Anti-il-33 antibody constructs and methods of use

Abstract

Antibody constructs that specifically bind to IL-33 and the use of the antibody constructs in the treatment of inflammatory and / or autoimmune diseases and disorders. The antibody constructs may be monospecific, biparatopic, bispecific or multispecific, including bispecific antibody constructs comprising at least one antigen-binding domain that binds to IL-33 and at least one antigen-binding construct that binds to another cytokine or cytokine receptor, such as IL-4Rα.

Description

ANTI-IL-33 ANTIBODY CONSTRUCTS AND METHODS OF USEFIELD

[0001] The present disclosure relates to the field of antibody therapeutics and, in particular, to monospecific and bispecific antibodies that bind to IL-33, including bispecific antibodies that bind to IL-33 and IL-4Ra, and methods of using such antibodies.BACKGROUND

[0002] Interleukin-33 (IL-33) is a member of the IL-1 family and plays an important role in tissue homeostasis and repair, type 2 immunity, allergic and non-allergic inflammation, viral infection, and cancer. IL-33 is an alarmin cytokine that is released upon cell damage or tissue injury and activates Myd88-dependent signalling pathways in cells expressing the ST2 (IL-1RL1) receptor.

[0003] IL-33 has been implicated in various human inflammatory diseases such as atopic dermatitis, asthma, chronic obstructive pulmonary disease, rheumatoid arthritis, sinusitis and psoriasis (see, for example, Miller, 2011, J Inflamm (Lond.), 8:22; Cayrol & Girard, 2022, Cytokine, 156: 155891).

[0004] The anti -IL-33 antibody drug itepekimab (Regeneron) is currently in clinical trials for treatment of chronic obstructive pulmonary disease (COPD) and asthma, and the anti -IL-33 antibody etokimab (AnaptysBio) is currently in clinical trials for treatment of asthma and chronic sinusitis.

[0005] This background information is provided for the purpose of making known information believed by the applicant to be of possible relevance to the present disclosure. No admission is necessarily intended, nor should be construed, that any of the preceding information constitutes prior art against the claimed invention(s).SUMMARY

[0006] Described herein are anti-IL-33 antibody constructs and methods of use. One aspect of the present disclosure relates to an antibody construct comprising one or more antigen-binding domains, wherein at least one of the antigen-binding domains is an IL-33 antigen-binding domain that specifically binds to human IL-33, the IL-33 antigen-binding domain comprising the CDR sequences (HCDR1, HCDR2, HCDR3) of the VH domain as set forth in any one of SEQ ID NOs:30, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 or 42, and the CDR sequences (LCDR1, LCDR2, LCDR3) of the VL domain as set forth in any one of SEQ ID NOs: 31, 43, 44, 45 or 46.

[0007] Another aspect of the present disclosure relates to a polynucleotide or set of polynucleotides encoding the antibody construct as described herein.

[0008] Another aspect of the present disclosure relates to an expression vector or set of expression vectors comprising a polynucleotide or set of polynucleotides encoding the antibody construct as described herein.

[0009] Another aspect of the present disclosure relates to a host cell comprising a polynucleotide or set of polynucleotides encoding the antibody construct as described herein or an expression vector or set of expression vectors comprising a polynucleotide or set of polynucleotides encoding the antibody construct as described herein.

[0010] Another aspect of the present disclosure relates to a method of preparing an antibody construct as described herein comprising transfecting a host cell with a polynucleotide or set of polynucleotides encoding the antibody construct as described herein or an expression vector or set of expression vectors comprising a polynucleotide or set of polynucleotides encoding the antibody construct as described herein, and culturing the host cell under conditions suitable for expression of the antibody construct.

[0011] Another aspect of the present disclosure relates to a bispecific antibody construct comprising an IL-33 antigen-binding domain that specifically binds to human IL-33, and a second target antigen-binding domain, that specifically binds to a second target antigen, wherein the second target antigen is other than IL-33, and wherein the IL-33 antigen-binding domain comprising the CDR sequences (HCDR1, HCDR2, HCDR3) of the VH domain as set forth in any one of SEQ ID NOs: 30, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 or 42, and the CDR sequences (LCDR1, LCDR2, LCDR3) of the VL domain as set forth in any one of SEQ ID NOs: 31, 43, 44, 45 or 46. In certain embodiments, the second target antigen is IL-4Ra.

[0012] Another aspect of the present disclosure relates to a bispecific antibody construct comprising an IL-33 antigen-binding domain that specifically binds to human IL-33 and an IL- 4Ra antigen-binding domain that specifically binds to human IL-4Ra, wherein the IL-33 antigenbinding domain comprises a VH domain comprising an HCDR1 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 3 or 6, an HCDR2 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 4 or 26, and an HCDR3 amino acid sequence comprising thesequence as set forth in SEQ ID NO: 5 or 8, and a VL domain comprising an LCDR1 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 16 or 29, an LCDR2 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 17 or 20, and an LCDR3 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 18, wherein the IL-4Ra antigenbinding domain comprises a VH domain comprising an HCDR1 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 53 or 56, an HCDR2 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 79 or 80, and an HCDR3 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 55 or 58, and a VL domain comprising an LCDR1 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 71 or 74, an LCDR2 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 72 or 75, and an LCDR3 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 73, and wherein the IL-33 antigen-binding domain and the IL-4Ra antigen-binding domain are both operably linked to a human IgG Fc region.

[0013] Another aspect of the present disclosure relates to a polynucleotide or set of polynucleotides encoding a bispecific antibody construct as described herein.

[0014] Another aspect of the present disclosure relates to an expression vector or set of expression vectors comprising a polynucleotide or set of polynucleotides encoding a bispecific antibody construct as described herein.

[0015] Another aspect of the present disclosure relates to a host cell comprising a polynucleotide or set of polynucleotides encoding a bispecific antibody construct as described herein or an expression vector or set of expression vectors comprising a polynucleotide or set of polynucleotides encoding a bispecific antibody construct as described herein.

[0016] Another aspect of the present disclosure relates to a method of preparing a bispecific antibody construct as described herein comprising transfecting a host cell with a polynucleotide or set of polynucleotides encoding the bispecific antibody construct or an expression vector or set of expression vectors comprising a polynucleotide or set of polynucleotides encoding the bispecific antibody construct, and culturing the host cell under conditions suitable for expression of the bispecific antibody construct.

[0017] Another aspect of the present disclosure relates to a pharmaceutical composition comprising an antibody construct or a bispecific antibody construct as described herein.

[0018] Another aspect of the present disclosure relates to an antibody construct or a bispecific antibody construct as described herein for use in therapy.

[0019] Another aspect of the present disclosure relates to a use of an antibody construct or a bispecific antibody construct as described herein in therapy.

[0020] Another aspect of the present disclosure relates to a use of an antibody construct or a bispecific antibody construct as described herein in the manufacture of a medicament.

[0021] Another aspect of the present disclosure relates to a method of treating an inflammatory or autoimmune disease in a subject comprising administering to the subject an effective amount of an antibody construct or a bispecific antibody construct as described herein.

[0022] Another aspect of the present disclosure relates to an antibody construct or a bispecific antibody construct as described herein for use in the treatment of an inflammatory or autoimmune disease in a subject.

[0023] Another aspect of the present disclosure relates to a use of an antibody construct or a bispecific antibody construct as described herein in the treatment of an inflammatory or autoimmune disease in a subject.

[0024] Another aspect of the present disclosure relates to a use of an antibody construct or a bispecific antibody construct as described herein in the manufacture of a medicament for the treatment of an inflammatory or autoimmune disease.BRIEF DESCRIPTION OF THE FIGURES

[0025] Figs. 1A-B show the inhibition of IL-33 mediated production of secreted embryonic alkaline phosphatase (SEAP) reporter in HEK-Blue™ IL-33 cells by bivalent anti -IL-33 antibody variants (full-sized antibodies) (Fig. 1A) and monovalent anti-IL-33 antibody variants (one-armed antibodies) (Fig. IB). v22277 is the negative control.

[0026] Figs. 2A-C show binding of anti -IL-33 x anti-IL-4Ra bispecific IgG4 antibody variants to IL-4Ra on human peripheral blood mononuclear cells (PBMCs) by flow cytometry compared to dupilumab (anti-IL-4Ra) and palivizumab (v36992; negative control). Fig. 2A CD3" CD14 CD15 CD16" classical monocytes; Fig. 2B CD3+CD4+T cells, and Fig. 2C CD3 CD19+B cells.

[0027] Figs. 3A-C show inhibition of IL-4 mediated production of the STAT6 inducible secreted embryonic alkaline phosphatase (SEAP) reporter in HEK-Blue™ IL-4 / IL-13 cells by anti-IL-33 xanti-IL-4Ra bispecific antibody variants. Fig. 3A shows inhibition by bispecific antibody variants with an IgGl FcKO backbone (v39982 and v36992 are negative controls); Figs. 3B and 3C show inhibition by bispecific antibody variants with an IgG4 backbone (v42104 and v39982 are negative controls).

[0028] Figs. 4A-C show inhibition of IL-33 mediated production of the secreted embryonic alkaline phosphatase (SEAP) reporter in HEK-Blue™ IL-33 cells by anti-IL-33 x anti-IL-4Ra bispecific antibody variants. Fig. 4A shows inhibition by bispecific antibody variants with an IgGl FcKO backbone (v40552 and dupilumab are negative controls); Figs. 4B and 4C show inhibition by bispecific antibody variants with an IgG4 backbone (v42104 and v39982 are negative controls).

[0029] Figs. 5A-D show inhibition of IL-4 mediated production of CCL17 (TARC) in peripheral blood mononuclear cells (PBMCs) by anti-IL-33 x anti-IL-4Ra bispecific antibody variants. Figs. 5A and 5B show inhibition by bispecific antibody variants with an IgGl FcKO backbone in PBMCs from two different donors (v39982 and v36992 are negative controls); Figs. 5C and 5D show inhibition by bispecific antibody variants with an IgG4 backbone in PBMCs from three different donors (v39982, v42104 and itepekimab are negative controls).

[0030] Figs. 6A-D show inhibition of IL-33 mediated IFN-y production in peripheral blood mononuclear cells (PBMCs) by anti-IL-33 x anti-IL-4Ra bispecific antibody variants. Figs. 6A and 6B show inhibition by bispecific antibody variants with an IgGl FcKO backbone in PBMCs from two different donors (v39982 and v36992 are negative controls); Figs. 6C and 6D show inhibition by bispecific antibody variants with an IgG4 backbone in PBMCs from three different donors (v39982, v42104 and dupilumab are negative controls).

[0031] Fig. 7 shows inhibition of IL-4 stimulated CD23 upregulation in peripheral blood mononuclear cells (PBMCs) by anti -IL-33 x anti-IL-4Ra bispecific IgGl and IgG4 antibody variants (v42104 is a negative control).

[0032] Fig. 8 shows inhibition of CCL2 gene expression in HEKa cells by an anti -IL-33 x anti- IL-4Ra bispecific IgG4 antibody variant, v42101, following combined cytokine stimulation of the IL-4, IL-13 and IL-31 pathways.

[0033] Figs. 9A-B show total antibody concentrations in serum after 3 mg / kg single intravenous dose of the anti-IL-33 x anti-IL-4Ra bispecific IgG4 antibody variants, v42100 and v42101 (Fig. 9A), and v42098 and v42099 (Fig. 9B), in Wistar Han rats.

[0034] Figs. 10A-B show total antibody concentrations in serum after 3 mg / kg single subcutaneous dose of the anti -IL-33 x anti-IL-4Ra bispecific IgG4 antibody variants, v42100 and v42101 (Fig. 10A), and v42098 and v42099 (Fig. 10B), in Wistar Han rats.

[0035] Figs. 11A-C show total antibody concentrations in serum after 5 mg / kg single intravenous dose of anti-IL-33 x anti-IL-4Ra bispecific antibody variants (IgGl backbone) in Tg32 mice. Fig. HA v41315 and v41246; Fig. HB v41316 and v41249; Fig. 11C v41317 and v41251.

[0036] Figs. 12A-C show total antibody concentrations in serum after 5 mg / kg single intravenous dose of anti-IL-33 x anti-IL-4Ra bispecific antibody variants in Tg32 mice. Fig. 12A v42100, v42103, v42101 (all IgG4 backbone) and v41316 (IgGl backbone); Fig. 12B v42098, v42102, v42099 (all IgG4 backbone) and v41317 (IgGl backbone); Fig. 12C dupilumab (anti-IL-4Ra) and itepekimab (anti-IL-33) controls.

[0037] Fig. 13 shows the serum pharmacokinetic (PK) profile from cynomolgus monkeys injected intravenously with 10 mg / kg of the anti -IL-33 x anti-IL-4Ra bispecific IgG4 antibody variant, v42101. (LLOQ: lower limit of quantification).

[0038] Fig. 14 shows the serum IgE levels from cynomolgus monkeys injected intravenously with 10 mg / kg of the anti-IL-33 x anti-IL-4Ra bispecific IgG4 antibody variant, v42101, or with an equal volume of saline (vehicle).

[0039] Figs. 15A-G show the results of treating an acute house dust mite (HDM) mouse model with 25, 10, 3 or 1 mg / kg of the anti-IL-33 x anti -IL-4Ra bispecific IgG4 antibody variant, v42101. Fig. 15A serum IgE levels; Fig. 15B lung hIL-4 levels; Fig. 15C lung eosinophil levels; Fig. 15D lung alveolar macrophage levels; Fig. 15E lung hIL-5 levels; Fig. 15F lung pathology evaluated by hemotoxylin and eosin staining of lung sections, and Fig. 15G total inflammation score. Data are represented as mean + / - SEM.

[0040] Figs. 16A-B show inhibition of IL-4 / IL-13 mediated production of the STAT6 inducible secreted embryonic alkaline phosphatase (SEAP) reporter in HEK-Blue™ IL-4 / IL-13 cells by anti- IL-33 x anti-IL-4Ra bispecific IgG4 antibody variants comprising an engineered anti-IL-4Ra paratope. Fig. 16A IL-4 stimulation, and Fig. 16B IL-13 stimulation. v42104 is a negative control and v42101 is the parental bispecific antibody variant.

[0041] Fig. 17 presents the CDR sequences for the chimeric parental anti-IL-33 antibody, v33006, and humanized variants based on the parental v33006 sequences (Table A). Differences compared to parental (v33006) CDR sequences are marked in bold and underlined.

[0042] Fig. 18 presents the CDR sequences for humanized anti-IL-4Ra antigen-binding domains comprised by bispecific anti-IL-33 x anti-IL-4Ra antibody variants (Table Bl).

[0043] Fig. 19 presents the CDR sequences for certain engineered anti-IL-4Ra antigen-binding domains comprised by bispecific anti-IL-33 x anti-IL-4Ra antibody variants (v42099, v43194, v43195, v43196, v43197, v43198, v43199 and v43200) (Table B2). Differences compared to parental anti-IL-4Ra (v38597) CDR sequences are marked in bold and underlined.

[0044] Fig. 20 presents the VH and VL sequences for humanized anti-IL-4Ra antigen-binding domains comprised by bispecific anti-IL-33 x anti-IL-4Ra antibody variants (Table Cl), and for certain engineered anti-IL-4Ra antigen-binding domains comprised by bispecific anti -IL-33 x anti- IL-4Ra antibody variants (v42099, v43194, v43195, v43196, v43197, v43198, v43199 and v43200) (Table C2).

[0045] Fig. 21 presents a table (Table D) summarizing the monospecific anti-IL-33 antibody variants described in the Examples.

[0046] Fig. 22 presents a table (Table E) summarizing the bispecific anti-IL-33 x anti-IL-4Ra antibody variants described in the Examples.

[0047] Fig. 23A-C show total antibody concentrations in serum after 5 mg / kg single intravenous dose of anti-IL-33 x anti-IL-4Ra bispecific antibody variants in Tg32-SCID mice. Fig. 23A v42101, v42103, dupilumab (anti-IL-4Ra) and itepekimab (anti-IL-33); Fig. 23B v43194, v43195, v43196 and v43197; Fig. 23C v43198, v43199 and v43200.

[0048] Fig. 24 shows the predicted half-life in humans for the anti-IL-33 x anti-IL-4Ra bispecific antibody variants v42101, v42103, v43194, v43195, v43196, v43197, v43198, v43199 and v43200 calculated from Tg32-SCID data by the allometric scaling approach.

[0049] Fig. 25 shows inhibition of CD23 upregulation in peripheral blood mononuclear cells (PBMCs) from healthy donors and chronic obstructive pulmonary disease (COPD) patients by anti-IL-33 x anti-IL-4Ra bispecific IgG4 antibody variants following IL-4 stimulation.

[0050] Fig. 26 shows inhibition of IL-33 mediated IFN-y production in peripheral blood mononuclear cells (PBMCs) from healthy donors and chronic obstructive pulmonary disease (COPD) patients by anti -IL-33 x anti-IL-4Ra bispecific antibody variants following IL- 12 stimulation.

[0051] Fig. 27A-B show the results of treating a chronic house dust mite (HDM) mouse model with 3, 1, 0.3 or 0.1 mg / kg of an anti-IL-33 OAA (v40570) or 1 mg / kg of the anti-IL-33 x anti-IL-4Ra bispecific IgGl antibody variant (v41251). Fig. 27 A hIL-33 mRNA transcript abundance in lungs of mice subjected to the chronic HDM model and treated as indicated; Fig. 27B hIL-33 cytokine protein concentration in lungs of mice subjected to the chronic HDM model and treated as indicated.

[0052] Fig. 28A-D show the ability of the anti -IL-33 x anti-IL-4Ra bispecific IgG4 antibody variant (v42101) to bind concurrently to IL-33 and IL-4Ra as assessed by an MSD assay, Fig. 28A capture by IL-33 and detection by an anti-human Fc biotinylated antibody, Fig. 28B capture by IL-33 and detection by an anti-human Fc biotinylated antibody, Fig. 28C capture by IL-33 and detection by biotinylated IL-4Ra, Fig. 28D capture by IL-4Ra and detection by biotinylated IL- 33. v40883 = dupilumab, v32657 = itepekimab, and v42104 = hemagglutinin-specific IgG4 antibody (negative control).

[0053] Fig. 29 shows inhibition of IL-33 and IL-4 mediated HLA-DR expression in PBMC- derived CD 14+CD 16+monocytes by the anti -IL-33 x anti-IL-4Ra bispecific IgG4 antibody variant (v42101) as measured by geometric mean fluorescent intensity (gMFI). Values shown for v42101, isotype control and itepekimab and dupilumab (alone and in combination) are for monocytes stimulated with both IL-33 and IL-4.

[0054] Fig. 30A-B shows the serum pharmacokinetic (PK) profile from cynomolgus monkeys injected intravenously with 20 mg / kg, 40 mg / kg or 100 mg / kg mg / kg (Fig. 30A), or subcutaneously with 40 mg / kg (Fig. 30B), of the anti-IL-33 x anti-IL-4Ra bispecific IgG4 antibody variant, v42101. (LLOQ: lower limit of quantification).

[0055] Fig. 31A-D shows the serum IgE levels from cynomolgus monkeys injected intravenously with 20 mg / kg (Fig. 31A), 40 mg / kg (Fig. 31B) or 100 mg / kg (Fig. 31C), or subcutaneously with 40 mg / kg (Fig. 31D), of the anti -IL-33 x anti-IL-4Ra bispecific IgG4 antibody variant, v42101.DETAILED DESCRIPTION

[0056] The present disclosure relates to antibody constructs that specifically bind to IL-33. The antibody constructs may be monospecific antibody constructs comprising one or more antigenbinding domains that bind to the same epitope on IL-33, or they may be biparatopic antibody constructs comprising two antigen-binding domains each of which binds to a different epitope on IL-33, or they may be bispecific or multispecific antibody constructs that comprise at least one antigen-binding domain that binds to IL-33 and at least one antigen-binding domain that binds toa target antigen other than IL-33. In certain embodiments, the bispecific or multispecific antibody constructs comprise at least one antigen-binding domain that binds to IL-33 and at least one antigen-binding construct that binds to another cytokine or cytokine receptor. Certain embodiments relate to bispecific antibody constructs comprising at least one antigen-binding domain that binds to IL-33 and at least one antigen-binding domain that binds to IL-4Ra.

[0057] IL-33 has been determined to be involved in a number of inflammatory and / or autoimmune diseases and disorders, such as atopic dermatitis, psoriasis, asthma, rhinosinusitis, chronic obstructive pulmonary disease (COPD), inflammatory bowel disease (IBD) and rheumatological diseases. Certain embodiments thus relate to the use of the anti-IL-33 antibody constructs of the present disclosure in the treatment of such IL-33 related inflammatory and / or autoimmune diseases and disorders.Definitions

[0058] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0059] As used herein, the term “about” refers to an approximately + / -10% variation from a given value. It is to be understood that such a variation is always included in any given value provided herein, whether or not it is specifically referred to.

[0060] The use of the word “a” or “an” when used herein in conjunction with the term “comprising” may mean “one,” but it is also consistent with the meaning of “one or more,” “at least one” and “one or more than one.”

[0061] As used herein, the terms “comprising,” “having,” “including” and “containing,” and grammatical variations thereof, are inclusive or open-ended and do not exclude additional, unrecited elements and / or method steps. The term “consisting essentially of’ when used herein in connection with a composition, use or method, denotes that additional elements and / or method steps may be present, but that these additions do not materially affect the manner in which the recited composition, method or use functions. The term “consisting of’ when used herein in connection with a composition, use or method, excludes the presence of additional elements and / or method steps. A composition, use or method described herein as comprising certain elements and / or steps may also, in certain embodiments consist essentially of those elements and / or steps,and in other embodiments consist of those elements and / or steps, whether or not these embodiments are specifically referred to.

[0062] A “complementarity determining region” or “CDR” is an amino acid sequence that contributes to antigen-binding specificity and affinity. “Framework” regions (FR) can aid in maintaining the proper conformation of the CDRs to promote binding between the antigen-binding region and an antigen. From N-terminus to C-terminus, both the light chain variable region (VL) and the heavy chain variable region (VH) of an antibody typically comprise the domains FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The three heavy chain CDRs are referred to herein as HCDR1, HCDR2, and HCDR3, and the three light chain CDRs are referred to as LCDR1, LCDR2, and LCDR3. CDRs provide the majority of contact residues for the binding of the antibody to the antigen or epitope. Often, the three heavy chain CDRs and the three light chain CDRs are required to bind antigen. However, as is known in the art, in some instances, just a single variable domain can confer binding specificity to the antigen, and in some cases, antigen-binding may also occur through a combination of a minimum of one or more CDRs selected from the VH and / or VL domains, for example HCDR3.

[0063] A number of different definitions of the CDR sequences are in common use, including those described by Kabat et al. (1983, Sequences of Proteins of Immunological Interest, NIH Publication No. 369-847, Bethesda, MD), by Chothia et al. (1987, J Mol Biol, 196:901-917), as well as the IMGT, AbM (University of Bath) and Contact (MacCallum, et al., 1996, J Mol Biol, 262(5):732-745) definitions. By way of example, CDR definitions according to Kabat, Chothia, IMGT, AbM and Contact are provided in Table 1 below. Accordingly, as would be readily apparent to one skilled in the art, the exact numbering and placement of CDRs may differ based on the numbering system employed. However, it is to be understood that the disclosure herein of a VH includes the disclosure of the associated (inherent) heavy chain CDRs (HCDRs) as defined by any of the known numbering systems. Similarly, disclosure herein of a VL includes the disclosure of the associated (inherent) light chain CDRs (LCDRs) as defined by any of the known numbering systems.Table 1: Common CDR Definitions11Either the Kabat or Chothia numbering system for antibody sequences may be used for HCDR2, HCDR3 and the light chain CDRs for all definitions except Contact, which uses Chothia numbering.2HCDR1 as shown is defined using Chothia numbering. The position in the Kabat numbering system that demarcates the end of the Chothia and IMGT CDR-H1 loop varies depending on the length of the loop because Kabat numbering places insertions outside of those CDR definitions at positions 35A and 35B. However, the IMGT and Chothia CDR-H1 loop can be unambiguously defined using Chothia numbering.

[0064] The term “identical” in the context of two or more polynucleotide or polypeptide sequences, refers to two or more sequences or subsequences that are the same. Sequences are “substantially identical” if they have a percentage of amino acid residues or nucleotides that are the same (for example, about 80%, about 85%, about 90%, about 95%, or about 98% identity, over a specified region) when compared and aligned for maximum correspondence over a comparison window or over a designated region as measured using one of the commonly used sequence comparison algorithms as known to persons of ordinary skill in the art or by manual alignment and visual inspection. For sequence comparison, typically test sequences are compared to a designated reference sequence. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters.

[0065] A “comparison window” refers to a segment of a sequence comprising contiguous amino acid or nucleotide positions, the length of which is typically determined based on the length of thetest sequence and may be, for example, from about 10 to 600 contiguous amino acid or nucleotide positions, or from about 10 to about 200, or from about 10 to about 150 contiguous amino acid or nucleotide positions over which the test sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. Methods of alignment of sequences for comparison are known to those of ordinary skill in the art. Optimal alignment of sequences for comparison can be conducted, for example, by the local homology algorithm of Smith & Waterman, 1970, Adv. Appl. Math., 2:482c; by the homology alignment algorithm of Needleman & Wunsch, 1970, J. Mol. Biol., 48:443; by the search for similarity method of Pearson & Lipman, 1988, Proc. Natl. Acad. Sci. USA, 85:2444, or by computerized implementations of these algorithms (for example, GAP, BESTFIT, FASTA or TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, Madison, WI), or by manual alignment and visual inspection (see, for example, Ausubel et al., Current Protocols in Molecular Biology, (1995 supplement), Cold Spring Harbor Laboratory Press). Examples of available algorithms suitable for determining percent sequence identity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al., 1997, Nuc. Acids Res., 25:3389-3402, and Altschul et al., 1990, J. Mol. Biol., 215:403-410, respectively. Software for performing BLAST analyses is publicly available through the website for the National Center for Biotechnology Information (NCBI).

[0066] The term “subject,” as used herein, refers to an animal, in some embodiments a mammal, which is the object of treatment, observation or experiment. The animal may be a human, a nonhuman primate, a companion animal (for example, dog, cat, or the like), farm animal (for example, cow, sheep, pig, horse, or the like) or a laboratory animal (for example, rat, mouse, guinea pig, non-human primate, or the like). In certain embodiments, the subject is a human.

[0067] It is contemplated that any embodiment described herein in relation to the IL-33 antibody constructs can be implemented with respect to any method, use or composition disclosed herein.

