D. et al. (1989) Nature 338, 649-651). The main difference that distinguishes the two receptors is that FcεRI binds to monomeric Cε, while FcεRII binds only to dimerized Cε, i.e., two Cε chains must be associated. IgE is glycosylated in vivo, but this is not required for its binding to FcεRI and FcεRRII. Binding is actually slightly stronger in the absence of glycosylation (Vercelli, D. et al. (1989) et al., supra).

[0076] Thus, binding to the Fcε receptor and associated effector functions is typically mediated by antibody heavy chain constant domains, particularly those domains that together form the Fc region of the antibody. The antibodies described herein typically contain at least a portion of an IgE antibody, e.g. For example, the antibody comprises one or more constant domains derived from IgE, preferably human IgE. In a specific embodiment, the antibody comprises one or more domains (derived from IgE) selected from Cε1, Cε2, Cε3, and Cε4. In one embodiment, the antibody comprises at least Cε2 and Cε3, more preferably at least Cε2, Cε3, and Cε4, preferably the domains are derived from human IgE. In one embodiment, the antibody comprises an epsilon (ε) heavy chain, preferably a human ε heavy chain.

[0077] The constant domains from human IgE, specifically the Cε1, Cε2, Cε3, and Cε4 domains, are set forth in SEQ ID NOs: 2, 3, 4, and 5, respectively. Nucleic acid sequences encoding these amino acid sequences can be deduced by one skilled in the art according to the genetic code. The amino acid sequences of other human and mammalian IgEs and their domains, including the human Cε1, Cε2, Cε3, and Cε4 domains and human ε heavy chain sequences, are known in the art and are available from publicly accessible databases. For example, a database of human immunoglobulin sequences is accessible from the International ImMunoGeneTics Information System (IMGT®) website at http: / / www.imgt.org. As an example, the sequences of various human IgE heavy (ε) chain alleles and their individual constant domains (Cε1-4) are accessible at http: / / www.imgt.org / IMGT_GENE-DB / GENElect?query=2+IGHE&species=Homo+sapiens. It is possible to access it.

[0078] The hybrid antibodies described herein typically bind to (e.g., human) Fcγ receptors, such as FcγRI (CD64), FcγRIIa, FcγRIIb, FcγRIIIa (CD 16a), and / or FcγRIIIb (CD16b). In some embodiments, the hybrid antibody binds to FcγRI (CD64) and / or FcγRIIIa ( In another embodiment, the hybrid antibody binds to FcγRI (CD64), FcγRIIIa (CD16a), and FcγRIIIb (CD16b). The hybrid antibody may also bind to a variant of FcγRIIIa (CD16a), such as human CD1 Preferably, the antibody is capable of binding to at least FcγRI or at least FcγRIIIa. More preferably, the hybrid antibody is capable of binding to and activating Fcγ receptors and / or activating cells of the immune system that express such receptors (including, for example, monocytes / macrophages and / or natural killer cells).

[0079] In some embodiments, the hybrid antibody may further bind to fetal Fc receptor (FcRn). The hybrid antibody may bind to FcRn in a pH-dependent manner. For example, the hybrid antibody may have higher affinity for FcRn at pH 6.0 than at pH 7.4. The fetal Fc receptor (FcRn) belongs to a broad and functionally diverse family of MHC molecules. Unlike classical MHC family members, FcRn possesses little diversity and cannot present antigens. Instead, through its ability to bind IgG and albumin with high affinity at low pH, it regulates the serum half-life of both these proteins. IgG enjoys a substantially longer serum half-life than similarly sized globular proteins, including IgE, which does not bind to FcRn (approximately 21 days for IgG and less than 2 days for IgG). In addition, FcRn plays an important role in immunity at mucosal and systemic sites through both its ability to influence the longevity of IgG and its participation in innate and adaptive immune responses.

[0080] FcRn is a major modifier of monoclonal antibody (mAb) efficacy It has emerged as a factor in the regulation of immune responses in the bloodstream (Chan AC, Carter PJ (2010) Nat. Rev. Immunol. 10:301-16; Weiner LM et al (2010) Nat. Rev. Immunol. 10:317-27). This directly relates to the persistence of therapeutic antibodies, which in turn may increase their localization to the target site. pH-dependent binding and FcRn-dependent recycling may be related to antibody function. Notably, proper release of IgG from cells requires limited binding at neutral pH, and increasing mAb affinity for FcRn at acidic pH correlates with increased half-life. Thus, IgG Fc modifications that optimize pH-dependent binding to FcRn can potentially be used to increase antibody half-life (see Dall'Acqua WF et al (2006) J. Biol. Chem. 281:23514-24; Yeung YA et al (2009) J. Immunol. 182:7663-1; Zalevsky J. et al (2010) Nat. Biotechnol. 28:157-9).

[0081] However, in other embodiments, it may be preferable to avoid FcRn binding, for example, if a shorter half-life of the antibody is desired. FcRn-binding ability can be conferred to the hybrid antibody by the presence of an IgG heavy chain constant domain, such as the IgG CH2 and CH3 domains described above. In some embodiments, it may be desirable for the antibody to be capable of binding to Fcγ receptors such as FcγRI (CD64), FcγRIIIa (CD16a), and / or FcγRIIIb (CD16b), but not FcRn. In such embodiments, the FcRn-binding ability of the antibody can be reduced or eliminated (compared to a native IgG antibody), for example, by amino acid substitutions at specific residues known to be involved in FcRn binding. Such residues include Ile253, His310, and His435 in the IgG heavy chain sequence (numbering is based on the EU numbering scheme, with reference to the IgG portion of the hybrid antibody sequence).

[0082] The antibodies described herein typically comprise at least a portion of an IgG antibody, such as one or more constant domains derived from an IgG (e.g., IgG1), preferably a human IgG. In certain embodiments, the antibody comprises one or more domains (derived from an IgG) selected from Cγ1, Cγ2, and Cγ3. In one embodiment, the antibody comprises at least Cγ2, more preferably at least Cγ2 and Cγ3, preferably the domains are derived from a human IgG1 antibody. In one embodiment, the antibody further comprises a hinge region derived from an IgG, e.g., an IgG1.

[0083] The constant domains derived from human IgG, in particular the Cγ2 and Cγ3 domains, are set forth in SEQ ID NOs: 10 and 11, respectively. Nucleic acid sequences encoding these amino acid sequences can be deduced by those skilled in the art according to the genetic code. The amino acid sequences of other human and mammalian IgG constant domains, including the human Cγ2 and Cγ3 domains and hinge sequences, are known in the art and are available from publicly accessible databases, as described above for IgE constant domains.

[0084] The amino acid sequences of one or more IgE domains and one or more IgG domains can be linked directly or via a suitable linker. Suitable linkers for joining polypeptide domains are well known in the art and can contain, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid residues. In some embodiments, the linker sequence can contain up to 20 amino acid residues.

[0085] Binding of hybrid antibodies to Fcε and Fcγ receptors can be assessed using standard techniques, for example, by determining antigen / antibody dissociation rates, by competitive radioimmunoassay, by enzyme-linked immunosorbent assay (ELISA), or by surface plasmon resonance (e.g., Biacore). Binding affinities can also be calculated using standard methods, for example, based on the Scatchard method described by Frankel et al., Mol. Immunol. 16:101-106, 1979.

[0086] In general, functional fragments of the sequences defined herein may be used in the present invention. A functional fragment is one that, when present in an antibody, retains the required activity. It can be of any length (e.g., at least 50, 100, 300, or 500 nucleotides, or at least 50, 100, 200, 300, or 500 amino acids), provided that it retains its ability to bind to Fcγ and / or Fcε receptors (e.g., binding to Fcγ and / or Fcε receptors).

[0087] Variants of the amino acid and nucleotide sequences described herein may also be used in the present invention, provided that the resulting antibody binds to both Fcγ and Fcε receptors. Typically, such variants will have a high degree of sequence identity with one of the sequences specified herein.

