Modified immunoglobulins with affinity for Fc gamma RIIb and methods of use thereof

Abstract

Disclosed is an immunotherapeutic protein comprising at least one heavy chain polypeptide derived from an IgG2 antibody, the heavy chain polypeptide comprising at least constant heavy domains 2 and 3 (CH2 and CH3) and a lower hinge, the sequence of the lower hinge comprising a mutation that allows the immunotherapeutic protein to bind to and / or activate FcγRIIb. The immunotherapeutic protein is suitable for use, for example, in a method for treating a disease or condition in which activation of FcγRIIb (i.e., for the recruitment of the inhibitory function of FcγRIIb) is beneficial, such as an allergic disease.

Description

[Technical field]

[0001] The present disclosure relates to immunotherapeutic proteins, including modified immunoglobulin molecules (i.e., antibodies), for use in methods of treating diseases or conditions. More specifically, the present disclosure describes mutant IgG2 antibodies that exhibit improved binding specificity and affinity to the human inhibitory receptor FcγRIIb. These antibodies can be used, for example, to treat diseases in which activation of FcγRIIb is beneficial, such as allergic diseases.

[0002] Priority document This application claims priority from Australian Patent Application No. 2020 / 904823, entitled "Modified Immunoglobulins and Methods of Use Thereof (1)", filed December 23, 2020, the entire contents of which are incorporated herein by reference. [Background technology]

[0003] Monoclonal antibodies (mAbs) have become one of the most important and successful types of therapy, revolutionizing the treatment of inflammatory diseases such as cancer and autoimmune diseases. Many mAbs engineered on the backbone of the IgG antibody class specifically exploit the powerful effector functions of the immune system by engaging both the target antigen via their variable Fab domain and the Fcγ receptor (FcγR) via their heavy chain containing the constant Fc fragment. In inflammatory diseases, engineered mAbs can potentially act by neutralizing inflammatory mediators, by neutralizing their receptors, or by engaging immunomodulatory receptors. One particular interest in the context of the present invention is the possibility of neutralizing the proinflammatory response in allergic reactions, the main mediator of which is allergen-specific IgE activation of its high affinity receptor, FcεRI.

[0004] An example of an engineered mAb approved for use in the treatment of allergic diseases is the IgG1-based mAb, omalizumab. This mAb targets the IgE / FcεRI pathway by neutralizing the interaction of IgE with FcεRI, thereby preventing the activation of basophils, key inflammatory cells in IgE-dependent allergic reactions (Gericke J et al., JEADV 29(9):1832-1836, 2014). Human basophils express both FcεRI as well as its regulator, the inhibitory receptor FcγRIIb (Kepley CL et al, J Allergy Clin Immunol 106(2):337-348, 2000). FcγRIIb is a potent checkpoint that regulates antibody-dependent inflammatory cell activation. FcγRIIb acts through immunoreceptor tyrosine-based activation motif (ITAM)-dependent signaling pathways of FcεRI and immunoreceptor tyrosine-based inhibitory motifs (ITIMs) that regulate activating IgG and IgA Fc receptors, namely FcγRI, FcγRIIa, FcγRIIIa and FcαRI. FcγRIIb also regulates B cell activation through the B cell antigen receptor (BCR). Targeting immune checkpoints such as FcγRIIb has emerged as a strategy to regulate leukocyte responses in disease (Kaplon H and JM Reichert, Mabs 11(2):219-238, 2019; Chenoweth AM et al., Immunol Cell Biol 98(4):287-304, 2020).However, unlike mAb targeting of checkpoints of T cell function (e.g., PD-1, PD-L1), FcγRIIb is specific in the context of mAb therapeutics due to its specificity for the Fc fragment of the mAb therapeutic, and since FcγRIIb requires co-crosslinking with ITAM-containing activating receptors, such as the activating FcR complex or the antigen receptor complex of B cells, also known as the B cell antigen receptor or BCR, to exert its inhibitory action (Getahun A and JC Cambier, Immunol Reviews 268(1):66-73, 2015; Chenoweth AM et al., 2020 (supra)), strategies using specific immunosuppressive antibodies that can exploit the normal physiological inhibitory role of FcγRIIb in allergic responses (coaggregating with FcεRI or coaggregating with the B cell antigen receptor BCR) provide a potentially useful method of targeting activating receptors such as the high affinity IgE receptor, FcεRI, or BCR.

[0005] In the work leading to the present disclosure, the inventors demonstrated that IgG2 antibodies express one allelic form of the low affinity activating FcγRIIa (i.e., the "His131 form," FcγRIIa-His 131Given that IgG2 scaffolds have a very restricted FcγR specificity binding only to FcγR receptors (FcγR1, FcγR2, FcγR3, FcγR4, FcγR5, FcγR6, FcγR7, FcγR8, FcγR9, FcγR10, FcγR11, FcγR12, FcγR13, FcγR14, FcγR15, FcγR16, FcγR17, FcγR18, FcγR20, FcγR21, FcγR32, FcγR33, FcγR44, FcγR56, FcγR60, FcγR72, FcγR8, FcγR9, FcγR10, FcγR11, FcγR12, FcγR15, FcγR16, FcγR21, FcγR17, FcγR22, FcγR33, FcγR44, FcγR56, FcγR10, FcγR11, FcγR23, FcγR14, FcγR25, FcγR36, FcγR44, FcγR56, FcγR10, FcγR21, FcγR15, FcγR22, FcγR36, FcγR44, FcγR56, FcγR10, FcγR21, FcγR12 ... However, although IgG2 antibodies are known not to bind FcγRIIb or other FcγRs, except for one allelic form of FcγRIIa mentioned above (Bruhns P et al, Blood 113(16):3716-3725, 2009, and Figure 1A below), the inventors were nevertheless surprisingly able to engineer an IgG2 scaffold to improve FcγRIIb specificity, affinity, and inhibitory potency. The approach employed is believed to have broader implications for the generation of immunotherapeutic proteins, including novel potent anti-inflammatory therapeutic mAbs and molecules. Summary of the Invention

[0006] Accordingly, in a first aspect, the disclosure provides a method of treating a disease or condition in a subject, where binding to and / or activation of FcγRIIb is beneficial in treating or preventing the disease or condition, the method comprising administering to the subject an effective amount of an immunotherapeutic protein comprising at least one heavy chain polypeptide derived from an IgG2 antibody, the heavy chain polypeptide comprising at least constant heavy domains 2 and 3 (i.e., CH2 and CH3) and a lower hinge, the sequence of the lower hinge comprising a mutation that enables the immunotherapeutic protein to bind to and / or activate FcγRIIb.

[0007] In some particularly preferred embodiments, the lower hinge sequence of the immunotherapeutic protein comprises an amino acid sequence selected from ELLGG (SEQ ID NO:1), EFLGG (SEQ ID NO:2), and EFEGG (SEQ ID NO:3).

[0008] In a second aspect, the disclosure provides the use of an immunotherapeutic protein as defined in the first aspect for treating a disease or condition in which binding to and / or activation of FcγRIIb is beneficial, including, for example, allergic diseases, autoimmune diseases and conditions, infectious diseases, and proliferative diseases.

[0009] In a third aspect, the disclosure provides the use of an immunotherapeutic protein as defined in the first aspect in the manufacture of a medicament for treating a disease or condition in which binding to and / or activation of FcγRIIb is beneficial, including, for example, allergic diseases, autoimmune diseases and conditions, other inflammatory diseases, infectious diseases, and proliferative diseases.

[0010] In a fourth aspect, the present disclosure provides a pharmaceutical composition or medicament comprising an immunotherapeutic protein as defined in the first aspect and a pharma- ceutically acceptable carrier, diluent and / or excipient. [Brief description of the drawings]

[0011] [Figure 1]1 shows the binding profiles of mutant IgG2 antibodies according to the present disclosure to human FcγR compared to IgG1, IgG2, IgG4, and mutant IgG4 antibodies. [Diagram 2] 1 shows inhibition of bee venom Api m 1 allergen-dependent basophil activation in washed blood lacking physiological levels of IgG from allergy patients using mutant IgG2 antibodies according to the present disclosure. [Diagram 3] 1 provides the results of a Basophil Activation Test (BAT) assay using variant IgG2 antibodies according to the present disclosure, showing inhibition of bee venom Api m 1 allergen-dependent basophil activation in whole blood containing physiological levels of IgG from allergy patients. [Figure 4] 4 shows the results of assays performed to determine whether mutant IgG2 antibodies containing the SELF mutation exhibit improved binding affinity for FcγRIIb and FcγRIIa-R131: (A) BLI analysis of binding of rsFcγRIIb captured on TNP-BSA to monomeric anti-TNP IgG1, IgG2 and the indicated mutants or IgG2 (mean ± SEM), (B) BLI analysis of binding of rsFcγRIIIa-R131 captured on TNP-BSA to monomeric anti-TNP IgG1-SELF, IgG2-SELF, IgG2-FLGG-SELF and IgG2-FEGG-SELF (mean ± SD). [Diagram 5] Results are provided demonstrating that mutant IgG2 antibodies according to the present disclosure have altered human FcγR binding profiles. Using cells expressing human FcγR, the avidity of conjugated IgG to low affinity human FcγR or affinity of unconjugated monomeric IgG to FcγRI was determined by flow cytometry: (A) FcγRIIb, (B) FcγRIIa-R131, (C) FcγRIIa-H131, (D) FcγRIIIa-F158, (E) FcγRIIIa-V158, and (F) FcγRI. Statistical comparisons were made between the IgG mutants and the relevant IgG WT scaffolds. *(p<0.5), **(p<0.1), ***(p<0.01), ****(p<0.0001), ns (not significant). [Figure 6]1 shows the results of FcγR binding specificity of monomeric mutant IgG2 antibodies according to the present disclosure, some of which contained a SELF mutation. [Figure 7] 1 provides the results of a BAT assay using mutant IgG2 antibodies according to the present disclosure (some with a SELF mutation) showing inhibition of anti-IgE-dependent basophil activation (using anti-IgE-TNP as an IgE-dependent stimulus) by the IgG variants in whole blood from healthy donors. [Figure 8] Results of experiments assessing inhibition of FcγRIIb expression and FcγRIIb-specific FcεRI activation are provided: (A) flow cytometric detection of FcγRIIb on IgE-positive basophils using F(ab')2 fragment of H2B6, an FcγRIIb-specific mAb, compared to buffer background (Bkg); (B) H2B6 F(ab')2 blockade of FcγRIIb prevents inhibition of basophil activation by IgG2-FLGG (7.5 μg / ml) in whole blood BAT; (C) mAb inhibition of IgE / FcεRI-dependent induced calcium mobilization in IIA1.6 cells co-expressing FcεRI(αβγ) complexes and inhibitory FcγRIIb. Cells were pretreated overnight with IgE and stimulated with anti-TNP antibodies pre-complexed with TNP-conjugated F(ab')2 anti-hIgE. Cells were stimulated with anti-IgE-TNP(F(ab')2) (20 μg / ml) and mAb (35 μg / ml). Calcium flux (340 / 380 nm) was measured over time and inhibition of calcium flux by mutant IgG2 antibodies was compared to parental wild-type IgG2, which does not bind FcγRIIb. Unstimulated baseline control was buffer alone (n=3). [Figure 9]We provide the results of experiments showing inhibition of antigen stimulation of B cells by IgG mAbs that detect the B cell antigen receptor complex. Anti-IgE mAbs bearing the variable domain of omalizumab with either an IgG4 backbone or an IgG2 backbone inhibit NIP(22)BSA stimulation of NIP-specific hu-IgE BCR-induced calcium flux in B cells co-expressing human FcγRIIb1. Modulation of IgE BCR had a hierarchy of IgG1 (omalizumab) ~ IgG4 > IgG2 that correlates with the rank of FcγRIIb binding activity of these IgG formats. [Figure 10] We provide the results of experiments showing inhibition of antigen stimulation of B cells by IgG2 mutant mAbs (according to the present disclosure) that bind to the B cell antigen receptor complex. Anti-IgE mAbs with the variable domain of omalizumab provided as IgG2 mutant antibodies according to the present disclosure inhibit antigen (i.e., NIP(22)BSA) stimulation of NP-specific hu-IgE BCR-induced calcium flux in B cells co-expressing human FcγRIIb1. The impact of mutations on IgE BCR modulation by IgG2 mutant mAbs had a hierarchy of FLGG-SELF>FLGG~FEGG-SELF>FEGG>IgG2, which broadly correlated with the rank of FcγRIIb binding activity of these mutations in the IgG2 format. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] The inventors have found that a functionally inactive IgG2 backbone can be used as a scaffold to incorporate mutations in the lower hinge sequence (e.g., effectively replacing the lower hinge with the IgG4 lower hinge sequence) and other mutations to improve binding specificity and affinity to the human inhibitory receptor FcγRIIb (including any or all of the mRNA splice variants known to those skilled in the art, i.e., FcγRIIb1, FcγRIIb2 and FcγRIIb3) (Getahun A and JC Cambier, 2015 (supra), and Chenoweth AM et al., 2020 (supra); Anania JC et al., Front Immunol 9:1809,2018). The resulting mutant IgG2 antibodies could potentially provide enhanced agonistic function of mAbs, for example, where FcγR "scaffolding" is required for therapeutic effect. They could also be useful for the development of non-agonistic therapeutic antibodies in situations where it is desirable to take advantage of the normal physiological inhibitory function of FcγRIIb, or antigen or immune complex clearance mediated by FcγRIIb (e.g., by endocytosis / internalization or "sweeping").