[0068] Particular features, structures and / or characteristics described in connection with an embodiment disclosed herein may be combined with features, structures and / or characteristics described in connection with another embodiment disclosed herein in any suitable manner to provide one or more further embodiments.

[0069] It is also to be understood that the positive recitation of a feature in one embodiment, serves as a basis for excluding the feature in an alternative embodiment. For example, where a list ofoptions is presented for a given embodiment or claim, it is to be understood that one or more option may be deleted from the list and the shortened list may form an alternative embodiment, whether or not such an alternative embodiment is specifically referred to.

[0070] As is known in the art, the amino acid residues for the immunoglobulin heavy and light chains may be numbered according to several conventions. Unless otherwise indicated, AbM numbering is used herein for the VH and VL domains, and EU numbering is used herein for the CL, CHI, CH2 and CH3 domains, and the hinge region.ANTI-IL-33 ANTIBODY CONSTRUCTS

[0071] The present disclosure relates to antibody constructs that specifically bind to human IL-33 (anti-IL-33 antibody constructs). In this context, the term “antibody construct” refers to a polypeptide or a set of polypeptides that comprises one or more antigen-binding domains, where each of the one or more antigen-binding domains specifically binds to an epitope or antigen. Where the antibody construct comprises two or more antigen-binding domains, each of the antigenbinding domains may bind the same epitope or antigen (i.e. the antibody construct is monospecific) or they may bind to different epitopes or antigens (i.e. the antibody construct is biparatopic, bispecific or multispecific). The antibody construct may further comprise a scaffold and the one or more antigen-binding domains can be fused or covalently attached to the scaffold, optionally via a linker, as described herein.

[0072] In accordance with the present disclosure, the anti-IL-33 antibody constructs comprise at least one antigen-binding domain that specifically binds to human IL-33 (an “IL-33 antigenbinding domain”). By “specifically binds” to IL-33, it is meant that the antigen-binding domain binds to human IL-33 and may bind to IL-33 from one or more other non-human species, but does not exhibit significant binding to any other antigen. Specific binding of an antigen-binding domain to a target antigen or epitope may be measured, for example, through an enzyme-linked immunosorbent assay (ELISA), a surface plasmon resonance (SPR) technique (employing, for example, a Biacore™ instrument) (see, for example, Liljeblad et al., 2000, Glyco J, 17:323-329), flow cytometry or a traditional binding assay (see, for example, Heeley, 2002, Endocr Res, 28:217- 229).

[0073] In certain embodiments, the anti -IL-33 antibody constructs of the present disclosure may also be capable of binding to IL-33 from one or more non-human species. In certain embodiments,the anti-IL-33 antibody constructs of the present disclosure are capable of binding to cynomolgus monkey IL-33.

[0074] The protein sequence for human IL-33 protein is known in the art and readily available from publicly accessible databases, such as GenBank or UniProtKB. For example, the sequence for human IL-33 is available under UniProt Nos. 095760-1 and Q2YEJ5 and is provided in Table 2 as SEQ ID NO: 1. The IL-33 sequence for the macaque (cynomolgus) monkey, Macaca fascicularis, is available under UniProt No. A0A2K5W3I1 and is provided in Table 2 as SEQ ID NO: 2.Table 2: Human and Macaque IL-33 Protein SequencesIL-33 Antigen-Binding Domains

[0075] The anti -IL-33 antibody constructs of the present disclosure comprise at least one antigenbinding domain that specifically binds to human IL-33 (an “IL-33 antigen-binding domain”), which is an immunoglobulin-based binding domain, such as an antigen-binding antibody fragment. Examples of an antigen-binding antibody fragment include, but are not limited to, a Fab fragment (Fab), a Fab’ fragment (Fab’), a single chain Fab (scFab), a single chain Fv (scFv) and a single domain antibody (sdAb).

[0076] A “Fab fragment” contains the constant domain of the light chain (CL) and the first constant domain of the heavy chain (CHI) along with the variable domains of the light and heavy chains (VL and VH, respectively). Fab' fragments differ from Fab fragments by the addition of a few amino acid residues at the C-terminus of the heavy chain CHI domain, including one or more cysteines from the antibody hinge region. A Fab fragment may also be a single-chain Fab molecule, i.e. a Fab molecule in which the Fab light chain and the Fab heavy chain are connected by a peptide linker to form a single peptide chain. For example, the C-terminus of the Fab light chain may be connected to the N-terminus of the Fab heavy chain in the single-chain Fab molecule.

[0077] An “scFv” includes a heavy chain variable domain (VH) and a light chain variable domain (VL) of an antibody in a single polypeptide chain. The scFv may optionally further comprise a polypeptide linker between the VH and VL domains which enables the scFv to form a desired structure for antigen binding. For example, an scFv may include a VL connected from its C- terminus to the N-terminus of a VH by a polypeptide linker. Alternately, an scFv may comprise a VH connected through its C-terminus to the N-terminus of a VL by a polypeptide linker (see review by Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994)).

[0078] An “sdAb” format refers to a single immunoglobulin domain. The sdAb may be, for example, of camelid origin. Camelid antibodies lack light chains and their antigen-binding sites consist of a single domain, termed a “VHH.” An sdAb comprises three CDR / hypervariable loops that form the antigen-binding site: CDR1, CDR2 and CDR3. sdAbs are fairly stable and easy to express, for example, as a fusion with the Fc chain of an antibody (see, for example, Harmsen & De Haard, 2007, Appl. Microbiol Biotechnol., TUff. 13-22).

[0079] In certain embodiments, the IL-33 antigen-binding domain is a Fab or an scFv. In some embodiments, the IL-33 antigen-binding domain is a Fab.

[0080] In those embodiments in which the anti -IL-33 antibody constructs comprise two or more antigen-binding domains, each additional antigen-binding domain may independently be an immunoglobulin-based domain, such as an antigen-binding antibody fragment, or a non- immunoglobulin-based domain, such as a non-immunoglobulin-based antibody mimetic, or other polypeptide or small molecule capable of specifically binding to its target, for example, a natural or engineered ligand. Non-immunoglobulin-based antibody mimetic formats include, for example,anticalins, fynomers, affimers, alphabodies, DARPins and avimers. The additional antigen-binding domains may bind to IL-33 or they may bind to a different antigen.

[0081] The present disclosure describes the identification of a rabbit antibody that specifically binds IL-33 (hl48C04; see Example 1), as well as a chimeric version of this antibody (v33006) and representative humanized versions of this antibody (variants v33068-v33111) (see Examples and Table D (Fig. 21). The CDR sequences of the parental chimeric antibody v33006 and of representative humanized versions of this antibody are shown in Fig. 17 (Table A). The VH and VL sequences of the parental rabbit antibody, hl48C04, and the chimeric antibody v33006 are shown in Table 1.1 (see Example 1). The VH and VL sequences of representative humanized versions of this antibody are shown in Table 1.2 (see Example 1).

[0082] In certain embodiments, the anti -IL-33 antibody constructs of the present disclosure comprise at least one IL-33 antigen-binding domain, where the IL-33 antigen-binding domain comprises the CDR sequences (HCDR1, HCDR2, HCDR3) of the VH domain as set forth in any one of SEQ ID NOs: 30, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 or 42. In some embodiments, the anti -IL-33 antibody constructs of the present disclosure comprise at least one IL-33 antigenbinding domain, where the IL-33 antigen-binding domain comprises the CDR sequences (HCDR1, HCDR2, HCDR3) of the VH domain as set forth in SEQ ID NO: 32 or 38.

[0083] In certain embodiments, the anti -IL-33 antibody constructs of the present disclosure comprise at least one IL-33 antigen-binding domain, where the IL-33 antigen-binding domain comprises the CDR sequences (LCDR1, LCDR2, LCDR3) of the VL domain as set forth in any one of SEQ ID NOs: 31, 43, 44, 45 or 46. In some embodiments, the anti-IL-33 antibody constructs of the present disclosure comprise at least one IL-33 antigen-binding domain, where the IL-33 antigen-binding domain comprises the CDR sequences (LCDR1, LCDR2, LCDR3) of the VL domain as set forth in SEQ ID NO: 45 or 46.

[0084] In certain embodiments, the anti -IL-33 antibody constructs of the present disclosure comprise at least one IL-33 antigen-binding domain, where the IL-33 antigen-binding domain comprises the CDR sequences (HCDR1, HCDR2, HCDR3) of the VH domain as set forth in any one of SEQ ID NOs: 30, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 or 42, and the CDR sequences (LCDR1, LCDR2, LCDR3) of the VL domain as set forth in any one of SEQ ID NOs: 31, 43, 44, 45 or 46. In some embodiments, the anti-IL-33 antibody constructs of the present disclosure comprise at least one IL-33 antigen-binding domain, where the IL-33 antigen-binding domaincomprises the CDR sequences (HCDR1, HCDR2, HCDR3) of the VH domain as set forth in SEQ ID NO: 32 or 38, and the CDR sequences (LCDR1, LCDR2, LCDR3) of the VL domain as set forth in SEQ ID NO: 45 or 46.

[0085] In certain embodiments, the anti -IL-33 antibody constructs of the present disclosure comprise at least one IL-33 antigen-binding domain, where the IL-33 antigen-binding domain comprises a VH domain comprising heavy chain CDR amino acid sequences (HCDR1, HCDR2 and HCDR3) comprising the sequences as set forth in SEQ ID NOs: 6, 47 and 8, respectively, and a VL domain comprising light chain CDR amino acid sequences (LCDR1, LCDR2 and LCDR3) comprising the sequences as set forth in SEQ ID NOs: 48, 20 and 18, respectively (see Table 3). SEQ ID NOs: 6, 47 and 8, and SEQ ID NOs: 48, 20 and 18 are consensus sequences based on the HCDR and LCDR sequences, respectively, of v33006 and representative humanized versions of this antibody when defined by the Kabat system.

[0086] In certain embodiments, the anti -IL-33 antibody constructs of the present disclosure comprise at least one IL-33 antigen-binding domain, where the IL-33 antigen-binding domain comprises a VH domain comprising heavy chain CDR amino acid sequences (HCDR1, HCDR2 and HCDR3) comprising the sequences as set forth in SEQ ID NOs: 13, 49 and 15, respectively, and a VL domain comprising light chain CDR amino acid sequences (LCDR1, LCDR2 and LCDR3) comprising the sequences as set forth in SEQ ID NOs: 21, 22 and 23, respectively (see Table 4). SEQ ID NOs: 13, 49 and 15, and SEQ ID NOs: 21, 22 and 23 are consensus sequences based on the HCDR and LCDR sequences, respectively, of v33006 and representative humanized versions of this antibody when defined by the Contact system.Table 3: Consensus CDR Sequences by KabatTable 4: Consensus CDR Sequences by Contact

[0087] In certain embodiments, the anti -IL-33 antibody constructs of the present disclosure comprise at least one IL-33 antigen-binding domain, where the IL-33 antigen-binding domain comprises a VH domain comprising heavy chain CDR amino acid sequences (HCDR1, HCDR2 and HCDR3) comprising the sequences as set forth in SEQ ID NOs: 6, 47 and 8, respectively, and a VL domain comprising light chain CDR amino acid sequences (LCDR1, LCDR2 and LCDR3) comprising the sequences as set forth in SEQ ID NOs: 48, 20 and 18, respectively, and where X1is DSV or DWA, and X2is R. In some embodiments, the anti-IL-33 antibody constructs of the present disclosure comprise at least one IL-33 antigen-binding domain, where the IL-33 antigenbinding domain comprises a VH domain comprising heavy chain CDR amino acid sequences (HCDR1, HCDR2 and HCDR3) comprising the sequences as set forth in SEQ ID NOs: 6, 47 and 8, respectively, and a VL domain comprising light chain CDR amino acid sequences (LCDR1, LCDR2 and LCDR3) comprising the sequences as set forth in SEQ ID NOs: 48, 20 and 18, respectively, and where X1is DSV, and X2is R.

[0088] In certain embodiments, the anti -IL-33 antibody constructs of the present disclosure comprise at least one IL-33 antigen-binding domain, where the IL-33 antigen-binding domain comprises a VH domain comprising heavy chain CDR amino acid sequences (HCDR1, HCDR2 and HCDR3) comprising the sequences as set forth in SEQ ID NOs: 13, 49 and 15, respectively,and a VL domain comprising light chain CDR amino acid sequences (LCDR1, LCDR2 and LCDR3) comprising the sequences as set forth in SEQ ID NOs: 21, 22 and 23, respectively, and where X3is VA or VS.

[0089] In certain embodiments, the anti -IL-33 antibody constructs of the present disclosure comprise at least one IL-33 antigen-binding domain, where the IL-33 antigen-binding domain comprises a VH domain comprising an HCDR1 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 3 or 6, an HCDR2 amino acid sequence comprising the sequence as set forth in any one of SEQ ID NOs: 4, 7, 24, 26 or 28, and an HCDR3 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 5 or 8. In some embodiments, the anti-IL-33 antibody constructs of the present disclosure comprise at least one IL-33 antigen-binding domain, where the IL-33 antigen-binding domain comprises a VH domain comprising an HCDR1 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 3 or 6, an HCDR2 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 4, 24 or 26, and an HCDR3 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 5 or 8.

[0090] In certain embodiments, the anti -IL-33 antibody constructs of the present disclosure comprise at least one IL-33 antigen-binding domain, where the IL-33 antigen-binding domain comprises a VL domain comprising an LCDR1 amino acid sequence comprising the sequence as set forth in any one of SEQ ID NOs: 16, 19 or 29, an LCDR2 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 17 or 20, and an LCDR3 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 18. In some embodiments, the anti-IL-33 antibody constructs of the present disclosure comprise at least one IL-33 antigen-binding domain, where the IL-33 antigen-binding domain comprises a VL domain comprising an LCDR1 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 16 or 29, an LCDR2 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 17 or 20, and an LCDR3 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 18.

[0091] In certain embodiments, the anti -IL-33 antibody constructs of the present disclosure comprise at least one IL-33 antigen-binding domain, where the IL-33 antigen-binding domain comprises a VH domain comprising an HCDR1 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 3 or 6, an HCDR2 amino acid sequence comprising the sequence as set forth in any one of SEQ ID NOs: 4, 7, 24, 26 or 28, and an HCDR3 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 5 or 8, and a VL domain comprising anLCDR1 amino acid sequence comprising the sequence as set forth in any one of SEQ ID NOs: 16, 19 or 29, an LCDR2 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 17 or 20, and an LCDR3 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 18.

[0092] In certain embodiments, the anti -IL-33 antibody constructs of the present disclosure comprise at least one IL-33 antigen-binding domain, where the IL-33 antigen-binding domain comprises a VH domain comprising either(i) an HCDR1 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 3 or 6, an HCDR2 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 4 or 24, and an HCDR3 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 5 or 8, or(ii) an HCDR1 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 3 or 6, an HCDR2 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 4 or 26, and an HCDR3 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 5 or 8, and

[0093] a VL domain comprising an LCDR1 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 16 or 29, an LCDR2 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 17 or 20, and an LCDR3 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 18.

[0094] In certain embodiments, the anti -IL-33 antibody constructs of the present disclosure comprise at least one IL-33 antigen-binding domain, where the IL-33 antigen-binding domain comprises:(a) a VH domain comprising an HCDR1 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 3 or 6, an HCDR2 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 4 or 7, and an HCDR3 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 5 or 8, and a VL domain comprising an LCDR1 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 16 or 19, an LCDR2 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 17 or 20, and an LCDR3 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 18, or(b) a VH domain comprising an HCDR1 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 3 or 6, an HCDR2 amino acid sequence comprising the sequenceas set forth in SEQ ID NO: 4 or 24, and an HCDR3 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 5 or 8, and a VL domain comprising an LCDR1 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 16 or 29, an LCDR2 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 17 or 20, and an LCDR3 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 18, or(c) a VH domain comprising an HCDR1 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 3 or 6, an HCDR2 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 4 or 26, and an HCDR3 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 5 or 8, and a VL domain comprising an LCDR1 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 16 or 29, an LCDR2 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 17 or 20, and an LCDR3 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 18, or(d) a VH domain comprising an HCDR1 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 3 or 6, an HCDR2 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 4 or 28, and an HCDR3 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 5 or 8, and a VL domain comprising an LCDR1 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 16 or 29, an LCDR2 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 17 or 20, and an LCDR3 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 18.

[0095] In certain embodiments, the anti -IL-33 antibody constructs of the present disclosure comprise at least one IL-33 antigen-binding domain, where the IL-33 antigen-binding domain comprises a VH domain comprising an HCDR1 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 3 or 6, an HCDR2 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 4 or 24, and an HCDR3 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 5 or 8, and a VL domain comprising an LCDR1 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 16 or 29, an LCDR2 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 17 or 20, and an LCDR3 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 18.

[0096] In certain embodiments, the anti -IL-33 antibody constructs of the present disclosure comprise at least one IL-33 antigen-binding domain, where the IL-33 antigen-binding domain comprises a VH domain comprising an HCDR1 amino acid sequence comprising the sequence asset forth in SEQ ID NO: 3 or 6, an HCDR2 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 4 or 26, and an HCDR3 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 5 or 8, and a VL domain comprising an LCDR1 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 16 or 29, an LCDR2 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 17 or 20, and an LCDR3 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 18.

[0097] In certain embodiments, the anti -IL-33 antibody constructs of the present disclosure comprise at least one IL-33 antigen-binding domain, where the IL-33 antigen-binding domain comprises a VH domain having a sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the VH sequence as set forth in any one of SEQ ID NOs: 30, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 or 42. In some embodiments, the anti-IL-33 antibody constructs of the present disclosure comprise at least one IL-33 antigen-binding domain, where the IL-33 antigen-binding domain comprises a VH domain having a sequence as set forth in any one of SEQ ID NOs: 30, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 or 42. In some embodiments, the anti-IL-33 antibody constructs of the present disclosure comprise at least one IL-33 antigen-binding domain, where the IL-33 antigen-binding domain comprises a VH domain having a sequence as set forth in any one of SEQ ID NOs: 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 or 42. In some embodiments, the anti -IL-33 antibody constructs of the present disclosure comprise at least one IL-33 antigenbinding domain, where the IL-33 antigen-binding domain comprises a VH domain having a sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the VH sequence as set forth in SEQ ID NO: 32 or 38. In some embodiments, the anti-IL-33 antibody constructs of the present disclosure comprise at least one IL-33 antigen-binding domain, where the IL-33 antigen-binding domain comprises a VH domain having the sequence as set forth in SEQ ID NO: 32 or 38.

[0098] In some embodiments, the anti -IL-33 antibody constructs of the present disclosure comprise at least one IL-33 antigen-binding domain, where the IL-33 antigen-binding domain comprises a VH domain having a sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the VH sequence as set forth in SEQ ID NO: 38. In some embodiments, the anti-IL-33 antibody constructs of the present disclosure comprise at least one IL-33 antigen-binding domain, where the IL-33 antigen-binding domain comprises a VH domain having the sequence as set forth in SEQ ID NO: 38.

[0099] In some embodiments, the anti -IL-33 antibody constructs of the present disclosure comprise at least one IL-33 antigen-binding domain, where the IL-33 antigen-binding domain comprises a VH domain having a sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the VH sequence as set forth in SEQ ID NO: 32. In some embodiments, the anti-IL-33 antibody constructs of the present disclosure comprise at least one IL-33 antigen-binding domain, where the IL-33 antigen-binding domain comprises a VH domain having the sequence as set forth in SEQ ID NO: 32.

[0100] In certain embodiments, the anti-IL-33 antibody constructs of the present disclosure comprise at least one IL-33 antigen-binding domain, where the IL-33 antigen-binding domain comprises a VL domain having a sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the VL sequence as set forth in any one of SEQ ID NOs: 31, 43, 44, 45 or 46. In some embodiments, the anti-IL-33 antibody constructs of the present disclosure comprise at least one IL-33 antigen-binding domain, where the IL-33 antigen-binding domain comprises a VL domain having the sequence as set forth in any one of SEQ ID NOs: 31, 43, 44, 45 or 46. In some embodiments, the anti-IL-33 antibody constructs of the present disclosure comprise at least one IL-33 antigen-binding domain, where the IL-33 antigen-binding domain comprises a VL domain having the sequence as set forth in any one of SEQ ID NOs: 43, 44, 45 or 46. In some embodiments, the anti-IL-33 antibody constructs of the present disclosure comprise at least one IL-33 antigen-binding domain, where the IL-33 antigen-binding domain comprises a VL domain having a sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the VL sequence as set forth in SEQ ID NO: 45 or 46. In some embodiments, the anti-IL-33 antibody constructs of the present disclosure comprise at least one IL-33 antigenbinding domain, where the IL-33 antigen-binding domain comprises a VL domain having the sequence as set forth in SEQ ID NO: 45 or 46.

[0101] In some embodiments, the anti -IL-33 antibody constructs of the present disclosure comprise at least one IL-33 antigen-binding domain, where the IL-33 antigen-binding domain comprises a VL domain having a sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the VL sequence as set forth in SEQ ID NO: 45. In some embodiments, the anti-IL-33 antibody constructs of the present disclosure comprise at least one IL-33 antigen-binding domain, where the IL-33 antigen-binding domain comprises a VL domain having the sequence as set forth in SEQ ID NO: 45.

[0102] In some embodiments, the anti -IL-33 antibody constructs of the present disclosure comprise at least one IL-33 antigen-binding domain, where the IL-33 antigen-binding domain comprises a VL domain having a sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the VL sequence as set forth in SEQ ID NO: 46. In some embodiments, the anti-IL-33 antibody constructs of the present disclosure comprise at least one IL-33 antigen-binding domain, where the IL-33 antigen-binding domain comprises a VL domain having the sequence as set forth in SEQ ID NO: 46.

[0103] In certain embodiments, the anti-IL-33 antibody constructs of the present disclosure comprise at least one IL-33 antigen-binding domain, where the IL-33 antigen-binding domain comprises a VH domain having a sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the VH sequence as set forth in any one of SEQ ID NOs: 30, 32,33, 34, 35, 36, 37, 38, 39, 40, 41 or 42, and a VL domain having a sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the VL sequence as set forth in any one of SEQ ID NOs: 31, 43, 44, 45 or 46. In some embodiments, the anti-IL-33 antibody constructs of the present disclosure comprise at least one IL-33 antigen-binding domain, where the IL-33 antigen-binding domain comprises a VH domain having the sequence as set forth in any one of SEQ ID NOs: 30, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 or 42, and a VL domain having the sequence as set forth in any one of SEQ ID NOs: 31, 43, 44, 45 or 46.

[0104] In certain embodiments, the anti-IL-33 antibody constructs of the present disclosure comprise at least one IL-33 antigen-binding domain, where the IL-33 antigen-binding domain comprises a VH domain having a sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the VH sequence as set forth in any one of SEQ ID NOs: 32, 33,34, 35, 36, 37, 38, 39, 40, 41 or 42, and a VL domain having a sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the VL sequence as set forth in any one of SEQ ID NOs: 43, 44, 45 or 46. In some embodiments, the anti-IL-33 antibody constructs of the present disclosure comprise at least one IL-33 antigen-binding domain, where the IL-33 antigen-binding domain comprises a VH domain having the sequence as set forth in any one of SEQ ID NOs: 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 or 42, and a VL domain having the sequence as set forth in any one of SEQ ID NOs: 43, 44, 45 or 46.

[0105] In certain embodiments, the anti-IL-33 antibody constructs of the present disclosure comprise at least one IL-33 antigen-binding domain, where the IL-33 antigen-binding domaincomprises a VH domain having a sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the VH sequence as set forth in SEQ ID NO: 32 or 38, and a VL domain having a sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the VL sequence as set forth in SEQ ID NO: 45 or 46. In some embodiments, the anti-IL-33 antibody constructs of the present disclosure comprise at least one IL-33 antigenbinding domain, where the IL-33 antigen-binding domain comprises a VH domain having the sequence as set forth in SEQ ID NO: 32 or 38, and a VL domain having the sequence as set forth in SEQ ID NO: 45 or 46.

[0106] In certain embodiments, the anti-IL-33 antibody constructs of the present disclosure comprise at least one IL-33 antigen-binding domain, where the IL-33 antigen-binding domain comprises a VH domain having a sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the VH sequence as set forth in SEQ ID NO: 32, and a VL domain having a sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the VL sequence as set forth in SEQ ID NO: 46. In some embodiments, the anti-IL-33 antibody constructs of the present disclosure comprise at least one IL-33 antigen-binding domain, where the IL-33 antigen-binding domain comprises a VH domain having the sequence as set forth in SEQ ID NO: 32, and a VL domain having the sequence as set forth in SEQ ID NO: 46.

[0107] In certain embodiments, the anti-IL-33 antibody constructs of the present disclosure comprise at least one IL-33 antigen-binding domain, where the IL-33 antigen-binding domain comprises:(i) a VH domain having the sequence as set forth in any one of SEQ ID NOs: 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 or 42, and a VL domain having the sequence as set forth in SEQ ID NO: 43, or(ii) a VH domain having the sequence as set forth in any one of SEQ ID NOs: 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 or 42, and a VL domain having the sequence as set forth in SEQ ID NO: 44, or(iii) a VH domain having the sequence as set forth in any one of SEQ ID NOs: 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 or 42, and a VL domain having the sequence as set forth in SEQ ID NO: 45, or(iv) a VH domain having the sequence as set forth in any one of SEQ ID NOs: 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 or 42, and a VL domain having the sequence as set forth in SEQ ID NO: 46, or(v) a VH domain having the sequence as set forth in SEQ ID NO: 32, and a VL domain having the sequence as set forth in any one of SEQ ID NOs: 43, 44, 45 or 46, or(vi) a VH domain having the sequence as set forth in SEQ ID NO: 33, and a VL domain having the sequence as set forth in any one of SEQ ID NOs: 43, 44, 45 or 46, or(vii) a VH domain having the sequence as set forth in SEQ ID NO: 34, and a VL domain having the sequence as set forth in any one of SEQ ID NOs: 43, 44, 45 or 46, or(viii) a VH domain having the sequence as set forth in SEQ ID NO: 35, and a VL domain having the sequence as set forth in any one of SEQ ID NOs: 43, 44, 45 or 46, or(ix) a VH domain having the sequence as set forth in SEQ ID NO: 36, and a VL domain having the sequence as set forth in any one of SEQ ID NOs: 43, 44, 45 or 46, or(x) a VH domain having the sequence as set forth in SEQ ID NO: 37, and a VL domain having the sequence as set forth in any one of SEQ ID NOs: 43, 44, 45 or 46, or(xi) a VH domain having the sequence as set forth in SEQ ID NO: 38, and a VL domain having the sequence as set forth in any one of SEQ ID NOs: 43, 44, 45 or 46, or(xii) a VH domain having the sequence as set forth in SEQ ID NO: 39, and a VL domain having the sequence as set forth in any one of SEQ ID NOs: 43, 44, 45 or 46, or(xiii) a VH domain having the sequence as set forth in SEQ ID NO: 40, and a VL domain having the sequence as set forth in any one of SEQ ID NOs: 43, 44, 45 or 46, or(xiv) a VH domain having the sequence as set forth in SEQ ID NO: 41, and a VL domain having the sequence as set forth in any one of SEQ ID NOs: 43, 44, 45 or 46, or(xv) a VH domain having the sequence as set forth in SEQ ID NO: 42, and a VL domain having the sequence as set forth in any one of SEQ ID NOs: 43, 44, 45 or 46.