[0088] Similarity between amino acid or nucleotide sequences is expressed in terms of the similarity between the sequences, otherwise referred to as sequence identity. Sequence identity is often measured in terms of percentage identity (or similarity or homology); the higher the percentage, the more similar the two sequences are. Homologs or variants of amino acid or nucleotide sequences will possess a relatively high degree of sequence identity when aligned using standard methods.

[0089] 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. USA 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. USA 85:2444, 1988. Altschul et al., Nature Genet. 6:119, 1994, presents a detailed consideration of sequence alignment methods and homology calculations.

[0090] NCBI's Basic Local Alignment Search Tool (BLAST) (Altschul et al., J. Mol. Biol. 215:403, 1990) is a sequence analysis platform. For use in connection with the programs blastp, blastn, blastx, tblastn, and tblastx, please refer to the National Center for Biotechnology Information (NCBI). Center for Biotechnology Information, Bethesda, Md. and on the internet. A description of how to use this program to determine sequence identity is available on the internet at the NCBI website.

[0091] Homologs and variants of the specific antibodies or domains thereof (e.g., VL, VH, CL, or CH domains) described herein typically have at least about 75%, e.g., at least about 80%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with the original sequence (e.g., a sequence defined herein), counted over, e.g., at least 20, 50, 100, 200, or 500 amino acid residues, or over a full-length alignment with the amino acid sequence of the antibody or domain thereof, using NCBI Blast 2.0, gapped blastp, set to default parameters. For comparison of amino acid sequences longer than about 30 amino acids, the Blast2 alignment function is employed using the default BLOSUM62 matrix set to default parameters (gap existence cost of 11, and per-residue gap cost of 1). When aligning short peptides (fewer than approximately 30 amino acids), the alignment should be performed using the Blast2 sequence function employing the PAM30 matrix set to default parameters (open gap 9, extension gap 1 penalty). Proteins with even greater similarity to the reference sequence will be assessed by increasing percentages of identity as assessed by this method, such as at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity. When less than the entire sequence is being compared for sequence identity, homologs and variants are typically considered to possess at least 80% sequence identity over a short window of 10-20 amino acids, and, depending on their similarity to the reference sequence, may possess at least 85%, or at least 90%, or 95% sequence identity. Methods for determining sequence identity over such short windows are available on the Internet at the NCBI website. Those skilled in the art will understand that these sequence identity ranges are provided for guidance only; it is entirely possible that strongly significant homologs can be obtained that fall outside the provided ranges.

[0092] Typically, a variant may contain one or more conservative amino acid substitutions compared to the original amino acid or nucleic acid sequence. Conservative substitutions are those substitutions that do not substantially affect or reduce the affinity of the antibody for Fcγ and / or Fcε receptors. For example, a human antibody that binds to Fcγ and / or Fcε may contain up to 1, up to 2, up to 5, up to 10, or up to 15 conservative substitutions compared to the original sequence (e.g., as defined above) and retain specific binding to Fcγ and / or Fcε receptors. The term conservative variation also includes the use of substituted amino acids in place of unsubstituted parent amino acids, provided that the antibody binds to Fcγ and / or Fcε. Non-conservative substitutions are those that reduce activity or binding to Fcγ and / or Fcε receptors.

[0093] Functionally similar amino acids that can be replaced 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).

[0094] The domains described above (e.g., one or more IgE and IgG constant domains) are typically present in the heavy chain of an antibody. Hybrid antibodies may further comprise one or more light chains in addition to one or more heavy chain sequences described herein. Antibodies are typically composed of heavy and light chains, each of which has a variable region referred to as a variable heavy (VH) region and a variable light (L) region. Together, the VH and VL regions are responsible for binding to the antigen recognized by the antibody. 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 (k). Thus, hybrid antibodies typically comprise two heavy chains and two light chains (e.g., joined by disulfide bonds) based on an IgE antibody, e.g., comprising an IgG hinge, CH2, and / or CH3 domain fused at the C-terminus of each heavy chain.

[0095] The hybrid antibodies described herein can specifically bind (i.e., via their variable domains or their complementarity-determining regions (CDRs)) to one or more target antigens useful for treating cancer. For example, hybrid antibodies can specifically bind to one or more cancer antigens (i.e., antigens selectively expressed or overexpressed on cancer cells). The novel combination of effector functions transduced by combined FcεR and Fcγ binding capabilities can enhance cytotoxicity, phagocytosis (e.g., ADCC and / or ADCP), and other cancer cell-killing functions of immune system cells (e.g., monocytes / macrophages and natural killer cells). Preferably, the hybrid antibodies can induce cytotoxicity (e.g., ADCC) and / or phagocytosis (ADCP), specifically against cancer cells. In particularly preferred embodiments, the hybrid antibodies induce enhanced phagocytosis by immune cells (e.g., ADCP of cancer cells by monocytes / macrophages or other effector cells, such as in the assay described in Example 8 below) compared to corresponding IgE and / or IgG antibodies. For example, a hybrid antibody may specifically bind to, e.g., EGF-R (epidermal growth factor receptor), VEGF (vascular endothelial growth factor), or erbB2 receptor (Her2 / neu). One example of an antibody containing a variable domain that selectively binds to Her2 / neu is trastuzumab (Herceptin).

[0096] In some embodiments, one or more of the variable domains and / or one or more of the CDRs, preferably at least three CDRs, or more preferably all six CDRs, are selected from the group consisting of the following antibodies: alemtuzumab (SEQ ID NOs: 27 to 32), atezolizumab (SEQ ID NOs: 33 to 38), avelumab (SEQ ID NOs: 39 to 45), bevacizumab (SEQ ID NOs: 46 to 51), blinatumomab, brentuximab, cemiplimab, certolizumab (SEQ ID NOs: 52 to 57), secukinumab, cetaxel ... The antibody may be derived from one or more of: ipilimumab (SEQ ID NOs: 58-63), denosumab, durvalumab (SEQ ID NOs: 64-69), efalizumab (SEQ ID NOs: 70-75), ipilimumab, nivolumab, obinutuzumab, ofatumumab, omalizumab (SEQ ID NOs: 76-81), panitumumab (SEQ ID NOs: 82-87), pembrolizumab, pertuzumab (SEQ ID NOs: 88-93), rituximab (SEQ ID NOs: 94-99), or trastuzumab (SEQ ID NOs: 100-105).

[0097] In such embodiments, the variable domain of the antibody may comprise one or more of the CDRs from one of the antibodies listed in Table 1, preferably at least three CDRs, or more preferably all six of the CDR sequences.

[0098] [Table 1]

[0099] In alternative embodiments, one or more of the variable domains and / or one or more CDRs, preferably at least three CDRs, or more preferably all six CDRs, are selected from the group consisting of the following antibodies: abciximab, adalimumab (SEQ ID NOs: 106-111), ... cucanumab, aducanumab, alefacept, alirocumab, anifrolumab, balstilimab, basiliximab (SEQ ID NOs: 112 to 117), belimumab (SEQ ID NOs: 118 to 123), benralizumab, bezlotoxumab, brodalumab, brolucizumab, burosumab, canakinumab, caplacizumab, crizanlizumab, daclizumab (SEQ ID NOs: 124 to 129) , daratumumab, dinutuximab, dostallimab, dupilumab, eculizumab, elotuzumab, emapalumab, emicizumab, eptinezumab, erenumab, etrolizumab, evinacumab, evolocumab, fremanezumab, galcanezumab, golimumab, guselkumab, ibalizumab, idarucizumab, inebilizumab, infliximab (SEQ ID NOs: 130-133) 5), isatuximab, ixekizumab, lanadelumab, leronlimab, marjetuximab, mepolizumab, mogamulizumab, muromonab, narsoplimab, natalizumab (SEQ ID NOs: 136 to 141), naxitamab, necitumumab, obiltoxaximab, ocrelizumab, omburtamab, palivizumab (SEQ ID NOs: 142 to 147), ramucirumab, ranibizumab (SEQ ID NOs: 148-153), reslizumab, risankizumab, romosozumab, sarilumab, satralizumab, secukinumab, spartalizumab, stimulimab, tafasitamab, tanezumab, teplizumab, teprotumumab, tildrakizumab, tocilizumab, toripalimab, ustekinumab, vedolizumab, or zalifrelimab.