[0013] Accordingly, in a first aspect, the disclosure provides a method of treating a disease or condition in a subject, where binding to and / or activation of FcγRIIb is beneficial in treating or preventing the disease or condition, the method comprising administering to the subject an effective amount of an immunotherapeutic protein comprising at least one heavy chain polypeptide derived from an IgG2 antibody, the heavy chain polypeptide comprising at least constant heavy domains 2 and 3 (i.e., CH2 and CH3) and a lower hinge, the sequence of the lower hinge comprising a mutation that enables the immunotherapeutic protein to bind to and / or activate FcγRIIb.

[0014] At least one heavy chain polypeptide of the immunotherapeutic protein may be any heavy chain polypeptide that a person skilled in the art would recognize as derived from an IgG2 antibody, for example because the CH2 and CH3 domains of the heavy chain polypeptide comprise an amino acid sequence that exhibits at least 95%, preferably at least 98%, identity to the sequence of the CH2 / CH3 domain of a wild type (WT) IgG2 antibody, or more preferably to the sequence of the CH2 / CH3 domain of a WT human IgG2 antibody, such as that provided by Wines BD et al., J Immunol 197(4):1507-1516, 2016 and GenBank Accession No: AH005273.2).

[0015] Those skilled in the art will readily appreciate that a heavy chain polypeptide derived from an IgG2 antibody may, for example, comprise a full-length heavy chain polypeptide (i.e., comprising a constant heavy region (CH) and a variable heavy (VH) region) or may comprise a fragment thereof comprising at least the CH2, CH3 and lower hinge. One preferred example of such a fragment is an Fc fragment corresponding to one of the heavy chain components of a fragment generated by papain digestion of an antibody (cleaving the polypeptide within the upper hinge sequence to generate an Fc fragment comprising two heavy chain bridge fragments each comprising the CH2, CH3 and lower and core hinge sequences). Similar heavy chain polypeptides may be prepared by digestion of an antibody with plasmin and human neutrophil elastase (NHE), also known to those skilled in the art as generating "Fc fragments", and such heavy chain polypeptides may suitably comprise the immunotherapeutic proteins of the method of the first aspect. Further examples of suitable heavy chain polypeptides may include all or a portion of the constant heavy domain 1 (CH1) of an IgG2 antibody, in addition to the CH2, CH3 and lower hinge, and / or the core hinge and / or upper hinge sequences.

[0016] In some embodiments, the immunotherapeutic protein may comprise a heavy chain polypeptide provided in the form of a fusion protein or protein conjugate. Those skilled in the art will appreciate that in a fusion protein, the heavy chain polypeptide is covalently linked (i.e., "fused") to a polypeptide or peptide partner (i.e., fusion partner) via a peptide bond or a short peptide linker sequence at the N-terminus or C-terminus of the fusion partner, whereas in a protein conjugate, the heavy chain polypeptide is linked to the polypeptide or peptide partner via a chemical linkage such as a disulfide bond or a disuccinimidyl suberate (DSS) (e.g., bis(sulfosuccinimidyl) suberate (BS)). 3It should be understood that the conjugate partner is covalently or non-covalently linked to the conjugate partner (which may be a polypeptide, peptide, or other chemical entity) via a crosslinker compound such as a homobifunctional crosslinker such as disuccinimidyl tartrate (DST), linked to an amine group, or a heterobifunctional crosslinker such as m-maleimidobenzoyl-N-hydroxysuccinimide ester (MDS) and N-(ε-maleimidocaproxy) succinimide ester (EMCS), or covalently or non-covalently linked to the conjugate partner by other non-covalent bonds such as hydrogen bonds. When the conjugate partner is a polypeptide, the protein conjugate may otherwise be considered a crosslinked protein. The conjugate partner may be conjugated to the heavy chain polypeptide at the N-terminus or C-terminus, or may otherwise be conjugated at any other suitable site on the heavy chain polypeptide (e.g., within the CH1 or upper hinge sequence, if included in the heavy chain polypeptide). Those skilled in the art will recognize that a fusion or conjugate partner may provide one or more useful activities or functions. For example, a fusion partner may improve protein recovery or expression (e.g., human serum albumin (HSA) and glutathione S-transferase (GST)), provide various affinity tags such as polyhistidine tags (His tags) or FLAG tags, or additional ability to bind to an antigen of interest. Other examples of fusion or conjugate partners include receptors (e.g., cytokine receptors, such as receptors for interleukins (e.g., IL-1 receptor) or receptors for cytokines of the TGF-β superfamily), or cell surface molecules and immune checkpoints (e.g., CTLA4 or PD1).

[0017] In some other embodiments, the immunotherapeutic protein may comprise at least one heavy chain polypeptide provided in a dimeric or multimeric form. For example, heavy chain polypeptides may have a natural tendency to form covalent dimers through one or more cysteine ​​(C) residues, especially when located within the hinge sequence (e.g., the core hinge sequence) (Yoo EM et al., J Immunol 170:3134-3138, 2003). Suitable techniques for producing multimeric forms of heavy chain polypeptides (e.g., including three, four, five, six, etc. copies of a heavy chain polypeptide) have been described and are well known to those skilled in the art (e.g., Fc multimeric forms (stradomers™) including linked multimerization domain (MD) sequences from the hinge region of human IgG2 or isoleucine zipper (ILZ) to the N-terminus or C-terminus of mouse IgG2a; Fitzpatrick EA et al., Front Immunol 11, article 496, 2020). Such dimeric or multimeric forms of the heavy chain polypeptide are preferably soluble (ie, in saline).

[0018] In yet some other embodiments, the immunotherapeutic protein comprises an IgG2 antibody comprising two full-length heavy chain polypeptides with two light chain polypeptides, particularly a mutated IgG2 antibody in which at least one of the two heavy chain polypeptides comprises a mutation in the lower hinge sequence that enables the IgG2 antibody to bind and / or activate FcγRIIb.

[0019] Wild-type (WT) human IgG2 antibodies exhibit no or virtually undetectable binding to FcγRIIb (see FIG. 1A). In contrast, the methods of the present disclosure may utilize mutant human IgG2 antibodies (i.e., IgG2 antibodies comprising mutations in the lower hinge sequence of at least one heavy chain polypeptide) that enable the IgG2 antibody to bind to and / or activate FcγRIIb. Mutations in the IgG2 lower hinge sequence may involve substitutions of the sequence or substitutions of one or more amino acids within the sequence at positions 233-236 (EU numbering system, Edelman GM et al., Proc Natl Acad Sci USA 63:78-85, 1969 and Kabat EA, Sequences of Proteins of Immunological Interest, 5 th ed., DIANE Publishing, PA, USA, 1991; however, for the avoidance of doubt, the IgG2 lower hinge sequence referred to herein may comprise an amino acid at an equivalent position to that of the IgG1 lower hinge sequence, which sequence is PVAG (SEQ ID NO: 14) in human IgG2 antibodies. Mutations in the IgG2 lower hinge sequence may further comprise the insertion or addition of amino acids, for example the mutated human IgG2 lower hinge (PVAG: SEQ ID NO: 14) sequence may be a 5 amino acid sequence in a mutated IgG2 antibody.

[0020] Preferably, in the immunotherapeutic protein, the lower hinge sequence comprises the following amino acid sequence: X 1 X 2 X 3 -GX 5 (SEQ ID NO:4) During the ceremony, X 1 is selected from proline (P) and glutamic acid (E); X 2 is selected from valine (V), leucine (L), and phenylalanine (F); X 3 is selected from leucine (L), alanine (A), and glutamic acid (E); X 5is selected from glycine (G) and proline (P) or is absent (i.e., the sequence is 1 X 2 X 3 -G: as SEQ ID NO: 31), However, the lower hinge does not consist of a wild-type IgG2 lower hinge sequence (for example, PVAG (SEQ ID NO: 14) of human IgG2 antibody).

[0021] In some particularly preferred embodiments, the lower hinge sequence of the mutant IgG2 antibody comprises an amino acid sequence selected from ELLGG (derived from human IgG1, SEQ ID NO: 1), EFLGG (derived from human IgG4, SEQ ID NO: 2), EFLGP (SEQ ID NO: 5) and EFEGG (SEQ ID NO: 3).

[0022] As mentioned above, the immunotherapeutic proteins of the present disclosure (e.g., mutant IgG2 antibodies) can bind to and / or activate FcγRIIb. If the immunotherapeutic proteins bind to and activate FcγRIIb, the immunotherapeutic proteins can act to induce FcγRIIb inhibitory function. Thus, in some embodiments, the methods of the present disclosure are particularly suitable for treating or preventing diseases or conditions in which the inhibitory effects of FcγRIIb are beneficial. Thus, in some preferred embodiments, the methods of the present disclosure are particularly directed to treating or preventing, for example, allergic diseases, where the binding and activation of FcγRIIb by the immunotherapeutic proteins mediates FcγRIIb-dependent inhibition of allergic basophil activation by IgE.

[0023] The disclosed methods are carried out for the treatment or prevention of allergic reactions, and the immunotherapeutic protein is preferably a protein comprising a mutant lower hinge sequence EFLGG (SEQ ID NO: 2) or ELLGG (SEQ ID NO: 1); in the examples described below, mutant IgG2 antibodies designated "IgG2-FLGG (SEQ ID NO: 6)" mAb and "IgG2-LLGG (SEQ ID NO: 7)" mAb were found to be the most potent inhibitors of IgE / FcεRI base affinity activation of mutant IgG2 antibodies tested, despite their relatively low affinity for FcγRIIb (note: they only bound as immune complexes).

[0024] In some preferred embodiments, the immunotherapeutic protein does not contain any further mutations in the heavy chain polypeptides (or at least in the constant heavy region of the heavy chain polypeptides), although in some other embodiments it may be advantageous to further contain one or more further mutations in at least one heavy chain polypeptide. For example, the immunotherapeutic protein (e.g., a mutant IgG2 antibody) may further comprise an amino acid substitution at position 267 and / or 328 (EU numbering) in the CH2 domain of at least one, and more preferably both, of the heavy chain polypeptides, such as the S267E and L328F substitutions, respectively, so-called "SELF" mutations. In the examples described below, it was found that a mutant IgG2 antibody (e.g., "IgG2-FEGG (SEQ ID NO: 8)-SELF" mAb) with an EFEGG (SEQ ID NO: 3) mutant lower hinge sequence and a SELF mutation bound to FcγRIIb in the most specific manner of the mutant IgG2 antibodies tested and retained inhibitory potency.

[0025] However, although the SELF mutation enhanced the interaction of the mutant IgG2 antibody with FcγRIIb, in vivo the specificity of such mutant IgG2 antibodies in a subject may be determined by the presence of the high / low responder polymorphism of FcγRIIa, since antibodies containing the SELF mutation have been found to have high affinity interactions with FcγRIIb and the activating receptor type FcγRIIa (only the "Arg131 form" and not the "His131 form"). Thus, in some embodiments where the method involves the administration of a mutant IgG2 antibody containing a SELF mutation, the method preferably involves the administration of FcγRIIa-H 131 Subjects homozygous for FcγRIIa-H 131 Subjects homozygous for FcγRIIa represent approximately 30% of the population; van der Pol WL and J van de Winkel, Immunogenetics 48:222-232, 1998). In such embodiments, the method may further comprise the step of selecting subjects by genotyping for the FcγRIIa high / low responder polymorphism. That is, in some embodiments, FcγRIIa-H is a genotype that is associated with a high / low responder polymorphism. 131 Subjects determined to be homozygous for may be selected for treatment by administering a mutant IgG2 antibody that contains a SELF mutation.