[0108] In certain embodiments, the anti-IL-33 antibody constructs of the present disclosure comprise at least one IL-33 antigen-binding domain, where the IL-33 antigen-binding domain comprises(i) a VH domain having the sequence as set forth in SEQ ID NO: 38, and a VL domain having the sequence as set forth in SEQ ID NO: 45, or(ii) a VH domain having the sequence as set forth in SEQ ID NO: 32, and a VL domain having the sequence as set forth in SEQ ID NO: 46.Formats

[0109] The anti -IL-33 antibody constructs of the present disclosure may have various formats. The minimal component of the anti -IL-33 antibody construct is an antigen-binding domain that binds to human IL-33. The anti-IL-33 antibody constructs may further optionally comprise one or more additional antigen-binding domains and / or a scaffold. In those embodiments in which the anti-IL-33 antibody construct comprises two or more antigen-binding domains, each additional antigen-binding domain may bind to the same epitope within IL-33, may bind to a different epitope within IL-33, or may bind to a different antigen. Thus, the anti-IL-33 antibody construct may be, for example, monospecific, biparatopic, bispecific or multispecific.

[0110] In certain embodiments, the anti-IL-33 antibody construct comprises at least one IL-33 antigen-binding domain and a scaffold, where the IL-33 antigen-binding domain is operably linked to the scaffold. The term “operably linked,” as used herein, means that the components described are in a relationship permitting them to function in their intended manner. Examples of suitable scaffolds are described below.

[0111] In certain embodiments, the anti -IL-33 antibody construct comprises two antigenbinding domains optionally operably linked to a scaffold, where at least one of the antigen-binding domains is an IL-33 antigen-binding domain. In some embodiments, the anti-IL-33 antibody construct may comprise three or four antigen-binding domains and optionally a scaffold, where at least one of the antigen-binding domains is an IL-33 antigen-binding domain. In these formats, when comprising a scaffold, at least a first antigen-binding domain is operably linked to the scaffold and the remaining antigen-binding domain(s) may each independently be operably linked to the scaffold or to the first antigen-binding domain or, when more than two antigen-binding domains are present, to another antigen-binding domain.

[0112] Anti -IL-33 antibody constructs that lack a scaffold may comprise a single IL-33 antigen-binding domain in an appropriate format, such as an sdAb, or they may comprise two or more antigen-binding domains optionally operably linked by one or more linkers, where at least one of the antigen-binding domains is an IL-33 antigen-binding domain. In such anti-IL-33 antibody constructs, the antigen-binding domains may be in the form of scFvs, Fabs, sdAbs, or a combination thereof. For example, using scFvs as the antigen-binding domains, formats such as atandem scFv ((scFv)2 or taFv) may be constructed, in which the scFvs are connected together by a flexible linker. scFvs may also be used to construct diabody formats, which comprise two scFvs connected by a short linker (usually about 5 amino acids in length). The restricted length of the linker results in dimerization of the scFvs in a head-to-tail manner. In any of the preceding formats, the scFvs may be further stabilized by inclusion of an interdomain disulfide bond. For example, a disulfide bond may be introduced between VL and VH through introduction of an additional cysteine residue in each chain (for example, at position 44 in VH and position 100 in VL) (see, for example, Fitzgerald et al., 1997, Protein Engineering, 10: 1221-1225), or a disulfide bond may be introduced between two VHs to provide a construct having a DART format (see, for example, Johnson etal., 2010, JMol. Biol., 399:436-449).

[0113] Similarly, formats comprising two sdAbs, such as VHs or VHHs, connected together through a suitable linker may be employed in some embodiments. Other examples of anti- IL-33 antibody construct formats that lack a scaffold include those based on Fab fragments, for example, Fab2 and F(ab’)2 formats, in which the Fab fragments are connected through a linker or an IgG hinge region.

[0114] Combinations of antigen-binding domains in different forms may also be employed to generate alternative scaffold-less formats. For example, an scFv or a sdAb may be fused to the C-terminus of either or both of the light and heavy chain of a Fab fragment resulting in a bivalent (Fab-scFv / sdAb) construct.

[0115] In certain embodiments, the anti -IL-33 antibody construct may be in an antibody format that is based on an immunoglobulin (Ig). In certain embodiments, the anti -IL-33 antibody construct may be based on an IgG class immunoglobulin, for example, an IgGl, IgG2, IgG3 or IgG4 immunoglobulin. In some embodiments, the anti-IL-33 antibody construct may be based on an IgGl or IgG4 immunoglobulin. In the context of the present disclosure, when an anti-IL-33 antibody construct is based on a specified immunoglobulin isotype, it is meant that the anti-IL-33 antibody construct comprises all or a portion of the constant region of the specified immunoglobulin isotype. For example, an anti-IL-33 antibody construct based on a given Ig isotype may comprise at least one IL-33 antigen-binding domain operably linked to an Ig scaffold, where the scaffold comprises an Fc region from the given isotype and optionally an Ig hinge region from the same or a different isotype. It is to be understood that the anti-IL-33 antibody constructs may also comprise hybrids of isotypes and / or subclasses in some embodiments. It is also to beunderstood that the Fc region and / or hinge region may optionally be modified to impart one or more desirable functional properties as is known in the art.

[0116] In some embodiments, the anti -IL-33 antibody constructs may be derived from two or more immunoglobulins that are from different species, for example, the anti-IL-33 antibody construct may be a chimeric antibody or a humanized antibody. The terms “chimeric antibody” and “humanized antibody” both refer generally to antibodies that combine immunoglobulin regions or domains from more than one species.

[0117] A “chimeric antibody” typically comprises at least one variable domain from a nonhuman antibody, such as a rabbit or rodent (for example, murine) antibody, and at least one constant domain from a human antibody. The human constant domain of a chimeric antibody need not be of the same isotype as the non-human constant domain it replaces. Chimeric antibodies are discussed, for example, in Morrison etal., 1984, Proc. Natl. Acad. Sci. USA, 81:6851-55, and U.S. Patent No. 4,816,567.

[0118] A “humanized antibody” is a type of chimeric antibody that contains minimal sequence derived from a non-human antibody. Generally, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a hypervariable region (CDR) of the recipient are replaced by residues from a hypervariable region (CDR) of a non-human species (donor antibody), such as mouse, rat, rabbit or non-human primate, having the desired specificity and affinity for a target antigen. This technique for creating humanized antibodies is often referred to as “CDR grafting.”

[0119] In some instances, additional modifications may be made to a humanized antibody to further refine antibody performance . For example, framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues, or the humanized antibodies may comprise residues that are not found in either the recipient antibody or the donor antibody. In general, a variable domain in a humanized antibody will comprise all or substantially all of the CDRs from a non-human immunoglobulin and all or substantially all of the FRs from a human immunoglobulin sequence. Humanized antibodies are described in more detail in Jones, et al., 1986, Nature, 321:522-525; Riechmann, etal., 1988, Nature, 332:323-329, and Presta, 1992, Curr. Op. Struct. Biol., 2:593-596, for example.

[0120] A number of approaches are known in the art for selecting the most appropriate human frameworks into which to graft the non-human CDRs. Early approaches used a limitedsubset of well-characterised human antibodies, irrespective of the sequence identity to the nonhuman antibody providing the CDRs (the “fixed frameworks” approach). More recent approaches have employed variable regions with high amino acid sequence identity to the variable regions of the non-human antibody providing the CDRs (“homology matching” or “best-fit” approach). An alternative approach is to select fragments of the framework sequences within each light or heavy chain variable region from several different human antibodies. CDR grafting may in some cases result in a partial or complete loss of affinity of the grafted molecule for its target antigen. In such cases, affinity can be restored by back-mutating some of the residues of human origin to the corresponding non-human ones. Methods for preparing humanized antibodies by these approaches are well-known in the art (see, for example, Tsurushita & Vasquez, 2004, Humanization of Monoclonal Antibodies, Molecular Biology of B Cells, 533-545, Elsevier Science (USA); Jones et al., 1986, Nature, 321:522-525; Riechmann et al., 1988, Nature, 332:323-329; Presta et al., 1997, Cancer Res, 57(20):4593-4599).

[0121] Alternatively, or in addition to, these traditional approaches, more recent technologies may be employed to further reduce the immunogenicity of a CDR-grafted humanized antibody. For example, frameworks based on human germline sequences or consensus sequences may be employed as acceptor human frameworks rather than human frameworks with somatic mutation(s). Another technique that aims to reduce the potential immunogenicity of non-human CDRs is to graft only specificity-determining residues (SDRs). In this approach, only the minimum CDR residues required for antigen-binding activity (the “SDRs”) are grafted into a human germline framework. This method improves the “humanness” (i.e. the similarity to human germline sequence) of the humanized antibody and thus may help reduce the risk of immunogenicity of the variable region. These techniques have been described in various publications (see, for example, Almagro & Fransson, 2008, Front Biosci, 13: 1619-1633; Tan, et al., 2002, J Immunol, 169: 1119-1125; Hwang, etal., 2005, Methods, 36:35-42; Pelat, etal., 2008, J Mol Biol, 384: 1400-1407; Tamura, et al., 2000, J Immunol, 164: 1432-1441; Gonzales, et al., 2004, Mol Immunol, 1:863-872, and Kashmiri, et al., 2005, Methods, 36:25-34).

[0122] In certain embodiments, the anti -IL-33 antibody construct of the present disclosure comprises humanized antibody sequences, for example, one or more humanized variable domains. In some embodiments, the anti-IL-33 antibody construct is a humanized antibody. Non-limiting examples of humanized antibodies based on the anti-IL-33 antibody h!48C04 (see Example 1 andTable D (Fig. 21)) are described herein (variants v33068-v33111; see Examples and Table D (Fig. 21)).

[0123] Humanized antibodies may also be “affinity matured” in order to improve binding to the target antigen. In vitro affinity maturation usually involves a diversification of the antibody base sequence, followed by stringent selections to isolate higher-affinity binders. Various affinity maturation techniques are known in the art (see, for example, Li, et al., 2023, Ini J Biological Macromolecules, 247: 125733, Kielczewska, et al., 2022, JBC, 298(2): 101533). In certain embodiments, the anti -IL-33 construct of the present disclosure may comprise humanized, affinity matured antibody sequences, for example, one or more humanized, affinity matured variable domains.Scaffolds

[0124] In certain embodiments, the anti-IL-33 antibody constructs of the present disclosure comprise one or more IL-33 antigen-binding domains operably linked to a scaffold. The antigenbinding domain(s) may be in one or a combination of the forms described above (for example, scFvs, Fabs and / or sdAbs). Examples of suitable scaffolds are described in more detail below and include, but are not limited to, immunoglobulin Fc regions, albumin, albumin analogues and derivatives, heterodimerizing peptides (such as leucine zippers, heterodimer-forming “zipper” peptides derived from Jun and Fos, IgG CHI and CL domains or bamase-barstar toxins), cytokines, chemokines or growth factors. Other examples include antibodies based on the DOCK- AND-LOCK™ (DNL™) technology developed by IBC Pharmaceuticals, Inc. and Immunomedics, Inc. (see, for example, Chang, et al., 2007, Clin. Cancer Res., 13:5586s-5591s).

[0125] A scaffold may be a peptide, polypeptide, polymer, nanoparticle or other chemical entity. Where the scaffold is a polypeptide, each antigen-binding domain of the anti-IL-33 antibody construct may be linked to either the N- or C-terminus of the polypeptide scaffold. Anti-IL-33 antibody constructs comprising a polypeptide scaffold in which one or more of the antigen-binding domains are linked to a region other than the N- or C-terminus, for example, via the side chain of an amino acid with or without a linker, are also contemplated in certain embodiments.

[0126] In embodiments where the anti-IL-33 antibody construct comprises a scaffold that is a peptide or polypeptide, the antigen-binding domain(s) may be linked to the scaffold by genetic fusion or chemical conjugation. Typically, when the scaffold is a peptide or polypeptide, the antigen-binding domain(s) are linked to the scaffold by genetic fusion. In some embodiments,where the scaffold is a polymer or nanoparticle, the antigen-binding domain(s) may be linked to the scaffold by chemical conjugation.

[0127] A number of protein domains are known in the art that comprise selective pairs of two different polypeptides and may be used to form a scaffold. An example is leucine zipper domains such as Fos and Jun that selectively pair together (Kostelny, et al., J Immunol, 148: 1547- 53 (1992); Wranik, et al., J. Biol. Chem., 287: 43331-43339 (2012)). Other selectively pairing molecular pairs include, for example, the bamase-barstar pair (Deyev, et al., Nat Biotechnol, 21: 1486-1492 (2003)), DNA strand pairs (Chaudri, et al., FEBS Letters, 450(l-2):23-26 (1999)) and split fluorescent protein pairs (International Patent Application Publication No. WO 2011 / 135040).

[0128] Other examples of protein scaffolds include immunoglobulin Fc regions, albumin, albumin analogues and derivatives, toxins, cytokines, chemokines and growth factors. The use of protein scaffolds in combination with antigen-binding moieties has been described (see, for example, Muller et al., 2007, J. Biol. Chem., 282: 12650-12660; McDonaugh et al., 2012, Mol. Cancer Ther., 11:582-593; Vallera et al., 2005, Clin. Cancer Res., 11:3879-3888; Song et al., 2006, Biotech. Appl. Biochem., 45: 147-154, and U.S. Patent Application Publication No. 2009 / 0285816).

[0129] For example, fusing antigen-binding moieties such as scFvs, diabodies or single chain diabodies to albumin has been shown to improve the serum half-life of the antigen-binding moieties (Muller et al., ibid.). Antigen-binding moieties may be fused at the N- and / or C-termini of albumin, optionally via a linker.

[0130] Derivatives of albumin in the form of heteromultimers that comprise two transporter polypeptides obtained by segmentation of an albumin protein such that the transporter polypeptides self-assemble to form quasi-native albumin have been described (see International Publication Nos. WO 2012 / 116453 and WO 2014 / 012082). As a result of the segmentation of albumin, the heteromultimer includes four termini and thus can be fused to up to four different antigen-binding moieties, optionally via linkers.

[0131] In certain embodiments, the anti -IL-33 antibody construct may comprise a protein scaffold. In some embodiments, the anti-IL-33 antibody construct may comprise a protein scaffold that is based on an immunoglobulin Fc region, an albumin or an albumin analogue or derivative.In some embodiments, the anti-IL-33 antibody construct may comprise a protein scaffold that is based on an immunoglobulin Fc region, for example, an IgG Fc region.Fc Regions

[0132] The terms “Fc region,” “Fc” or “Fc domain” as used herein refer to a C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region. The term includes native sequence Fc regions and variant Fc regions. Unless otherwise specified herein, numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also called the EU index, as described in Kabat, et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991).

[0133] In certain embodiments, the anti-IL-33 antibody constructs of the present disclosure may comprise a scaffold that is based on an immunoglobulin Fc region. The Fc region may be dimeric and composed of two Fc polypeptides or alternatively, the Fc region may be composed of a single polypeptide. In certain embodiments, the anti -IL-33 antibody construct comprises a dimeric Fc region.

[0134] An “Fc polypeptide” in the context of a dimeric Fc refers to one of the two polypeptides forming the dimeric Fc domain, i. e. a polypeptide comprising one or more C-terminal constant regions of an immunoglobulin heavy chain that is capable of stable self-association. When referring to the polypeptides forming a dimeric Fc region, the terms “first Fc polypeptide” and “second Fc polypeptide” may be used interchangeably provided that the Fc region comprises one first Fc polypeptide and one second Fc polypeptide.

[0135] An Fc region may comprise a CH3 domain or it may comprise both a CH3 and a CH2 domain. For example, in certain embodiments, each Fc polypeptide of a dimeric IgG Fc region may comprise an IgG CH2 domain sequence and an IgG CH3 domain sequence. In such embodiments, the CH3 domain comprises two CH3 sequences, one from each of the two Fc polypeptides of the dimeric Fc region, and the CH2 domain comprises two CH2 sequences, one from each of the two Fc polypeptides of the dimeric Fc region. In certain embodiments, the Fc region comprises a CH3 domain, a CH2 domain and an IgG hinge region, with each Fc polypeptide comprising a CH3 domain sequence, a CH2 domain sequence and an IgG hinge region sequence.

[0136] In some embodiments, the anti-IL-33 antibody construct may comprise a scaffold that is an IgG Fc region. In some embodiments, the anti-IL-33 antibody construct may comprise ascaffold that is a human IgG Fc region. In some embodiments, the anti-IL-33 antibody construct may comprise a scaffold that is an IgGl or IgG4 Fc region. In some embodiments, the anti-IL-33 antibody construct may comprise a scaffold that is a human IgGl or IgG4 Fc region. In some embodiments, the anti-IL-33 antibody construct may comprise a scaffold that is a human IgGl or IgG4 Fc region comprising a CH3 domain, a CH2 domain and an IgG hinge region.

[0137] In certain embodiments, the anti -IL-33 antibody construct may comprise a scaffold based on an IgG Fc region, which is a homodimeric Fc region, comprising a first Fc polypeptide and a second Fc polypeptide, each comprising a CH3 sequence, and optionally a CH2 sequence and in which the amino acid sequences of the first and second Fc polypeptides are the same. In certain embodiments, the homodimeric Fc region further comprises an IgG hinge region.

[0138] In certain embodiments, the anti -IL-33 antibody construct may comprise a scaffold based on an IgG Fc region, for example an IgGl or IgG4 Fc region, which is a heterodimeric Fc region, comprising a first Fc polypeptide and a second Fc polypeptide, each comprising a CH3 sequence, and optionally a CH2 sequence and in which the amino acid sequences of the first and second Fc polypeptides are different. In certain embodiments, the heterodimeric Fc region further comprises an IgG hinge region. Heterodimeric Fc regions may be particularly useful in those embodiments in which the anti -IL-33 antibody construct comprises two or more different antigenbinding domains, for example, when the anti -IL-33 antibody construct is biparatopic, bispecific or multispecific.

[0139] In some embodiments, the anti-IL-33 antibody construct may comprise a scaffold based on an Fc region, for example an IgGl or IgG4 Fc region, which comprises two CH3 sequences, at least one of which comprises one or more amino acid modifications. In some embodiments, the anti-IL-33 antibody construct may comprise a scaffold based on an Fc region which comprises two CH3 sequences and two CH2 sequences, at least one of the CH2 sequences comprising one or more amino acid modifications. In some embodiments, the anti-IL-33 antibody construct may comprise a scaffold based on an Fc region which comprises two CH3 sequences and two CH2 sequences, where at least one of the CH3 sequences comprises one or more amino acid modifications and at least one of the CH2 sequences comprises one or more amino acid modifications.

[0140] In some embodiments, the anti -IL-33 antibody construct may comprise a heterodimeric Fc region comprising a modified CH3 domain, where the modified CH3 domain isan asymmetrically modified CH3 domain comprising one or more asymmetric amino acid modifications. As used herein, an “asymmetric amino acid modification” refers to a modification, such as a substitution or an insertion, in which an amino acid at a specific position on a first CH3 or CH2 sequence is different to the amino acid on a second CH3 or CH2 sequence at the same position. These asymmetric amino acid modifications can be a result of modification of only one of the two amino acids at the same respective amino acid position on each sequence, or different modifications of both amino acids at the same respective position on each of the first and second CH3 or CH2 sequences. Each of the first and second CH3 or CH2 sequences of a heterodimeric Fc may comprise one or more than one asymmetric amino acid modification.

[0141] In some embodiments, the anti -IL-33 antibody construct may comprise a heterodimeric Fc comprising a modified CH3 domain, where the modified CH3 domain comprises one or more amino acid modifications that promote formation of the heterodimeric Fc over formation of a homodimeric Fc. In some embodiments, one or more of the amino acid modifications are asymmetric amino acid modifications.

[0142] Amino acid modifications that may be made to the CH3 domain of an Fc in order to promote formation of a heterodimeric Fc are known in the art and include, for example, those described in International Publication No. WO 96 / 027011 (“knobs into holes”), Gunasekaran et al., 2010, J Biol Chem, 285, 19637-46 (“electrostatic steering”), Davis etal., 2010, Prot Eng Des Sei, 23(4): 195-202 (strand exchange engineered domain (SEED) technology) and Labrijn et al., 2013, Proc Natl Acad Sci USA, 110(13):5145-50 (Fab-arm exchange). Other examples include approaches combining positive and negative design strategies to produce stable asymmetrically modified Fc regions as described in International Publication Nos. WO 2012 / 058768 and WO 2013 / 063702. In certain embodiments, the anti -IL-33 antibody construct may comprise a scaffold based on a modified Fc region as described in International Publication No. WO 2012 / 058768 or WO 2013 / 063702.

[0143] Table 5 provides the amino acid sequences of a human IgGl Fc region (SEQ ID NO: 50) and a human IgG4 Fc region (SEQ ID NO: 51). Table 6 shows CH3 domain amino acid amino acid substitutions that promote formation of a heterodimeric Fc as described in International Patent Publication Nos. WO 2012 / 058768 and WO 2013 / 063702.

[0144] In certain embodiments, the anti -IL-33 antibody construct may comprise a heterodimeric Fc scaffold based on an IgGl or IgG4 Fc region having a modified CH3 domaincomprising the amino acid substitutions of any one of Variant 1, Variant 2, Variant 3, Variant 4 or Variant 5, as shown in Table 6.Table 5: Amino Acid Sequence of the Human IgGl and IgG4 Fc Regions (CH2 and CH3 Domains)Table 6: CH3 Domain Amino Acid Substitutions Promoting Heterodimer Formation

[0145] In those embodiments in which the anti-IL-33 antibody construct comprises an IgG4 Fc region as a scaffold, the Fc region may comprise one or more amino acid mutations to decrease or eliminate Fab arm exchange and / or to improve stability. Examples of such amino acid mutations include the amino acid substitutions Y219C, G220C, S228P and R409K (see, forexample, Handlogten, et al., 2020, MAbs, 12(1): 1779974; Namisaki, et al., 2020, PLoS ONE, 15(3): e0229027). In certain embodiments, the anti-IL-33 antibody construct may comprise an Fc scaffold based on an IgG4 Fc region comprising the amino acid substitution S228P (EU numbering; S24 IP Kabat numbering), where the Fc region may be homodimeric or heterodimeric. In certain embodiments, the anti-IL-33 antibody construct may comprise an Fc scaffold based on an IgG4 Fc region comprising the amino acid substitution R409K, where the Fc region may be homodimeric or heterodimeric. In certain embodiments, the anti-IL-33 antibody construct may comprise an Fc scaffold based on an IgG4 Fc region comprising the amino acid substitutions S228P and R409K, where the Fc region may be homodimeric or heterodimeric. In some embodiments, the anti-IL-33 antibody construct may comprise a heterodimeric IgG4 Fc region comprising the amino acid substitution R409K and amino acid substitutions of any one of Variant1, Variant 2, Variant 3, Variant 4 or Variant 5, as shown in Table 6. In some embodiments, the anti -IL-33 antibody construct may comprise a heterodimeric IgG4 Fc region comprising the amino acid substitutions S228P and R409K and amino acid substitutions of any one of Variant 1, Variant2, Variant 3, Variant 4 or Variant 5, as shown in Table 6.

[0146] In some embodiments, the anti-IL-33 antibody construct may comprise a scaffold based on an Fc region comprising two CH3 sequences and two CH2 sequences, at least one of the CH2 sequences comprising one or more amino acid modifications that affect the binding of Fc receptors (FcRs) to the Fc, such as receptors of the FcyRI, FcyRII and FcyRIII subclasses. In some embodiments, the anti-IL-33 antibody construct comprises a scaffold based on an IgG Fc having a modified CH2 domain, wherein the modification of the CH2 domain results in altered binding to one or more of the FcyRI, FcyRII and FcyRIII receptors.

[0147] A number of amino acid modifications to the CH2 domain that selectively alter the affinity of the Fc for different Fey receptors are known in the art (see, for example, Lu, et al. , 2011 , J Immunol Methods, 365(1-2): 132-41; Stavenhagen, et al. 2007, Cancer Res 67(18):8882-90; Nordstrom JL, et al., 2011, Breast Cancer Res, 13(6):R123; Stewart, etal., 2011, Protein Eng Des Sei., 24(9):671-8; Shields, et al., 2001, J Biol Chem, 276(9):6591-604; Lazar, et al., 2006, Proc Natl Acad Sci USA, 103(11):4005-10; Chu, et al., 2008, Mol Immunol, 45(15):3926-33, and International Publication No. WO 2021 / 232162). Amino acid modifications that result in increased binding and amino acid modifications that result in decreased binding can each be useful in certain indications. For example, increasing binding affinity of an Fc for FcyRIIIa (an activating receptor)may result in increased antibody dependent cell-mediated cytotoxicity (ADCC), which in turn results in increased lysis of the target cell. Decreased binding to FcyRIIb (an inhibitory receptor) likewise may be beneficial in some circumstances. In certain indications, a decrease in, or elimination of, ADCC and complement-mediated cytotoxicity (CDC) may be desirable. In such cases, modified CH2 domains comprising amino acid modifications that result in increased binding to FcyRIIb or amino acid modifications that decrease or eliminate binding of the Fc region to all Fey receptors (“knock-out” variants) may be useful.

[0148] In certain embodiments, the anti-IL-33 antibody construct comprises a scaffold based on an IgG Fc having a modified CH2 domain, in which the modified CH2 domain comprises one or more amino acid modifications that result in decreased or eliminated binding of the Fc region to all Fey receptors (i.e. a “knock-out” variant). Various publications describe strategies that have been used to engineer antibodies to produce “knock-out” variants (see, for example, Strohl, 2009, Curr Opin Biotech 20:685-691, and Strohl & Strohl, “Antibody Fc engineering for optimal antibody performance" In Therapeutic Antibody Engineering, Cambridge: Woodhead Publishing, 2012, pp 225-249). These strategies include reduction of effector function through modification of glycosylation, use of IgG2 / IgG4 scaffolds, or the introduction of mutations in the hinge or CH2 domain of the Fc (see also, U.S. Patent Publication No. 2011 / 0212087, International Publication No. WO 2006 / 105338, U.S. Patent Publication No. 2012 / 0225058, U.S. Patent Publication No. 2012 / 0251531 and Strop et al., 2012, Mol. Biol., 420: 204-219).