[0100] In such embodiments, the antibody variable domain may comprise one or more, preferably at least three, or more preferably all six of the CDR sequences from one of the antibodies listed in Table 2.

[0101] [Table 2]

[0102] In other embodiments, one or more of the variable domains and / or one or more, preferably at least three, or more preferably all six CDRs of the CDR sequences may be derived from an anti-HMW-MAA antibody. In one embodiment, one or more of the variable domains and / or one or more, preferably at least three, or more preferably all six CDRs of the CDR sequences may be derived from an anti-HMW-MAA antibody described in WO 2013 / 050725 (SEQ ID NOS: 161 and 162 for the variable domains and SEQ ID NOS: 154-159 for the CDRs). HMW-MAA refers to high molecular weight melanoma-associated proteoglycan 4 (CSPG4), also known as chondroitin sulfate proteoglycan 4 (CSPG4) or melanoma chondroitin sulfate proteoglycan (MCSP). Refers to antigen, see for example Uniprot Q6UVK1.

[0103] In such embodiments, the antibody variable domain may comprise one or more of the CDR sequences, preferably at least three CDRs, or more preferably all six of the CDR sequences, as defined in Table 3. In other embodiments, one or more of the antibody variable domains comprise one or more of the variable domain sequences listed in Table 3.

[0104] [Table 3]

[0105] In some embodiments, the hybrid antibody binds to the target antigen with a dissociation constant (Kd) of less than 1 μM, preferably less than 1 nM. For example, in one embodiment, the hybrid antibody binds to the target antigen with a dissociation constant (Kd) of less than 1×10 -9 It binds to human Her2 or HMW-MAA with a Kd of (1 nM) or lower.

[0106] Provided herein are compositions comprising a carrier and one or more hybrid antibodies that bind to Fcγ and Fcε receptors or functional fragments thereof. The compositions may be prepared in unit dosage form for administration to a subject. The amount and timing of administration are at the discretion of the treating physician to achieve the desired goal. The antibodies may be formulated for systemic or local (e.g., intratumoral) administration. In one example, the antibodies are formulated for parenteral administration, such as intravenous administration.

[0107] Compositions for administration can comprise a solution of the antibody or functional fragment thereof dissolved in a pharmaceutically acceptable carrier, such as an aqueous carrier. A variety of aqueous carriers can be used, such as buffered saline. These solutions are sterile and generally free of undesirable matter. These compositions can be sterilized by conventional, well-known sterilization techniques. The compositions can contain pharmaceutically acceptable auxiliary substances required to approximate physiological conditions, such as pH adjusting and buffering agents, toxicity adjusting agents, and the like, e.g., sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, etc. The concentration of the antibody in these formulations can vary widely and will be selected primarily based on fluid volume, viscosity, body weight, and the like, according to the particular mode of administration selected and the needs of the subject.

[0108] A typical dosage of a pharmaceutical composition for intravenous administration contains about 0.1 to 15 mg of antibody per kg body weight of the subject per day. Dosages of 0.1 up to about 100 mg per kg per day can be used, particularly when the agent is administered to an isolated site, such as into a body cavity or into the lumen of an organ, and not into the circulatory or lymphatic system. Actual methods for preparing administrable compositions are known or will be apparent to those skilled in the art, and are described in Remington's Pharmaceuticals, Inc. Science, 19th ed., Mack Publishing Company, Easton, Pa. (1995) and other publications. It will be described in detail.

[0109] Antibodies can be provided in lyophilized form and hydrated with sterile water before administration, but they can also be provided in sterile solutions of known concentrations. The antibody solution is then added to an infusion bag containing 0.9% sodium chloride, USP, and typically administered at a dosage of 0.5 to 15 mg / kg of body weight. Antibodies can be administered by slow infusion rather than by intravenous push or bolus. In one example, a higher loading dose is administered, with subsequent maintenance doses administered at lower levels. For example, an initial loading dose of 4 mg / kg can be infused over approximately a 90-minute period, followed by weekly maintenance doses of 2 mg / kg infused over a 30-minute period for 4 to 8 weeks, if the previous dose was well tolerated.

[0110] The antibodies (or functional fragments thereof) described herein can be administered to slow or inhibit the growth of cells, such as cancer cells. In these applications, a therapeutically effective amount of the antibody is administered to a subject in an amount sufficient to inhibit the growth, replication, or metastasis of cancer cells, or to inhibit a sign or symptom of cancer. In some embodiments, the antibody is administered to a subject to inhibit or prevent the progression of metastasis, or to reduce the size or number of metastases, such as micrometastases in regional lymph nodes (Goto et al., Clin. Cancer Res. 14(11):3401-3407, 2008).

[0111] The therapeutically effective amount of antibody will depend on the severity of the disease and the general state of the patient's health. A therapeutically effective amount of antibody is one that provides either a subjective relief of symptoms or an objectively identifiable improvement as noted by a clinician or other qualified observer. These compositions can be administered in conjunction with another chemotherapeutic agent, either simultaneously or sequentially.

[0112] Many chemotherapeutic agents are currently known in the art, hi one embodiment, the chemotherapeutic agent is selected from the group consisting of antimitotic agents, alkylating agents, antimetabolites, intercalating antibiotics, growth factor inhibitors, cell cycle inhibitors, enzymes, topoisomerase inhibitors, anti-survival agents, biological response modifiers, antihormones, e.g., antiandrogens, and anti-angiogenic agents.

[0113] All documents cited herein are hereby incorporated by reference in their entirety. The invention will now be described in more detail by the following non-limiting examples. [Example]

[0114] IgG-mediated antibody-dependent cellular cytotoxicity (ADCC) occurs when antibodies bound to target pathogens or cells simultaneously bind to FcγRIIIa on natural killer cells (NK cells). This occurs when NK cells can bind to antibodies. These cells release a cocktail of factors (e.g., granzymes, perforin) that result in the destruction of antibody-opsonized pathogens / target cells. FcγRIIIa binds to IgG in the CH2 domain proximal to the hinge region. (Figure 8; see Sondermann et al (2000) Nature 406: 267-73).

[0115] Comparison of the FcγRIIIa binding region of IgG with the CH3 and CH4 domains of IgE As can be seen in Figure 6, the CH2 and CH3 domains of IgG occupy a very similar 3D space as the CH3 and CH4 domains of IgE.

[0116] A combination of amino acid sequence alignment, secondary structure prediction, and structural inspection of IgG and IgE, shown in Figure 6, allowed for targeted mutations in the IgG CH2 domain to be incorporated into the homologous region of the IgE scaffold to accommodate FcγRIIIa binding. This resulted in the design of several variant IgE molecules that exhibited the same binding activity. These variants were expressed and subjected to receptor binding and tumor cell killing assays (both with NK cells and conventional effector cells for IgE).

[0117] Asn29 in the CH2 domain of IgG is required for FcγRIIIa binding and NK cell activation. Glycosylation at position 7 is also known to be required. This indicates that potentially complex conformational epitopes may be required for IgE. Therefore, the highly variable glycosylation of IgE limits the inference of the FcγRIIIa binding site on IgE. This can be difficult to achieve through examination of amino acid sequence alignments and structural homology modeling.

[0118] In the following examples, FcγR binding (e.g., FcγRIIIa binding) is achieved by fusing at least the IgG hinge, CH2, and CH3 domains to the C-terminus of the heavy chain of an antibody. It is demonstrated that ) can be conferred on IgE antibodies. [Example]

[0119] FcγR binding site transplantation In this example, an IgE variant was created in which an IgG hinge and an IgG CH2-CH3 domain pair were fused to an IgE framework at the C-terminus (Figure 2A). The IgE antibody is based on trastuzumab IgE, e.g., as disclosed in Karagiannis et al., Cancer Immunol Immunother. (2009) Jun;58(6):915-30.

[0120] Another IgE variant was created in which the IgG hinge and CH2 domains were fused to the C-terminus of trastuzumab IgE.