[0026] When the immunotherapeutic protein comprises a mutated IgG2 antibody, the mutated IgG2 antibody typically comprises a monoclonal antibody (mAb), preferably a human mAb or a humanized mAb. Such antibodies can be produced according to any of the standard methodologies known to those skilled in the art. For example, those skilled in the art can easily prepare a mutated IgG2 antibody suitable for use in the disclosed method by using standard molecular biology techniques to generate a construct, which comprises a polynucleotide sequence encoding the variable heavy (VH) and light (VL) region sequences of a suitable antibody (e.g., one that comprises an antigen-binding region that binds to an antigen of interest) and the constant heavy (CH) region from an IgG2 antibody (e.g., as described in Wines BD et al., 2016 (supra)), and the polynucleotide sequence encoding the CH region is incorporated by standard molecular biology techniques, such as site-directed mutagenesis, polynucleotide sequence changes that encode the lower hinge sequence mutations described above (and SELF mutations, if desired). The construct is introduced into suitable host cells (e.g., human kidney (HEK) host cells), cultured according to standard culture protocols, and the expressed mutant IgG2 antibody is purified from the culture supernatant using, for example, any of the known suitable methodologies for purification (e.g., affinity chromatography).

[0027] Treatment of diseases through activation of FcγRIIb In some embodiments, the methods of the disclosure may be used to treat a disease or condition in a subject, where activation of FcγRIIb (i.e., due to recruitment of inhibitory effects) is beneficial in the treatment or prevention of the disease or condition.

[0028] In some examples of such embodiments, the immunotherapeutic protein may target the ITAM signaling receptor complex by comprising a binding domain, such as an antigen binding region (e.g., a Fab region) that recognizes a component of a potential signaling complex, such as (a) an antigen (e.g., an allergen or autoantigen bound to an antibody such as IgG, IgE, or IgA bound to a receptor), (b) an antibody bound to an activating receptor, (c) an antibody (ligand) binding domain of an activating receptor, or (d) a subunit of an activating receptor (e.g., an Fc receptor common gamma chain), while in other examples, the immunotherapeutic protein may target the ITAM signaling receptor complex by comprising a binding domain, such as an antigen binding region (e.g., a Fab region) that recognizes a component of a potential signaling complex, such as (a) an antigen (e.g., an allergen or autoantigen bound to an antibody such as IgG, IgE, or IgA bound to a receptor), (b) an antibody bound to an activating receptor, (c) an antibody (ligand) binding domain of an activating receptor, or (d) a subunit of an activating receptor (e.g., an Fc receptor common gamma chain). The immunotherapeutic protein may target, for example, (a) an antigen bound to an immunoglobulin component of the BCR complex (e.g., an antigen of an infectious agent such as an allergen, an autoantigen, an antigen of a bacterial or viral pathogen, or an antigen from a transplanted tissue or organ), or (b) a component of the B cell antigen receptor (BCR) complex, including a subunit of the BCR complex (e.g., a membrane immunoglobulin of the BCR complex such as IgM, IgD, IgG, IgE, or IgA) or an associated Ig-α or β chain (e.g., CD79a or CD79b, or CD19, CD21, or CD81).

[0029] In all such examples, the immunotherapeutic protein provides the necessary co-crosslinking of the ITAM-containing activating receptor with the inhibitory receptor FcγRIIb, recruiting the inhibitory action of FcγRIIb, and as will be appreciated from the above discussion, this can be achieved in a number of different ways that vary considerably in the targets of the immunotherapeutic proteins, thus enabling their potential use for the treatment or prevention of a wide range of different diseases or conditions.

[0030] More specifically, in some examples where the immunotherapeutic protein is intended to be used for the treatment or prevention of a disease or condition by binding to and activating FcγRIIb, the immunotherapeutic protein may comprise a binding domain such as an antigen binding region targeted to an antigen of interest present in an immune complex bound to FcεRI via the Fc portion of an immunoglobulin (i.e., an antigen complexed with IgE, IgG or IgA), or in an activating FcγR (e.g., FcγRI, FcγRIIa, FcγRIIc, and FcγRIIIa or FcγRIIIb, otherwise known as CD64, CD32a, CD32c, CD16a, and CD16b, respectively), or in the activating receptor FcαRI (CD89). Thus, the antigen of interest may be selected from, for example, allergens (e.g., bee venom) for the use of immunotherapeutic proteins for the treatment or prevention of allergic diseases, autoantigens (e.g., autoantigens associated with systemic lupus erythematosus (SLE) or multiple sclerosis (MS)) for the use of immunotherapeutic proteins for the treatment or prevention of autoimmune diseases, antigens associated with other inflammatory diseases such as immune complex vasculitis, antigens from transplanted tissues or organs allowing the use of immunotherapeutic proteins for the treatment or prevention of antibody-mediated transplant rejection, and antigens of infectious agents such as antigens of bacterial or viral pathogens (antigens of SARS-CoV-2 or dengue virus).

[0031] Alternatively, in some instances where the immunotherapeutic protein is intended to be used for the treatment or prevention of a disease or condition by binding to FcγRIIb and activating FcγRIIb, the immunotherapeutic protein may comprise a binding domain such as an antigen-binding region that targets an immunoglobulin present in an immune complex bound to FcεRI via the Fc portion of the immunoglobulin, or an activating FcγR (e.g., FcγRI, FcγRIIa, FcγRIIc, and FcγRIIIa or FcγRIIIb), or an activating receptor FcαRI. That is, when an antigen is complexed with IgE, IgG, or IgA, the immunotherapeutic protein may be targeted to the heavy or light chain of IgE, IgG, or IgA, resulting in co-crosslinking of FcγRIIb with the activating Fc receptor. Thus, such targeted immunotherapeutic proteins can also be used for the treatment or prevention of allergic diseases (e.g., when an allergen is conjugated to a targeted immunoglobulin), autoimmune diseases such as SLE and MS (e.g., when an autoantigen is conjugated to a targeted immunoglobulin by an immunotherapeutic protein), other inflammatory diseases such as immune complex vasculitis (e.g., when a relevant antigen is conjugated to a targeted immunoglobulin), antibody-mediated transplant rejection (e.g., when an antigen from a transplanted tissue or organ is conjugated to a targeted immunoglobulin), as well as infectious diseases (e.g., when an antigen of an infectious agent is conjugated to a targeted immunoglobulin) and even proliferative diseases (e.g., when a cancer antigen is conjugated to a targeted immunoglobulin by a therapeutic protein).

[0032] Furthermore, in some examples where the immunotherapeutic protein is intended to be used for treating or preventing a disease or condition through binding to and activating FcγRIIb, the immunotherapeutic protein may comprise a binding domain, such as an antigen binding region, targeted to an activating Fc receptor or one or more subunits thereof, such as an immunoglobulin Fc binding subunit or associated subunits required for expression and / or signaling, regardless of whether an immune complex is bound to the activating Fc receptor (or one or more subunits) (e.g., the binding domain may be targeted to any of the subunits of FcεRI, which is composed of the ligand binding chain FcεRIα subunit, and the associated FcεRIβ and γ subunits). Thus, such targeted immunotherapeutic proteins can also be used for the treatment or prevention of allergic diseases (e.g., when immune complexes containing allergens bind to activated FcR), autoimmune diseases such as SLE and MS (e.g., when immune complexes containing autoantigens bind to activated FcR), other inflammatory diseases such as immune complex vasculitis (e.g., when immune complexes containing related antigens bind to activated FcR), antibody-mediated transplant rejection (e.g., when immune complexes containing antigens from a transplanted tissue or organ bind to activated FcR), and infectious diseases (e.g., when immune complexes containing antigens of an infectious agent bind to activated FcR).

[0033] Furthermore, in some instances where the immunotherapeutic protein is intended to be used for the treatment or prevention of a disease or condition by binding to and activating FcγRIIb, the immunotherapeutic protein may comprise a binding domain, such as an antigen-binding region, targeted to an antigen of interest bound to the B cell receptor complex (BCR) (i.e., the antigen is bound to membrane IgE, IgG or IgA on the surface of the B cell). Thus, such targeted immunotherapeutic proteins can be used for the treatment or prevention of allergic diseases (e.g., when the immunotherapeutic protein binds to an antigen bound to membrane immunoglobulin of the BCR), such that co-crosslinking of FcγRIIb to the BCR containing the bound antigen results in activation of FcγRIIb, thereby recruiting the FcγRIIb inhibitory effect that stops antibody production (and / or B cell proliferation). Similarly, such immunotherapeutic proteins can be used for the treatment or prevention of autoimmune diseases such as SLE and MS, other inflammatory diseases such as immune complex vasculitis, antibody-mediated transplant rejection, and infectious diseases.

[0034] Furthermore, in some instances where the immunotherapeutic protein is intended to be used for the treatment or prevention of a disease or condition by binding to and activating FcγRIIb, the immunotherapeutic protein may comprise a binding domain, such as an antigen-binding region, that is targeted to an activating receptor other than an Fc receptor, such as the B cell antigen receptor (BCR) complex. For example, the immunotherapeutic protein may be targeted to a membrane immunoglobulin of the BCR complex (e.g., membrane IgE, IgG, or IgA on the surface of a B cell), for example, by targeting a variable domain of the membrane immunoglobulin (e.g., the immunotherapeutic protein may be an anti-idiotypic IgG2 antibody). The membrane immunoglobulin of the targeted BCR may or may not comprise a bound antigen. Thus, such targeted immunotherapeutic proteins can be used for the treatment or prevention of allergic diseases (e.g., when the immunotherapeutic protein binds to membrane immunoglobulins of the BCR complex with or without bound allergen), such that co-crosslinking of FcγRIIb to the BCR results in activation of FcγRIIb, thereby recruiting the FcγRIIb inhibitory effect that stops antibody production (and / or B cell proliferation).Similarly, such immunotherapeutic proteins can be used for the treatment or prevention of autoimmune diseases such as SLE and MS, other inflammatory diseases such as immune complex vasculitis, antibody-mediated transplant rejection, and infectious diseases. In addition, immunotherapeutic proteins targeted to non-Fc type activating receptors such as BCR can also be used for the treatment or prevention of proliferative diseases, particularly lymphoproliferative diseases (LPDs), such as leukemias (e.g., acute lymphoblastic leukemia (ALL) and chronic lymphocytic leukemia (CLL)), lymphomas (e.g., B-cell lymphomas and T-cell lymphomas), and X-linked proliferative diseases, where the ability of the immunotherapeutic proteins to effect co-crosslinking of FcγRIIb to BCR can in turn cause activation of FcγRIIb, thereby recruiting FcγRIIb inhibitory effects to stop the proliferation of cancerous cells.

[0035] Additionally, in some instances where the immunotherapeutic protein is intended to be used for the treatment or prevention of a disease or condition through binding to and activation of FcγRIIb, the immunotherapeutic protein may comprise a binding domain, such as an antigen-binding region, targeted to the B cell antigen receptor (BCR) complex or one or more subunits thereof, e.g., a membrane immunoglobulin or associated subunit required for expression and / or signaling (e.g., the binding domain may be a subunit of the BCR complex, which is composed of antigen-binding membrane immunoglobulins (e.g., IgM, IgD, IgG, IgE, or IgA), as well as associated Ig-α or β chains (e.g., CD79a or CD79b) or other associated proteins (e.g., CD19, CD21, or CD81). The immunotherapy protein may be targeted to any of the units. The membrane immunoglobulin of the targeted BCR may or may not contain a bound antigen. Thus, such targeted immunotherapeutic proteins can also be used for the treatment or prevention of allergic diseases (e.g., when an allergen may or may not be bound to the BCR complex), autoimmune diseases such as SLE and MS (e.g., when an autoantigen is bound to the BCR complex), other inflammatory diseases such as immune complex vasculitis (e.g., when a related antigen is bound to the BCR complex), antibody-mediated transplant rejection (e.g., when a related antigen from a transplanted tissue or organ is bound to the BCR complex), and infectious diseases (e.g., when a related antigen of an infectious agent is bound to the BCR complex).