[0149] Examples of mutations that may be introduced into the hinge or CH2 domain of an IgGl Fc to produce a “knock-out” variant include the amino acid substitutions L234A / L235A and L234A / L235A / D265S, which are typically introduced into both chains of the CH2 domain. In certain embodiments, the anti-IL-33 antibody construct comprises a scaffold based on an IgGl Fc having a modified CH2 domain that comprises the amino acid substitutions L234A / L235 A / D265 S in both chains of the CH2 domain.

[0150] In certain embodiments, the anti-IL-33 antibody constructs described herein may comprise a scaffold based on an IgG Fc in which native glycosylation has been modified. As is known in the art, glycosylation of an Fc may be modified to increase or decrease effector function. For example, mutation of the conserved asparagine residue at position 297 (N297) to alanine, glutamine, lysine or histidine (i.e. N297A, Q, K or H) results in an aglycoslated Fc that lacks alleffector function (Bolt et al., 1993, Eur. J. Immunol., 23:403-411; Tao & Morrison, 1989, J. Immunol., 143:2595-2601).

[0151] Other amino acid mutations in the CH2 domain that may be useful include amino acid mutations that result in increased binding to the neonatal Fc receptor (FcRn). Increased binding to FcRn may improve the in vivo half-life of the antibody construct. Amino acid substitutions in the CH2 domain that enhance binding to FcRn include, for example, the amino acid substitutions M252Y / S254T / T256E (“YTE mutations”) in both chains of the CH2 domain. In certain embodiments, the anti-IL-33 antibody constructs comprise YTE mutations.

[0152] In certain embodiments, the anti -IL-33 antibody constructs comprise an IgGl Fc region and comprise knock-out mutations and YTE mutations. In certain embodiments, the anti- IL-33 antibody constructs comprise an IgG4 Fc region and comprise YTE mutations.

[0153] In certain embodiments, the anti-IL-33 antibody constructs have the format of a full-size antibody (FSA). In some embodiments, the anti-IL-33 antibody constructs have the format of an IgG FSA, for example, an IgGl or IgG4 FSA. In some embodiments, the anti-IL-33 antibody construct is a FSA comprising a first heavy chain sequence (Hl), a second heavy chain sequence (H2), a first light chain sequence (LI) and a second light chain sequence (L2), where Hl pairs with LI and H2 pairs with L2. In some embodiments, the anti-IL-33 antibody construct is a monospecific FSA with a homodimeric Fc and comprises Hl, H2, LI and L2 sequences, where Hl and H2 have the same amino acid sequence, and LI and L2 have the same amino acid sequence. In some embodiments, the anti-IL-33 antibody construct is a monospecific FSA with a heterodimeric Fc and comprises Hl, H2, LI and L2 sequences, where Hl and H2 have different amino acid sequences, and LI and L2 have the same amino acid sequence. In some embodiments, the anti-IL-33 antibody construct is a biparatopic, bispecific or multispecific FSA with a heterodimeric Fc and comprises Hl, H2, LI and L2 sequences, where Hl and H2 have different amino acid sequences, and LI and L2 have different amino acid sequences.

[0154] In certain embodiments, the anti-IL-33 antibody construct is a FSA having a set of Hl, H2, LI and L2 sequences comprising the Hl, H2, LI and L2 amino acid sequences as set forth in Tables F and G for any one of variants v33006 and v33068-v33111. In certain embodiments, the anti-IL-33 antibody construct is a FSA having a set of Hl, H2, LI and L2 sequences comprising the Hl, H2, LI and L2 amino acid sequences as set forth in Tables F and G for any one of variants v33068-v33111. As is known in the art, expression of antibody heavy chain sequences in certaincell lines or from certain expression vector may result in the inclusion of a C-terminal lysine residue on one or both of the heavy chains. Accordingly, certain embodiments of the present disclosure relate to anti-IL-33 antibody constructs that are FSAs having a set of Hl, H2, LI and L2 sequences comprising the Hl, H2, LI and L2 amino acid sequences as set forth in Tables F and G for any one of variants v33006 and v33068-v33111, in which one or both of the Hl and H2 sequences comprise a C-terminal lysine.BISPECIFIC ANTIBODY CONSTRUCTS

[0155] Certain embodiments of the present disclosure relate to bispecific antibody constructs that specifically bind to IL-33 and to a second target antigen, where the second target antigen is not IL-33. The bispecific antibody constructs may be, for example, bivalent, trivalent or tetravalent. Higher valencies are also contemplated in certain embodiments. In some embodiments, the bispecific antibody constructs are bivalent and comprise a first antigen-binding domain that binds to IL-33 (the IL-33 antigen-binding domain) and a second antigen-binding domain that binds to a target antigen other than IL-33 (the second target antigen-binding domain). In some embodiments, the bispecific antibody constructs are trivalent or tetravalent and comprise one or two antigen-binding domains that bind to IL-33 (the IL-33 antigen-binding domain) and one or two antigen-binding domains that bind to a target antigen other than IL-33.

[0156] In accordance with the present disclosure, the at least one IL-33 antigen-binding domain comprised by the bispecific antibody constructs may be the IL-33 antigen-binding domain of any one of the embodiments described above.

[0157] The bispecific antibody constructs further comprise at least one antigen-binding domain that binds to the second target antigen. In some embodiments, the second target antigen is a cytokine or a cytokine receptor. In some embodiments, the second target antigen is a cytokine or cytokine receptor associated with an inflammatory or autoimmune disorder. In some embodiments, the second target antigen is a cytokine or cytokine receptor associated with asthma or chronic obstructive pulmonary disease (COPD), for example, IL- 13, IL-4 or IL-4Ra. In some embodiments, the second target antigen is IL-4Ra.

[0158] The second target antigen-binding domain may be derived from a known antibody that binds to the second target antigen, for example, from a known antibody that binds to IL- 13, IL-4 or IL-4Ra, or it may be derived from an antibody against the second target antigen generatedby standard antibody generation techniques. Examples of known antibodies that bind to IL-13 include, but are not limited to, lebrikizumab, tralokinumab, dectrekumab, anrukinzumab, cendakimab, romilkimab and GSK679586. Examples of known antibodies that bind to IL-4 include, but are not limited to, pascolizumab, romilkimab and GSK2434735. Examples of known antibodies that bind IL-4Ra include, but are not limited to, dupilumab, stapokibart, rademikibart, AMG317 and NM26-2198. The VH and VL sequences for these known antibodies may be obtained from publicly accessible databases, such as the Therapeutic Antibody Database (Tabs).

[0159] In certain embodiments, the second target antigen-binding domain comprised by the bispecific antibody constructs binds to IL-4Ra (an “IL-4Ra antigen-binding domain”).

[0160] In certain embodiments, the second target antigen-binding domain comprised by the bispecific antibody constructs is an immunoglobulin-based binding domain, such as an antigenbinding antibody fragment. Examples of an antigen-binding antibody fragment include, but are not limited to, a Fab fragment (Fab), a Fab’ fragment (Fab’), a single chain Fab (scFab), a single chain Fv (scFv) and a single domain antibody (sdAb). In certain embodiments, the second target antigen-binding domain is a Fab or an scFv.IL-4Ra Antigen-Binding Domains

[0161] In certain embodiments, the bispecific antibody constructs are anti-IL-33 x anti-IL- 4Ra bispecific antibodies comprising at least one IL-33 antigen-binding domain as described in any one of the embodiments defined above and at least one IL-4Ra antigen-binding domain. In these embodiments, the at least one IL-4Ra antigen-binding domain comprised by the bispecific antibody construct is capable of specifically binding human IL-4Ra. In certain embodiments, the at least one IL-4Ra antigen-binding domain comprised by the bispecific antibody construct may be capable of binding to human IL-4Ra and to an IL-4Ra from one or more non-human species. In certain embodiments, the at least one IL-4Ra antigen-binding domain comprised by the bispecific antibody construct is capable of binding to human IL-4Ra and cynomolgus monkey IL- 4Ra.

[0162] The protein sequences of IL-4Ra from various sources are known in the art and readily available from publicly accessible databases, such as GenBank or UniProtKB. Examples of human IL-4Ra sequences include those provided under NCBI reference numbers P24394, NP_000409.1, NP_001244335.1 and NP_001244336.1. IL-4Ra is a transmembrane protein with a 207 amino acid extracellular domain, a 24 amino acid transmembrane segment, and a 569 aminoacid cytoplasmic domain. IL-4Ra antigen-binding domains typically bind to the extracellular domain of IL-4Ra. The amino acid sequence of the extracellular domain of an exemplary human IL-4Ra protein is provided in Table 7 as SEQ ID NO: 52 (amino acids 24-232 of NCBI Accession No. P24394).Table 7: Amino Acid Sequence of the Extracellular Domain of Human IL-4Rot

[0163] In certain embodiments, the one or more IL-4Ra antigen-binding domain comprised by the bispecific antibody constructs is an antigen-binding antibody fragment, such as a Fab fragment (Fab), a Fab’ fragment (Fab’), a single chain Fab (scFab), a single chain Fv (scFv) and a single domain antibody (sdAb). In certain embodiments, the IL-4Ra antigen-binding domain is a Fab or an scFv. In some embodiments, the IL-4Ra antigen-binding domain is a Fab.

[0164] In certain embodiments, the IL-4Ra antigen-binding domain(s) comprised by the bispecific antibody constructs of the present disclosure may be derived from a known anti-IL-4Ra antibody, such as dupilumab, stapokibart, rademikibart, AMG317 or NM26-2198. In some embodiments, the bispecific antibody constructs may comprise one of the IL-4Ra antigen-binding domains described in the Examples provided herein, for example, the IL-4Ra binding domain of any one of the variants v38597, v38504, v43194, v43195, v43196, v43197, v43198, v43199 or v43200.

[0165] The CDR sequences of the IL-4Ra antigen-binding domains comprised by variants v38597, v38504, v43194, v43195, v43196, v43197, v43198, v43199 and v43200 described in the Examples herein are provided in Fig. 18 (Table Bl) and Fig. 19 (Table B2) and the VH and VL sequences are provided in Fig. 20 (Tables Cl and C2).

[0166] In certain embodiments, the bispecific antibody constructs of the present disclosure comprise at least one IL-4Ra binding domain, where the IL-4Ra binding domain comprises the heavy chain CDR sequences (HCDR1, HCDR2, HCDR3) and the light chain CDR sequences(LCDR1, LCDR2, LCDR3) of the IL-4Ra binding domain of any one of variants v38597, v38504,V43194, v43195, v43196, v43197, v43198, v43199 and v43200.

[0167] In certain embodiments, the bispecific antibody constructs of the present disclosure comprise at least one IL-4Ra antigen-binding domain, where the IL-4Ra antigen-binding domain comprises the CDR sequences of the VH domain as set forth in any one of SEQ ID NOs: 90, 91, 93, 94, 95, 96, 97, 98 or 99. In some embodiments, the bispecific antibody constructs comprise at least one IL-4Ra antigen-binding domain, where the IL-4Ra antigen-binding domain comprises the CDR sequences of the VH domain as set forth in SEQ ID NO: 90 or 93. In certain embodiments, the bispecific antibody constructs of the present disclosure comprise at least one IL-4Ra antigenbinding domain, where the IL-4Ra antigen-binding domain comprises the CDR sequences of the VL domain as set forth in any one of SEQ ID NOs: 92, 100, 101, 102, 103, 104, 105 or 106. In some embodiments, the bispecific antibody constructs comprise at least one IL-4Ra antigenbinding domain, where the IL-4Ra antigen-binding domain comprises the CDR sequences of the VL domain as set forth in SEQ ID NO: 92.

[0168] In certain embodiments, the bispecific antibody constructs of the present disclosure comprise at least one IL-4Ra antigen-binding domain, where the IL-4Ra antigen-binding domain comprises the CDR sequences of the VH domain as set forth in any one of SEQ ID NOs: 90, 91, 93, 94, 95, 96, 97, 98 or 99, and the CDR sequences of the VL domain as set forth in any one of SEQ ID NOs: 92, 100, 101, 102, 103, 104, 105 or 106. In some embodiments, the bispecific antibody constructs comprise at least one IL-4Ra antigen-binding domain, where the IL-4Ra antigen-binding domain comprises the CDR sequences of the VH domain as set forth in SEQ ID NO: 90 or 93, and the CDR sequences of the VL domain as set forth in SEQ ID NO: 92.

[0169] In certain embodiments, the bispecific antibody constructs of the present disclosure comprise at least one IL-4Ra antigen-binding domain, where the IL-4Ra antigen-binding domain comprises a VH domain comprising an HCDR1 amino acid sequence comprising the sequence as set forth in any one of SEQ ID NOs: 53, 56, 66 or 67, an HCDR2 amino acid sequence comprising the sequence as set forth in any one of SEQ ID NOs: 54, 57, 79, 80, 84, 85, 86 or 87, and an HCDR3 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 55 or 58. In some embodiments, the bispecific antibody constructs comprise at least one IL-4Ra antigenbinding domain, where the IL-4Ra antigen-binding domain comprises a VH domain comprising an HCDR1 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 53 or 56, anHCDR2 amino acid sequence comprising the sequence as set forth in any one of SEQ ID NOs: 54, 57, 79, 80, 84, 85, 86 or 87, and an HCDR3 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 55 or 58. In some embodiments, the bispecific antibody constructs comprise at least one IL-4Ra antigen-binding domain, where the IL-4Ra antigen-binding domain comprises a VH domain comprising an HCDR1 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 53 or 56, an HCDR2 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 79 or 80, and an HCDR3 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 55 or 58.

[0170] In certain embodiments, the bispecific antibody constructs of the present disclosure comprise at least one IL-4Ra antigen-binding domain, where the IL-4Ra antigen-binding domain comprises a VL domain comprising an LCDR1 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 71 or 74, an LCDR2 amino acid sequence comprising the sequence as set forth in any one of SEQ ID NOs: 72, 75 or 88, and an LCDR3 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 73. In some embodiments, the bispecific antibody constructs comprise at least one IL-4Ra antigen-binding domain, where the IL-4Ra antigenbinding domain comprises a VL domain comprising an LCDR1 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 71 or 74, an LCDR2 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 72 or 75, and an LCDR3 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 73.

[0171] In certain embodiments, the bispecific antibody constructs of the present disclosure comprise at least one IL-4Ra antigen-binding domain, where the IL-4Ra antigen-binding domain comprises a VH domain comprising an HCDR1 amino acid sequence comprising the sequence as set forth in any one of SEQ ID NOs: 53, 56, 66 or 67, an HCDR2 amino acid sequence comprising the sequence as set forth in any one of SEQ ID NOs: 54, 57, 79, 80, 84, 85, 86 or 87, and an HCDR3 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 55 or 58, and a VL domain comprising an LCDR1 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 71 or 74, an LCDR2 amino acid sequence comprising the sequence as set forth in any one of SEQ ID NOs: 72, 75 or 88, and an LCDR3 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 73. In some embodiments, the bispecific antibody constructs of the present disclosure comprise at least one IL-4Ra antigen-binding domain, where the IL-4Ra antigen-binding domain comprises a VH domain comprising an HCDR1 amino acid sequencecomprising the sequence as set forth in SEQ ID NO: 53 or 56, an HCDR2 amino acid sequence comprising the sequence as set forth in any one of SEQ ID NOs: 54, 57, 79, 80, 84, 85, 86 or 87, and an HCDR3 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 55 or 58, and a VL domain comprising an LCDR1 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 71 or 74, an LCDR2 amino acid sequence comprising the sequence as set forth in any one of SEQ ID NOs: 72, 75 or 88, and an LCDR3 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 73. In some embodiments, the bispecific antibody constructs of the present disclosure comprise at least one IL-4Ra antigen-binding domain, where the IL-4Ra antigen-binding domain comprises a VH domain comprising an HCDR1 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 53 or 56, an HCDR2 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 79 or 80, and an HCDR3 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 55 or 58, and a VL domain comprising an LCDR1 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 71 or 74, an LCDR2 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 72 or 75, and an LCDR3 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 73.

[0172] In certain embodiments, the bispecific antibody constructs of the present disclosure comprise at least one IL-4Ra antigen-binding domain, where:(a) the IL-4Ra antigen-binding domain comprises a VL domain comprising an LCDR1 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 71 or 74, an LCDR2 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 72 or 75, and an LCDR3 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 73, and a VH domain comprising:(i) an HCDR1 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 53 or 56, an HCDR2 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 54 or 57, and an HCDR3 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 55 or 58, or(ii) an HCDR1 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 66 or 67, an HCDR2 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 54 or 57, and an HCDR3 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 55 or 58, or(iii) an HCDR1 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 53 or 56, an HCDR2 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 79 or 80, and an HCDR3 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 55 or 58, or(iv) an HCDR1 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 53 or 56, an HCDR2 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 79 or 84, and an HCDR3 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 55 or 58, or(v) an HCDR1 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 53 or 56, an HCDR2 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 79 or 85, and an HCDR3 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 55 or 58, or(vi) an HCDR1 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 53 or 56, an HCDR2 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 79 or 86, and an HCDR3 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 55 or 58, or(vii) an HCDR1 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 53 or 56, an HCDR2 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 79 or 87, and an HCDR3 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 55 or 58, or(b) the IL-4Ra antigen-binding domain comprises a VL domain comprising an LCDR1 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 71 or 74, an LCDR2 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 72 or 88, and an LCDR3 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 73, and a VH domain comprising an HCDR1 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 53 or 56, an HCDR2 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 79 or 80, and an HCDR3 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 55 or 58.

[0173] In certain embodiments, the bispecific antibody constructs of the present disclosure comprise at least one IL-4Ra antigen-binding domain, where the IL-4Ra antigen-binding domain comprises a VH domain having a sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%,98%, 99%, or 100% identical to the VH sequence as set forth in any one of SEQ ID NOs: 90, 91, 93, 94, 95, 96, 97, 98 or 99. In some embodiments, the bispecific antibody constructs comprise at least one IL-4Ra antigen-binding domain, where the IL-4Ra antigen-binding domain comprises a VH domain having a sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the VH sequence as set forth in SEQ ID NO: 90. In some embodiments, the bispecific antibody constructs comprise at least one IL-4Ra antigen-binding domain, where the IL-4Ra antigen-binding domain comprises a VH domain having a sequence as set forth in any one of SEQ ID NOs: 90, 91, 93, 94, 95, 96, 97, 98 or 99.

[0174] In certain embodiments, the bispecific antibody constructs of the present disclosure comprise at least one IL-4Ra antigen-binding domain, where the IL-4Ra antigen-binding domain comprises a VL domain having a sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the VL sequence as set forth in any one of SEQ ID NOs: 92, 100, 101, 102, 103, 104, 105 or 106. In some embodiments, the bispecific antibody constructs comprise at least one IL-4Ra antigen-binding domain, where the IL-4Ra antigen-binding domain comprises a VL domain having a sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the VL sequence as set forth in SEQ ID NO: 92. In some embodiments, the bispecific antibody constructs comprise at least one IL-4Ra antigen-binding domain, where the IL-4Ra antigen-binding domain comprises a VL domain having a sequence as set forth in any one of SEQ ID NOs: 92, 100, 101, 102, 103, 104, 105 or 106.

[0175] In certain embodiments, the bispecific antibody constructs of the present disclosure comprise at least one IL-4Ra antigen-binding domain, where the IL-4Ra antigen-binding domain comprises a VH domain having a sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the VH sequence as set forth in any one of SEQ ID NOs: 90, 91, 93, 94, 95, 96, 97, 98 or 99, and a VL domain having a sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the VL sequence as set forth in any one of SEQ ID NOs: 92, 100, 101, 102, 103, 104, 105 or 106. In some embodiments, the bispecific antibody constructs comprise at least one IL-4Ra antigen-binding domain, where the IL-4Ra antigen-binding domain comprises a VH domain having a sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the VH sequence as set forth in SEQ ID NO: 90, and a VL domain having a sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the VL sequence as set forth in SEQ ID NO: 92. In someembodiments, the bispecific antibody constructs comprise at least one IL-4Ra antigen-binding domain, where the IL-4Ra antigen-binding domain comprises a VH domain having a sequence as set forth in any one of SEQ ID NOs: 90, 91, 93, 94, 95, 96, 97, 98 or 99, and a VL domain having a sequence as set forth in any one of SEQ ID NOs: 92, 100, 101, 102, 103, 104, 105 or 106. In some embodiments, the bispecific antibody constructs comprise at least one IL-4Ra antigenbinding domain, where the IL-4Ra antigen-binding domain comprises a VH domain having a sequence as set forth in SEQ ID NO: 90 or 93, and a VL domain having a sequence as set forth in SEQ ID NO: 92.Bispecific Formats

[0176] The bispecific antibody constructs described herein comprise at least one IL-33 antigen-binding domain and at least one second target antigen-binding domain, for example, an IL-4Ra antigen-binding domain. In certain embodiments, the bispecific antibody constructs may further comprise a scaffold, such as a scaffold as described in any one of the embodiments defined above, and the antigen-binding domains are linked directly or indirectly (for example, via a linker or via one of the antigen-binding domains) to the scaffold. In certain embodiments, the bispecific antibody constructs are bivalent (i.e. comprise two antigen-binding domains), trivalent (i.e. comprise three antigen-binding domains) or tetravalent (i.e. comprise four antigen-binding domains).

[0177] In certain embodiments, the bispecific antibody constructs may lack a scaffold and thus comprise two or more antigen-binding domains optionally operably linked by one or more linkers. In such antibody constructs, the antigen-binding domains may be in the form of scFvs, Fabs, sdAbs, or a combination thereof. For example, using scFvs as the antigen-binding domains, formats such as a tandem scFv ((scFv)2 or taFv) may be constructed, in which the scFvs are connected together by a flexible linker. scFvs may also be used to construct diabody formats, which comprise two scFvs connected by a short linker (usually about 5 amino acids in length). The restricted length of the linker results in dimerization of the scFvs in a head-to-tail manner. In any of the preceding formats, the scFvs may be further stabilized by inclusion of an interdomain disulfide bond. For example, a disulfide bond may be introduced between VL and VH through introduction of an additional cysteine residue in each chain (for example, at position 44 in VH and position 100 in VL) (see, for example, Fitzgerald et al., 1997 , Protein Engineering, 10: 1221-1225),or a disulfide bond may be introduced between two VHs to provide a construct having a DART format (see, for example, Johnson et al., 2010, J Mol. Biol., 399:436-449).

[0178] Similarly, formats comprising two sdAbs, such as VHs or VHHs, connected together through a suitable linker may be employed in some embodiments. Other examples of antibody construct formats that lack a scaffold include those based on Fab fragments, for example, Fab2 and F(ab’)2 formats, in which the Fab fragments are connected through a linker or an IgG hinge region.

[0179] Combinations of antigen-binding domains in different forms may also be employed to generate alternative scaffold-less formats. For example, an scFv or a sdAb may be fused to the C-terminus of either or both of the light and heavy chain of a Fab fragment resulting in a bivalent (Fab-scFv / sdAb) construct.

[0180] In certain embodiments, the bispecific antibody constructs may be in an antibody format that is based on an immunoglobulin (Ig), such as an IgG class immunoglobulin, and thus comprise an IgG Fc region as a scaffold. In certain embodiments, the bispecific antibody constructs may be based on an IgGl, IgG2, IgG3 or IgG4 immunoglobulin. In some embodiments, the bispecific antibody constructs may be based on an IgGl or IgG4 immunoglobulin. In the context of the present disclosure, when an antibody construct is based on a specified immunoglobulin isotype, it is meant that antibody construct comprises all or a portion of the constant region of the specified immunoglobulin isotype. For example, a bispecific antibody construct based on a given Ig isotype comprises an Ig scaffold to which at least one antigen-binding domain is operably linked, where the scaffold comprises an Fc region from the given isotype and optionally an Ig hinge region from the same or a different isotype. It is to be understood that the bispecific antibody constructs may also comprise hybrids of isotypes and / or subclasses in some embodiments. It is also to be understood that the Fc region and / or hinge region may optionally be modified to impart one or more desirable functional properties as is known in the art. Examples of such modified Fc and hinge regions are described above.

[0181] In certain embodiments, the bispecific antibody constructs are bivalent, trivalent or tetravalent and comprise a scaffold based on an IgGl or IgG4 Fc region. Examples of such scaffolds are described in detail above. In some embodiments, the bispecific antibody constructs are bivalent and comprise a scaffold based on an IgGl or IgG4 Fc region. In some embodiments, the bispecific antibody constructs are bivalent and comprise a scaffold based on an IgG4 Fc region.In those embodiments in which the bispecific antibody construct comprises an Fc region, the Fc region comprises a first Fc polypeptide and a second Fc polypeptide.

[0182] As would be appreciated by one of skill in the art, the bispecific antibody constructs may be constructed in various formats. For example, each of the antigen-binding domains comprised by the bispecific antibody may be in a different format (for example, Fab, scFv), or they may all be in the same format. Where the bispecific antibody construct comprises an Fc region, each antigen-binding domain may be operably linked to the N-terminus of an Fc polypeptide, to the C-terminus of an Fc polypeptide, or to one of the other antigen-binding domains.

[0183] In certain embodiments, the bispecific antibody constructs are bivalent and comprise one IL-33 antigen-binding domain and one second target antigen-binding domain (for example, an IL-4Ra antigen-binding domain) and an IgG Fc region, where (a) both antigenbinding domains are in Fab format, or (b) both antigen-binding domains are in scFv format, or (c) one antigen-binding domain is in Fab format and the other is in scFv format. In some embodiments, in such bivalent bispecific antibody constructs, the IgG Fc region is an IgGl or IgG4 Fc region. In some embodiments, in such bivalent bispecific antibody constructs, one antigen-binding domain is linked to the N-terminus of one Fc polypeptide and the other antigen-binding domain is linked to the N-terminus of the other Fc polypeptide. In certain embodiments, in such bivalent bispecific antibody constructs, each of the antigen-binding domains is linked to the N-terminus of its respective Fc polypeptide via an IgG hinge region.