[0121] Further variant IgE antibodies were generated in which one or more loops in the Cε3 domain of IgE were replaced by one or more FcγR-binding loops from the Cγ2 domain of an IgG antibody, where the replaced loops in the Cε3 domain of IgE show structural homology to the FcγR-binding loops in the Cγ2 domain of IgG.

[0122] method Cloning: Wild-type (WT) trastuzumab with flanking restriction enzyme sites for cloning into the Abzena pANT dual Ig expression vector system for human heavy and kappa light chains. DNA sequences corresponding to both the trastuzumab IgE constant domain and, separately, the IgE containing IgG FcγR binding loop 1 + loop 2 + loop 3 were synthesized. The heavy chain, which also contains the trastuzumab VH, was cloned between the Mlu I and Kpn I restriction sites. Tuzumab Vk was cloned between the Pte I and BamH I restriction sites. Individual loop variants were constructed using specific primers and pulled-through PCR to generate IgE with one or two IgG1 loops in all possible combinations, resulting in a total of six additional constructs (1, 2, 3, 1+2, 1+3, 2+3).

[0123] To generate the IgE-IgG1-CH2 and CH2-CH3 fusion variants, specific primers were used to amplify WT IgE, while removing the stop codon at the end of IgE CH4 and amplifying either the separately synthesized IgG1 CH2 or IgG1 CH2-CH3 in separate reactions. Pull-through PCR was used to combine both fragments and amplify them at the MluI and KpnI restriction sites for cloning into the dual expression vector. Figure 7 shows stylized schematic representations of the two fusion variants, Figure 7a illustrating IgE-IgG1-CH2 and Figure 7b illustrating IgE-IgG1-CH2-CH3.

[0124] array: The following hybrid antibody molecules were constructed: IgE containing IgG FcγR loop 1; IgE containing IgG FcγR loop 2; IgE containing IgG FcγR loop 3; IgE containing IgG FcγR loop 1 + loop 2; IgE containing IgG FcγR loop 1 + loop 3; IgE containing IgG FcγR loop 2 + loop 3; and IgE containing IgG FcγR loop 1 + loop 2 + loop 3

[0125] In addition, the following fusion proteins were constructed: IgE+IgG1 hinge-CH2 IgE+IgG1 hinge-CH2-CH3

[0126] The sequence for wild-type trastuzumab IgE was as follows:

[0127] WT IgE_VH:

[0128] TIFF2026016524000009.tif28168

[0129] WT IgE_VL:

[0130] TIFF2026016524000010.tif33170

[0131] WT IgE_CH1:

[0132] TIFF2026016524000011.tif19170

[0133] WT IgE_CH2:

[0134] TIFF2026016524000012.tif26170

[0135] WT IgE_CH3 (altered loops are underlined):

[0136] TIFF2026016524000013.tif31170

[0137] WT IgE_CH4:

[0138] TIFF2026016524000014.tif27170

[0139] IgE loop 1:

[0140] TIFF2026016524000015.tif14146

[0141] IgE loop 2:

[0142] TIFF2026016524000016.tif13146

[0143] IgE loop 3:

[0144] TIFF2026016524000017.tif14146

[0145] The sequence for wild type IgG was as follows:

[0146] WT IgG_hinge:

[0147] TIFF2026016524000018.tif14146

[0148] WT IgG_CH2 (altered loops are italicized and underlined):

[0149] TIFF2026016524000019.tif25170

[0150] WT IgG_CH3:

[0151] TIFF2026016524000020.tif25170

[0152] IgG FcγR binding loop 1:

[0153] TIFF2026016524000021.tif9170

[0154] IgG FcγR binding loop 2:

[0155] TIFF2026016524000022.tif9170

[0156] IgG FcγR binding loop 3:

[0157] TIFF2026016524000023.tif8170

[0158] The sequences for the hybrid molecules were as follows: Each hybrid molecule further comprises a wild-type IgE_VH, an IgE_CH1, an IgE_CH2, and an IgE_CH4 (ie, SEQ ID NOs: 1, 2, 3, and 5).

[0159] IgE_CH3 containing IgG FcγR binding loop 1:

[0160] TIFF2026016524000024.tif30170

[0161] IgE_CH3 containing IgG FcγR binding loop 2:

[0162] TIFF2026016524000025.tif26170

[0163] IgE_CH3 containing IgG FcγR binding loop 3:

[0164] TIFF2026016524000026.tif25170

[0165] IgE_CH3 containing IgG FcγR binding loop 1 + loop 2:

[0166] TIFF2026016524000027.tif25170

[0167] IgE_CH3 containing IgG FcγR binding loop 1 + loop 3:

[0168] TIFF2026016524000028.tif25170

[0169] IgE_CH3 containing IgG FcγR binding loop 2 + loop 3:

[0170] TIFF2026016524000029.tif27168

[0171] IgE_CH3 containing IgG FcγR binding loop 1 + loop 2 + loop 3:

[0172] TIFF2026016524000030.tif27168

[0173] The sequences for the fusion proteins were as follows: Each fusion protein further comprises a wild-type IgE_VH, an IgE_CH1, an IgE_CH2, and an IgE_CH3 (ie, SEQ ID NOs: 1, 2, 3, and 4).

[0174] IgE_CH4+IgG1 Hinge-CH2 (containing RS linker):

[0175] TIFF2026016524000031.tif44168

[0176] IgE_CH4+IgG1 hinge-CH2-CH3 (containing RS linker)

[0177] TIFF2026016524000032.tif57170

[0178] The full amino acid sequence of the IgE heavy chain + IgG1 hinge_CH2 construct is shown below:

[0179] TIFF2026016524000033.tif109170

[0180] The full amino acid sequence of the IgE heavy chain + IgG1 hinge-CH2-CH3 construct is shown below:

[0181] TIFF2026016524000034.tif115170

[0182] All constructs were verified by sequencing. DNA was prepared and analyzed using the OC-400 processor. CHO cells were transiently transfected using the MaxCyte STX® electroporation system with a fusion assembly (MaxCyte, Gaithersburg, USA). Supernatants were harvested 7-10 days after transfection.

[0183] The antibody (i.e., comprising the variant heavy chain described above and a kappa light chain derived from trastuzumab IgE) was screened using CaptureSelect (registered trademark) against an IgG1 CH2-CH3 fusion. IgE affinity matrix (ThermoFisher, Loughborough, The antibody was purified from cell culture supernatant using either a Mab Select Sure column (GE Healthcare, Little Chalfont, UK) or a Mab Select Sure column (GE Healthcare, Little Chalfont, UK). The eluted fraction was buffer exchanged into PBS and analyzed using an extinction coefficient (E) based on the predicted amino acid sequence. c(0.1%) ) with A 280nm The mixture was filter sterilized before quantitation by HPLC.

[0184] Fusion proteins containing IgE with IgG1 hinge-CH2-CH3 (SEQ ID NOs: 24 and 26; see Figure 2A) were purified using a 1 mL MabSelect Prism A column. The purified product was purified using PBS to yield 11 mg of total protein (4.09 mg / ml in a volume of approximately 2.7 ml; see Figure 2B). SDS-PAGE was performed with 1 μg of protein loaded per lane (see Figure 2C). [Example]

[0185] Binding of fusion proteins to CD64 (FcγRI) To accurately determine the kinetics of fusion proteins to CD64 (FcγRI), single-cycle kinetic analysis was performed on purified antibodies. The assay principle is shown in Figure 3. A Biacore T200 (serial number 19) running Biacore T200 Control software V2.0.1 and Evaluation software V3.0 (GE Healthcare, Uppsala, Sweden) was used. All single-cycle kinetic experiments were performed in HBS-P+ running buffer (pH 7.4) (GE Healthcare, Little Chilli). The run was performed at 25°C using a 1000kJ / series PCR system (Ifont, UK).