[0036] Allergic diseases Mast cells and basophils are two important effector cells in the pathogenesis of allergic diseases, and both express the high affinity IgE receptor FcεRI. However, they differ in their expression profile with basophils expressing the inhibitory receptor FcγRIIb, an IgG receptor that can inhibit FcεRI signaling and reduce basophil activation. Because the immunotherapeutic proteins of the disclosure, such as mutant IgG2 antibodies, can be targeted to bind allergens (in immune complexes bound to activating FcR on the surface of basophils) and also bind FcγRIIb resulting in co-crosslinking to activating FcR, thereby activating FcγRIIb (i.e., recruiting FcγRIIb inhibitory function; more specifically, binding of mutant IgG2 antibodies to FcγRIIb mediates FcγRIIb-dependent inhibition of allergic basophil activation by IgE), the methods of the disclosure enable the treatment or prevention of allergic diseases and conditions such as severe hay fever, atopic dermatitis, food allergies such as peanut allergies, and allergies to toxins (e.g., bee venom). The subset of human mast cells expressing FcγRIIb may also act to regulate allergic diseases and conditions (e.g., food allergy, Burton OT et al., J Allergy Clin Immunol 141(1):189-201.e3,2018) through its expression on a subset of human mast cells (Burton OT et al., Front Immunol 9:1244. doi: 10.3389 / fimmu 2018). Thus, the immunotherapeutic proteins of the present disclosure, such as mutant IgG2 antibodies, may mobilize FcγRIIb inhibitory function on mast cells. Desirably, the mutant IgG2 antibodies may also exhibit no or little binding capacity to FcγRI (which can induce potent mast cell activation) to avoid undesirable mast cell activation.

[0037] Autoimmune Diseases and Conditions For example, by promoting activation of FcγRIIb receptors present in an affected subject by co-crosslinking FcγRIIb to activating FcR via bound immune complexes that include an autoantigen, the methods of the disclosure allow for the beneficial treatment and / or prevention of autoimmune diseases and conditions, such as those described above. However, reduced expression and / or signaling activity of FcγRIIb in a subject (e.g., due to polymorphisms in the promoter and transmembrane domains of FcγRIIb that affect receptor expression and signaling) has been associated with increased susceptibility to autoimmune diseases and conditions, including systemic lupus erythematosus (SLE), Goodpasture's syndrome, immune thrombocytopenia (ITP), and rheumatoid arthritis (RA) (Floto RA et al., Nat Med 11:1056-1058, 2005; Li X et al., Arthritis Rheum 48:3242-3252, 2003; and Radstake TR et al., Arthritis Rheum 54:3828-3837, 2006). Thus, in some embodiments where the methods of the disclosure are intended for use with subjects suffering from or prone to an autoimmune disease or condition, the methods may further comprise the step of selecting the subject by genotyping for relevant polymorphisms in the promoter and transmembrane domain of FcγRIIb (e.g., polymorphisms in the promoter region of FCGR2B (Su K et al., J Immunol 172:7186-7191, 2004, and Blank MC et al., Hum Genet 117:220-227, 2005), and the I232T polymorphism in the transmembrane domain of FcγRIIb (Kyogoku C et al., Arthritis Rheum 46:1242-1254, 2002)).

[0038] infectious disease Immunotherapeutic proteins such as mutant IgG2 antibodies according to the present disclosure can be used to target, for example, the regulation of ITAM receptor-based signaling of the BCR on B lymphocytes (i.e., by targeting ITAM receptors on cells that co-express FcγRIIb (ITIM receptors). That is, in B cells, FcγRIIb inhibition of the BCR is a key immune checkpoint for regulating antibody production (Lehmann B et al., Expert Rev Clin Immunol 8:243-254, 2012), possibly through apoptotic elimination of autoreactive B cells during somatic hypermutation (Pearse RN et al., Immunity 10:753-760, 1999), restricting the selective antigen specificity of the humoral immune system and directing B cell production towards the appropriate antibody repertoire. This can, of course, be beneficial in "fighting" infections with bacterial pathogens (e.g., Neisseria meningitides, Streptococcus pneumoniae, Haemophilus influenzae, and Methicillin-Resistant Staphylococcus aureus ("Golden Staph")) or viruses (e.g., Hepatitis C (HCV), and Human Immunodeficiency Virus-1 (HIV-1)).

[0039] Proliferative disorders The BCR of B lymphocytes has been shown to be involved in the pathogenesis of various B cell-derived lymphoid cancers, and increasing evidence suggests that antigen-independent self-association of the BCR is a key feature in many B cell neoplasm types, such as chronic lymphocytic leukemia (CLL), heavy chain disease (HCD), and activated B cell-like subtype diffuse large B cell lymphoma (ABC DLBCL) (Duhren-von Minden M et al., Nature 463(7415):309-312, 2012; Corcos D et al., Current Biology 5(10):1140-1148, 1995; and Davis RE et al., Nature 463(7277):88-92, 2010). Thus, the methods of the present disclosure may also be applied to the prevention and / or treatment of proliferative diseases, particularly lymphoid cancers of B cell origin, where the mutant IgG2 antibody can be used to activate FcγRIIb on B cells, for example, by co-crosslinking FcγRIIb to the BCR via the relevant antigen complexed to the BCR or a subunit of the BCR (i.e. targeted by the mutant IgG2 antibody), resulting in inhibition of BCR signaling.

[0040] Treating Diseases Through FcγRIIb-Mediated Endocytosis / Internalization ("Sweeping") In some other embodiments, the methods of the present disclosure may be used for the treatment of a disease or condition in a subject, where clearance of immune complexes (e.g., "small" soluble complexes including opsonized viruses, proteins (e.g., cytokines), and toxins; Iwayanagi Y et al., J Immunol 195:3198-3205, 2015, and Mates JM et al., Front Immunol 8:35, 2017) from the circulation by FcγRIIb present on the surface of leukocytes and some other non-hematopoietic cell types (e.g., hepatic sinusoidal endothelial cells (LSECs)) is beneficial in the treatment or prevention of the disease or condition. Mechanistically distinct from phagocytosis, which is carried out by activating Fc receptor-type and phagocytes (e.g., FcγRIII) and removes large immune complexes, including those large in bacteria, parasites, and cancerous cells, there is considerable interest in developing new therapeutics centered around this phenomenon (see, e.g., Iwayanagi Y et al., 2015 (supra), and Chenoweth AM et al., 2020 (supra)). Thus, in some examples of such embodiments, immunotherapeutic proteins such as mutant IgG2 antibodies can be targeted to, for example, antigens (e.g., viral antigens), or other proteins or chemicals such as immunoglobulins, hormones, metabolites, cytokines, or toxins, to enable the formation of small soluble immune complexes. Binding of the immunotherapeutic protein to the FcγRIIb receptor of LSECs can then mediate sweeping clearance of immune complexes from the circulation.Thus, such immunotherapeutic proteins can be used, for example, for the treatment or prevention of infectious diseases (e.g., viral infections such as SARS-CoV-2 virus infections, or infectious diseases characterized by the production of toxins such as endotoxins), and endocrine disorders such as Cushing's syndrome (Buliman et al., J Med Life 9:12-18, 2016), as well as inflammatory diseases characterized by, for example, cytokine overexpression, such as TNF receptor-associated periodic syndrome (TRAPS), characterized by overexpression of IL-1β from circulating monocytes during disease flares (Bachetti T et al., Ann Rheum Dis 72:1044-1052, 2013), psoriasis, characterized by the production of IL-17 or IL-23, rheumatoid arthritis, characterized by the production of cytokines such as TNF or IL-1β, autoimmune diseases, characterized by the production of autoantibodies, and allergic diseases, characterized by the production of IgE.

[0041] Treating disease with FcγRIIb-mediated scaffold formation In some other embodiments, the methods of the present disclosure may be used to treat a disease or condition in a subject, where enhanced agonistic function of the immunotherapeutic protein may be achieved by FcγRIIb "scaffolding," where no signal is generated in the effector cell, but "hypercrosslinking" of an opsonizing antibody (e.g., a mutant IgG2 antibody of the present disclosure) by FcγRIIb on one cell generates a signal in the conjugated target cell that may result in beneficial therapeutic effects, such as, for example, induction of apoptosis or activation in the agonistic proliferation of cells and / or their cytokine secretion (Chenoweth AM et al., 2020 (supra)). Thus, in some examples of such embodiments, an immunotherapeutic protein such as a mutant IgG2 antibody may be targeted to, for example, a cancer antigen present on the surface of a cancerous cell (e.g., CD20), or a cell surface antigen (e.g., CTLA4) present on an immune cell (e.g., T cell). Thus, such immunotherapeutic proteins may be used, for example, for the treatment or prevention of proliferative and autoimmune diseases.

[0042] From the above, it will be apparent that the immunotherapeutic proteins, preferably variant IgG2 antibodies, of the present disclosure typically comprise an antigen-binding region that specifically binds to a target, such as an antigen of interest, or an immunoglobulin, such as an antibody bound to an activating Fc receptor, an immunoglobulin of a BCR complex, or a subunit of an activating Fc receptor complex or a BCR complex. Thus, for example, where the method of the present disclosure is intended for use with a subject suffering from an allergic disease, the antigen-binding region of a variant IgG2 antibody according to the present disclosure may specifically bind to an antigen that is an allergen (e.g., bee venom allergen Api m 1, and peanut allergens Ara h 1, Ara h 2, Ara h 3, and Ara h 6). Similarly, in the context of the method of the present disclosure intended for use with a subject suffering from or prone to an autoimmune disease or condition, the antigen-binding region of a variant IgG2 antibody may specifically bind to an antigen that is an autoantigen (e.g., one of the common anti-Sm / RNP, anti-Ro / La, and anti-dsDNA autoantigens of SLE). Furthermore, if the method is intended for use in the treatment of an infectious disease, the antigen-binding region of the mutated IgG2 antibody may specifically bind to a pathogenic antigen (e.g., bound within the BCR complex) or may otherwise specifically bind to a membrane immunoglobulin of the BCR complex that contains the pathogenic antigen. Similarly, the antigen-binding region of a mutated IgG2 antibody intended for use in the treatment of a proliferative disease may specifically bind to a cancer antigen bound to the BCR complex, or alternatively, in a method involving FcγRIIb-mediated scaffolding, the antigen-binding region of a mutated IgG2 antibody may specifically bind to a cancer antigen present on the surface of a cancer cell (e.g., cell surface antigens that are differentially expressed and / or present on cancer cells, such as CD20 and CD52 antigens found on the surface of CLL cells, and mucins (e.g., MUC-1) that are overexpressed in some breast and pancreatic cancers, resulting in apoptosis of the cancer cell.

[0043] In some embodiments, a variant IgG2 antibody suitable for use in the methods of the disclosure may be provided as an immunoconjugate, in which the antibody is conjugated to another molecule, such as a molecule that provides additional capability of binding to an antigen of interest (e.g., a bispecific IgG2 antibody that comprises an antigen binding region with a first binding specificity and is linked to another molecule with a second binding specificity) or a molecule that provides other functionality (e.g., a detectable molecule such as a dye or a therapeutically important molecule such as a complementary drug molecule).

[0044] In some other embodiments, mutant IgG2 antibodies suitable for use in the methods of the present disclosure may be provided in dimeric or multimeric forms (e.g., three, four, five, six (hexamers), etc. copies of the mutant IgG2 antibody). Standard methodologies for producing dimeric and multimeric forms of antibodies (e.g., by stradomers™, or self-association of Fc regions of adjacent antibody molecules) are well known to those of skill in the art (see, e.g., Diebolder CA et al., Science 343(6176):1260-1263, 2014).

[0045] The methods of the present disclosure will typically be applied to the treatment of a disease or condition in a human subject. However, the subject may also be selected from, for example, livestock animals (e.g., cows, horses, pigs, sheep and goats), companion animals (e.g., dogs and cats), and exotic animals (e.g., non-human primates, tigers, elephants, etc.).