[0184] In certain embodiments, the bispecific antibody constructs are bivalent and comprise one IL-33 antigen-binding domain and one second target antigen-binding domain (for example, an IL-4Ra antigen-binding domain) and an IgG Fc region, where both antigen-binding domains are in Fab format, and one antigen-binding domain is linked to the N-terminus of one Fc polypeptide and the other antigen-binding domain is linked to the N-terminus of the other Fc polypeptide. In some embodiments, in such bivalent bispecific antibody constructs, the IgG Fc region is an IgGl or IgG4 Fc region. In certain embodiments, in such bivalent bispecific antibody constructs, each of the antigen-binding domains is linked to the N-terminus of its respective Fc polypeptide via an IgG hinge region.

[0185] In those embodiments in which the bispecific antibody constructs comprise a scaffold based on an IgGl or IgG4 Fc region, the Fc region will typically comprise a heterodimeric Fc comprising amino acid substitutions in the CH3 domain that promote formation of aheterodimeric Fc over a homodimeric Fc, as described above. In certain embodiments in which the bispecific antibody constructs comprise a scaffold based on an IgGl or IgG4 Fc region, the Fc region comprises a modified CH3 domain comprising the amino acid substitutions of any one of Variant 1, Variant 2, Variant 3, Variant 4 or Variant 5, as shown in Table 6.

[0186] In addition, in those embodiments in which the bispecific antibody constructs are bivalent and comprise one IL-33 antigen-binding domain and one second target antigen-binding domain and an IgG Fc region, where both antigen-binding domains are in Fab format, the CHI and CL domains of the IL-33 antigen-binding domain and the CHI and CL domains of the second target antigen-binding domain may comprise sets of mutations to drive the correct pairing between the heavy and light chains of the IL-33 antigen-binding domain and between the heavy and light chains of the second target antigen-binding domain. Examples of sets of mutations that may be used in this context include those described in International Patent Publication Nos. WO 2014 / 082179, WO 2015 / 181805 and WO 2017 / 059551. In certain embodiments, the bispecific antibody constructs comprise sets of mutations to drive the correct pairing between the heavy and light chains of the IL-33 antigen-binding domain and between the heavy and light chains of the second target antigen-binding domain. In some embodiments, the bispecific antibody constructs comprise sets of mutations to drive the correct pairing between the heavy and light chains of the IL-33 antigen-binding domain and between the heavy and light chains of the second target antigenbinding domain, where the sets of mutations are Set 1 or Set 2 as shown in Table 8.Table 8: Sets of CH1 / CL Mutations to Promote Correct Pairing* Numbering of amino acid positions according to EU

[0187] In certain embodiments, the bispecific antibody constructs are bivalent and comprise one IL-33 antigen-binding domain and one IL-4Ra antigen-binding domain. In some embodiments, the bispecific antibody constructs are bivalent and comprise one IL-33 antigenbinding domain and one IL-4Ra antigen-binding domain, where the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 sequences of the IL-33 antigen-binding domain and the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 sequences of the IL-4Ra antigen-binding domain have amino acid sequences as set forth for the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of the respective IL-33 antigen-binding domain and IL-4Ra antigen-binding domain of any one of variants v41240, v41243 , v41245 , v41246, v41249, v41251 , v41315, v41316, v41317, v42098, v42099, v42100, v42101, v42102, v42103, v43194, v43195, v43196, v43197, v43198, v43199 or v43200 (see Tables E and G). In some embodiments, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 sequences of the IL-33 antigen-binding domain and the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 sequences of the IL-4Ra antigen-binding domain have amino acid sequences as set forth for the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of the respective IL-33 antigen-binding domain and IL-4Ra antigen-binding domain of any one of variants v41245 , v41246, v41249, v41251 , v41315 , v41316, v41317, v42098, v42099, v42100, v42101, v42102, v42103, v43194, v43195, v43196, v43197, v43198, v43199 or v43200 (see Tables F and G).

[0188] In some embodiments, the bispecific antibody constructs are bivalent and comprise one IL-33 antigen-binding domain and one IL-4Ra antigen-binding domain, where the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 sequences of the IL-33 antigen-binding domain and the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 sequences of the IL-4Ra antigen-binding domain have amino acid sequences as set forth for the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of the respective IL-33 antigen-binding domain and IL-4Ra antigenbinding domain of variant v42099 or variant v42101.

[0189] In some embodiments, the bispecific antibody constructs are bivalent and comprise one IL-33 antigen-binding domain and one IL-4Ra antigen-binding domain, where the VH and VL sequences of the IL-33 antigen-binding domain and the VH and VL sequences of the IL-4Ra antigen-binding domain comprise amino acid sequences as set forth for the VH and VL sequences of the respective IL-33 antigen-binding domain and IL-4Ra antigen-binding domain of any one ofvariants v41245, v41246, v41249, v41251, v41315, v41316, v41317, v42098, v42099, v42100, v42101, v42102, v42103, v43194, v43195, v43196, v43197, v43198, v43199 or v43200 (see Tables E (Fig, 22), F and G). In some embodiments, the bispecific antibody constructs are bivalent and comprise one IL-33 antigen-binding domain and one IL-4Ra antigen-binding domain, where the VH and VL sequences of the IL-33 antigen-binding domain and the VH and VL sequences of the IL-4Ra antigen-binding domain comprise amino acid sequences as set forth for the VH and VL sequences of the respective IL-33 antigen-binding domain and IL-4Ra antigen-binding domain of of variant v42099 or variant v42101.

[0190] In certain embodiments, the bispecific antibody constructs have the format of a full- size antibody (FSA), for example, an IgGl or IgG4 FSA. In some embodiments, the bispecific antibody construct is a FSA comprising a first heavy chain sequence (Hl), a second heavy chain sequence (H2), a first light chain sequence (LI) and a second light chain sequence (L2), where Hl pairs with LI and H2 pairs with L2, and where Hl and H2 have different amino acid sequences, and LI and L2 have different amino acid sequences.

[0191] In certain embodiments, the bispecific antibody construct is an FSA having a set of Hl, H2, LI and L2 sequences comprising the Hl, H2, LI and L2 amino acid sequences as set forth in Tables F and G for any one of variants v41240, v41243, v41245, v41246, v41249, v41251, v41315, v41316, v41317, v42098, v42099, v42100, v42101, v42102, v42103, v43194, v43195, v43196, v43197, v43198, v43199 or v43200. In some embodiments, the bispecific antibody construct is an FSA having a set of Hl, H2, LI and L2 sequences comprising the Hl, H2, LI and L2 amino acid sequences as set forth in Tables F and G for any one of variants v41245, v41246, v41249, v41251, v41315, v41316, v41317, v42098, v42099, v42100, v42101, v42102, v42103, v43194, v43195, v43196, v43197, v43198, v43199 or v43200. In some embodiments, the bispecific antibody construct is an FSA having a set of Hl, H2, LI and L2 sequences comprising the Hl, H2, LI and L2 amino acid sequences as set forth in Tables F and G for variant v42099 or variant v42101.

[0192] As is known in the art, expression of antibody heavy chain sequences in certain cell lines or from certain expression vector may result in the inclusion of a C-terminal lysine residue on one or both of the heavy chains. Accordingly, certain embodiments of the present disclosure relate to bispecific antibody constructs that are FSAs having a set of Hl, H2, LI and L2 sequences comprising the Hl, H2, LI and L2 amino acid sequences as set forth in Tables F and G for anyone of variants v41240, v41243, v41245, v41246, v41249, v41251, v41315, v41316, v41317, v42098, v42099, v42100, v42101, v42102, v42103, v43194, v43195, v43196, v43197, v43198, v43199 or v43200, in which one or both of the Hl and H2 sequences comprise a C-terminal lysine.METHODS OF PREPARING ANTIBODY CONSTRUCTS

[0193] The anti-IL-33 antibody constructs and bispecific antibody constructs described herein may be produced using standard recombinant methods known in the art (see, for example, U.S. Patent No. 4,816,567 and “Antibodies: A Laboratory Manual, ” 2ndEdition, Ed. Greenfield, Cold Spring Harbor Laboratory Press, New York, 2014).

[0194] Typically, for recombinant production of an antibody construct, a polynucleotide or set of polynucleotides encoding the antibody construct is generated and inserted into one or more vectors for further cloning and / or expression in a host cell. Polynucleotide(s) encoding the antibody construct may be produced by standard methods known in the art (see, for example, Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, New York, 1994 & update, and “Antibodies: A Laboratory Manual,” 2ndEdition, Ed. Greenfield, Cold Spring Harbor Laboratory Press, New York, 2014). As would be appreciated by one of skill in the art, the number of polynucleotides required for expression of the antibody construct will be dependent on the format of the construct, including whether or not the antibody construct comprises a scaffold. For example, for a monospecific antibody in full-size antibody format with two Fab antigen-binding domains, a set of two polynucleotides will be required: one polynucleotide encoding the heavy chain and one polynucleotide encoding the light chain. Whereas for a bispecific antibody construct in a full-size antibody format with two Fab antigen-binding domains, a set of four polynucleotides will be required: two polynucleotides each encoding a heavy chain and two polynucleotides each encoding a light chain. When multiple polynucleotides are required, they may be incorporated into one vector or into more than one vector.

[0195] Generally, for expression, the polynucleotide or set of polynucleotides is incorporated into an expression vector or vectors together with one or more regulatory elements, such as transcriptional elements, which are required for efficient transcription of the polynucleotide. Examples of such regulatory elements include, but are not limited to, promoters, enhancers, terminators, and polyadenylation signals. One skilled in the art will appreciate that the choice of regulatory elements is dependent on the host cell selected for expression of the antibodyconstruct and that such regulatory elements may be derived from a variety of sources, including bacterial, fungal, viral, mammalian or insect genes. The expression vector may optionally further contain heterologous nucleic acid sequences that facilitate expression or purification of the expressed protein. Examples include, but are not limited to, signal peptides and affinity tags such as metal-affinity tags, histidine tags, avidin / streptavidin encoding sequences, glutathione-S- transferase (GST) encoding sequences and biotin encoding sequences. The expression vector may be an extrachromosomal vector or an integrating vector.

[0196] Suitable host cells for cloning or expression of the antibody constructs include various prokaryotic or eukaryotic cells as known in the art. Eukaryotic host cells include, for example, mammalian cells, plant cells, insect cells and yeast cells (such as Saccharomyces or Pichia cells). Prokaryotic host cells include, for example, E. coli, A. salmonicida or B. subtilis cells.

[0197] In certain embodiments, the antibody construct may be produced in bacteria, in particular when glycosylation and Fc effector function are not needed, as described for example in U.S. Patent Nos. 5,648,237; 5,789,199, and 5,840,523, and in Charlton, Methods in Molecular Biology, Vol. 248, pp. 245-254, B.K.C. Lo, ed., Humana Press, Totowa, N.J., 2003.

[0198] Eukaryotic microbes such as fdamentous fungi or yeast may be suitable expression host cells in certain embodiments, in particular fungi and yeast strains whose glycosylation pathways have been “humanized” resulting in the production of an antibody construct with a partially or fully human glycosylation pattern (see, for example, Gemgross, 2004, Nat. Biotech. 22: 1409-1414, and Li et al., 2006, Nat. Biotech. 24:210-215).

[0199] Suitable host cells for the expression of glycosylated antibody constructs are usually eukaryotic cells. For example, U.S. Patent Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978 and 6,417,429 describe PLANTIBODIES™ technology for producing antigen-binding constructs in transgenic plants. Mammalian cell lines adapted to grow in suspension may be particularly useful for expression of antibody constructs. Examples include, but are not limited to, monkey kidney CV1 line transformed by SV40 (COS-7), human embryonic kidney (HEK) line 293 or 293 cells (see, for example, Graham et al., \9T1,J. Gen Virol., 36:59), baby hamster kidney cells (BHK), mouse sertoli TM4 cells (see, for example, Mather, 1980, Biol Reprod, 23:243-251), monkey kidney cells (CV1), African green monkey kidney cells (VERO-76), human cervical carcinoma (HeLa) cells, canine kidney cells (MDCK), buffalo rat liver cells (BRL 3A), humanlung cells (W138), human liver cells (Hep G2), mouse mammary tumour (MMT 060562), TRI cells (see, for example, Mather et al., 1982, Annals N.Y. Acad Sci, 383:44-68), MRC 5 cells, FS4 cells, Chinese hamster ovary (CHO) cells (including DHFR CHO cells, see Urlaub et al., 1980, Proc Natl Acad Sci USA, Tl.MAG), and myeloma cell lines (such as Y0, NSO and Sp2 / 0). Exemplary mammalian host cell lines suitable for production of antibody constructs are reviewed in Yazaki & Wu, Methods in Molecular Biology , Vol. 248, pp. 255-268 (B.K.C. Lo, ed., Humana Press, Totowa, N.J., 2003).

[0200] In certain embodiments, the host cell used to produce the antibody constructs may be a transient or stable higher eukaryotic cell line, such as a mammalian cell line. In some embodiments, the host cell may be a mammalian HEK293T, CHO, HeLa, NSO or COS cell line, or a cell line derived from any one of these cell lines. In some embodiments, the host cell may be a stable cell line that allows for mature glycosylation of the antibody construct.

[0201] The host cells comprising the expression vector(s) encoding the antibody construct may be cultured using routine methods to produce the antibody construct. Alternatively, in some embodiments, host cells comprising the expression vector(s) encoding the antibody construct may be used therapeutically or prophylactically to deliver the antibody construct to a subject, or polynucleotides or expression vectors may be administered to a cell from a subject ex vivo and the cell then returned to the body of the subject.

[0202] Typically, the antibody constructs are purified after expression. Proteins may be isolated or purified in a variety of ways known to those skilled in the art (see, for example, Protein Purification: Principles and Practice, 3rdEd., Scopes, Springer-Verlag, NY, 1994). Standard purification methods include chromatographic techniques, including ion exchange, hydrophobic interaction, affinity, sizing (or gel filtration), and reverse-phase, carried out at atmospheric pressure or at high pressure using systems such as HPLC or UPLC. Additional purification methods include electrophoretic, immunological, precipitation, dialysis and chromatofocusing techniques. Ultrafiltration and diafiltration techniques, in conjunction with protein concentration, are also useful. As is well known in the art, a variety of natural proteins bind Fc and antibodies, and these proteins may be used for purification of certain antibody constructs. For example, the bacterial proteins A and G bind to the Fc region. Likewise, the bacterial protein L binds to the Fab region of some antibodies. Purification may also be enabled by a particular fusion partner. For example, antibodies may be purified using glutathione resin if a GST fusion is employed,Ni+2affinity chromatography if a His-tag is employed or immobilized anti-flag antibody if a flagtag is used. The degree of purification necessary will vary depending on the use of the antibody constructs. In some instances, no purification may be necessary.

[0203] In certain embodiments, the antibody constructs are substantially pure. The term “substantially pure” (or “substantially purified”) when used in reference to an antibody construct described herein, means that the antibody construct is substantially or essentially free of components that normally accompany or interact with the protein as found in its naturally occurring environment, such as a native cell, or a host cell in the case of recombinantly produced construct. In certain embodiments, an antibody construct that is substantially pure is a protein preparation having less than about 30%, less than about 25%, less than about 20%, less than about 15%, less than about 10%, or less than about 5% (by dry weight) of contaminating protein.

[0204] Certain embodiments of the present disclosure relate to a method of making an anti- IL-33 antibody construct or a bispecific antibody construct as described herein comprising culturing a host cell into which one or more polynucleotides encoding the antibody construct, or one or more expression vectors encoding the antibody construct, have been introduced, under conditions suitable for expression of the antibody construct, and optionally recovering the antibody construct from the host cell or from host cell culture medium.Post-Translational Modifications

[0205] In certain embodiments, the antibody constructs described herein may comprise one or more post-translational modifications. Such post-translational modifications may occur in vivo, or they be conducted in vitro after isolation of the antibody construct from the host cell.

[0206] Post-translational modifications include various modifications as are known in the art (see, for example, Proteins - Structure and Molecular Properties, 2nd Ed., T. E. Creighton, W. H. Freeman and Company, New York, 1993; Post-Translational Covalent Modification of Proteins, B. C. Johnson, Ed., Academic Press, New York, pgs. 1-12, 1983; Seifter et al., 1990, Meth. Enzymol., 182:626-646, and Rattan et al., 1992, Aww. N.Y. Acad. Sci., 663:48-62). In those embodiments in which the antibody construct comprises one or more post-translational modifications, the construct may comprise the same type of modification at one or several sites, or it may comprise different modifications at different sites.

[0207] Examples of post-translational modifications include glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting / blocking groups, formylation,oxidation, reduction, proteolytic cleavage or specific chemical cleavage by cyanogen bromide, trypsin, chymotrypsin, papain, V8 protease or NaBHr.

[0208] Other examples of post-translational modifications include, for example, addition or removal of N-linked or O-linked carbohydrate chains, chemical modifications of N-linked or O-linked carbohydrate chains, processing of N-terminal or C-terminal ends, attachment of chemical moieties to the amino acid backbone, and addition or deletion of an N-terminal methionine residue resulting from prokaryotic host cell expression.

[0209] Post-translational modifications may also include modification with a detectable label, such as an enzymatic, fluorescent, luminescent, isotopic or affinity label to allow for detection and isolation of the protein. Examples of suitable enzyme labels include, but are not limited to, horseradish peroxidase, alkaline phosphatase, beta-galactosidase and acetylcholinesterase. Examples of suitable prosthetic group complexes include, but are not limited to, streptavidin / biotin and avidin / biotin. Examples of suitable fluorescent materials include, but are not limited to, umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride and phycoerythrin. Examples of luminescent materials include luminol, and bioluminescent materials such as luciferase, luciferin and aequorin. Examples of suitable radioactive materials include iodine, carbon, sulfur, tritium, indium, technetium, thallium, gallium, palladium, molybdenum, xenon and fluorine.

[0210] Additional examples of post-translational modifications include acylation, ADP- ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cysteine, formation of pyroglutamate, gamma-carboxylation, GPI anchor formation, hydroxylation, iodination, methylation, myristylation, pegylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to proteins such as arginylation, and ubiquitination.POLYNUCLEOTIDES, VECTORS AND HOST CELLS

[0211] Certain embodiments of the present disclosure relate to an isolated polynucleotide or a set of polynucleotides encoding an anti -IL-33 antibody construct or bispecific antibody construct described herein.

[0212] The terms “nucleic acid,” “nucleic acid molecule” and “polynucleotide” are used interchangeably herein and refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogues thereof. Non-limiting examples of polynucleotides include a gene, a gene fragment, messenger RNA (mRNA), cDNA, recombinant polynucleotides, plasmids, vectors, isolated DNA, isolated RNA, nucleic acid probes, and primers.

[0213] A polynucleotide that “encodes” a given polypeptide is a polynucleotide that is transcribed (in the case of DNA) and translated (in the case of mRNA) into a polypeptide in vivo when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence are determined by a start codon at the 5' (amino) terminus and a translation stop codon at the 3' (carboxy) terminus. A transcription termination sequence may be located 3' to the coding sequence.

[0214] Certain embodiments of the present disclosure relate to vectors (such as expression vectors) comprising one or more polynucleotides encoding an anti -IL-33 antibody construct or a bispecific antibody construct as described herein. The polynucleotide (s) may be comprised by a single vector or by more than one vector. In some embodiments, the polynucleotides are comprised by a multicistronic vector.

[0215] Certain embodiments of the present disclosure relate to host cells comprising polynucleotide(s) encoding an anti -IL-33 antibody construct or a bispecific antibody construct as described herein or one or more vectors comprising the polynucleotide(s). In some embodiments, the host cell is eukaryotic, for example, a Chinese Hamster Ovary (CHO) cell, a human embryonic kidney (HEK) cell or a lymphoid cell (e.g. Y0, NS0, Sp20 cell).PHARMACEUTICAL COMPOSITIONS

[0216] For therapeutic use, the anti-IL-33 antibody construct or bispecific antibody construct may be provided in the form of pharmaceutical compositions comprising the antibodyconstruct and a pharmaceutically acceptable carrier or diluent. The compositions may be prepared by known procedures using well-known and readily available ingredients.

[0217] Pharmaceutical compositions may be formulated for administration to a subject by, for example, parenteral, oral (including, for example, buccal or sublingual), topical, rectal or vaginal routes, or by inhalation or spray. “Parenteral” administration may be subcutaneous injection, or intradermal, intra-articular, intravenous, intramuscular, intravascular, intrastemal or intrathecal injection or infusion. The pharmaceutical composition will typically be formulated in a format suitable for administration to the subject, for example, as a syrup, elixir, tablet, troche, lozenge, hard or soft capsule, pill, suppository, oily or aqueous suspension, dispersible powder or granule, emulsion, injectable or solution. Pharmaceutical compositions may be provided as unit dosage formulations.

[0218] In certain embodiments, pharmaceutical compositions comprising the antibody constructs may be formulated for parenteral administration by infusion or in a unit dosage injectable form, for example as lyophilized formulations or aqueous solutions.

[0219] Pharmaceutically acceptable carriers are generally nontoxic to recipients at the dosages and concentrations employed. Examples of such carriers include, but are not limited to, buffers such as phosphate, citrate, and other organic acids; antioxidants such as ascorbic acid and methionine; preservatives such as octadecyldimethylbenzyl ammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butyl alcohol, benzyl alcohol, alkyl parabens (such as methyl or propyl paraben), catechol, resorcinol, cyclohexanol, 3 -pentanol and m-cresol; low molecular weight (less than about 10 residues) polypeptides; proteins such as serum albumin or gelatin; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates such as glucose, mannose or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes such as Zn-protein complexes, and non-ionic surfactants such as polyethylene glycol (PEG).

[0220] In certain embodiments, pharmaceutical compositions comprising the antibody constructs may be in the form of a sterile injectable aqueous or oleaginous solution or suspension. Such suspensions may be formulated using suitable dispersing or wetting agents and / or suspending agents that are known in the art. The sterile injectable solution or suspension may comprise theantibody construct in a non-toxic parentally acceptable diluent or solvent. Acceptable diluents and solvents that may be employed include, for example, 1,3-butanediol, water, Ringer’s solution or isotonic sodium chloride solution. In addition, sterile, fixed oils may be employed as a solvent or suspending medium. For this purpose, various bland fixed oils may be employed, including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid find use in the preparation of injectables. Adjuvants such as local anesthetics, preservatives and / or buffering agents may also be included in the injectable solution or suspension.

[0221] In certain embodiments, pharmaceutical compositions comprising the antibody constructs may be formulated for parenteral administration to a subject, for example a human. Typically, compositions for parenteral administration are solutions in sterile isotonic aqueous buffer. Where necessary, the composition may also include a solubilizing agent and / or a local anaesthetic such as lignocaine to ease pain at the site of the injection. Generally, the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water-free concentrate in a hermetically sealed container such as an ampoule or sachet indicating the quantity of active agent. Where the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.

[0222] Other pharmaceutical compositions and methods of preparing pharmaceutical compositions are known in the art and are described, for example, in “Remington: The Science and Practice of Pharmacy" (formerly “Remingtons Pharmaceutical Sciences”) Gennaro, A., Lippincott, Williams & Wilkins, Philadelphia, PA (2000).METHODS OF USE

[0223] Certain aspects of the present disclosure relate to the therapeutic use of the anti -IL- 33 antibody constructs and bispecific antibody constructs described herein.

[0224] Certain embodiments relate to use of the anti-IL-33 antibody constructs and bispecific antibody constructs in the treatment of an inflammatory or autoimmune disease. Some embodiments relate to methods of treating a subject having an inflammatory or autoimmune disease comprising administering an effective amount of the anti-IL-33 antibody construct or bispecific antibody construct to the subject.

[0225] Examples of inflammatory or autoimmune diseases that may be treated with the anti-IL-33 antibody constructs or bispecific antibody constructs in certain embodiments include, but are not limited to, atopic dermatitis, psoriasis, asthma, rhinosinusitis, chronic obstructive pulmonary disease (COPD) (emphysema-dominant, airway-dominant and / or mixed type), inflammatory bowel disease (IBD) and rheumatological diseases.

[0226] The terms “treat” and “treatment” and grammatical variations thereof as used herein, refer to an intervention performed with the intention of alleviating the symptoms associated with, or altering the pathology of, a disease, disorder or condition. Thus, the terms include in various embodiments one or more of alleviation, moderation, reduction or curing of a disease, disorder or condition.

[0227] The dosage of the anti -IL-33 antibody construct or bispecific antibody construct to be administered is not subject to defined limits, but it will be a therapeutically effective amount. A “therapeutically effective amount” refers to that amount of an antibody construct described herein which, when administered to a subject, is sufficient to effect a treatment of the particular indication.PHARMACEUTICAL KITS

[0228] Certain embodiments relate to pharmaceutical kits (or articles of manufacture) comprising an anti-IL-33 antibody construct or a bispecific antibody construct as described herein.

[0229] The kit typically will comprise a container holding the antibody construct and a label and / or package insert on or associated with the container. The label or package insert contains instructions customarily included in commercial packages of therapeutic products, providing information about the indications, usage, dosage, administration, contraindications and / or warnings concerning the use of such therapeutic products. The label or package insert may further include a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, for use or sale for human or animal administration. In some embodiments, the container may have a sterile access port. Lor example, the container may be an intravenous solution bag or a vial having a stopper that may be pierced by a hypodermic injection needle.

[0230] In addition to the container holding the antibody construct, the kit may optionally comprise one or more additional containers comprising other components of the kit. for example,a pharmaceutically acceptable buffer (such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer’s solution or dextrose solution), other buffers or diluents.

[0231] Suitable containers include, for example, bottles, vials, syringes, intravenous solution bags, and the like. The containers may be formed from a variety of materials such as glass or plastic. If appropriate, one or more components of the kit may be lyophilized or provided in a dry form, such as a powder or granules, and the kit can additionally contain a suitable solvent for reconstitution of the lyophilized or dried component(s).

[0232] The kit may further include other materials desirable from a commercial or user standpoint, such as filters, needles, and syringes.

[0233] The following Examples are provided for illustrative purposes and are not intended to limit the scope of the claimed invention in any way.EXAMPLES

[0234] The practice of the present disclosure will employ, unless otherwise indicated, conventional methods of protein chemistry, biochemistry, recombinant DNA techniques and pharmacology, within the skill of the art. Such techniques are explained fully in the literature. See, e.g., T.E. Creighton, Proteins: Structures and Molecular Properties (W.H. Freeman and Company, 1993); A.L. Lehninger, Biochemistry (Worth Publishers, Inc., current addition); Sambrook, et al., Molecular Cloning: A Laboratory Manual (2nd Edition, 1989); Methods In Enzymology (S. Colowick and N. Kaplan eds., Academic Press, Inc.); Remington 's Pharmaceutical Sciences, 18th Edition (Easton, Pennsylvania: Mack Publishing Company, 1990).EXAMPLE 1: PREPARATION AND CHARACTERIZATION OF HUMANIZED ANTI- IL-33 ANTIBODIES1.1 Antibody Generation

[0235] Antibodies that specifically bind IL-33 were generated by immunizing rabbits with human IL-33 as described below.