[0186] At the start of each cycle, His-tagged CD64 diluted to a final concentration in running buffer (HBS-P+ buffer) was loaded onto an anti-HIS antibody capture chip (CM5 (catalog no. 28995056); GE Healthcare, Little Chalfont, UK) to approximately 60 RU at a flow rate of 30 μl / min, coupled with approximately 9000 RU of anti-His antibody using standard amine chemistry. The surface was then allowed to stabilize. To minimize any potential mass transport limitations, single-cycle kinetic data were acquired using purified antibody as the analyte at a flow rate of 30 μL / min. A five-point, 3-fold dilution range of antibody from 0.411 nM to 33.33 nM was used, with no regeneration between each concentration. The association phase for five injections of increasing antibody concentrations was monitored for 200 s each time, and a single dissociation phase was measured for 300 s after the final injection of analyte. Regeneration of the anti-HIS antibody capture surface was performed using two injections of 10 mM glycine-HCl pH 1.5. Reference channel F c Signal from 1 to F c Correct for differences in non-specific binding to the reference surface by subtracting it from that of the global R max The parameters were used in a one-to-one binding model. Figure 6 is a schematic representation of the scientific principles behind the assay.

[0187] Langmuir (1:1) binding analysis was the model chosen for kinetic evaluation. The model describes a 1:1 interaction at the surface:

[0188]

number

[0189] In the formula: k a is the association rate constant (M -1 s -1 ) and k d is the dissociation rate constant (s -1 )

[0190] The closeness of the data fit is judged in terms of a chi-squared value that describes the deviation between the experimental and fitted curves:

[0191]

number

[0192] In the formula: r f is the fitted value at a given point; r x is the experimental value at the same point; n is the number of data points; and p is the number of fitted parameters.

[0193] The fitting algorithm seeks to minimize the chi-square.

[0194] result As shown in Figure 4 and Table 4 below, binding of IgE-CH2CH3 (SEQ ID NO: 26) to FcγRI (CD64) is similar to that of wild-type IgG.

[0195] [Table 4] [Example]

[0196] Hybrid IgE variant binding High affinity FcγRI (CD64) and low affinity FcγRIIIA (CD 16A) To test the binding of hybrid IgE variants to the receptor, wild-type IgE was used as a negative control and CHO supernatants were screened prior to variant selection and purification.

[0197] Figure 8 shows the IgE in the supernatant bound to a streptavidin chip using CaptureSelect Assay step using biotin anti-IgE antibody captured on a Biacore chip Illustrated schematic diagram: Only Fc2 was used for capture, with Fc1 used as a reference.

[0198] Antibodies were loaded to the same level. Single injections of CD64 (25 nM) and CD16A (1 μM) were used. The concentrations used were based on the affinity of binding to IgG1.

[0199] CD64 Single-Cycle Kinetic Biacore® Analysis of Purified Proteins To accurately determine the kinetics of selected variants to CD64, single-cycle kinetic analysis was performed on purified antibodies. Biacore T200 Control software V2.0.1 and Evaluation software V3.0 (GE Healthcare, Uppsala, Sweden) were used for the run. Kinetic experiments were performed on a Biacore T200 (serial number 1909913) running HBS-P+ running buffer (pH 7.4) (GE Healthcare, Little Chalfont, UK) at 25°C.

[0200] At the start of each cycle, His-tagged CD64 diluted to a final concentration in running buffer (HBS-P+ buffer supplemented with 150 mM NaCl) was loaded onto an anti-HIS antibody capture chip (GE Healthcare, Little Chalfont, UK) at a flow rate of 10 μl / min to approximately 60 RU or approximately 20 RU. The surface was then allowed to stabilise. Single-cycle kinetic data were acquired using purified antibody as the analyte at a flow rate of 30 μL / min to minimize any potential mass transport limitations. A five-point, three-fold dilution range of antibody from 0.411 nM to 33.33 nM was used, with no regeneration between concentrations. The association phase for five injections of increasing antibody concentrations was monitored for 200 seconds each, and a single dissociation phase was measured for 300 seconds after the final injection of analyte. Regeneration of the anti-HIS antibody capture surface was performed using two injections of 10 mM glycine-HCl pH 1.5. Reference channel F c Signal from 1 to F c Correct for differences in non-specific binding to the reference surface by subtracting it from that of the global R max The parameters were used in a one-to-one binding model.

[0201] Biacore® Screening (CD64 and CD16A (176Val)): CD64 (Sino Biological Co., Ltd., Catalog No. CT009-H08H) and CD16A (176Val) (Sino Biological Co., Ltd., Cat. No. 10389-H08H1) To assess the binding of all variants, Biacore kinetic analysis was performed at a single concentration on supernatants from transfected CHO cell cultures. Biacore T200 Control Software V2.0.1 and Evaluation Software V3.0 (GE Healthcare, Up Kinetic experiments were performed on a Biacore T200 (serial number 1909913) running a 1000kJ / ml buffer (Bio-Rad, Radsala, Sweden). All kinetic experiments were performed in HBS-EP+ running buffer ( pH 7.4) (GE Healthcare, Little Chalfont, UK) The run was performed at 5°C. CaptureSelect biotin anti-IgE antibody (Thermo, Cat. No. 7103542500) was applied to a pre-loaded streptavidin chip (GE Healthcare, Little Chalfont, UK) c Antibodies were loaded onto channel 2. Antibodies were captured at a flow rate of 10 μl / min to give an immobilization level (RL) of approximately 400 RU. Binding data were acquired using either 25 nM CD64 for 150 seconds or 1 μM CD16A (176Val) for 30 seconds as analytes at a flow rate of 10 μl / min. Reference channel F c The signal from 1 (without antibody) is F c This was subtracted from 2 to correct for differences in non-specific binding to the reference surface. Regeneration of the anti-IgE antibody capture surface was performed using one injection of glycine pH 2.0.

[0202] result Figure 9 shows results from a manual run against 25 nM CD64 (FcγRI). As can be seen, the CaptureSelect biotin anti-IgE antibody conjugate was able to bind to all of the antibody variants tested, suggesting that the receptor does not bind to an epitope present in any of the shuffled loops. However, antibody variants containing loop 2 (green) appeared to bind less stably. Only IgE fusions containing the IgG CH2 and IgG CH2-CH3 domains (SEQ ID NOs: 25 and 26) were able to bind CD64, although the off-rate for fusions containing only CH2 appeared to be much faster.

[0203] Figure 10 shows the results of a manual run against 1 μM CD16A (FcRγIIIA) (176Val). The figure shows results from the study, which demonstrates that under the specific conditions of the experiment, only the IgE fusion protein with the IgG CH2-CH3 domain (SEQ ID NO: 26) appeared to be able to bind to CD16A.

[0204] In further investigations, similar techniques can be used to compare IgE-CH2-CH3 with IgG1 and IgE against the full panel of Fcγ and Fcε receptors. [Example]

[0205] Binding to Fc epsilon RI alpha (FcεRIα) The goal of this experiment was to examine the binding of purified wild-type IgE and IgE CH2-CH3 to FcεRIα. Herceptin (trastuzumab) was used as a control. The principle of the assay is shown in Figure 11.

[0206] As shown in Figure 12 and Table 5 below, wild-type IgE and IgE CH2-CH3 (SEQ ID NO: 26) bound similarly to the FcεRIα receptor. No binding of Herceptin to FcεRIα was observed.

[0207] [Table 5]

[0208] The above studies demonstrate that variant IgE antibodies containing IgG CH2 and CH3 domains bind to gamma and epsilon Fc receptors. In further studies, the antibodies may be assessed for targeting both IgG and IgE effector cells for tumor cell killing in vitro. In vivo comparisons of hybrid IgE versus wild-type IgE versus IgG may also be performed.

[0209] Unless otherwise specified, all terms, including technical and scientific terms, used in disclosing the present invention All terms used herein have meanings commonly understood by one of ordinary skill in the art to which this invention pertains. By way of further guidance, term definitions may be included to better understand the teachings of the present invention. [Example]

[0210] Anti-HMW-MAA hybrid antibody In a further example, another IgE variant is created in which an IgG hinge and an IgG CH2-CH3 domain pair are fused at the C-terminus to an IgE framework (as in Example 1). The IgE antibody is based on the anti-HMW-MAA antibody disclosed, for example, in WO 2013 / 050725.