[0046] In a second aspect, the disclosure provides the use of an immunotherapeutic protein as defined in the first aspect for treating a disease or condition in which binding to and / or activation of FcγRIIb is beneficial, including, for example, allergic diseases, autoimmune diseases and conditions, infectious diseases, and proliferative diseases.

[0047] In a third aspect, the disclosure provides the use of an immunotherapeutic protein as defined in the first aspect in the manufacture of a medicament for the treatment of a disease or condition in which activation of FcγRIIb is beneficial, including, for example, allergic diseases, autoimmune diseases and conditions, other inflammatory diseases, infectious diseases, and proliferative diseases.

[0048] In a fourth aspect, the present disclosure provides a pharmaceutical composition or medicament comprising an immunotherapeutic protein as defined in the first aspect and a pharma- ceutically acceptable carrier, diluent and / or excipient.

[0049] In this specification, a number of terms are used that are familiar to those of ordinary skill in the art. Nevertheless, for the sake of clarity, some of these terms are defined below.

[0050] As used herein, the term "IgG2 antibody" refers to any heavy chain polypeptide comprising an IgG2 constant heavy (CH) region (i.e., the CH1, CH2 and CH3 domains are IgG2 CH1, CH2 and CH3 domains, each domain independently comprising an amino acid sequence that is identical to the wild-type sequence, or exhibits at least 95%, preferably at least 98%, identity to the wild-type sequence, e.g., as the wild-type human sequence previously described in detail in Wines BD et al., 2016 (supra)), such that the antibody comprises an "IgG2 backbone" that comprises an IgG2 Fc domain, including antibodies that comprise chimeric heavy chain polypeptides, e.g., in which the variable heavy chain (VH) region is derived from an antibody of another immunoglobulin class or subclass (e.g., IgA, IgE, IgG1, etc.), which may or may not be derived from the same species as the IgG2 CH region (e.g., an IgG2 antibody may comprise a heavy chain polypeptide comprising a human IgG2 CH region and a murine IgE VH region). It should further be understood that an "IgG2 antibody" may comprise a light chain polypeptide comprising a constant light (CL) region and / or a variable light (VL) region derived from IgG2 or any other immunoglobulin class or subclass, or that the light chain polypeptide is chimeric, where one of the regions is derived from an antibody from one immunoglobulin class or subclass and the other of the regions is derived from an antibody from another immunoglobulin class or subclass (e.g., an IgG2 antibody is a human Ig Κ The antibody may comprise a light chain polypeptide comprising a CL region and a VL region derived from IgE.

[0051] The term "target" and its derivatives such as "targeted" and "targeting" will be well understood by those skilled in the art depending on the context in which the term is used. For example, "target" will be understood to refer to something that an action or process is directed at. For example, when used herein in the context of an antibody characteristic / activity, "target antigen" will be understood to refer to the antigen that the antibody binds to, and similarly, "targeting" something will be understood to mean that an antibody (or other immunotherapeutic molecule) is prepared to bind to that something (e.g., an antigen, immune checkpoint, receptor, etc.).

[0052] As used herein, the term "% identity" between two amino acid sequences refers to the percentage of sequence identity that is understood to be calculated using a mathematical algorithm such as that described in Karlin S and SF Altschul, Proc Natl Acad Sci USA 87:2264-2268, 1990 and modified as in Karlin S and SF Altschul, Proc Natl Acad Sci USA 90:5873-5877, 1993. Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul SF et al., J Mol Biol 215:403-410, 1990. BLAST protein searches can be performed with the XBLAST program using default parameters (see ncbi.nlm.nih.gov / BLAST / ). To determine the percentage of sequence identity between two amino acid sequences, a mathematical algorithm can align the sequences for optimal comparison purposes and calculate the percent identity between the sequences as a function of the number of identical positions shared by the sequences (i.e., percent identity = number of identical positions / total number of positions (e.g., overlapping positions) x 100).

[0053] As used herein, the term "treating" includes prevention and alleviation of established symptoms of a disease or condition. Thus, the act of "treating" a disease or condition includes (1) preventing or delaying the appearance of clinical symptoms of a disease or condition that develop in a subject suffering from or prone to the disease or condition, (2) inhibiting the disease or condition (i.e., in the case of maintenance treatment, preventing, reducing, or delaying the onset of the disease or condition or its recurrence, or at least one of its clinical or subclinical symptoms), and (3) relieving or attenuating the disease or condition (i.e., causing regression of the disease or condition or at least one of its clinical or subclinical symptoms).

[0054] As used herein, the phrase "manufacturing a medicament" includes the use of one or more immunotherapeutic proteins at any stage in the manufacture of a medicament, either directly as defined in the first aspect or comprising one or more immunotherapeutic proteins as defined in the first aspect.

[0055] The term "effective amount" is an amount sufficient to produce a beneficial or desired clinical result. An effective amount can be administered in one or more administrations. Typically, an effective amount is sufficient to treat or otherwise alleviate, ameliorate, stabilize, reverse, delay, or slow the progression of a disease or condition. By way of example only, an effective amount of an immunotherapeutic protein, such as a mutant IgG2 antibody, may include from about 0.1 to about 250 mg / kg body weight per day, more preferably from about 0.1 to about 100 mg / kg body weight per day, and even more preferably from about 0.1 to about 25 mg / kg body weight per day. However, notwithstanding the above, it will be understood by those skilled in the art that an effective amount may vary and may depend on a variety of factors, including the age, weight, sex, and / or health of the subject being treated, the activity of the particular protein, the metabolic stability and duration of action of the particular protein, the route and time of administration of the particular protein, the rate of excretion of the particular protein, and, for example, the severity of the disease or condition being treated.

[0056] Immunotherapeutic proteins may be administered in combination with one or more additional agents for the treatment of the particular disease or condition being treated. For example, immunotherapeutic proteins may be combined with other agents for treating allergic diseases (e.g., antihistamines (administered intravenously (iv), including beta-agonist agents such as cortisone and / or albuterol)) or, in the context of treating proliferative diseases, immunotherapeutic proteins may be combined with other agents for treating cancer (e.g., anti-tumor agents such as cisplatin, gemcitabine, cytosine arabinoside, doxorubicin, epirubicin, taxoids including taxol, topoisomerase inhibitors such as etoposide, cytostatic agents such as tamoxifen, aromatase inhibitors (e.g., anastrozole), and inhibitors of growth factor function (e.g., antibodies such as the anti-erbB2 antibody trastuzumab (Herceptin™)).

[0057] When used in combination with other agents, the immunotherapeutic protein may be administered in the same pharmaceutical composition or in separate pharmaceutical compositions. When administered in separate pharmaceutical compositions, the immunotherapeutic protein and the other agent may be administered simultaneously or sequentially in any order (e.g., within seconds, or minutes, or within a few hours (e.g., 2-48 hours)).

[0058] The immunotherapeutic protein may be formulated into a pharmaceutical composition together with a pharma- ceutically acceptable carrier, diluent, and / or excipient. Examples of suitable carriers and diluents are well known to those skilled in the art and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, PA 1995. Examples of excipients suitable for the various different forms of pharmaceutical compositions described herein can be found in the Handbook of Pharmaceutical Excipients, 2001, pp. 211-215, 1995. ndEdition, (1994), Edited by A Wade and PJ Weller. Examples of suitable carriers include lactose, starch, glucose, methylcellulose, magnesium stearate, mannitol, sorbitol, and the like. Examples of suitable diluents include ethanol, glycerol, and water. The selection of carriers, diluents, and / or excipients can be made with regard to the intended route of administration and standard pharmaceutical practice.

[0059] The pharmaceutical composition comprising the immunotherapeutic protein defined in the first aspect may further comprise any suitable binder, lubricant, suspending agent, coating agent, and solubilizing agent. Examples of suitable binders include starch, gelatin, natural sugars such as glucose, anhydrous lactose, free lactose, beta-lactose, corn sweeteners, natural and synthetic gums such as acacia, tragacanth or sodium alginate, carboxymethylcellulose and polyethylene glycol. Examples of suitable lubricants include sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride, and the like. Preservatives, stabilizers, and even dyes may be provided in the pharmaceutical composition. Examples of preservatives include sodium benzoate, sorbic acid, and esters of p-hydroxybenzoic acid. Antioxidants and suspending agents may also be used.

[0060] The pharmaceutical composition comprising the immunotherapeutic protein defined in the first aspect is typically adapted for intravenous or subcutaneous administration.Accordingly, the pharmaceutical composition may be injected into a subject and may comprise a solution or emulsion prepared from a sterile or sterilizable solution.The pharmaceutical composition may be formulated in unit dosage form (i.e., in the form of a unit dose, or a separate portion comprising a multiple unit or subunit of a unit dose).

[0061] The methods, uses, and pharmaceutical compositions of the disclosure are further described below with reference to the following non-limiting examples. EXAMPLES

[0062] Example 1 Methods and Materials Antibodies and Reagents F(ab’)2 fragments of rabbit anti-human IgE (Dako Agilent, Santa Clara, CA <United States of America) were produced by pepsin digestion as described in Current Protocols in Immunology, Andrew SM and JA Titus, Chapter 2: Unit 2.8, 2001. Briefly, rabbit antibodies were dialyzed against digestion buffer (0.2 M NaOAc, pH 4.0), and then an equal volume of pepsin (0.1 mg / ml) (Sigma-Aldrich, St Louis, MO, United States of America) in digestion buffer was added and incubated overnight at 37°C. Digestion was stopped by adding 10% (v / v) of 2 M Tris base (pH 8.0), and then the digest was dialyzed against phosphate-buffered saline (PBS, pH 8.0). Hapten 2,4,6-trinitrophenyl (TNP) was conjugated to F(ab’)2 fragments of rabbit anti-human IgE (anti-IgE-TNP), bee venom allergen (Api m 1-TNP), or bovine serum albumin (BSA-TNP) by incubating with 10% 2,4,6-trinitrobenzenesulfonic acid (Sigma-Aldrich) in water, diluted 1 / 20 with 0.1 M borate (pH 7.0) for 90 minutes at room temperature, and then dialyzed against PBS (pH 7.0).

[0063] Human Donors Healthy donors and allergic patients were recruited and blood samples were collected. Patients presenting with bee venom allergy were tested for IgE reactivity against relevant allergens by ImmunoCAP (Phadia, Uppsala, Sweden).

[0064] Expression of Cell Surface FcγR Human FcγR was expressed in the mouse B cell line IIA1.6 lacking endogenous mouse Fc receptors. Human FcγRIIa-H 131, FcγRIIa-R 131 and FcγRIIb expressing cells have been described previously (e.g., Powell MS et al., J Immunol 176(12):7489-7494, 2006; Ramsland PA et al., J Immunol 187(6):3208-3217, 2011; and Trist HM et al., J Immunol 192(2):792-803, 2014). Human FcγRIIIa (GenBank: Accession NP_001121065) allelic form V 158 and F 158As well as human FcγRI (Allen JM and B Seed, Science 243(4889):378-381, 1989) expressing cell lines were generated as described for FcγRIIa (Powell et al., 2006, supra). Briefly, receptor cDNAs were separately cloned into the Gateway entry plasmid pENTR1A (Invitrogen Corporation, Waltham, MA, United States of America) using standard molecular biology techniques, and the Gateway LR was subsequently cloned into the Gateway-compatible pMXI expression vector containing a neomycin resistance cassette (Wines B et al., J Biol Chem 279(25):26339-26345, 2004). Retroviruses were generated using the Phoenix packaging cell line (Powell et al., 2006, supra) and used to transduce the IIA1.6 cell line to express FcγRIIIa or FcγRI. IIA1.6 cells, which already express the human FcRγ chain (Wines et al., 2004, supra), were then transduced with either FcγRI or FcγRIII retroviruses to generate cell lines co-expressing FcRγ / FcγRI or FcRγ / FcγRIII. Receptor expression on the transduced cell populations was assessed using biotinylated Fab or F(ab')2 fragments of receptor-specific antibodies and streptavidin-APC (1 / 500). The anti-Fc receptor antibodies used were FcγRIIa (IV-3 Fab-biotin fragment), FcγRIIb (H2B6 F(ab')2-biotin), FcγRI (32.2 F(ab')2-biotin), FcγRIIIa (3G8 F(ab')2-biotin), and FcRγ-EGFP (green fluorescent protein) and were detected in the FITC channel.