[0236] Two New Zealand White rabbits were subcutaneously immunized with recombinant human IL-33 (AdipoGen Life Sciences, San Diego, CA; Cat. No. AG-40B-0160- 0000), after which blood was drawn. Anti-human IL-33 antibody titers were determined by flowcytometry using streptavidin beads (Spherotech Inc., Green Oaks, IL) coated with IL-33 antigen. Test bleed sera mounted a significant response again human IL-33.

[0237] Immunized rabbits were sacrificed and the spleens harvested. Splenocytes for each rabbit were used for B cell enrichment and sorted on a FACSAria™ (Becton, Dickinson & Co., Franklin Lakes, NJ) into wells containing lysis buffer with a modified protocol based on the Selected Lymphocyte Antibody Method (SLAM) (Babcook et al., 1996, Proc Natl Acad Set USA, 93(15):7843-7848).

[0238] Total RNA from wells containing a single B cell was used as template with SuperScript™ III (Thermo Fisher Scientific Corp., Waltham, MA) and oligo-dT20 (Integrated DNA Technologies, Inc., Coralville, IA) to transcribe cDNA from mRNA. Initial PCR of heavy and light chain antibody-coding sequences was performed using primers and methods modified from Babcook etal., \996, ProcNatlAcadSci USA, 93(15):7843-7848, von Boehmer etal., 2016, NatProtoc., 11(10): 1908 and Peng et al., 2017, J. Mol. Biol, 429(19):2954-2973, with cDNA as the nucleic acid template. A subsequent PCR reaction was then performed on these unique sequences using V-segment family and J-segment family-specific primers and the resulting amplicons were cloned into pTT5-based expression plasmids (National Research Council of Canada). Unique heavy chain sequences and light chain sequences emerging from a single well sample were co-expressed in Expi293F cells (Thermo Fisher Scientific, Waltham, MA).

[0239] Cell supernatants containing secreted antibodies were assessed forblocking human IL-33Ra receptor in a receptor ligand assay. The wells having supernatant that blocked human IL- 33Ra receptor (and therefore contained anti-IL-33 antibody) were selected for sequencing.

[0240] Heavy and light chain PCR amplicons were sequenced using NGS-based Amplicon-EZ and analyzed for unique antibody-coding sequences. The following rabbit antihuman IL-33 antibody VH and VL sequences were identified for the antibody referred to below as hl48C04.Table 1.1: Rabbit VH and VL Sequences for Anti-Human IL-33 Antibody (hl48C04)

[0241] These rabbit VH and VL sequences were used to prepare a rabbit-human chimeric IgGl / kappa antibody construct (v33006) as follows. Coding sequences for antibody variable regions were cloned in frame into a human IgGl expression vector or a human C kappa expression vector (based on the pTT5 vector). The human IgGl constant region starts at alanine Kabat-114, and human C kappa constant region starts at arginine Kabat-108. The activities of the resultant recombinant chimeric antibody construct were confirmed in specificity binding assays.1.2 Humanization

[0242] The rabbit VH and VL sequences from chimeric antibody construct v33006 were aligned against human immunoglobulin germline sequences to select basis germline sequences for humanization. Human germline IGHV3-23*02 with IGHJ4*01 was selected for VH humanization. Human germline IGKV1-39*O1 with IGLJ4*01 was selected for VL humanization. The CDR sequences by AbM definition for CDRH1, CDRH2 and IMGT definition for CDRH3 from v33006 were swapped into the selected human germline frameworks to create a basis humanized construct. Several areas of the basis construct were identified for back mutation or deletion to the original rabbit parental sequence to minimize potential disruption to antigen binding. Humanization produced eleven new candidate humanized VH sequences and four new candidate humanized VL sequences. Recombinant human IgGl -based monoclonal antibodies containing each combination of the candidate VH and VL humanized sequences were expressed in ExpiCHO™ cells and purified by Protein A affinity chromatography using MabSelect™ SuRe™ resin (GE HealthCare). The purified variants were screened for activity by reporter gene assay (RGA) using human IL-33 and cynomolgus IL-33 as described in Example 2. Forty-four constructs represented by 11 humanized VH and 4 humanized VL sequences (see Tables 1.2 and 1.3) demonstrated similar activity compared to the parental antibody v33006. Binding was assessed using KinExA™ (Sapidyne Instruments) for representative variants (see Table 1.8).Table 1.2: Humanized Anti-IL-33 VH and VL Domain SequencesTable 1.3: VH and VL Composition of Humanized Anti-IL-33 Antibody Constructs (Full Size Antibody (FSA))1.4 Preparation of Humanized Anti-IL33 Antibody Constructs

[0243] Each of the 44 humanized antibody variants listed in Table 1.3 was produced in full-size antibody (FSA) IgGl format containing two identical full length heavy chains and two identical kappa light chains. Each of the humanized VH domain sequences was appended to a human CHl-hinge-CH2-CH3 domain sequence of IGHGl*01 (SEQ ID NO: 107; see Table 1.4)to provide 11 different heavy chain sequences and each of the humanized VL domain sequences was appended to the human kappa CL domain sequence of IGKC*01 (SEQ ID NO: 108; see Table 1.4) to provide 4 light chain sequences.

[0244] In addition to the FSA variants, the 14 humanized antibody variants listed in Table 1.5 were produced in one-armed antibody (OAA) format containing one full length heavy chain (Heavy Chain B), one truncated heavy chain (Heavy Chain A) and one kappa light chain (Light Chain). The full-length heavy chain (Heavy Chain B) contained the human CHl-hinge-CH2-CH3 domain sequence of IGHGl*01 with the mutations: T350V_T366L_K392L_T394W. Each of the humanized VH domain sequences listed in Table 1.5 was appended to the CHl-hinge-CH2-CH3 domain sequence of Heavy Chain B. The truncated heavy chain (Heavy Chain A) contained hinge- CH2-CH3 domain sequence of IGHGl*01 with the mutations: T350V_L351Y_F405A_Y407V. The Fc domain formed by Heavy Chain A and Heavy Chain B is referred to herein as “HetFc.” The Light Chain contained the human kappa CL domain sequence of IGKC*01 (SEQ ID NO: 108; see Table 1.4). The OAA antibody variants also included the following CH2 amino acid substitutions in both chains: L234A, L235A and D265S. These CH2 domain substitutions knock out FcyR binding and are referred to herein as “FcKO.” Amino acid residues in the Fc domain are numbered according to the EU index.Table 1.4: Sequences of Wild-Type IgGl Heavy Chain and Kappa Light Chain

[0245] All sequences were reverse translated to DNA, codon optimized for mammalian expression and gene synthesized.

[0246] Heavy chain vector inserts comprising a signal peptide (artificially designed sequence: MRPTWAWWLFLVLLLALWAPARG [SEQ ID NO: 109] (Barash et al., 2002, Biochem. Biophys Res. Comm., 294:835-842) and the heavy chain clone terminating at residue G446 (EU numbering) of the CH3 domain were ligated into a pTT5 vector to produce heavy chain expression vectors. Light chain vector inserts comprising the same signal peptide were ligated into a pTT5 vector to produce light chain expression vectors. The resulting heavy and light chain expression vectors were sequenced to confirm correct reading frame and sequence of the coding DNA.Table 1.5: VH and VL Composition of Selected Humanized Anti-IL-33 Antibody Constructs (One Armed Antibody (OAA))* FcKO indicates that the Fc region contained the mutations L234A_L235A_D265S in both chains, which knock out FcyR binding

[0247] For FSA preparation, gene products were cloned (by GenScript Biotech Corporation, Piscataway, NJ) into the pTT5 mammalian expression vector (NRC-BRI, Canada) and expressed in CHO cells. Briefly, CHO cells were transfected in exponential growth phase (1.5 to 2 million cells / mL) with aqueous 1 mg / mL 25 kDa polyethylenimine (PEI) using an PEEDNA ratio of 2.5 : 1. The DNA was transfected in an equal DNA ratio of heavy chain (HC) to light chain(LC). Transfected cells were harvested after 5-6 days, culture medium was collected after centrifugation at 4000 rpm and clarified using a 0.45 gm filter.

[0248] For OAA preparation, gene products were cloned (by GenScript Biotech Corporation, Piscataway, NJ) into the pTT5 mammalian expression vector (NRC-BRI, Canada) and transiently expressed in ExpiCHO™ (Thermo Fisher Scientific, Waltham, MA) cells using ExpiFectamine™ CHO Transfection Kit (Thermo Fisher Scientific, Cat. No. A29129). Transfected cells were cultured in ExpiCHO™ expression medium (Thermo Fisher Scientific, Cat No. A2910001) as per the “ExpiCHO™ Expression System User Guide” (Thermo Fisher Scientific, 2018) for the ExpiCHO™ Max Titer protocol. Transfected cells were harvested after 7 days post transfection, culture medium was collected after centrifugation at 4000 rpm and clarified using a 0.22 gm filter.

[0249] Each clarified culture medium was loaded onto a MabSelect™ SuRe™ protein A resin (GE HealthCare) in a gravity column and washed with 10 column volumes of PBS buffer at pH 7.2 - 7.4. Antibody was eluted with 10 column volumes of 0.1 M citrate buffer at pH 3.6 and neutralized with 1 M TRIS at pH 9. The amount of antibody variant was then quantified based on A280 nm (NanoDrop™ Spectrophotometer; Thermo Fisher Scientific).

[0250] The purity of each antibody variant was assessed by electrophoresis under nonreducing and reducing conditions using the High Throughput Protein Express assay and Caliper LabChip® GXII or GXII Touch HT (Perkin Elmer, Waltham, MA). Procedures were carried out according to HT Protein Express LabChip® User Guide version 2 with the following modifications. Antibody samples, at either 2gl or 5 pl (concentration range 5-2000 ng / gl), were added to separate wells in 96 well plates (BioRad, Hercules, CA; Cat. No. HSP9601B) along with 7gl of HT Protein Express Sample Buffer (Perkin Elmer; Cat. No. 760328). Antibody samples were then denatured at 70°C for 15 mins. The LabChip® instrument was operated using the HT Protein Express Chip (Perkin Elmer, Waltham, MA) and the Ab-200 assay setting.

[0251] Species homogeneity of the humanized antibody variants and parental rabbithuman chimeric antibody variant were assessed by UPLC-SEC. UPLC-SEC was performed on Agilent Technologies 1290 Infinity with a diode array detector (DAD) using the Agilent Technologies AdvanceBio SEC300A column (7.8 x 150 mm, 2.7 gm) or Waters Acquity™ BEH200 SEC column (2.5 mL, 4.6 x 150 mm, 1.7 gm) at 25°C. The mobile phase was 200 mM KPOr + 200 mM KC1, pH 7.4 and the flow rate was 1 mL / min for the Agilent column and 0.4mL / min for the Waters column. Total run time for each injection was 7 min with a total injection of lOug of protein sample. Elution was monitored by UV absorbance in the range 190-400 nm and chromatograms were extracted at 280 nm. Peak integration was performed using OpenLAB CDS ChemStation software (Agilent Technologies, Inc., Santa Clara, CA).

[0252] The purified variants were assessed by non-reducing and reducing High Throughput Protein Express assay using Caliper LabChip GXII and UPLC-SEC using a Waters Acquity™ BEH200 SEC column (2.5 mL, 4.6x150 mm, stainless steel, 1.7 pm particles) (Waters LTD, Mississauga, ON) set to 30°C or 25 °C and mounted on a Waters Acquity™ UPLC H-Class Bio system with a PDA detector.

[0253] Endotoxin levels were determined by the limulus amebocyte lysate (LAL) assay using the Endosafe™ Portable Test System (PTS) (Charles River, Wilmington, MA).

[0254] The yields for the 44 humanized FSA variants ranged from approximately 6.6 - 9.9 mg (from 100 mL culture) and were purified to >98 % purity (see Table 1.6). The yields for the 14 humanized OAA variants ranged from approximately 1.2 - 4.2 mg (from 50 mL culture) and were purified to >98 % purity (see Table 1.7).Table 1.6: Production Titers, Yields and Purity of Humanized Anti-IL-33 Full Size Antibody(FSA) VariantsTable 1.7: Production Titers, Yields and Purity of Humanized Anti-IL-33 One ArmedAntibody (OAA) Variants1.5 Characterization of Anti-IL-33 Antibody Variants1.5.1 Binding to Human IL- 33

[0255] The equilibrium binding affinity of the humanized antibody variants to human IL- 33 was determined KinExA™ (Sapidyne Inc., Boise, ID). Briefly, human IL-33 (Sino Biological, Inc., Beijing, China; Cat. No. 10368-HNAE) was immobilized on azlactone beads. Antibody variants were used as the constant binding partner (CBP) and antigen (hu IL-33) was used as thetitrant. The antibody variants were tested at a constant concentration of 1 pM and 10 pM or 5 pM and 50 pM. The titrant, human IL-33 was titrated by 2-fold serial dilutions of 400 pM. The antibody / antigen mixture was incubated at 25 °C for 4 days and assessed using goat anti-human IgG (H+L) conjugated with Alexa Fluor™ 647 detection with data analyzed using the KinExA™ software. The results are shown in Table 1.8.Table 1.8: Binding Affinity (KD) of Representative Anti-IL-33 Antibody Variants for Binding to Human IL-331.5.2 Thermal Stability Assessment of Anti-IL-33 Antibody Variants

[0256] Thermal stability of the humanized antibody variants was assessed by differential scanning fluorimetry (DSF) as described below.

[0257] All DSF experiments were carried out using a CFX96 Touch™ Real-Time PCR instrument (Bio-Rad Laboratories, Inc., Hercules, CA). The proteins were diluted to 1 mg / mL and 10 pg was loaded into 35 pL buffer in 96 well plates with 5 pL of 40x Invitrogen™ SYPRO™ Orange Protein Gel Stain (5,000X concentrate in DMSO) (Thermo Fisher Scientific) and measured with a scan rate of 0.5 °C / min from 25 °C to 95 °C. Data was analyzed using Microsoft Excel with the PBS, pH 7.4 buffer background subtracted. The results for representative antibody variants are shown in Tables 1.9 and 1.10, which show the maximum melting temperatures (Tm) for each of the peaks in the thermograms of the tested antibody variants. The results demonstrate that the tested antibody variants unfold with melting temperatures expected for conventional IgGl antibody constructs.Table 1.9: Thermal Melting Temperature (Tm) for Representative Anti-IL-33 Full SizeAntibody (FSA) VariantsTable 1.10: Thermal Melting Temperature (Tm) Assessed for Representative Anti-IL-33 OneArmed Antibody (OAA) Variants1.5.3 Accelerated Stability Assessment of Anti-IL-33 Full Size Antibody Variants

[0258] The antibody variants were stored at 4 °C and 40 °C at Img / mL concentration in PBS pH 7.4 buffer for 14 days. The purity of the samples was assessed after the storage period by UPLC-SEC and compared to the purity on Day 0. The results are shown in Table 1.11. None of the tested antibody variants displayed an increase in high molecular weight impurities (HMWs) under either of the test conditions. The changes in % monomer purity observed after 14 days at 40 °C were due to formation of low molecular weight impurities (LMWs).Table 1.11: Purity Assessment of Representative Humanized Anti-IL-33 Full Size AntibodyVariants After Storage at 4 °C and 40 °C for 14 Days1.5.4 Assessment of Non-specific Interactions and Self-interaction Propensity of Antibody Variants

[0259] The potential propensity of the antibody variants for non-specific interaction and self-interaction were assessed by non-specific ELISA (NS-ELISA) and affinity-capture selfinteraction nanoparticle spectroscopy (AC-SINS), respectively, as described below. Two knownanti -IL-33 antibodies (itepekimab and etokimab) were included in the assays. Trastuzumab (anti- HER2) was used as a negative control (low propensity) and lenzilumab (anti-GM-CSF) was used as a positive control (high propensity).

[0260] ELISA-based non-specific binding to several coating materials was adapted as previously described (Jain, et al., 2017, Proc Natl Acad Set USA, 114(5):944-949). Non-specific ELISA (NS-ELISA) was standardized in a Coming® 96-well EIA / RIA Easy Wash™ Clear Flat Bottom Polystyrene High Bind Microplate coated with 50 pL of heparin diluted in 50 mM sodium carbonate pH 9.6 to a final concentration of 250 pg / mL. The plate was incubated for 2 days at room temperature. Insulin and keyhole limpet hemocyanin (KLH) were diluted in 50 mM sodium carbonate pH 9.6 to a final concentration of 5 pg / mL. ssDNA and dsDNA were diluted in PBS pH7.4 to a final concentration of 10 pg / mL. 50 pL of insulin, KLH, dsDNA and ssDNA were added to a 96 well plate, followed by the incubation at 37°C for 2 hrs, removal and blocking with 200 pL of PBS pH7.4, 0.1% Tween 20, for 1 hr at room temperature with shaking at 200 rpm. The plate was washed 3 times with PBS pH7.4, 0.1% Tween 20 and 50 pL of each test article at 100 nM (15 pg / mL) in PBS pH7.4, 0.1% Tween 20 were added in duplicate to the wells and incubated for 1 hr at room temperature with shaking at 200 rpm. Plates were washed three times with PBS pH7.4, 0.1% Tween 20, and 50 pL of 50 ng / mL anti-human IgG horseradish peroxidase (HRP) was added to each well. Plates were incubated for 1 hr at room temperature, with shaking at 200 rpm. Plates were washed three times with PBS pH7.4, 0.1% Tween 20, and 100 pL of 3, 3', 5,5'- tetramethylbenzidine (TMB) substrate (Cell Signaling Technology, Inc., Danvers, MA; Cat No. 7004P6) was added to each well. Reactions were stopped after approximately 10 minutes by adding 100 pL of 1 M HC1 to each well, and absorbance was read at 450 nm.

[0261] Binding scores were calculated as the ratio of the ELISA signal of the antibody to the signal of a well containing buffer instead of the primary antibody. The results are shown in Table 1. 12 and Table 1.13. All the antibody variants tested showed binding scores similar to those for the negative control and the known anti -IL-33 antibody itepekimab. The binding scores for the antibody variants were lower than those for the positive control and the known anti -IL-33 antibody etokimab.Table 1.12: Assessment of Non-specific Interactions of the Anti-IL-33 Full Size AntibodyVariantsTable 1.13: Assessment of Non-specific Interactions of the Anti-IL-33 One Armed AntibodyVariants

[0262] The AC-SINS method was adapted as previously described (Liu, et al., 2014, MAbs, 6(2):483-92) and standardized in a 384-well plate format.

[0263] Briefly, 20 nm gold nanoparticles (Ted Pella, Inc., #15705) washed with 0.22 pm filtered milli Q water were coated with a mixture of 80% AffmiPure™ goat anti -human IgG (H+L) (Jackson ImmunoResearch Laboratories, Inc., Cat. No. 109-005-088) as capture antibody and 20% ChromPure™ goat IgG, whole molecule (Jackson ImmunoResearch Laboratories, Inc., Cat. No. 005-000-003) that were buffer exchanged into 20 mM sodium acetate pH 4.3 and diluted to 0.4 mg / mL. The mixture of gold nanoparticles, capture antibody and non-capture antibody was incubated in the dark for 18 hr at room temperature. Sites unoccupied on the gold nanoparticles were blocked with 1 pM thiolated polyethylene glycol (2 kDa) in 20 mM sodium acetate, pH 4.3 to a final concentration of 0.1 pM, followed by 1 hr incubation at room temperature. The coated nanoparticles were then concentrated by centrifugation at 21,000 x g for 7 min, at 8°C. 95% of the supernatant was removed and the gold pellet was resuspended in the remaining buffer. 5 pL of concentrated nanoparticles were added to 45 pL of test articles at 0.05 mg / mL in PBS pH 7.4 in a 384-well plate and incubated for 4 h at room temperature in the dark. The absorbance was readfrom 450-700 nm and was used to identify the wavelength at max absorbance. The wavelength at max absorbance of the average blank (PBS alone) was subtracted from that of the test article to determine the antibody AC-SINS score (A X). The results are shown in Table 1.14. All the tested antibody constructs had AC-SINS scores similar to those of the negative control and the known anti-IL-33 antibody itepekimab. The AC-SINS scores for all tested antibody variants were lower than those of the positive control and the known anti -IL-33 antibody etokimab.Table 1.14: Assessment of Self-interaction Propensity of the Anti-IL-33 Full Size and One- Armed Antibody VariantsEXAMPLE 2: FUNCTIONAL CHARACTERIZATION OF ANTI- IL-33 ANTIBODY VARIANTS - IL-33 REPORTER GENE ASSAY

[0264] To determine the impact of selected humanized anti -IL-33 antibody variants fromExample 1 on IL-33 activation of NF-KB and AP-1 pathways, the antibody variants were assessed for inhibition of IL-33 mediated production of secreted embryonic alkaline phosphatase (SEAP) reporter in HEK-Blue™ IL-33 cells as described below. HEK-Blue™ IL-33 cells express the IL- 33 receptor and are transfected with an NF-KB / AP-1 inducible SEAP reporter. Binding of IL-33 to its receptor triggers a signalling cascade leading to NF-KB / AP-1 activation and subsequent production of SEAP. The anti-IL-33 antibodies, etokimab and itepekimab, were included as positive controls, and palivizumab (v22277) was included as a negative control.

[0265] Test articles were serially diluted starting at 20 000 pM in DMEM + 10% heat- inactivated fetal bovine serum (ThermoFisher Scientific, Waltham, MA) in a 384-well black flat bottom assay plate (ThermoFisher Scientific, Waltham, MA). 250 pM IL-33 (SinoBiologics, Beijing, China) was added, followed by 12500 HEK-Blue™ IL-33 cells (InvivoGen, San Diego, CA). After 24 hr at 37°C, 5% CO2, SEAP production was assessed by incubating supernatant with QUANTI-Blue™ solution (InvivoGen, San Diego, CA) and measuring OD620nm on a Synergy™ plate reader (BioTek, Winooski, VT).

[0266] The results are shown in Fig. 1A (full-sized antibody variants) and Fig. IB (one- armed antibody variants). All humanized anti-IL-33 antibody variants in either monovalent or bivalent format showed similar inhibition of IL-33 mediated production of NF-KB and AP-1 inducible SEAP reporter in HEK-Blue™ IL-33 cells. The inhibitory activity of the anti-IL-33 antibody variants was similar to that of the benchmark anti-IL-33 antibody controls (itepekimab and etokimab) and to the parental antibody variants (v33006 - bivalent and v40564 - monovalent).EXAMPLE 3: PREPARATION AND CHARACTERIZATION OF BISPECIFIC ANTLIL- 33 x ANTI-IL-4Ra ANTIBODIES (IgGl BACKBONE)

[0267] Bivalent, bispecific anti-IL-33 x anti-IL-4Ra antibody variants comprising an IgGl Fc region were produced as described below.

[0268] Bispecific anti -IL-33 x anti-IL-4Ra antibody variants that are monovalent for each antigen were prepared in a format in which both the IL-33 and IL-4Ra antigen binding domains are Fabdomains and in which the Fc region is a heterodimeric Fc region. These bispecific antibody variants comprise four different chains - 2 heavy chains (heavy chain A and heavy chain B) and 2 light chains (light chain A and light chain B) as described in Table 3.1.

[0269] The heavy chains comprised by the bispecific antibody variants described in Table 3. 1 are human IgGl heavy chains that comprise amino acid substitutions in the CHI domain that promote correct heavy and light chain pairing and amino acid substitutions in the CH3 domain that promote formation of a heterodimeric Fc over a homodimer Fc.

[0270] The CHI domain amino acid substitutions promoting correct heavy and light chain pairing are:(i) HetFabl:Chain A: L145E / K147T / Q175E (HetFabl HCA) and Chain B: L145R (HetFabl HCB), or(ii) HetFab2:Chain A: Q175R (HetFab2 HCA) and Chain B: A141W / L145E / K147T / Q175E (HetFab2 HCB).

[0271] The CH3 domain amino acid substitutions promoting heterodimeric Fc formation are:Chain A: T350V / L351Y / F405A / Y407V (HetFcA) and Chain B: T350V / T366L / K392L / T394W (HetFcB).

[0272] Fc regions comprising the HetFcA and HetFcB CH3 domains are referred to as “Het Fc.” Numbering of amino acids in the CHI domain and in the Fc region (CH2 and CH3 domains) is according to the EU index.

[0273] The Fc region of the bispecific antibody variants described in Table 3.1 also comprises the following amino acid substitutions in the CH2 domain of both heavy chains which eliminate (“knock out”) FcyR binding: L234A / L235A / D265S. These CH2 amino acid substitutions are referred to herein as “FcKO.”

[0274] In addition, certain of the bispecific antibody variants further comprise amino acid substitutions in the CH2 domain that enhance binding to the neonatal Fc receptor (FcRn). These amino acid substitutions, referred to herein “YTE mutations” or “YTE,” are: M252Y / S254T / T256E in both Fc polypeptide chains.

[0275] The light chains comprised by the bispecific antibody variants described in Table 3. 1 are human kappa light chains which comprise amino acid substitutions in the CL domain thatpromote formation of correct heavy and light chain pairs. The CL domain amino acid substitutions are:(i) HetFabl:Chain A: Q124R / T178R (HetFabl LCA) and Chain B: Q124E / V133E (HetFabl LCB), or(ii) HetFab2:Chain A: Q124E / L135W / T178E / T180E (HetFab2 LCA) and Chain B: F 116 A / Q 124R / L 135 V / T 178R (HetFab2 LCB) .

[0276] Numbering of amino acids in the CL domain is according to the EU index.

[0277] The genes encoding the antibody heavy and light chains were constructed via gene synthesis using codons optimized for human / mammalian expression. The anti-IL-33 Fab domain sequences were generated from the VH and VL sequences of the humanized anti-IL-33 antibody variants v33096 or v33101 described in Example 1. The anti-IL-4Ra Fab domain sequences were generated from the VH and VL sequences of one of the anti-IL-4Ra monoclonal antibody variants v38597 or v38504. The VH and VL sequences for these anti-IL-4Ra antibody variants are provided in Table 3.2 and Fig. 20 (Table Cl). The CDR sequences for these anti-IL-4Ra antibody variants are provided in Fig. 18 (Table Bl).Table 3.1: Description of Bispecific Anti- IL-33 x Anti-IL-4Ra Bispecific Antibodies (IgGl)Table 3.2: VH and VL Sequences of Anti-IL-4Ra Antibody Variants* NOTE: SEQ ID NO: 110 = SEQ ID NO: 90; SEQ ID NO: 111 & 113 = SEQ ID NO: 92; SEQ ID NO: 112 = SEQID NO: 913.1 Production of Bispecific Anti-IL-33 x Anti-IL-4Ra Antibody Variants

[0278] The bispecific antibody variants were expressed and characterized following generally the protocols described in International Patent Publication No. WO 2015 / 109131.