[0211] Another anti-HMW-MAA IgE antibody variant is created in which the IgG hinge and CH2 domains are fused to the C-terminus of the anti-HMW-MAA antibody.

[0212] Further variant anti-HMW-MAA IgE antibodies are generated in which one or more loops in the Cε3 domain of IgE are replaced by one or more FcγR-binding loops from the Cγ2 domain of an IgG antibody. The replaced loops in the Cε3 domain of IgE show structural homology to the FcγR-binding loops in the Cγ2 domain of IgG.

[0213] Antibodies are produced and purified as described in Example 1. Antibody binding assays are tested as described in Examples 2-4.

[0214] The sequence for HMW-MAA IgE is as follows:

[0215] TIFF2026016524000039.tif9131

[0216] TIFF2026016524000040.tif27165

[0217] TIFF2026016524000041.tif10165

[0218] TIFF2026016524000042.tif18165

[0219] Alternative variable domain sequences for HMW-MAA IgE are as follows:

[0220] TIFF2026016524000043.tif11163

[0221] TIFF2026016524000044.tif27163

[0222] TIFF2026016524000045.tif10163

[0223] TIFF2026016524000046.tif16163

[0224] The constant domain sequences of the HMW-MAA IgE antibody (comprising an IgG hinge and IgG CH2-CH3 domain (or IgG CH2 domain) fused to an IgE framework) are as shown in Example 1 above, i.e., SEQ ID NOs: 2-4, as well as SEQ ID NO: 23 or SEQ ID NO: 24. [Example]

[0225] Production of heterodimeric IgE Construction of IgE-IgG-Fc (IGEG) fusion protein The DNA sequence corresponding to the WT IgE constant domain was codon-optimized for CHO expression and synthesized with flanking restriction enzyme sites for cloning into the pANT dual Ig expression vector system for the human heavy chain and kappa light chain (GeneArt, ThermoFisher Scientific, Loughborough, UK). The heavy chain, which also contains the trastuzumab VH, was cloned between the Mlu I and Kpn I restriction sites. The separately synthesized trastuzumab Vk was cloned between the BssH II and BamH I restriction sites upstream of the kappa constant region.

[0226] To generate the IgE-IgG (IGEG) fusion, specific primers were used to amplify WT IgE, while removing the stop codon at the end of the IgE CH4 and amplifying the separately synthesized IgG1 hinge-CH2-CH3 in a separate reaction. Pull-through PCR was used to combine both fragments and introduce MluI and KpnI restriction sites for cloning into the dual expression vector. A BsmBI restriction site was then introduced into the FW4 region of the trastuzumab VH by site-directed mutagenesis (Quikchange, Agilent). , which, together with Mlu I, allowed swapping of the VH region (see for a schematic representation of the vector , see Figure 13).

[0227] To remove potential free cysteine ​​residues within the IgG hinge region, BsmBI-containing IgE Primers were designed to introduce the Cys220Ser amino acid substitution by site-directed mutagenesis using the -IgG construct as a template (numbering is based on the EU numbering scheme, with reference to the IgG portion of the IGEG sequence). The Cys220Ser mutation is shown in blue in the sequence below.

[0228] To eliminate the ability of the IgG portion of IgE G to bind to FcRn, amino acid substitutions Ile253Ala, His310Ala, and His435Ala were made at three residues normally involved in FcRn binding (numbering is based on the EU numbering scheme, with reference to the IgG portion of the IgE G sequence). Primers were designed to generate BsmBI-containing IgE-IgG constructs. Site-directed mutagenesis (Agilent Quikchange) was performed using the fragments (containing either Cys220 or Ser220) as templates.

[0229] To generate the HMW-MAA(CSPG4) series of constructs, HMW-MAA VH and VK were synthesized (GeneArt) and cloned into the IGEG vector. The AA VH was cloned between the MluI and BsmBI restriction sites, and the HMW-MAA Vk was cloned between the BssH II and BamH I restriction sites.

[0230] All constructs were confirmed by Sanger sequencing.

[0231] The sequences were as follows (underlined indicates variable domain sequence, standard text is IgE Fc sequences are shown, italics indicate IgG-derived sequences, and bold indicates specific mutations).

[0232] Trastuzumab IgE / IGEG variant sequences

[0233] TIFF2026016524000047.tif10160

[0234] TIFF2026016524000048.tif86162

[0235] TIFF2026016524000049.tif9162

[0236] TIFF2026016524000050.tif114168

[0237] TIFF2026016524000051.tif9170

[0238] TIFF2026016524000052.tif115170

[0239] TIFF2026016524000053.tif11170

[0240] TIFF2026016524000054.tif119169

[0241] TIFF2026016524000055.tif17169

[0242] TIFF2026016524000056.tif119170

[0243] TIFF2026016524000057.tif10170

[0244] TIFF2026016524000058.tif35170

[0245] HMW-MAA IgE / IGEG variant sequences

[0246] TIFF2026016524000059.tif10170

[0247] TIFF2026016524000060.tif86169

[0248] TIFF2026016524000061.tif10169

[0249] TIFF2026016524000062.tif119169

[0250] TIFF2026016524000063.tif12164

[0251] TIFF2026016524000064.tif118166

[0252] TIFF2026016524000065.tif10166

[0253] TIFF2026016524000066.tif119168

[0254] TIFF2026016524000067.tif17168

[0255] TIFF2026016524000068.tif114170

[0256] TIFF2026016524000069.tif10170

[0257] TIFF2026016524000070.tif32170

[0258] CHO transient expression of IgE-IgG (IGEG) variants Endotoxin-free DNA encoding the various IGEG constructs was transiently co-transfected into Freestyle® CHO-S cells (ThermoFisher, Loughborough, UK) using an OC-400 processing assembly and the MaxCyte STX® electroporation system (MaxCyte, Gaithersburg, USA). After cell harvest, cells were pooled and cultured at 3 × 10 in CD Opti-CHO medium (ThermoFisher) containing 8 mM L-glutamine (ThermoFisher) and 1 × hypoxanthine-thymidine (ThermoFisher). 6 The cells were diluted to 100 cells / mL. 24 hours after transfection, The culture temperature was reduced to 32°C and 30% (of the starting volume) of Efficient Feed B (ThermoFisher), 3.3% FunctionMAX® TiterEnhancer (ThermoFisher), and 1 mM sodium butyrate (Sigma, Dorset, UK) were added. Cultures were fed on day 7 by the addition of (current volume) CHO CD Efficient Feed B (ThermoFisher) and 1.65% FunctionMAX® TiterEnhancer (ThermoFisher). All transfections were cultured for up to 14 days before supernatants were harvested.

[0259] Purification and analysis of IGEG variants After culture harvest, antibody supernatants were filtered to remove remaining cell debris and supplemented with 10x PBS to neutralize the pH. The majority of IGEG purifications (including dFcRn IGEG) were performed using IgE CaptureSelect® affinity resin (ThermoFisher Scientific) in a batch binding mode. The affinity resin was equilibrated in PBS pH 7.2 and then incubated with each sample for 2 hours at room temperature with rotation, followed by a series of PBS washes. All samples were eluted in 50 mM sodium citrate, 50 mM sodium chloride pH 3.5, and buffer exchanged into PBS pH 7.2. Samples were analyzed by measuring the extinction coefficient (E) based on the predicted amino acid sequence. c(0.1%) ) to OD 280nm was quantified by.

[0260] Selected IGEG constructs (e.g., trastuzumab IGEGs containing either Cys220 or Ser220) were purified using Protein A to demonstrate retention of Protein A binding. After culture harvest, antibody supernatants were filtered to remove remaining cell debris and supplemented with 10x PBS to neutralize the pH. Subsequently, the antibody was purified using a 1 mL Hitrap MabSelect PrismA column (Cytiva, Little Chalfo) pre-equilibrated with PBS pH 7.2. Antibodies were purified from the supernatant using a chromatographic method (nt, UK). After sample loading, the column was washed with PBS pH 7.2 and the protein was eluted with 0.1 M sodium citrate pH 3.0. Fractions were collected and the pH was adjusted with 1 M Tris-HCl pH 9.0, followed by buffer exchange into PBS pH 7.2. Samples were analyzed by the extinction coefficient (E) based on the predicted amino acid sequence. c(0.1%) ) to OD 280nm was quantified by.