[0065] Generation of anti-TNP human IgG and mutant anti-TNP human IgG plasmid constructs Chimeric anti-TNP human IgG antibody constructs consisting of variable heavy (VH) and light (VL) region sequences of the murine anti-trinitrophenyl (anti-TNP) antibody TIB142 and sequences from the constant heavy (CH) region from human IgG subclasses have been described in detail previously - IgG1 (Patel D et al., J Immunol 184(11):6283-6292, 2010), hIgG2 and hIgG4 (Wines BD et al., 2016 (supra)). This includes stabilization of the core hinge of the IgG4-based mutants by converting the CPSC sequence of normal IgG4 to CPPC, which prevents half molecule exchange. All chimeric antibody sequences were subcloned into pCR3 vector. Mutations were made in the IgG constant heavy chain (CH) cDNA sequence using standard molecular biology techniques and are listed in Table 1. [Table 1]

[0066] The cDNA sequences encoding the constant regions of the IgG heavy chain polypeptide (CH) for IgG2-FEGG-SELF and IgG2-FLGG-SELF are provided in Table 2 and are shown ligated to the cDNA sequence for the variable heavy (VH) region. The cDNA sequence for the variable light (VL) region of the murine (anti-TNP) antibody is also provided. [Table 2] JPEG2024500945000003.jpg211169 JPEG2024500945000004.jpg205169 JPEG2024500945000005.jpg210169 JPEG2024500945000006.jpg78169 JPEG2024500945000007.jpg142169 JPEG2024500945000008.jpg110169

[0067] Production of anti-human IgE and mutant anti-human IgE plasmid constructs The anti-human IgE antibody construct contained synthetic DNA encoding the variable heavy (VH) and light chain region sequences of the therapeutic mAb omalizumab (ThermoFisher, GeneArt, Waltham, MA, United States of America). The H chain construct contained the VH cDNA sequence and constant domain sequences for IgG4 and IgG2 variants. DNA for IgG heavy and light chains were subcloned into the expression vectors pCR3 or pcDNA3.4. Mutations in the IgG2 constant heavy (CH) cDNA sequence that affect binding are listed in Table 1.

[0068] The cDNA sequences encoding the constant regions of the IgG heavy chain polypeptide (CH) or the anti-IgE light chain (CL), IgG4, and IgG2-FEGG-SELF and IgG2-FLGG-SELF are provided in Table 3 and are shown ligated to the cDNA sequence for the variable heavy (VH) region. The cDNA sequence for the anti-IgE antibody light chain is also provided. [Table 3] JPEG2024500945000010.jpg210169 JPEG2024500945000011.jpg210169 JPEG2024500945000012.jpg211169 JPEG2024500945000013.jpg210169 JPEG2024500945000014.jpg32169 JPEG2024500945000015.jpg196169 JPEG2024500945000016.jpg208169 JPEG2024500945000017.jpg226169 JPEG2024500945000018.jpg210169 JPEG2024500945000019.jpg51169 JPEG2024500945000020.jpg172169 JPEG2024500945000021.jpg66169

[0069] Expression and production of human IgG and mutant IgG proteins by Expi293 cells Human IgG and mutant human IgG were produced in Expi293 human embryonic kidney cells as previously described (Wines et al., 2016, supra). Briefly, Expi293 cells were maintained in Expi293 expression medium (Gibco, Waltham, MA, United States of America) for both cell growth and protein production. Cells were co-transfected with IgG heavy chain plasmid (15ug) and light chain plasmid (15μg) diluted in Opti-MEM I reduced serum medium (Gibco) using the Expifectamine transfection kit (Life Technologies Corporation, Carlsbad, CA, United States of America) and then cultured for 4 days. Culture supernatants were clarified by centrifugation and filtered through 0.2 μm filters, after which IgG was purified by affinity chromatography using a Hi-Trap HP Protein A column (GE Healthcare Life Sciences, Marlborough, MA, United States of America) and eluted with 0.1 M citric acid (pH 3.5), followed by neutralization with 1 M Tris-HCl (pH 9.0) and dialysis against PBS (pH 7.5). Any aggregates were removed by subsequent gel filtration on a Superose 6 10 / 300GL column (GE Healthcare Biosciences), and monomeric IgG peak fractions were collected. Antigen-binding activity of all antibody preparations was tested on BSA-TNP by ELISA as described (Wines et al., 2016 (op. cit.)).

[0070] Flow cytometric measurement of IgG binding to cell surface FcγR Antibody FcγR binding was measured using either immune complexes or monomeric IgG. Immune complexes were generated by incubating anti-TNP antibodies (parent or mutant anti-TNP IgG) with TNP-BSA in a 2:1 ratio (40 μg / ml:20 μg / ml) for 30 min at 37° C., followed by 10 min at 4° C. For flow cytometry binding analysis, complexes or monomeric IgG were incubated at the indicated concentrations with 25 μl of FcγR-expressing cells (5×10 6 Cells were added to 50 μl of PBS / 0.5% BSA and 10,000 viable cells were analyzed in at least triplicate experiments. Monomeric IgG binding (MFI) was fitted to a single binding site model to determine binding affinity (K A Similarly, immune complex binding was measured using the apparent affinity (K A app )

[0071] Measuring the affinity of IgG:FcR interactions using Bio-Layer Interferometry (OCTET) TNP-BSA was reacted with EZ-link™ biotinylation reagent (ThermoFisher Scientific) according to the manufacturer's instructions. The resulting TNP-BSA-biotin (5 μg / ml, 30 sec) was captured to approximately 0.8 nm on a streptavidin BLI probe using Octet Red96 (ForteBio; Molecular Devices LLC, San Jose, CA, United States of America) and then loaded with anti-TNP IgG (4 μg / ml, 75 sec). A baseline was established (60 sec), followed by rsFcγRIIa-R. 131 Association and dissociation of a concentration series of rsFcγRIIb (15 nM to 20 μM) was performed (90 s). 10 mM HCl was used for regeneration after each initial reaction TNP-BSA-biotin capture and subsequent binding cycles. Sensograms were fitted to a 1:1 Langmuir binding model or the binding response at the end of the association cycle was fitted to steady-state affinity.

[0072] Production and purification of honeybee venom allergen: Api m 1 The major allergen from honeybee (Apis mellifera) venom: phospholipase A2 (Api m 1) (GenBank X16709, allergen name: Api m 1) was produced in the suspension-adapted insect cell line Spodoptera frugiperda Sf21 according to the manufacturer's instructions (Gibco). Briefly, Sf21 cells were maintained at 27°C in Sf-900 II SFM medium for growth, virus production, and protein production. A cDNA encoding full-length Api m 1 with a 3' hexa-His tag was cloned into the donor plasmid pFastBac and then transfected into DH10Bac E. coli. The resulting bacmid DNA was purified from DH10Bac E. coli cells and transfected into Sf21 cells. The recombinant baculovirus was then used to infect Sf21 cells, and the secreted Api m 1 was purified from the cell culture supernatant by Talon Superflow Metal Affinity chromatography (Clontech, Mountain View, CA, United States of America).

[0073] Basophil Activation Test (BAT) BAT assays were performed as previously described (Drew AC et al., J Immunol 173(9):5872-5879, 2004) using one of two cell sources: whole (raw) blood or blood washed twice with DMEM / 0.1% BSA. Basophils were stimulated with haptenized (TNP) rabbit F(ab')2 anti-human IgE (anti-IgE-TNP) or haptenized bee venom allergen (Api m 1-TNP) in the presence or absence of IgG mAb. Briefly, 100 μl of heparinized human whole blood from either healthy donors or allergic patients was incubated with 20 μl of stimulation buffer (133 mM NaCl, 20 mM Hepes, 7 mM CaCl2, 5 mM KCl, 3.5 mM MgCl2, 1 mg / ml BSA, 20 μl / ml heparin, 2 ng / ml IL3, pH 7.4) for 10 min at 37° C. Samples were then stimulated for 20 min at 37° C. by adding 100 μl of either anti-hIgE-TNP (20 μg / ml) or Api m 1-TNP (4 μg / ml) that had been precomplexed with anti-TNP hIgG (30 min at 37° C.). Background stimulation was determined by adding 100 μl of stimulation buffer alone. Positive controls for stimulation included either N-formyl-Met-Leu-Phe (fMLP, 9ug / ml) (Sigma-Aldrich) or intact rabbit anti-human IgE (10μg / ml) (Dako Agilent, Santa Clara, CA, United States of America). Assays were terminated by incubation on ice for 5 minutes followed by the addition of normal goat serum (10μl) (Sigma-Aldrich). Stimulation responses were quantified by flow cytometry.Therefore, after stimulation, cells were stained for 40 min on ice by adding mouse anti-human CD63-PE (2 ul / test) (BD Biosciences, Franklin Lakes, NJ, United States of America), mouse anti-human IgE-FITC (3 ul / test) (eBioscience, San Diego, CA, United States of America) and mouse anti-human CD203c-APC (5 ul / test) (Miltenyi Biotec, Auburn, CA, United States of America). After staining, red blood cells (RBCs) were lysed by incubation with 2 ml of lysis solution (154 mM NH4Cl, 10 mM KHCO2, 0.8 mM EDTA) for 10 min twice at room temperature and centrifugation (250 x g, 5 min). The cell pellet was washed with 3 ml of wash buffer (133 mM NaCl, 20 mM Hepes, 5 mM KCl, 0.27 mM EDTA, pH 7.3) and resuspended for flow cytometry analysis in 200 μl of wash buffer containing 7-aminoactinomycin D (2 μl / test) (BD Biosciences) to exclude non-viable cells.

[0074] Cells were analyzed on a FACS CantoII cytometer and fluorescence data was analyzed using Flowlogic analysis software. The gating strategy used was as follows: Washed blood or whole blood cells were gated on forward and side scatter to include the basophil population, followed by live cell gating based on the exclusion of 7AAD positive cells. Basophils were identified as high IgE expression (FITC high) and CD203c (APC positive) cells, which were then used to set a CD63 negative gate (stimulation buffer alone) and a CD63 positive gate (stimulated with anti-human IgE or fMLP). Activated basophils were identified as cells within the CD63 positive gate. The % inhibition of basophil activation was calculated as the % reduction in CD63 positive cells induced by Api m 1-TNP or anti-IgE-TNP:IgG complexes compared to Api m 1-TNP or anti-IgE-TNP stimulation alone.

[0075] Blockade of FcγRIIb inhibitory effects in the basophil activation test (BAT) The interaction of FcγRIIb with anti-IgE-TNP:IgG complexes in the BAT was blocked by incubation of cells with the F(ab')2 fragment of H2B6, an FcγRIIb-specific blocking mAb, prior to the addition of anti-IgE-TNP:IgG in the BAT as described above. Briefly, 10 μl of anti-H2B6 F(ab')2 (final concentration 7.5 μg / ml) or stimulation buffer alone was added to 90 μl of washed blood and incubated on ice for 30 min, after which the BAT was performed with anti-IgE-TNP or anti-IgE-TNP:IgG (final concentration 5 μg / ml) and % basophil activation was measured.

[0076] Cell surface co-expression of human high affinity FcεRI complexes with inhibitory hFcγRIIb Cells expressing FcεRI complexes (FcεRIα, β, γ) together with inhibitory FcγRIIb were generated by transduction of IIA1.6 cells with codon-optimized cDNAs encoding each FcR or subunit interrupted by an intervening picornavirus ribosomal skipping 2A peptide (Szymczak AL et al., Nat Biotechnol 22(5):589-594, 2004). The sequence was FcεRIα-P2A-FcεRIβ-T2A-FcεRIγ-F2A-FcγRIIb-translation stop. This was synthesized as a sequence-verified 2863 bp synthetic DNA (ThermoFisher, GeneArt, Waltham, MA, United States of America) with flanking Gateway attB1 and attB2 sites. The synthetic DNA was subcloned into the Gateway-adapted murine leukemia virus expression vector pMXI-neo. Transient transfection of the Phoenix packaging line and infection of the FcR-deficient murine IIA1.6 cell line were performed as previously described (Powell et al., 2006, supra).