[0279] The final gene products were sub-cloned into a mammalian expression vector(typically, the mammalian expression vector pTT5 (NRC-BRI, Canada)) and expressed in CHO-KI cells. The cells were transfected in exponential growth phase (1.5 to 2 million cells / mL). In order to determine the optimal concentration range for forming heterodimers, the DNA was transfected in optimal DNA ratios of the heavy chain A (HC-A), heavy chain B (HC-B), and light chain A (LC-A) and light chain B (LC-B) that allow for heterodimer formation (for example, HC- A:HC-B:LC-A:LC-B ratios of 15: 15:53: 17 or 15: 15:35:35 or 8:22:35:35). Transfected cells were harvested after 5-6 days and the culture medium collected after centrifugation at 4000 rpm and clarification using a 0.45 pm filter. The clarified culture medium was loaded onto a MabSelect™ SuRe™ Protein A column (GE Healthcare) and eluted with 0. 1 M citrate or 0. 1 M acetate buffer at pH 3.0 with the pooled fractions containing the antibody variant neutralized with 1 M TRIS at pH 9 or 11. The amount of antibody variant was then quantified based on A280 nm (NanoDrop™ Spectrophotometer; Thermo Fisher Scientific).

[0280] The antibody variants were further purified by chromatography using a POROS™ X cation exchange column with a salt gradient of 20 mM sodium acetate, 0- 1 M NaCl buffer, pH 5.5, or a SP (sulfopropyl) HP (high performance) column with a salt gradient of 20 mM sodium acetate, 0-150 mM NaCl buffer, pH 5.5, or a Capto™ Butyl ImpRes hydrophobic interaction column with a gradient of0-100 % 50 mM phosphate buffer pH 6.5 with 0- 1 M ammonium sulfate . Fractions of eluted antibody variant were collected based on absorbance at A280 nm and the fractions were assessed by non-reducing SDS-PAGE. Fractions corresponding to the purified antibody variant were collected, buffer exchanged into 20 mM histidine, pH 6.0, concentrated to 10 mg / mL or 20 mg / mL, and stored at -80°C.

[0281] The apparent purity and yield of the final antibody variant was estimated by HPLC- SEC and LC / MS following protocols as described in International Patent Publication No. WO 2015 / 109131. All antibody variants were expressed and purified to > 99% heterodimer purity without contaminating homodimers (see Table 3.3).Table 3.3: Post Purification Yield and Purity for Anti-IL-33 x Anti-IL-4Ra Bispecific Antibody Variants (IgGl)1As determined by HPLC-SEC2As determined by LCMS intensity3.2 Characterization of Bispecific Anti-IL-33 x Anti-IL-4Ra Antibody Variants (IgGl)3.2.1 Thermal Stability of Bispecific Antibody Variants

[0282] The thermal stability of the bispecific anti -IL-33 x anti-IL-4Ra bispecific antibody variants was assessed by differential scanning calorimetry (DSC) and differential scanning fluorimetry (DSF) as described below.

[0283] All DSC experiments were carried out using a NanoDSC calorimeter (TA Instruments). The proteins were diluted to 1 mg / mL with 0.95 mL loaded into the 96 well plates and measured with a scan rate of l°C / min from 25 °C to 95 °C. Data was analyzed using the NanoAnalyze software (TA Instruments) with the 20 mM histidine, pH 6.0 buffer background subtracted.

[0284] All DSF experiments were carried out using a CFX96 Touch™ Real-Time PCR instrument (BioRad Laboratories, Inc., Hercules, CA). The proteins were diluted to 1 mg / mL and lOpg loaded into 35pL buffer in the 96 well plates with 5pL of 40x Invitrogen™ SYPRO™ Orange Protein Gel Stain (5,000X Concentrate in DMSO) (Thermo Fisher Scientific) and measured with a scan rate of 0.5°C / min from 25°C to 95°C. Data was analyzed using Microsoft Excel with the 20 mM histidine, pH 6.0 buffer background subtracted.

[0285] The results are shown in Table 3.4, which shows the maximum melting temperatures (Tm) for each of the peaks in the thermograms of the bispecific antibody variants. The results demonstrate that the tested antibody variants exhibited thermostability profiles comparable to those of conventional IgGl antibodies.Table 3.4: Thermostability of Anti-IL-33 x Anti-IL-4Ra Bispecific Antibody Variants (IgGl)3.2.2 Accelerated Stability of Anti-IL-33 x Anti-IL-4Ra Bispecific Antibody Constructs

[0286] The accelerated stability of the bispecific anti -IL-33 x anti-IL-4Ra antibody variants was evaluated by assessing the purity after storage at 40°C for 4 weeks. The bispecific antibody variants were stored at 20 mg / mL in 20 mM histidine pH 6.0 buffer and the purity was assessed by UPLC-SEC.

[0287] The results are shown in Table 3.5 and demonstrate that the bispecific antibody variants display minimal changes in % monomer purity as measured by UPLC-SEC. Some decrease in purity was observed after 4 weeks at 40°C for certain samples with an increase in low molecular weight species (LMWs) but not in high molecular weight species (HMWs).Table 3.5: Purity of Anti-IL-33 x Anti-IL-4Ra Bispecific Antibody Variants Assessed by UPLC-SEC after storage at 40°C for 4 Weeks3.2.3 Stability of Anti-IL-33 x Anti-IL-4Ra Bispecific Antibody Variants after Long Term Storage at -80 J

[0288] The stability of the bispecific anti -IL-33 x anti-IL-4Ra antibody variants was evaluated by assessing the purity after storage at -80°C for 8 weeks. The bispecific antibody variants were stored at 20 mg / mL in 20 mM histidine pH 6.0 buffer and the purity was assessed by UPLC-SEC.

[0289] The results are shown in Table 3.6 and show the bispecific antibody variants displayed no significant changes in % monomer purity as measured by UPLC-SEC.Table 3.6: Purity of Anti-IL-33 x Anti-IL-4Ra Bispecific Antibody Variants Assessed by UPLC-SEC after Storage at -80°C for 8 Weeks3.2.4 Stability of Anti-IL-33 x Anti-IL-4Ra Bispecific Antibody Variants after 5 Cycles of Freeze- Thaw

[0290] The stability of the bispecific anti -IL-33 x anti-IL-4Ra antibody variants was evaluated by assessing the purity after 1 or 5 cycles of freeze-thaw (5xF / T). Each cycle consisted of freezing at -80°C for 30 mins followed by thawing at 4°C for 30 mins. The bispecific antibody variants were at 20 mg / mL in 20 mM histidine pH 6.0 buffer and the purity was assessed by UPLC- SEC.

[0291] The results are shown in Table 3.7 and show that the bispecific antibody variants displayed no significant changes in % monomer purity as measured by UPLC-SEC after 1 or 5 freeze-thaw cycles.Table 3.7: Purity of Anti-IL-33 x Anti-IL-4Ra Bispecific Antibody Variants Assessed by UPLC-SEC after 1 or 5 Freeze- Thaw Cycles3.2.2 Assessment of Non-specific Interactions and Self-interaction Propensity of Bispecific Antibody Variants

[0292] The potential propensity of the bispecific antibody variants for non-specific interaction and self-interaction were assessed by non-specific ELISA (NS-ELISA) and affinitycapture self-interaction nanoparticle spectroscopy (AC-SINS), respectively, as described in Example 1. Trastuzumab (anti-HER2) was used as a negative control (low propensity) and lenzilumab (anti-GM-CSF) was used as a positive control (high propensity).

[0293] The results are shown in Table 3.8 (NS-ELISA) and Table 3.9 (AC-SINS) and indicate that all bispecific antibody variants showed a propensity for both non-specific interaction and self-interaction that was higher than that shown by the negative control, but still lower than that shown by the positive control. As the propensities for the bispecific antibody variants were increased when compared to the corresponding monospecific antibody variants (see Example 1), the increase is likely attributable to the anti-IL-4Ra arm comprised by the bispecific antibody variants.Table 3.8: Assessment of Non-specific Interactions of Anti-IL-33 x Anti-IL-4Ra Bispecific Antibody Variants (IgGl)Table 3.9: Assessment of Self-interaction Propensity of Anti-IL-33 x Anti-IL-4Ra Bispecific Antibody Variants (IgGl)EXAMPLE 4: PREPARATION AND CHARACTERIZATION OF BISPECIFIC ANTI-IL- 33 x ANTI-IL-4Ra ANTIBODIES (IgG4 BACKBONE) #1

[0294] Bivalent, bispecific anti-IL-33 x anti-IL-4Ra antibody variants comprising an IgG4 Fc region were produced as described below.

[0295] Bispecific anti -IL-33 x anti-IL-4Ra antibody variants that are monovalent for each antigen were prepared in a format in which both the IL-33 and IL-4Ra antigen binding domains are Fab domains and in which the Fc region is a heterodimeric Fc region. These bispecific antibody variants comprise four different chains - 2 heavy chains (heavy chain A and heavy chain B) and 2 light chains (light chain A and light chain B) as described in Table 4.1.

[0296] The heavy chains comprised by the bispecific antibody variants described in Table 4.1 are human IgG4 heavy chains that comprise amino acid substitutions in the CHI domain that promote correct heavy and light chain pairing and amino acid substitutions in the CH3 domain that promote formation of a heterodimeric Fc over a homodimer Fc. The heavy chains also contained the human IgG4 hinge domain which further comprised amino acid substitution S228P.

[0297] The CHI domain amino acid substitutions promoting correct heavy and light chain pairing are:HetFab 1:Chain A: L145E / K147T / Q175E (HetFab HCA) and Chain B: L145R (HetFab HCB)

[0298] The CH3 domain amino acid substitutions promoting heterodimeric Fc formation are:Chain A: T350V / L351Y / F405A / Y407V / R409K (HetFcA) and Chain B: T350V / T366L / K392L / T394W / R409K (HetFcB) .

[0299] Fc regions comprising the HetFcA and HetFcB CH3 domains are referred to as “Het Fc.” Numbering of amino acids in the CHI domain and in the Fc region (CH2 and CH3 domains) is according to the EU index.

[0300] In addition, certain of the bispecific antibody variants further comprise amino acid substitutions in the CH2 domain that enhance binding to the neonatal Fc receptor (FcRn). These amino acid substitutions, referred to herein “YTE mutations” or “YTE,” are: M252Y / S254T / T256E in both Fc polypeptide chains.

[0301] The light chains comprised by the bispecific antibody variants described in Table 4. 1 are human kappa light chains which comprise amino acid substitutions in the CL domain thatpromote formation of correct heavy and light chain pairs. The CL domain amino acid substitutions are:HetFab 1:Chain A: Q124R / T178R (HetFab LCA) and Chain B: Q124E / V133E (HetFab LCB)

[0302] Numbering of amino acids in the CL domain is according to the EU index.

[0303] In the bispecific antibody variants, the VH and VL sequences of each of the anti-IL- 33 Fab domain and the anti-IL-4Ra Fab domain were coupled to human IgG4 CHI and CL sequences, respectively.

[0304] The anti-IL-33 Fab domain sequence was generated from the VH and VL sequences of the humanized anti-IL-33 antibody variant v33096 or v33101 described in Example 1. The anti- IL-4Ra Fab domain sequences were generated from the anti-IL-4Ra monoclonal antibody v38597 (described in Example 3) or from the VH and VL sequences of the anti-IL-4Ra monoclonal antibody v38597-Y54A. The Vnand VL sequences of v38597-Y54A are identical to those of v38597 except that an amino acid substitution, Y 54A, was made in CDRH2 of the VH domain to potentially reduce hydrophobicity. The Vnand VL sequences of v38597-Y54A are provided in Table 4.2.Table 4.1: Description of Anti-IL-33 x Anti-IL-4Ra Bispecific Antibody Variants (IgG4)Table 4.2: VH and VL Sequences of Anti-IL-4Ra Antibody Variant v38597-Y54A* NOTE: SEQ ID NO: 114 = SEQ ID NO: 93; SEQ ID NO: 111 = SEQ ID NO: 924.1 Production of bivalent and bispecific IL-33 x IL-4Ra Antibody Constructs

[0305] The bispecific antibody variants were expressed and characterized following the protocols described in International Patent Publication No. WO 2015 / 109131.

[0306] Briefly, the genes encoding the bispecific antibody heavy and light chains were constructed via gene synthesis using codons optimized for human / mammalian expression. The final gene products were sub-cloned into a mammalian expression vector and expressed in CHO- K1 cells. The cells were transfected in exponential growth phase (1.5 to 2 million cells / mL). In order to determine the optimal concentration range for forming heterodimers, the DNA was transfected in optimal DNA ratios of the heavy chain A (HC-A), heavy chain B (HC-B), and light chain A (LC-A) and light chain B (LC-B) that allow for heterodimer formation (for example, HC- A:HC-B:LC-A:LC-B ratios of 15: 15:53: 17 or 15: 15:35:35 or 8:22:35:35). Transfected cells were harvested after 5-6 days and the culture medium collected after centrifugation at 4000 rpm and clarification using a 0.45 pm filter. The clarified culture medium was loaded onto a MabSelect™ SuRe™ Protein A column (GE Healthcare) and eluted with 0. 1 M citrate or 0. 1 M acetate buffer at pH 3.0 and the pooled fractions containing the antibody variant were neutralized with 1 M TRISat pH 9 or 11. The amount of antibody variant was then quantified based on A280 nm (NanoDrop™ Spectrophotometer; Thermo Fisher Scientific).

[0307] The antibody constructs were further purified by chromatography using a POROS™ X cation exchange column with a salt gradient of 20 mM sodium acetate, 0-1 M NaCl buffer, pH 5.5, or a SP (sulfopropyl) HP (high performance) column with a salt gradient of 20 mM sodium acetate, 0-150 mM NaCl buffer, pH 5.5, or a Capto™ Butyl ImpRes hydrophobic interaction column with a gradient of 0-100 % 50 mM phosphate buffer pH 6.5 with 0-1 M ammonium sulfate. Fractions of eluted antibody variant were collected based on absorbance at A280 nm and the fractions were assessed by non-reducing SDS-PAGE. Fractions corresponding to the purified antibody variant were collected, buffer exchanged into 20 mM histidine, pH 6.0, concentrated to 10 mg / mL or 20 mg / mL, and stored at -80°C.

[0308] The apparent purity and yield of the final antibody variant was estimated by HPLC-SEC and LC / MS following generally the protocols as described in International Patent Publication No. WO 2015 / 109131. All antibody constructs were expressed and purified to > 99 % heterodimer purity without contaminating homodimers (see Table 4.3).Table 4.3: Post Purification Yield and Purity for Anti-IL-33 x Anti-IL-4Ra BispecificAntibody Variants (IgG4)1As determined by HPLC-SEC (size-exclusion chromatography HPLC)2As determined by LCMS intensity4.2 Characterization of Anti-IL-33 x Anti-IL-4Ra Bispecific Antibody Variants (IgG4 backbone)4. .1 Thermal Stability of Bispecific Antibody Variants

[0309] The thermal stability of the bispecific anti -IL-33 x anti-IL-4Ra bispecific antibody variants was assessed by differential scanning calorimetry (DSC) and differential scanning fluorimetry (DSF) as described below.

[0310] All DSC experiments were carried out using a NanoDSC calorimeter (TA Instruments). The proteins were diluted to 1 mg / mL with 1 mL loaded into the 96 well plates and measured with a scan rate of l°C / min from 25°C to 90°C. Data was analyzed using the NanoAnalyze software (TA Instruments) with the 20 mM histidine, pH 6.0 buffer background subtracted.

[0311] All DSF experiments were carried out using a CFX96 Touch™ Real-Time PCR instrument (BioRad Laboratories, Inc., Hercules, CA). The proteins were diluted to 1 mg / mL and lOpg loaded into 35pL buffer in the 96 well plates with 5pL of 40x Invitrogen™ SYPRO™ Orange Protein Gel Stain (5,000X Concentrate in DMSO) (Thermo Fisher Scientific) and measured with a scan rate of 0.5°C / min from 25°C to 95°C. Data was analyzed using Microsoft Excel with the 20 mM histidine, pH 6.0 buffer background subtracted.

[0312] The results are shown in Table 4.4, which shows the maximum melting temperatures (Tm) for each of the peaks in the thermograms of the bispecific antibody variants. All the bispecific antibody variants exhibited thermostability profiles comparable to those of the corresponding IgGl bispecific variants in Example 3.Table 4.4: Thermostability of Anti-IL-33 x Anti-IL-4Ra Bispecific Antibody Variants (IgG4)* ND = not determined4.2.2 Stability of Anti-IL-33 x Anti-IL-4Ra Bispecific Antibody Variants

[0313] The bispecific antibody variants were tested for (i) freeze-thaw stability, (ii) long term storage stability at -80°C, and (iii) stability in an accelerated stress test, as described in Example 3.

[0314] The results of the freeze-thaw stability testing are shown in Table 4.5 (antibody concentration: 20 mg / mL) and Table 4.6 (antibody concentration: 150 mg / mL). None of the bispecific antibody variants showed a significant change in purity after 5 cycles of freeze-thaw at either concentration.

[0315] The results of the long-term storage stability testing are shown in Table 4.7. The bispecific antibody variants showed only minimal change in purity after 9 weeks storage at -80°C using an antibody concentration of 20 mg / mL.

[0316] The results of the accelerated stress test are shown in Table 4.8 (antibody concentration: 20 mg / mL, 4-week incubation at 37°C) and Table 4.9 (antibody concentration: 150 mg / mL, 4-week incubation at 40°C). The bispecific antibody variants showed minimal change in purity under either set of conditions.Table 4.5: Purity (%) of Bispecific Antibody Variants After 5 Cycles of Freeze / Thaw (5x F / T) Treatment at 20 mg / mL in 20 mM Histidine pH 6.0 bufferTable 4.6: Purity (%) of Bispecific Antibody Variants After 5 Cycles of Freeze / Thaw (5x F / T) at 150 mg / mL in 20 mM Histidine pH 6.0 bufferTable 4.7: Purity (%) of Bispecific Antibody Variants After 9 Weeks Storage at -80°C at 20 mg / mL in 20 mM Histidine pH 6.0 bufferTable 4.8: Purity (%) of Bispecific Antibody Variants After 4 Weeks Storage at 37°C at 20 mg / mL in 20 mM Histidine pH 6.0 bufferTable 4.9: Purity (%) of Bispecific Antibody Variants After 4 weeks Storage at 40°C at 150 mg / mL in 20 mM Histidine pH 6.0 buffer4.2.3 Assessment of Non-specific Interactions and Self-interaction Propensity of Bispecific Antibody Variants

[0317] The potential propensity of the bispecific antibody variants for non-specific interaction and self-interaction were assessed by non-specific ELISA (NS-ELISA) and affinitycapture self-interaction nanoparticle spectroscopy (AC-SINS), respectively, as described in Example 1. Trastuzumab (anti-HER2) was used as a negative control (low propensity) and lenzilumab (anti-GM-CSF) was used as a positive control (high propensity).

[0318] The results are shown in Table 4.10 (NS-ELISA) and Table 4.11 (AC-SINS). The NS-ELISA results indicate that all bispecific antibody variants showed a propensity for nonspecific interaction that was higher than that shown by the negative control, with the exception of dsDNA, which showed similar values to the negative control. All values for the bispecific antibody variants were lower than those shown by the positive control. The AC-SINS results indicate that all bispecific antibody variants showed a propensity for both non-specific interaction and selfinteraction that was higher than that shown by the negative control, but still lower than that shown by the positive control. As the propensities for the bispecific antibody variants were increased when compared to the corresponding monospecific antibody variants (see Example 1), the increase is likely attributable to the anti-IL-4Ra arm comprised by the bispecific antibody variants.Table 4.10: Assessment of Non-specific Interactions of the Anti- IL-33 x Anti-IL-4Ra Bispecific Antibody Variants (IgG4)Table 4.11: Assessment of Self-interaction Propensity of the Anti-IL-33 x Anti-IL-4RaBispecific Antibody Variants (IgG4)EXAMPLE 5: CELLULAR BINDING OF ANTLIL-33 x ANTI-IL-4Ra BISPECIFIC ANTIBODY VARIANTS (IgG4 BACKBONE)

[0319] To test the ability of the bispecific antibody variants to bind human IL-4Ra, selected bispecific antibody variants were assessed for binding to PBMCs by flow cytometry as described below. The anti-IL-4Ra antibody dupilimab was used as a positive control for IL-4Ra binding and a human-IgG isotype (v36992) was used as a non-specific negative control.

[0320] The variants tested were: v41793, v 41798, v41795 and v41799. These variants are identical to v42098, v42099, v42100 and v42101 (see Table 4.1), respectively, except they include the mutation N31A (AbM numbering) in the anti-IL-33 arm. This mutation does not affect the binding of the anti-IL-4Ra arm, which is being tested in this assay.

[0321] Flow cytometry was performed on human PBMCs (StemCell Technologies, Vancouver Canada) or cynomolgus PBMCs (BioIVT, Westbury, NY). Briefly, PBMCs were thawed and plated in 96 well v-bottom plates at IxlO5cells / well. Antibody variants were serially diluted in flow cytometry staining buffer (5% FBS, 2.5mM EDTA, PBS). Cells were stained with Fixable Viability Dye eFluor™ 506 (Thermo Fisher Scientific, Waltham, MA) for 15 minutes in PBS. Cells were washed once with PBS, washed once with flow cytometry staining buffer, then incubated with antibody variants at 4°C for 1 hour in flow cytometry staining buffer to allow binding. Cells were washed and stained for 30 minutes with fluorescently-conjugated antibodies against human (CD3, CD4, CD 14, CD 15, CD 16, CD 19) cell surface markers to allow phenotyping of cells by flow cytometry. An anti-human-IgG4 antibody (Southern Biotech, Birmingham, AL) was included in the antibody panel to detect antibody variant binding to PBMCs. Cells were washed with flow cytometry staining buffer, fixed for 10 minutes in BD Cytofix™ Fixation Buffer (BD Biosciences, San Francisco, CA), washed with flow cytometry staining buffer, resuspended in flow cytometry staining buffer, and analyzed by flow cytometry on an LSR Fortessa™ X-20 flow cytometer (BD Biosciences). Geometric mean fluorescent intensity (gMFI) of anti-human- IgG was used to assess binding of constructs on CD4+T cell (CD3+CD 19" CD4+), B cell (CD3‘ CD19+) and monocyte (CD-CD14 CD15 CD16+ / ) populations as defined by phenotyping antibody panel staining.

[0322] The results are shown in Fig. 2A-C. Bispecific antibody variants were shown to bind IL-4Ra across multiple cell types known to express IL-4Ra (T cells, B cells, and monocytes) as shown by the presence of an anti-human-IgG positive population. Furthermore, the gMFI ofanti-human-IgG staining increased as bispecific antibody variant concentration increased, while the isotype control gMFI did not, indicating specific binding of the bispecific antibody variants to their target.EXAMPLE 6: FUNCTIONAL CHARACTERIZATION OF ANTLIL-33 x ANTI-IL-4Ra BISPECIFIC ANTIBODY VARIANTS (IgGl AND IgG4 BACKBONE) - IL-4 REPORTER GENE ASSAY

[0323] To determine the impact of the anti-IL-33 x anti-IL-4Ra bispecific antibody variants on IL-4 activation of the STAT6 signalling pathway, selected variants were assessed for inhibition of IL-4 mediated production of the STAT6 inducible secreted embryonic alkaline phosphatase (SEAP) reporter in HEK-Blue™ IL-4 / IL-13 cells as described below. The variants tested were:

[0324] IgGl backbone: v41245, v41246, v41249, v41251, v41315, v41316 and v41317 (see Table 3.1)

[0325] IgG4 backbone: v42103, v42100, v42101, v42102, v42098 and v42099 (see Table 4.1)

[0326] An anti-IL-4Ra antibody (dupilumab) in monovalent and bivalent format, a tetravalent anti-IL-4Ra x anti-IL-31 bispecific antibody (NM26-2198), an anti-RSV IgGl FcKO antibody (v39982), an anti-RSV IgG4 antibody (v36992) and an IgG4 isotype antibody (v42104) were used as controls.

[0327] Briefly, test articles were serially diluted starting at 20 000 pM in DMEM + 10% heat-inactivated fetal bovine serum (ThermoFisher Scientific, Waltham, MA) and plated into a 384-well black flat bottom assay plate. 125 pM IL-4 (R&D Systems, Minneapolis, MN) was added, followed by 5000 - 12500 HEK-Blue™ IL-4 / IL-13 cells (InvivoGen, San Diego, CA). After 24 hr at 37°C, 5% CO2, SEAP production was assessed by incubation of supernatant with QUANTI-Blue™ solution (InvivoGen, San Diego, CA) and measuring OD620nm on a Synergy™ plate reader (BioTek Instruments, Inc., Winooski, VT).

[0328] The results are shown in Fig. 3A-C. All bispecific antibody variants tested blocked IL-4 mediated production of STAT6 inducible SEAP reporter in HEK-Blue™ IL-4 / IL-13 cells. The level of inhibitory activity was agnostic of Fc format (IgGl KO vs IgG4), and the presence or absence of the YTE mutations and the Y54A mutation. All bispecific antibody variants showedsimilar or superior inhibitory activity compared to the bivalent anti-IL-4Ra antibody control (dupilumab) and the tetravalent anti-IL-4Ra x IL-31 bispecific antibody control (NM26-2198).EXAMPLE 7: FUNCTIONAL CHARACTERIZATION OF ANTI-IL-33 x ANTI-IL-4Ra BISPECIFIC ANTIBODY VARIANTS (IgGl AND IgG4 BACKBONE) - IL-33 REPORTER GENE ASSAY

[0329] To determine the impact of the anti-IL-33 x anti-IL-4Ra bispecific antibody variants on IL-33 activation of NF-KB and AP-1 pathways, selected bispecific antibody variants were assessed for inhibition of IL-33 mediated production of the NF-KB / AP-1 inducible secreted embryonic alkaline phosphatase (SEAP) reporter in HEK-Blue™ IL-33 cells as described below. The variants tested were:

[0330] IgGl backbone: v40570, v41245, v41246, v41249, v4I25I, v4I3I5, v41316 and v41317 (see Table 3.1)

[0331] IgG4 backbone: v42103, v42100, v42101, v42102, v42098 and v42099 (see Table 4.1)

[0332] An anti-IL-4Ra antibody (dupilumab) in monovalent and bivalent format, an anti- IL-33 antibody (itepekimab) in monovalent and bivalent format, anti-RSV IgGl FcKO antibodies (v39982 and v40552) and an IgG4 isotype antibody (v42104) were used as controls.