[0261] All IGEG antibody variants were analyzed on a HiLoad® 26 / 60 Superdex® 200 pg preparative SEC column (GE Healthcare, L) using PBS pH 7.2 as the mobile phase. The peak fractions from the purification containing the monomeric protein were pooled, concentrated, and analyzed by the extinction coefficient (E) based on the predicted amino acid sequence. c(0.1%) ) with A 280nm The mixture was filter sterilized before quantitation by HPLC.

[0262] The purified material was then analyzed by analytical SE-HPLC and SDS-PAGE using a Dionex Ultimate 3000RS HPLC system (ThermoFisher Scientific, Hemel Hempstead, VA, USA). Two Acquity UPLC Protein BEH SEC Columns connected to a 1000-well plate (Hempstead, UK) 00Å, 1.7 μm, 4.6 mm × 150 mm (Waters, Elstree, UK) and Acquity UPLC Protein BEH SEC guard column 30 × 4.6 mm, 1.7 μm, 200 Analytical SEC was performed using a HPLC column chromatography column (Waters, Elstree, UK). The method consisted of an isocratic elution over 10 minutes, with a mobile phase of 0.2 M potassium phosphate pH 6.8, 0.2 M potassium chloride. The flow rate was 0.35 mL / min. Detection was by UV absorbance at 280 nm. After purification, all IGEG antibody variants were shown to contain greater than 95% monomeric species.

[0263] Single-cycle kinetic analysis of IGEG variants to cognate antigens Analysis of the binding of HMW-MAA IGEG variants to their cognate antigens by Biacore analysis was not possible due to the lack of conformationally appropriate antigens. Instead, binding was analyzed by flow cytometry.

[0264] To assess the binding of all purified trastuzumab IGEG variants to the human Her2 antigen, single-cycle kinetic analysis was performed on the purified antibodies. Kinetic experiments were performed at 25°C on a Biacore T200 running Biacore T200 Control software V2.0.1 and Evaluation software V3.0 (Cytiva, Uppsala, Sweden). See Figure 14 for a schematic of the process.

[0265] HBS-EP+ (Cytiva, Uppsala, Sweden) supplemented with 1% BSA (Sigma, Dorset, UK) was used as the running buffer and for ligand and analyte dilution. Purified antibodies were diluted to 10 μg / mL in the running buffer. At the start of each cycle, anti-Fab antibodies (a mixture of anti-kappa and anti-lambda antibodies) were added. ) CM5 sensor chip (Cytiva, Little Chalfont, UK) c 2. F c 3, and F c The antibody was loaded onto the column at 4. The antibody was captured at a flow rate of 10 μl / min, resulting in an immobilization level (R) of approximately 45 RU. L ) was applied. The surface was then allowed to settle.

[0266] To minimize any potential mass transport effect, recombinant human Her2 antigen (Sino (Biological Co., Beijing, China) was injected at a flow rate of 40 μl / min. Single-cycle kinetic data were acquired using the antigen as the analyte to be analyzed. A four-point, three-fold dilution range of 1.1 nM to 30 nM of antigen in running buffer was used, with no regeneration between concentrations. The association phase was monitored for 240 s for each of four injections of increasing concentrations of antigen, and a single dissociation phase was measured for 600 s after the final injection of antigen. Regeneration of the sensor chip surface was performed using two injections of 10 mM glycine pH 2.1.

[0267] Reference Channel F c The signal from 1 (no captured antibody) is F c 2. F c 3, and F c 4 to correct for differences in bulk effects and nonspecific binding to the reference surface. The signal from each antibody blank run (antibody captured but no antigen) was subtracted to correct for differences in surface stability (see Figure 15). Each trastuzumab construct tested showed similar binding to human Her2 (Table 6).

[0268] [Table 6]

[0269] Assessment of IGEG variant binding to human Fc receptors The binding of purified IGEG to high and low affinity Fc gamma receptors and high affinity Fc epsilon receptors was evaluated on a Biacore T200 run at a flow rate of 30 μl / min. A Biacore T200 (serial number 10 ... All human Fc gamma receptors (low affinity receptor hFcγRIIIa (176F and 176F)) were assessed by single-cycle analysis using the ELISA kit (product number 1909913). hFcγRI (both 76V polymorphisms) and hFcγRIIIb (along with hFcγRI) were obtained from Sino Biological Co., Ltd. (Beijing, China), and hFcεR1 was obtained from R&D Systems Co., Ltd. (Minn The FcRs were obtained from Biosciences (Belfast, VA, USA). Standard amine chemistry was used to capture FcRs onto a pre-coupled CM5 sensor chip using a His capture kit (Cytiva, Uppsala, Sweden). A schematic detailing the assay used to assess antibody binding to Fc gamma receptors can be found in Figure 16.

[0270] At the start of each cycle, His-tagged Fc receptors diluted in HEPES-buffered saline containing 0.05% v / v surfactant P20 (HBS-P+) were loaded to the indicated RU levels (Table 7). A five-point, 3-fold dilution range of test antibodies was used for each receptor tested, with no regeneration between concentrations. The target RU loaded for each Fc receptor, the concentration range used for each test antibody, along with the association and dissociation times used for test antibody binding, are shown in Table 7. In all cases, antibodies were passed over the chip at increasing concentrations, followed by a single dissociation step. After dissociation, the chip was regenerated using two injections of glycine pH 1.5. Reference channel F c The signal from 1 (blank) was calculated by the receptor-loaded F c The results were subtracted from those of the reference surface to correct for differences in nonspecific binding to the reference surface. High affinity interactions were analyzed using a 1:1 fit (see Figures 17a and 17b for example data), while low affinity interactions were analyzed using a steady-state model (see Figures 17c and 17d for example data). Table 8 shows a summary of the data obtained. The IgE variants bound to both the Fc gamma and Fc epsilon receptors tested. The IgG control bound to the Fc gamma receptor but not to the Fc epsilon receptor, while conversely, the IgE control bound to the Fc epsilon receptor but not to the Fc gamma receptor tested.

[0271] [Table 7]

[0272] [Table 8]

[0273] Assessment of IGEG variant binding to human FcRn Binding of purified antibodies to FcRn was assessed using Biacore T200 Evaluation software V3.0.1 (Up hFcRn (Sino Biological Co., Beijing, China) was coupled to acetic acid using standard amine coupling. Purified HMW-MAA antibodies were coupled onto a Series S CM5 (carboxymethylated dextran) sensor chip (Cytiva, Uppsala, Sweden) at 10 μg / mL in sodium chloride, pH 5.5. Purified HMW-MAA antibodies were titrated in 7 two-fold dilutions from 31.25 nM to 2000 nM in PBS containing 0.05% polysorbate 20 (P20) at pH 6.0, or in 4 three-fold dilutions from 250 nM to 2000 nM in PBS containing 0.05% polysorbate 20 (P20) at pH 7.4. Antibodies were passed over the chip in increasing concentrations at a flow rate of 30 μl / min at 25°C. Injection times were 40 s per concentration, and dissociation times were 75 s. After a single dissociation, the chip was regenerated with 0.1 M Tris, pH 8.0. Figure 18 shows an overview of the assay used to assess antibody binding to FcRn. The interaction was analyzed using a steady-state model (see Figures 19a-19d for example data). Table 9 shows a summary of the data obtained. IgE variants bound to FcRn at pH 6.0, except for those in which the FcRn binding site was removed (dFcRn) and those that were unable to bind to FcRn. The IgG control ended up at FcRn as expected, while IgE did not show any binding to FcRn.