[0077] IgE-induced calcium mobilization IIA1.6 cells co-expressing inhibitory hFcγRIIb and hFcεRI (α, β, γ) complexes were sensitized with IgE by overnight incubation with 0.5 μg / ml IgE (JW8 / 5 / 13 mouse / human chimeric anti-NP IgE) (Bruggemann M et al., J Exp Med 166:1351-1361, 1987). The next day, cells were stimulated with 20 μg / ml anti-IgE-T alone or in complex with anti-TNP IgG mAb (final concentration, 35 μg / ml) and calcium mobilization was measured (Anania JC et al., 2018 (ibid.)).

[0078] Cell surface co-expression of chimeric anti-NP IgE BCR and inhibitory hFcγRIIb Reporter cells expressing JW8 / 5 / 13 mouse / human chimeric anti-NP (4-hydroxy-3-nitrophenylacetyl) IgE BCR containing mouse V domains (and human cell surface IgE heavy chains containing transmembrane and cytoplasmic domains) and light chains, and co-expressing inhibitory FcγRIIb, were generated by transduction of IIA1.6 cells with codon-optimized cDNAs encoding a single polyprotein containing light chains, IgE heavy chains, and FcγRIIb1 separated by picornavirus ribosomal skipping P2A and F2A peptides (Szymczak AL et al., Nat Biotechnol 22(5):589-594, 2004). Thus, the DNA sequence encoding the polyprotein was composed of anti-NP IgE light chain cDNA, then P2A peptide, then IgE heavy chain, then F2A peptide, then FcγRIIb1, and then translation stop. The mouse / human chimeric anti-NP heavy chain sequence contained the appropriately spliced ​​JW8 / 5 / 13 anti-NP IgE DNA sequence (Bruggemann μ et al., J Exp Med 166:1351-1361, 1987) with sequence accession number X63693.1 (H. sapiens germline alternatively spliced ​​IgE heavy chain DNA). It was synthesized as sequence-verified synthetic DNA (ThermoFisher, GeneArt) with flanking Gateway attB1 and attB2 sites. The synthetic DNA was subcloned into the Gateway-compatible murine leukemia virus expression vector pMXI-neo. Transient transfection of the Phoenix packaging line and infection of the FcR-deficient murine IIA1.6 B cell line were performed as previously described (Powell et al., 2006, supra).

[0079] IgE B cell receptor (BCR)-induced calcium mobilization IIA1.6 cells co-expressing anti-NP surface IgE and inhibitory hFcγRIIb were loaded with the calcium indicator Fura-2 and incubated with the IgE-specific therapeutic mAb omalizumab or variants, i.e. omalizumab formatted as IgG4, IgG2, IgG2-FEGG, IgG2-FLGG, IgG2-FEGG-S267E-L328F, IgG2-FLGG-S267E-L328F- (1 μg / ml), followed by the NP-associated antigen NIP(22)BSA (bovine serum albumin derivatized with an average of 22 4-hydroxy-3-iodo-5-nitrophenylacetyl groups per BSA molecule). Calcium mobilization was measured (Anania JC et al., 2018 (op. cit.)).

[0080] result Mutant IgG2 antibodies with modified Fc generate potent specific basophil inhibitors. Using IgG2 as a scaffold for sequence elements from IgG4 and IgG1 (Table 1), a series of inhibitory mAbs were developed focusing on the lower hinge region, which differs between IgG subclasses and is a key contact with FcγR. In particular, the VAG of the IgG2 lower hinge was replaced with either FLGG from IgG4 (IgG2-FLGG) or LLGG from IgG1 (IgG2-LLGG). The mutant IgG2 mAbs were evaluated for their FcγR binding specificity against different human FcγRs expressed on the cell surface. The results are shown in Figure 1.

[0081] The interaction of the mutant IgGs with human FcγRs on the cell surface was assessed by flow cytometry and compared to the binding of parental IgG2 or IgG4, as well as to IgG1, the "universal" ligand for all human FcγRs. Binding to low affinity human FcγRs (FcγRIIb, FcγRIIa, and FcγRIII) was performed using immune complexes (Figure 1A-E). Binding to the high affinity FcγRI was measured using monomeric IgG (Figure 1F). Each of the parental IgG molecules showed the expected specificity (i.e., complexed IgG1 bound to all receptors, IgG2 bound only to the inhibitory FcγRIIb, FcγRIIa-H).131 (Uncomplexed monomeric IgG1 and IgG4, but not IgG2, bound to the high affinity FcγRI.)

[0082] The IgG2-LLGG or IgG2-FLGG mAb FcγR binding profiles were found to be significantly different from IgG2 and, in the case of IgG2-FLGG, also from IgG4 (Figures 1A-E). The IgG2-LLGG mAb showed a specificity profile comparable to the parental IgG1 (Figures 1A-E). Thus, replacement of only the lower hinge in IgG2 was sufficient to confer IgG1-like binding, including binding to inhibitory FcγRIIb. Moreover, the receptor binding profile of IgG2-FLGG is distinct from both parental IgGs (Figure 1). Notably, this mutant mAb exhibits significantly enhanced binding to inhibitory FcγRIIb as well as broader specificity compared to IgG4, binding both to FcγRIIIa allelic forms that do not bind either IgG2 or IgG4, and to FcγRIIa-R, which is normally also a poor binder of IgG2 and IgG4. 131 It also bound avidly to FcγRIIa-H. 131 Binding to FcγRI is likely a contribution from the IgG2 backbone. However, similar to monomeric IgG4, monomeric IgG2-FLGG bound to the high affinity FcγRI, which does not bind IgG2 (FIG. 1F).

[0083] Mutant IgG2 antibodies inhibit Api m 1 allergen-induced basophil activation The mutant IgG2 mAbs were also evaluated for their ability to mediate FcγRIIb-dependent inhibition of allergic basophil activation by IgE. In particular, IgG2-FLGG, IgG4-LLGG and IgG2-LLGG antibodies were compared with parental IgG2, IgG4 and IgG1 for their ability to modulate FcεRI activation of basophils from IgE+ atopic individuals (honeybee venom allergic patients). Basophils in washed blood were stimulated with phospholipase A2 (Api m 1-TNP), a major honeybee venom allergen, in the presence of anti-TNP IgG2 or IgG4 mAbs. The results are shown in Figure 2.

[0084] Most surprisingly, an almost complete inhibition of Api m 1-induced basophil activation was mediated by IgG2-LLGG and IgG2-FLGG (81% and 85% inhibition, respectively), whereas, as expected, the parental IgG2, which does not bind FcγRIIb, did not inhibit the Api m 1 response. Surprisingly, the parental IgG4 and IgG1, both of which avidly bind FcγRIIb, showed a relatively weak inhibition, achieving only 42% and 45% inhibition, respectively, at the highest concentration used (2 μg / ml).

[0085] Mutations in the lower hinge improve mAb specificity for FcγRIIb Specificity for FcγRIIb interactions was further improved by additional mutations in the lower hinge (Figure 1) and assessed for potency in the BAT assay using washed blood (i.e., plasma-free) or whole blood (i.e., containing physiological levels of IgG), the results of which are shown in Figures 2, 3, and 7.

[0086] First, L 235A point mutation in E was introduced into the FLGG lower hinge sequence of the parental IgG4-WT and IgG2-FLGG mAbs to generate IgG4-FEGG and IgG2-FEGG, respectively. This mutation has been commonly described to eliminate FcγR binding (Alegre ML et al., J Immunol 148(11):3461-3468, 1992) and has been used to inactivate FcγR binding in some antibodies (Reddy MP et al., J Immunol 164(4):1925-1933, 2000). Here, however, binding to FcγRIIb was found to be retained.

[0087] L in IgG2-FEGG and IgG4-FEGG 235 The E mutation eliminated FcγRI binding of the original unmodified IgG2-FLGG and IgG4-WT to nearly the baseline level of the parent IgG2-WT (Figure 1F). Other scaffold-dependent differences were also evident. On the IgG2 scaffold, the IgG2-FEGG and IgG2-FLGG mutants not only bound FcγRIIb but also FcγRIIa-H 131 and R 131 Both IgG2-FEGG mAbs displayed similar low affinity FcγR binding profiles, including the ability to readily bind both alleles (Fig. 1A-E), with binding of IgG2-FEGG to both alleles of FcγRIIIa being only relatively slightly reduced (Fig. 1D, E). However, this contrasts sharply with the effect of the same mutations on the IgG4 backbone in the IgG4-FEGG mAb (Fig. 1), where binding was strongly reduced in the context of the SELF mutation (Fig. 5F).

[0088] The variants were then evaluated in washed blood BAT (Figure 2), where, despite the apparent preferential and improved binding to FcγRIIb (see Figure 1A), IgG4-FEGG showed a surprisingly weak level of inhibition of basophil activation (42%), which was essentially equivalent to IgG4-WT (45%). In contrast, the equivalent sequence on an IgG2 backbone, the IgG2-FEGG antibody, retained the more potent inhibition seen with the original IgG2-FLGG variant and also with IgG2-LLGG (Figure 2).

[0089] Modification of CH2 for improved mAb affinity and inhibitory potency To further improve FcγRIIb specificity, two additional residues in CH2 of the Fc domain were modified (see Table 1). In particular, the two mutant S 267 E and L 328 F(SELF) (Chu SY et al., Mol Immunol 45(15):3926-3933, 2008) was introduced into IgG2 and IgG4 based mAbs, IgG2-FLGG, IgG2-FEGG and IgG4-FEGG, as well as the parental IgG4 antibody. The antibodies were then tested for specificity (see Figures 4-6) and inhibitory potency in allergen-specific (Figure 3) and anti-IgE BATs (Figure 7). Several effects became evident: introduction of the SELF mutation significantly increased the affinity (>70-fold increase) and altered the specificity of binding of both monomeric IgG (Figure 6) and conjugated IgG (Figure 5). Monomeric IgG2-FLGG-SELF and IgG2-FEGG-SELF, respectively, increased the affinity of binding to the cell surface (K A 89 and 32 x 10 6 M -1 ) (Figure 6A), and BLI analysis (K A 103 and 29 x 10 6 M -1 ) (Figure 4 and Table 4), which is a 70-120-fold increased affinity compared to the equivalent mAb without the SELF mutation (Figure 4 and Table 4). The IgG1-SELF mutants showed high affinity binding as previously reported (Chu SY et al., Mol Immunol 45:3926-3933, 2008). Notably, these antibodies with SELF mutations preferentially bind to FcγRIIa-R rather than to the inhibitory FcγRIIb on cells. 131 (as confirmed by BLI analysis, as shown in Figure 6A,B, and Figure 4 and Table 4).

[0090] This newly acquired high affinity binding was also reflected in a large increase in immune complex avidity (Figure 5). In addition, the unexpected immune complex binding to the FcγRIII form of the original IgG2-FLGG and IgG2-FEGG mAbs (Figure 1D,E) was eliminated by the SELF mutations (Figure 5). The impact of including the SELF mutation on the IgG2 backbone was also evident in the equivalent IgG4 mAbs (Figure 5), where the binding of FcγRIIb and FcγRIIa-R 131 Increased binding to FcγRIIa-H was also observed, but 131 No increase in binding to

[0091] Despite the large changes in affinity and specificity of interaction with low affinity FcγR induced by the SELF mutations, the interaction with FcγRI was not affected, and IgG2-FLGG, IgG2-FLGG-SELF showed identical binding to the IgG1-SELF mutant antibody (Figures 6F and 7D). Moreover, importantly, it was found that the SELF mutations in IgG2-FEGG-SELF and IgG4-FEGG-SELF did not abolish the ablation of FcγRI binding by the L235E mutation observed in the original IgG2-FEGG or Ig4-FEGG (Figure 1F). Thus, this combination of antibody mutations resulted in not only a more restricted specificity but also tighter binding to the inhibitory FcγRIIb. [Table 4]

[0092] Inhibition of basophil activation in whole blood from allergic patients and healthy donors The mutant IgG2 mAbs were also evaluated for their inhibitory potency in whole blood, i.e., in the presence of physiological levels of IgG, using either the allergen Api m 1-T (Figure 3) or anti-IgE-TNP (Figure 7), two distinct IgE-dependent stimuli. Compared to the corresponding IgG4 variants, the IgG2-based mAbs (Figure 7A) were more potent inhibitors of allergen-induced activation than the IgG4-mAbs (Figure 7B). Indeed, IgG4, IgG4-FEGG, and IgG4-LLGG (Figure 2), which inhibited activation in washed blood, showed very low inhibition in whole blood (8%, 8%, and 13% inhibition, respectively). In contrast, despite the presence of physiological IgG, IgG2-FLGG, which contains the lower hinge of IgG4 and binds FcγRIIb with low affinity, retained substantial inhibition (54%, IC 50 = 1.6 μg / ml). In addition, the results showed that the potency of the IgG2 mAb was significantly increased by the inclusion of the SELF mutation in the CH2 region. For example, the binding of IgG2-FLGG-SELF and IgG2-FEGG-SELF was IC 50 = 0.24 μg / ml and 0.38 μg / ml. Interestingly, mutant IgG2 mAbs with SELF mutations were substantially more potent than their IgG4 lower hinge equivalents (e.g., IgG4-SELF IC 50 = 1.1 μg / ml compared to IgG2-FLGG-SELF with IgG4 lower hinge (IC 50 = 0.24 μg / ml), indicating that the nature of the IgG backbone is a key factor determining the inhibitory potency of these engineered antibodies.