[0274] [Table 9]

[0275] UNcle biostability platform analysis of IGEG variants IGEG variants were synthesized using the UNcle biostable platform (Unchained Labs, P Proteins were analyzed for thermal stability using a thermostability gradient assay (Tm and Tagg) using a denaturation profile (Figure 1). Thermal gradient stability experiments (Tm and Tagg) are well-established methods for ranking proteins and formulations for stability. The denaturation profile of a protein provides information about its thermal stability and represents a structural "fingerprint" for assessing structural modifications and formulation buffer changes. A widely used measure of the thermal structural stability of a protein is the temperature at which it unfolds from the native state to the denatured state. For many proteins, this unfolding process occurs over a narrow temperature range, and the midpoint of this transition is called the "melting temperature" or "Tm." To determine the melting temperature of a protein, UNcle was used to measure the melting temperature of a protein by using Sypro Orange (a fluorescent dye that shows the temperature at which the exposed surface of the protein changes when the protein undergoes a conformational change). The fluorescence of the ATP (which binds to the hydrophobic region) is measured.

[0276] Samples for each variant were formulated in PBS and Sypro Orange at a final concentration of 0.8 mg / mL. 9 μL of each sample mixture was loaded in duplicate into UNi microcuvettes. The sample was subjected to a thermal ramp from 25 to 95 °C using a ramp rate of 0.3 °C / min and excitation at 473 nm. Full emission spectra were collected from 250 to 720 nm, and the area under the curve between 510 and 680 nm was used to determine the inflection point (T 開始 and T m ) was calculated. 473 Monitoring static light scattering (SLS) at 100 nm revealed that Protein aggregation can be detected, and T agg(onset of aggregation) was calculated from the resulting SLS profiles. Data analysis was performed using UNcle® software version 4.0 and is summarized in Table 10. Tm1 values ​​were generally consistent within each set of variants and between the IgE and IgE G variants (Figure 20a); however, the IgE G variants showed significantly improved static light scattering profiles compared to the equivalent IgE variants alone (Figure 20b).

[0277] [Table 10] [Example]

[0278] Assessment of IGEG variant binding to A375 cells Binding of the HMW-MAA(CSPG4) antibody variants detailed in Example 6 to HMW-MAA was assessed using A375 cell HMW-MAA.

[0279] method Harvesting A375 cells A375 cells were cultured using standard methods. When A375 cells became confluent, they were harvested. Briefly, cells were washed with PBS and then incubated with TrypLE® at 37°C for 10 minutes to detach the cells from the flask. The cells were resuspended in 10 mL of medium and centrifuged at 250 g for 3 minutes. The cells were then resuspended in 1 mL FAC The cells were then resuspended in S buffer and counted on a Cellometer® to determine cell number and viability. After this, the cells were diluted to 1 x 10 per mL using FACS buffer. 6 The cells were diluted to 100 μL per well and 100 μL of this cell suspension was seeded onto the plate.

[0280] Binding assay Binding of purified IGEG to A375 cells (ATCC, Virginia, US) was measured using an Attune® NxT Acoustic Focusing Cytometer running Attune software V3.1.2. (ThermoFisher Scientific, Loughborough, UK) A375 cells were incubated with primary antibodies (described in Example 6) for 30 minutes at 4°C, followed by 10 μg / ml of FITC-conjugated goat anti-human IgG or IgE secondary antibody (Vector Laboratories, California, USA). This was followed by a further 30 min incubation with FACS buffer at 4°C. Cells were washed, resuspended in FACS buffer, and then acquired on an Attune® NxT Acoustic Focusing Cytometer. FlowJo® software version 10 (Becton, Dickinson and Company, New Jersey, US) and GraphPad Prism 8 (GraphPad Software Data were analyzed using the Quantitative Analysis System (Quantitative Analysis System, Inc., California, US).

[0281] result As demonstrated in Figures 21a and 21b, all HMW-MAA antibodies and variants bound to A375 cells. [Example]

[0282] ADCC and ADCP assays Assays were performed to determine the effect of the described antibodies on the levels of both antibody-dependent cell-mediated phagocytosis (ADCP) and antibody-dependent cell-mediated cytotoxicity (ADCC), the two main mechanisms by which immune effector cells can kill tumor cells. The trastuzumab antibody variants described in Example 6 were compared to the trastuzumab IgE and Herceptin IgG antibodies.

[0283] method ADCC and ADCP assays were performed using U-937 effector cells and SK-BR-3 target cells using methods similar to those known in the art (see, e.g., Three-color flow cytometric method to measure antibody-dependent tumor cell killing by cytotoxicity and phagocytosis. J Immunol Methods. 2007 Jun 30;323(2):160-71).

[0284] The day before the assay, Her2-expressing tumor cells (SK-BR-3) were stained. To do this, SK-BR-3 cells were detached from the plate using TrypLE and resuspended in complete RPMI medium (RPMI 1640 medium supplemented with pen / strep and 10% HI FBS). After washing, 1 × 10 cells were added to serum-free HBSS. 0.75 μL of 0.5 mM carboxyfluorescein succinimidyl ester (CSFE) in HBSS was added. 6 The solution was added individually and the cells were incubated for 10 minutes at 37° C. After washing, the cells were seeded and incubated overnight.

[0285] The next day, U-937 effector cells were passaged, counted using trypan blue, and resuspended in complete RPMI medium to a concentration of 1.5 × 10 per mL. 6 CFSE-labeled SK-BR-3 cells were detached by TrypLE treatment, washed, counted, and resuspended in complete RPMI medium to provide 0.5 × 10 cells per mL. 6 Then, trastuzumab IgE, Herceptin IgG, trastuzumab-IGEG, trastuzumab-IGEG-C220S, and IgG isotype antibodies detailed in Example 6 were added to the cells at a starting concentration of 120 nM. The antibody dilutions were diluted to a concentration of 0.01% and then serially diluted six-fold. 25 μL of each antibody dilution was added in duplicate to a 96-well plate along with 50 μL of SK-BR-3 cell suspension (equivalent to 25,000 cells) and 25 μL of U-937 effector cell suspension (equivalent to 37,500 cells). Appropriate control wells lacking CSFE staining, U-397 cells, SK-BR-3 cells, viable SK-BR-3 cells (replaced with heat-shocked SK-BR-3 cells), or one or more of the test antibodies were included in the assay. Plates were then incubated at 37°C for 3 hours, centrifuged, washed twice with FACS buffer (PBS + 2% FCS), and resuspended in 100 μL FACS buffer containing 2 μL CD89 APC-conjugated labeled antibody. Control wells were resuspended in FACS buffer alone. After 30 minutes at 4°C, the plates were centrifuged and washed twice again with FACS buffer, after which the cells were resuspended in 100 μL FACS buffer containing propidium iodide (PI) stain (5 μL per 100 μL). Control wells were resuspended in FACS buffer and incubated for 15 minutes at room temperature.

[0286] 50,000 cells / tube were then acquired on an Attune® NxT Acoustic Focusing Cytometer. Compensation was set up using control wells. R1, R2 and R3 gating was applied in the analysis software (Flow Jo) (Figure 22). Cell counts were obtained for each gate, and then calculations were performed to determine cytotoxic (ADCC) or phagocytic (ADCP) activity.

[0287] result As demonstrated in Figure 23, the trastuzumab-IGEG (IGEG-CH2CH3) antibody appears to induce higher levels of phagocytosis than the Herceptin IgG and trastuzumab IgE antibodies across all concentrations tested (120 to 7.5 nM). The trastuzumab-IGEG-C200S (IGEG-CH2CH3-C220S) antibody appears to induce higher levels of phagocytosis than the Herceptin IgG and trastuzumab IgE antibodies. In addition, the results demonstrate that trastuzumab IgE, Herceptin IgG, and both IGEG antibodies had comparable effects on cytotoxicity.

[0288] This application claims priority from UK Patent Application No. 1914165.4, filed October 1, 2019, UK Patent Application No. 1917059.6, filed November 22, 2019, and UK Patent Application No. 2008248.3, filed June 2, 2020, the contents of which are incorporated herein by reference. All publications mentioned in the above specification are incorporated herein by reference. Various modifications and variations of the described embodiments of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes 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 hybrid antibody that binds to Fcε and Fcγ receptors.

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