[0093] To ensure that efficacy was not specific to the allergen system, the mutant IgG2 mAbs were also tested in a second IgE / FcεRI-dependent system: anti-IgE-TNP stimulated whole blood basophils were assayed, revealing that the relative potency of antibody-mediated inhibition was significantly greater than that seen with Api m 1-T allergen stimulation, i.e.

number

number

[0094] The inhibitory function of mutant IgG2 antibodies is compromised by FcγRIIb blockade. The expected expression of inhibitory FcγRIIb receptors on blood basophils (Kepley CL et al., J Allergy Clin Immunol 106:337-348, 2000) was confirmed by flow cytometry (see FIG. 8A). The FcγRIIb dependency of inhibition of IgE / FcεRI basophil activation by IgG mAbs was assessed by blocking FcγRIIb expressed on basophils using H2B6, an FcγRIIb-specific mAb (FIG. 8B). Pretreatment of whole blood with H2B6 F(ab')2 fragments prior to stimulation with anti-IgE-TNP in the presence of IgG2-FLGG significantly reduced the potency of IgG2-FLGG (FIG. 8B).

[0095] Inhibition of FcεRI-induced calcium mobilization The mutant IgG2 antibodies were also tested for FcγRIIb-dependent regulation of IgE:FcεRI-induced calcium mobilization (Figure 8C). Cells co-expressing FcγRIIb and FcεRI (αβγ2) complexes were sensitized with IgE and stimulated with anti-IgE-TNP in the presence of parental anti-TNP IgG2 or IgG2-based anti-TNP mutant mAbs (Figure 8C). Inhibition of IgE / FcεRI calcium mobilization by mAbs correlated with their affinity for FcγRIIb and their potency of inhibition on both allergen- or anti-IgE-induced activation of basophils (Figures 3 and 8). IgG2-FLGG-SELF and IgG2-FEGG-SELF antibodies showed the greatest inhibition of calcium mobilization, causing a similar reduction in the magnitude and kinetics of the response. Interestingly, IgG2-FLGG antibodies, which did not contain the SELF mutation, inhibited IgE / FcεRI Ca mobilization. 2+ There was still a substantial reduction in response (FIG. 8C), consistent with its inhibition of basophil activation in whole blood (FIG. 3).

[0096] Surface IgE inhibits B cell antigen receptor-induced calcium mobilization The therapeutic mAb omalizumab was reformatted as IgG4 and IgG2 antibodies and these were tested for FcγRIIb-dependent regulation of antigen (NIP22BSA)-induced calcium mobilization (FIGS. 9 and 10).

[0097] IIA1.6 B cells, co-expressing FcγRIIb and NP-specific cell surface IgE BCR, were treated with therapeutic mAb omalizumab (IgG1 mAb) or omalizumab variant mAb (provided with an IgG4 backbone or as a variant IgG2 antibody according to the present disclosure) and then the BCR was stimulated with the NP-associated antigen NIP(22)BSA antigen. IgG1 omalizumab treatment strongly inhibited subsequent calcium mobilization by antigen (2nd injection, NIP(22)BSA, FIG. 9A). IgG2 variant anti-IgE treatment only partially inhibited subsequent calcium mobilization by NIP(22)BSA antigen (2nd injection, FIG. 9B). Similar to omalizumab, but unlike its IgG2 counterpart, IgG4 formatted anti-IgE strongly inhibited antigen-stimulated calcium mobilization (FIG. 9C). These suppressive activities correlate with the ability of IgG1 and IgG4 to engage inhibitory FcγRIIb1, whereas IgG2 is unable to bind FcγRIIb1.

[0098] The effect of BCR-targeting mAbs on antigen stimulation was evaluated on IIA1.6 B cells co-expressing FcγRIIb1 using an anti-IgE mAb containing the variable domain of omalizumab with an IgG2 backbone (Figure 10). Treatment of B cells co-expressing IgE BCR and human FcγRIIb1 with the IgG2 form of omalizumab resulted in a partial inhibition of antigen (NIP)-specific hu-IgE BCR-induced calcium flux compared to the buffer control (2nd injection, NIP(22)BSA, Figure 10A). This partial inhibition is independent of FcγRIIb1, since IgG2 does not bind to FcγRIIb. However, treatment with omalizumab provided as an IgG2-FLGG antibody significantly inhibited antigen-specific hu-IgE BCR-induced calcium flux (2nd injection, Figure 10B). Omalizumab delivered as an IgG2-FEGG mAb also inhibited antigen-induced calcium flux (second injection, FIG. 10C), but less potently than the IgG2-FLGG formatted mAb. Omalizumab delivered as an IgG2-FLGG-SELF mAb also inhibited antigen-stimulated responses (second injection, FIG. 10D), but inhibition by the IgG2-FEGG-SELF format (FIG. 10E) was comparable to that of the IgG-FLGG mAb. Overall, IgE BCR modulation had a hierarchy of FLGG-SELF>FLGG~FEGG-SELF>FEGG>IgG2, which broadly correlated with the rank order of FcγRIIb binding activity of these mutations in this IgG2 format.

[0099] Consideration Targeting immune checkpoints has emerged as an important strategy to regulate leukocyte responses in disease. FcγRIIb is one of the earliest immune checkpoints described (Hibbs ML et al., Proc Natl Acad Sci USA 83:6980-6984, 1986). Its regulation of ITAM-dependent signaling pathways utilized by FcεRI, other activating FcRs, and the B cell antigen receptor regulates antibody-dependent leukocyte functions in innate and adaptive immunity. This includes inhibition of IgE-dependent basophil activation (Cady CT et al., Immunol Lett 130(1-2):57-65, 2010) and the B cell antigen receptor (Amigorena S et al., Science 256:1808-1812, 1992). In the work described in this example, it was found that a "functionally inactive" human IgG2 can be used as a scaffold to develop mAbs with FcγR specificity / affinity modified to exploit the inhibitory potency of FcγRIIb by mutating the lower hinge sequence. Indeed, in this way, it was found possible to exploit the inhibitory potency of FcγRIIb to modulate the activating receptor FcεRI and thereby inhibit IgE / FcεRI allergen or anti-IgE activation of human basophils. Furthermore, providing an anti-IgE mAb as such a mutant IgG2 antibody was effective in inhibiting antigen stimulation of surface IgE B cell receptors. Such mutant IgG2 antibodies therefore offer considerable promise as the basis of novel mAb therapeutics for treating or preventing allergic reactions.

[0100] Throughout this specification and the claims that follow, unless the context dictates otherwise, the words "comprise" and "include," as well as variations such as "comprising" and "including," will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers.

[0101] The reference to any prior art in this specification is not, and should not be construed as, any form of suggestion that such prior art forms part of the common general knowledge.

[0102] It will be understood by those skilled in the art that the methods and uses of the immunotherapeutic proteins (e.g., mutant IgG2 antibodies) and compositions disclosed herein are not limited by the specific applications described. The methods, uses, and compositions are not limited in their preferred embodiments with respect to the specific elements and / or features described or depicted herein. It will also be understood that the methods and uses of the immunotherapeutic proteins and compositions disclosed herein are not limited to one or more of the disclosed embodiments, but are capable of numerous rearrangements, variations, and substitutions without departing from the scope of the present disclosure as described and defined by the following claims.

Claims

1. An immunotherapeutic protein comprising at least one heavy chain polypeptide derived from an IgG2 antibody, said heavy chain polypeptide comprising at least constant heavy domains 2 and 3 (CH2 and CH3) and a lower hinge, wherein the sequence of the lower hinge comprises a mutation that enables said immunotherapeutic protein to bind to and / or activate FcγRIIb.

2. The immunotherapeutic protein of claim 1 , wherein the heavy chain polypeptide is a heavy chain component of an Fc fragment or the immunotherapeutic protein is a mutated IgG2 antibody.

3. 3. The immunotherapeutic protein of claim 1 or 2, wherein the mutation comprises a substitution in the lower hinge sequence or a substitution of one or more amino acids in the lower hinge sequence at positions 233-236 (EU numbering).

4. the lower hinge sequence comprises the amino acid sequence: X １ X ２ X ３ -G-X ５ or During the ceremony, X １ is selected from proline (P) and glutamic acid (E); X ２ is selected from valine (V), leucine (L), and phenylalanine (F); X ３ is selected from leucine (L), alanine (A), and glutamic acid (E); ５ is selected from glycine (G) and proline (P) or is absent; The immunotherapeutic protein of claim 1 or 2, wherein the lower hinge does not consist of a wild-type IgG2 lower hinge sequence.

5. The immunotherapeutic protein of any one of claims 1 to 4, wherein the lower hinge sequence comprises an amino acid sequence selected from the group consisting of ELLGG, EFLGG, EFLGP, and EFEGG.

6. The immunotherapeutic protein of any one of claims 1 to 5, comprising at least one of the following features: i) the immunotherapeutic protein binds to and activates FcγRIIb, recruiting FcγRIIb inhibitory function; ii) the immunotherapeutic protein binds to FcγRIIb and induces FcγRIIb-mediated endocytosis / internalization ("sweeping"); iii) the immunotherapeutic protein binds to FcγRIIb and induces FcγRIIb-mediated scaffolding; iv) the immunotherapeutic protein further comprises an S267E and / or an L328F amino acid substitution (EU numbering) within the CH2 region of at least one of the heavy chain polypeptides; and v) The immunotherapeutic protein is a human or humanized monoclonal antibody (mAb).

7. The immunotherapeutic protein of any one of claims 1 to 6, wherein the lower hinge sequence comprises the amino acid sequence EFLGG.

8. 7. The immunotherapeutic protein of any one of claims 1 to 6, wherein the lower hinge sequence comprises the amino acid sequence EFEGG, and the immunotherapeutic protein further comprises a S267E and / or a L328F amino acid substitution (EU numbering) in the CH2 region of the at least one heavy chain polypeptide.

9. An immunotherapeutic protein described in any one of claims 1 to 8 for treating a disease or condition in a subject, wherein binding to and / or activation of FcγRIIb is beneficial in treating or preventing the disease or condition.

10. The immunotherapeutic protein of claim 9, wherein the disease or condition is selected from allergic diseases, autoimmune diseases and conditions, other inflammatory diseases, infectious diseases, and proliferative diseases.

11. The immunotherapeutic protein of claim 10, wherein the disease or condition to be treated is an allergic disease and the immunotherapeutic protein comprises an antigen-binding region that specifically binds to an allergen.

12. The immunotherapeutic protein of claim 11, wherein the immunotherapeutic protein mediates FcγRIIb-dependent inhibition of allergic basophil activation by IgE.

13. The immunotherapeutic protein of any one of claims 9 to 12, wherein the disease or condition to be treated is cancer and the immunotherapeutic protein comprises an antigen-binding region that specifically binds to a cancer antigen.

14. The immunotherapeutic protein is (a) antigen, (b) an antibody bound to an activating receptor; (c) the antibody (ligand) binding domain of an activating receptor; (d) a subunit of an activating receptor; (e) an antigen bound to the immunoglobulin component of the BCR complex; or (f) an immunotherapeutic protein according to any one of claims 1 to 13, comprising an antigen-binding region that specifically binds to a subunit of the BCR complex or an associated Ig-α or β chain.

15. A pharmaceutical composition or medicament comprising an immunotherapeutic protein according to any one of claims 1 to 14 and a pharma- ceutically acceptable carrier, diluent and / or excipient.

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