[0333] Test articles were serially diluted starting at 20 000 pM in DMEM + 10% heat- inactivated fetal bovine serum (ThermoFisher Scientific, Waltham, MA) in a 384-well black flat bottom assay plate. 250 pM IL-33 (SinoBiologics, Beijing, China) was added, followed by 12 500 HEK-Blue™ IL-33 cells (InvivoGen, San Diego, CA). After 24 hr at 37°C, 5% CO2, SEAP production was assessed by incubating supernatant with QUANTI-Blue™ solution (InvivoGen, San Diego USA) and measuring OD620nm on a Synergy™ plate reader (BioTek Instruments, Inc., Winooski, VT).

[0334] The results are shown in Fig. 4A-C. All tested bispecific antibody variants showed similar inhibition of IL-33 mediated production of NF-KB and AP-1 inducible SEAP reporter in HEK-Blue™ IL-33 cells. The level of inhibitory activity was agnostic of Fc format (IgGl KO vs IgG4), and the presence or absence of the YTE mutations and the Y54A mutation. All bispecific antibody variants showed comparable activity to bivalent anti -IL-33 control (itepekimab), when taking into account the bivalency of the itepekimab control compared to the monovalency of theanti -IL-33 arm of the bispecific antibody variants. (Note that in the experiment shown in Fig. 4A, itepekimab showed much lower activity than usually observed in these assays (compare with Fig. 4B and 4C)).EXAMPLE 8: FUNCTIONAL CHARACTERIZATION OF ANTI-IL-33 x ANTI-IL-4Ra BISPECIFIC ANTIBODY VARIANTS (IgGl AND IgG4 BACKBONE) - IL-4 INDUCED CCL17 PRODUCTION

[0335] Selected anti-IL-33 x anti-IL-4Ra bispecific antibody variants were assessed for inhibition of IL-4 mediated production of CCL17 (also known as thymus and activation regulated chemokine (TARC)) in peripheral blood mononuclear cells (PBMCs). The variants tested were:

[0336] IgGl backbone: v41240, v41243, v41245, v41246, v41249, v41251, v41315, v41316 and v41317 (see Table 3.1)

[0337] IgG4 backbone: v42103, v42100, v42101, v42102, v42098 and v42099 (see Table 4.1)

[0338] A bivalent anti-IL-4Ra antibody (dupilumab), a bivalent anti-IL-33 antibody (itepekimab), an anti-RSV IgGl FcKO antibody (v39982), an anti-RSV IgG4 antibody (v36992) and an IgG4 isotype antibody (v42104) were used as controls.

[0339] Test articles were serially diluted starting at 20 000 pM in RPMI + 10% heat inactivated fetal bovine serum) (ThermoFisher Scientific, Waltham, MA) and plated in a 384-well black flat bottom assay plate. 300 pM human IL-4 (R&D Systems, Minneapolis, MN) was added followed by 40 000 freshly thawed PBMCs (Stemcell, Vancouver, Canada). After 24 hours at 37°C, 5% CO2, CCL17 levels in the supernatants were quantified with U-PLEX™ human TARC assay (Meso Scale Discovery, Rockville, MD) following manufacturer’s instructions.

[0340] The results are shown in Fig. 5A-D. All tested bispecific antibody variants demonstrated blockade of IL-4 mediated CCL17 production in PBMCs. The level of inhibitory activity was agnostic of Fc format (IgGl KO vs IgG4), and the presence or absence of the YTE mutations and the Y54A mutation. All bispecific antibody variants showed comparable inhibitory activity to the bivalent anti-IL-4Ra antibody control (dupilumab).EXAMPLE 9: FUNCTIONAL CHARACTERIZATION OF ANTLIL-33 x ANTI-IL-4Ra BISPECIFIC ANTIBODY VARIANTS (IgGl AND IgG4 BACKBONE) - IL-33 PBMC ASSAY (IFN-y)

[0341] Selected anti-IL-33 x anti-IL-4Ra bispecific antibody variants were assessed for inhibition of IL-33 mediated interferon-y (IFN-y) production in IL-12 stimulated peripheral blood mononuclear cells (PBMCs). The variants tested were:

[0342] IgGl backbone: v41240, v41243, v41245, v41246, v41249, v41251, v41315, v41316 and v41317 (see Table 3.1)

[0343] IgG4 backbone: v42103, v42100, v42101, v42102, v42098 and v42099 (see Table 4.1)

[0344] Two bivalent anti-IL-33 antibodies (itepekimab and etokimab), an anti-RSV IgGl FcKO antibody (v39982), an anti-RSV IgG4 antibody (v36992) and an IgG4 isotype antibody (v42104) were used as controls.

[0345] Test articles were serially diluted starting from 20 000 pM in assay media, RPMI + 10% heat inactivated fetal bovine serum) (ThermoFisher Scientific, Waltham, MA) in a 384-well black flat bottom assay plate. 50 pM oxidation resistant human IL-33 (AdipoGen Life Sciences, San Diego, CA) was added, followed by 40 000 freshly thawed PBMCs (Stemcell, Vancouver, Canada) in assay media containing 10 ng / mL IL- 12 (R&D Systems, Minneapolis, MN). After 24 hr at 37°C, 5% CO2, IFN-y levels in the supernatants were quantified with U-PLEX™ human IFN- y assay (Meso Scale Discovery, Rockville, MD) following manufacturer’s instructions.

[0346] The results are shown in Figs. 6A-D. All bispecific antibody variants demonstrated blockade of IL-33 mediated IFN-y production in IL-12 stimulated PBMCs. The level of inhibitory activity was agnostic of Fc format (IgGl KO vs IgG4), and the presence or absence of the YTE mutations and the Y 54A mutation. All IgGl bispecific antibody variants showed inhibitory activity comparable to that shown by the bivalent anti-IL33 benchmark control etokimab and superior to that shown by the bivalent anti-IL33 benchmark control itepekimab (see Figs. 6A and 6B). All IgG4 bispecific antibody variants showed inhibitory activity superior to that shown by the bivalent anti-IL33 benchmark control itepekimab (see Figs. 6C and 6D).EXAMPLE 10: FUNCTIONAL CHARACTERIZATION OF ANTLIL-33 x ANTI-IL-4Ra BISPECIFIC ANTIBODY VARIANTS (IgGl AND IgG4 BACKBONE) - IL-4 PBMC (CD23) ASSAY

[0347] To test the functional impact of blockade on IL-4Ra signalling by the bispecific antibodies, selected anti-IL-33 x anti-IL-4Ra bispecific antibody variants were assessed for inhibition of CD23 upregulation by PBMCs following IL-4 stimulation. Flow cytometry was used to assess CD23 expression following IL-4 stimulation as described below. Dupilumab was used as a positive control for blocking Il-4Ra signalling, while an anti-hemagglutinin (HA) IgG4 antibody (v42104) was used as a non-specific negative control.

[0348] The antibody variants tested were: v41316 and v41317 (IgGl backbone, see Table 3.1), v42103, v42102, v42100, v42098, v42101 and v42099 (IgG4 backbone, see Table 4.1).

[0349] Human PBMCs (StemCell, Vancouver Canada) were plated at 2xl05cells / well in 96 well round-bottom plates and incubated with serially diluted concentrations of antibody variants for 30 minutes to allow binding. IL-4 (R&D Systems, Minneapolis, MN) was added to each well to a final concentration of 2ng / mL and cells were incubated at 37°C for 48 hours in 10% FBS RPMI (ThermoFisher Scientific, Waltham, MA). Following incubation, cells were stained with Fixable Viability Dye eFluor™ 506 (Thermo Fisher Scientific, Waltham, MA) for 15 minutes in PBS, washed with PBS, washed with flow cytometry staining buffer (5% FBS, 2.5mM EDTA, PBS), and stained in flow cytometry staining buffer for 30 minutes with fluorescently-conjugated antibodies against human cell surface markers (CD3, CD14, CD16, CD19, CD23, CD27) to allow phenotyping of cells by flow cytometry. Cells were washed twice with flow cytometry staining buffer and fixed for 10 minutes in BD Cytofix™ Fixation Buffer (BD Biosciences, San Francisco, CA). Cells were washed and resuspended in flow cytometry staining buffer then analyzed by flow cytometry on an LSR Fortessa™ X-20 flow cytometer (BD Biosciences). Geometric mean fluorescent intensity (gMFI) of CD23 on various immune cell populations as defined by phenotyping antibodies (B-cells: CD19+CD3" CD27+ / ", T-cells: CD3+CD19", Monocytes: CD14+CD16+ / ) was used to assess inhibition of IL-4Ra signalling.

[0350] The results are shown in Fig. 7. The bispecific antibody variants were shown to limit changes to cellular phenotypes resulting from IL-4Ra stimulation by IL-4 as evidenced by decreasing CD23 expression as antibody variant concentration increases. Specifically, naive B- cells and monocytes, which have been shown to increase CD23 expression following IL-4Rastimulation, were both shown to have reduced CD23 expression which was dependent on antibody variant concentration. This effect was not seen in cells treated with isotype control showing that the effect was mediated by inhibition of IL-4Ra stimulation by the antibody variants. Furthermore, constructs with an IgG4 backbone (v42098, v42099, v42100, v42101, v42102 and v42103) outperformed constructs with a shared binding sequence but an IgGl backbone (v41316 and v41317) suggesting that the IgG4 backbone provides increased efficacy in preventing activation of key cell populations which respond to IL-4Ra stimulation.EXAMPLE 11: FUNCTIONAL CHARACTERIZATION OF ANTLIL-33 x ANTI-IL-4Ra BISPECIFIC ANTIBODY VARIANTS (IgGl AND IgG4 BACKBONE) - IL-13 / 33 (CCL2 PRODUCTION)

[0351] The anti-IL-33 x anti-IL-4Ra bispecific antibody variant v42101 (see Table 4.1) was assessed for the ability to inhibit CCL2 gene expression following combined cytokine stimulation of the IL-4, IL-13 and IL-33 pathways as described below. An anti-IL-4Ra monospecific antibody (dupilumab) and an anti -IL-33 monospecific antibody (itepekimab) were used as controls.

[0352] Briefly, 9x104HEKa cells (ATCC, Manassas, VA) were plated in a flat-bottom 24 well plate in assay media (1: 1 mix of DMEM and Ham’s F12K (both from Sigma-Aldrich, St. Louis, MO), and 10% FBS) and incubated at 37°C for 24 hours. All cytokines were prepared separately in assay media for a final concentration in cell cultures of 50nM per cytokine. Prior to adding to cell cultures, antibody variants were added into IL-33 cytokine (R&D Systems, Minneapolis, MN) preparations and incubated for 30 minutes at room temperature to allow binding of cytokine by the antibody variant. Media was aspirated from cell cultures and ImL of cytokineantibody variant mixtures were added to applicable wells. Cell cultures were incubated for 30 minutes at room temperature to allow the antibody variant to bind IL-4Ra. After 30 minutes, IL- 13 (R&D Systems, Minneapolis, MN) was added to wells for a final concentration of 50nM. Cells were incubated at 37°C for 6 hours, then media was aspirated out of the wells and cells were washed twice with cold PBS. RNA was isolated from the cells via RNeasy™ Mini Kit columns (Qiagen, Hilden, Germany) following manufacturer’s instructions and cDNA was generated from the isolated RNA using a High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Waltham, MA) following manufacturer’s instructions. qPCR was then performed using TaqMan™Gene Expression Assay primers and TaqMan™ Universal PCR Master Mix (Applied Biosystems, Waltham, MA) following manufacturer’s instructions, and a QuantStudio™ 3 Real-Time PCR machine (ThermoFisher Scientific, Waltham, MA) to quantify the amount of CCL2 and ubiquitin C (UBC) mRNA in the original isolated RNA sample. CCL2 mRNA production was used as a proxy for measuring cell stimulation after combined cytokine exposure. Ubiquitin C (UBC) was used as the housekeeping gene. Relative quantification of CCL2 mRNA was determined by calculating the double delta Ct (threshold cycle) for each sample. Briefly, the Ct of CCL2 cDNA was subtracted from the Ct of UBC cDNA from the same sample to give the delta Ct. This value was then subtracted by the delta Ct of unstimulated control samples to provide the delta delta Ct value. Taking the natural log of the negative of this value gives the fold change in CCL2 gene expression in experimental samples as compared to unstimulated controls.

[0353] The results are shown in Fig. 8. Cells incubated with v42101 show a lower level of CCL2 mRNA production compared to cells incubated with either cytokine alone, cytokine combinations, monospecific antibody controls or combinations of the monospecific antibody controls. This suggests the v42101 can inhibit CCL2 mRNA production by blocking stimulation through both IL-4Ra signalling and by sequestering free-floating IL-33, preventing cells which express IL-4Ra from receiving direct IL-33 stimulation.EXAMPLE 12: PHARMACOKINETICS OF BISPECIFIC ANTIBODY VARIANTS (IgG4 BACKBONE) IN A RAT MODEL

[0354] The pharmacokinetics (PK) of the anti-IL-33 x anti-IL-4Ra bispecific antibody variants, v42098, v42099, v42100 and v42101 (see Table 4.1), were assessed in a rat pharmacokinetics (PK) model as described below.

[0355] Wistar Han rats were treated intravenously (IV) or subcutaneously (SC) at 3 mg / kg with v42098, v42099, v42100, v42101, anti-IL-4Ra control antibody (dupilumab) or anti-IL-33 control antibody (itepekimab). Serum samples were collected at 15 minutes, 2 hrs, 8hrs, day 1, day 2, day 3, day 5, day 7, day 10, day 14 and day 21 post injection. Levels of antibody in the serum were quantified by a Meso Scale Discovery (MSD) based PK assay using a biotinylated antihuman IgG-Fc antibody (Thermo Fisher Scientific, Waltham, MA) as the capture antibody and a SULFO-TAG™-conjugated anti-human IgG-Fc antibody (Fortis Life Sciences, Waltham, MA) as the detection antibody. The magnitudes of electrochemiluminescence signals correlating with theantibody levels were read by the MESO SECTOR™ S 600MM instrument (Meso Scale Discovery, Rockville, MD) and analyzed by the DISCOVERY WORKBENCH 4.0 Analysis Software (Meso Scale Discovery). PK parameters were generated from non-compartmental analysis (NCA) using the Phoenix WinNonlin™ software (Certara, Radnor, PA). (NCA calculation method: Linear log Trapezoidal, Half-life calculation method: Time range day 7-21).

[0356] The results are shown in Fig. 9A-B and Fig. 10A-B, and in Tables 12.1 and 12.2. Figs. 9A-B and 10A-B show the serum concentrations of the anti-IL-33 x anti-IL-4Ra antibody variants and benchmark controls for intravenous (IV) and subcutaneous (SC) dosing, respectively. Tables 12.1 and 12.2 provide summaries of PK parameters for intravenous (IV) and subcutaneous (SC) dosing, respectively.Table 12.1: NCA of Total Serum Antibody Concentrations After 3 mg / kg Single IV Dose in Wistar Han RatsTable 12.2: NCA of Total Serum Antibody Concentrations After 3 Mg / Kg Single SC Dose in Wistar Han RatsEXAMPLE 13: ASSESSMENT OF HALF-LIFE OF BISPECIFIC ANTIBODY VARIANTS (IgGl AND IgG4 BACKBONE) IN A TG2 MOUSE MODEL #1

[0357] The pharmacokinetics (PK) of selected anti-IL-33 x anti-IL-4Ra bispecific antibody variants were evaluated in Tg32 mice (Jackson Laboratories, Bar Harbor, ME). The bispecific antibody variants tested were:

[0358] IgGl backbone: v41246, v41249, v41251, v41315, v41316 and v41317 (see Table3.1)

[0359] IgG4 backbone: v42098, v42099, v42100, v42101, v42102 and v42103 (see Table4.1)

[0360] The anti -IL-33 antibody itepekimab and the anti-IL-4Ra antibody dupilumab were used as controls.

[0361] Mice were injected intravenously with 5.0 mg / kg of test antibody. Serum samples were collected at 10 minutes (or 1 hour), 24 hours, day 8, day 11, day 14 (or day 15), day 18 and day 21 post-injection. Levels of antibody in the serum were quantified by a Meso Scale Discovery (MSD) based PK assay using a biotinylated anti-human IgG-Fc antibody (Thermo Fisher Scientific, Waltham, MA) as the capture antibody and a SULFO-TAG™-conjugated anti-human IgG-Fc antibody (Fortis Life Sciences, Waltham, MA) as the detection antibody. The magnitudes of electrochemiluminescence signals correlating with the antibody levels were read by a MESO SECTOR™ S 600MM instrument (Meso Scale Discovery, Rockville, MD) and analyzed by the DISCOVERY WORKBENCH 4.0 Analysis Software (Meso Scale Discovery). PK parameters were generated from non-compartmental analysis (NCA) using the Phoenix WinNonlin™ software (Certara, Radnor, PA). (NCA calculation method: Linear log Trapezoidal, Half-life calculation method: Time range day 7-21 or 8-21).

[0362] The results are shown in Figs. 11A-C and 12A-C, and in Tables 13.1 and 13.2. Figs. 11A-C and 12A-C show the serum concentrations of representative anti-IL-33 x anti-IL-4Ra bispecific antibody variants and benchmark controls. Tables 13.1 (IgGl backbone) and 13.2 (IgG4 backbone) provide summaries of PK parameters for all tested variants.Table 13.1: NCA of Total Serum Antibody Concentrations After 5 Mg / Kg Single IV Dose in Tg32 MiceTable 13.2: NCA of Total Serum Antibody Concentrations After 5 Mg / Kg Single IV Dose in Tg32 MiceEXAMPLE 14: PHARMACOKINETIC / PHARMACODYNAMIC STUDY OF A BISPECIFIC ANTIBODY VARIANT (IgG4 BACKBONE) IN CYNOMOLGUS MONKEYS #1

[0363] Activity and pharmacokinetics (PK) of the anti-IL-33 x anti-IL-4Ra bispecific antibody variant v42101 (see Table 4. 1) was assessed in vivo in cynomolgus monkeys as described below.

[0364] Two cynomolgus monkeys were injected intravenously with 10 mg / kg of test antibody or an equal volume of saline. Blood was collected pre-dose, and Omin (after injection), 3h, 8h, 24h, 48h, 96h, 168h, 240h, 336h, 408h, 504h, 576h, 672h, 744h, 840h, 912h, and 984h post injection and was analyzed for serum PK by an anti-human IgG ELISA assay. Serum IgE levels were monitored by ELISA assay at pre-dose and at 168h, 336h, 504h, and 672h post dose. Blood samples were collected throughout the study to monitor clinical chemistry, hematology and coagulation parameters.

[0365] Results

[0366] Treatment with v42101 was well tolerated in monkeys. No adverse responses were observed throughout the study. Analysis for serum PK showed that v42101 had antibodylike PK (see Fig. 13).

[0367] To determine the activity of v42101, serum IgE levels were monitored. Treatment with a single dose of v42101 was sufficient to decrease serum IgE levels up to 28 days post dosing (see Fig. 14).EXAMPLE 15: IN VIVO EFFICACY STUDY OF A BISPECIFIC ANTIBODY VARIANT (IgG4 BACKBONE) IN AN ACUTE HOUSE DUST MITE MOUSE MODEL

[0368] The anti-inflammatory activity of the anti -IL-33 x anti-IL-4Ra antibody variant v42101 (see Table 4.1) was assessed in vivo in an acute house dust mite mouse (HDM) model as described below. The anti-IL-4Ra monospecific antibody dupilumab was used as a positive control, and an anti-hemagglutinin (HA) IgG4 antibody and a no HDM treatment group were used as negative controls. The experiments were run by GemPharmatech Co., Ltd. (Nanjing, China).

[0369] C57B1 / 6 background human IL-4 / IL-4Ra knock-in, mouse IL-4 / IL-4Ra knockout mice (Biocytogen, Waltham, MA) were treated with 50 ug of house dust mite or saline intranasallyunder isofluorane anesthesia three times weekly for 4 consecutive weeks. Three days prior to initiation of HDM treatment, mice were treated with test antibody or an equal volume of saline solution at doses of 1, 3, 10, or 25 mg / kg by subcutaneous injection twice weekly. Body weights and the general health and welfare of animals were monitored three times a week. 28- or 29-days after HDM treatment initiation, mice were sacrificed. Serum was collected for detection of circulating total IgE levels. Lung tissue was divided for downstream analysis. A portion of lung tissue was homogenized to determine local concentrations of human IL-4 (hIL-4) by ELISA and levels of human IL-5 (hIL-5) by Luminex™ assay. An additional portion of the lung was processed for characterization of lung resident immune cells. To differentiate the circulating versus tissue resident cells, mice were injected with an anti-CD45 antibody conjugated to Brilliant Violet™ 605 dye 5 minutes prior to sacrifice. Immune cells were identified by flow cytometry using the cellular surface markers: CD45, CD1 lb, Ly6G, Ly6C, CD3, CD4, CD8, ST2, CD19, MERTK and Siglec- E.

[0370] To determine if v42101 can reduce systemic allergy-associated responses, serum IgE concentrations were quantified following treatment. The results are shown in Fig. 15A. Treatment with 25 mg / kg and 10 mg / kg v42101 was sufficient to reduce serum IgE concentrations to levels similar to those observed in the no HDM control group. The reduction of IgE was similar to that observed following treatment of 25 mg / kg, 10 mg / kg and 3 mg / kg dupilumab.

[0371] To specifically monitor the immune response in the lung, hIL-4 and hIL-5 levels were quantified at endpoint. The results are shown in Fig. 15B (hIL-4) and Fig, 15E (hIL-5). Treatment with 25 mg / kg and 10 mg / kg v42101 decreased levels of hIL-4 and hIL-5 to levels similar to those observed in the no HDM control group. This decrease was also observed in mice treated with 25 mg / kg, 10 mg / kg and 3 mg / kg dupilumab. The dose response difference observed with the serum IgE and lung hIL-4 and hIL-5 levels may be attributed to the bivalency of dupilumab compared to the monovalency of the anti-IL-4Ra arm of the v42101 bispecific antibody.

[0372] In addition to hIL-4 and hIL-5 quantification, the tissue resident immune cells in the lung following treatment were also characterized. The results are shown in Fig. 15C and Fig. 15D. Treatment with 25 mg / kg and 10 mg / kg v42101 was sufficient to decrease eosinophil numbers (see Fig. 15C) and increase alveolar macrophage numbers (see Fig. 15D) in the lung,with immune cell numbers resembling those observed in mice treated with the same doses of dupilumab and in the no HDM control group.

[0373] Lung pathology following treatment was evaluated by hemotoxylin and eosin staining. The results are shown in Fig. 15F. Lung sections from mice receiving irrelevant antibody along with HDM showed severe inflammation in the lungs (Fig. 15F, second row) compared to no HDM controls (Fig. 15F, top row) thus validating the model. Specifically, alveolar wall thickening and bronchial epithelial hyperplasia resulted in the loss of airway (white) space while large amounts of inflammatory cell infiltrates can be seen in the form of multiple granulomas (dark grey) within the lung tissue. Lumen stenosis can also be seen, with far less space in the larger airway passages in HDM treated mice as compared to untreated controls. In comparison, mice receiving HDM and treated with either dupilumab (Fig. 15F, bottom row) or v42101 (Fig. 15F, third row) showed much less severe physiological changes to lung architecture as compared to those seen in mice receiving irrelevant antibody controls.

[0374] A total inflammation score was determined for each treatment by evaluation of alveolar wall thickening, bronchial epithelial hyperplasia lumen stenosis and inflammatory cell infdtration. The total inflammation scores are summarized in Fig. 15G and show that treatment with 25 mg / kg and 10 mg / kg v42101 decreased the total inflammation score in mice.

[0375] Taken together, these data indicate that the anti-IL-33 x anti-IL-4Ra bispecific antibody variant v42101 can reduce pathogenic immune processes associated with asthma in a manner similar to the clinically validated monoclonal antibody benchmark, dupilumab.EXAMPLE 16: PREPARATION AND CHARACTERIZATION OF ANTI-IL-33 x ANTI- IL-4Ra BISPECIFIC ANTIBODIES (IgG4 BACKBONE) #2

[0376] Additional anti -IL-33 x anti-IL-4Ra bispecific antibody variants were prepared using engineered versions of the IL-4Ra paratope of variant v38597 described in Example 4 and the IL-33 paratope of variant v33101 (H1L4; see Tables 1.2 & 1.3). The engineered versions of the IL-4Ra paratopes included additional mutations to modulate the total charge and / or charge asymmetry in the VH and VL domains, with a view to improving the pharmacokinetic properties of the bispecific antibody variants. The engineered IL-4Ra paratopes are described in Table 16.1. The CDR sequences of the engineered IL-4Ra paratopes are shown in Fig. 19 (Table B2) and the VH and VL sequences are shown in Fig. 20 (Table C2).

[0377] Bispecific antibody variants that are monovalent for each antigen were prepared in a format in which both the IL-33 and IL-4Ra antigen binding domains are Fab domains and in which the Fc region is a heterodimeric IgG4 Fc region as described in Example 4. The heavy chains (heavy chain A and heavy chain B) and light chains (light chain A and light chain B) of each of the bispecific antibody variants are described in Table 16.2. All bispecific antibody variants comprised the HetFabl, HetFc and S228P mutations described in Example 4. In addition, the bispecific antibody variants further comprise the YTE mutations (M252Y / S254T / T256E) as noted in Table 16.2.Table 16.1: IL-4Ra Paratope Mutational Designs** All amino acid positions are numbered using AbM numbering.Table 16.2: Description of Anti-IL-33 x Anti-IL-4Ra Bispecific Antibody Variants (IgG4)16.1 Preparation of Bispecific Anti-IL-33 x Anti-IL-4Ra Antibody Variants

[0378] The antibody variants shown in Table 16.2 were expressed in ExpiCHO™ cells at a 200 m culture volume. Generally, the final gene products were sub-cloned into the mammalian expression vector pTT5 (NRC-BRI, Canada) or other mammalian expression vector. Cells were transfected in exponential growth phase (1.5 to 2 million cells / mL) with aqueous 1 mg / mL 25 kDa polyethylenimine (PEI) using an PEEDNA ratio of 2.5 : 1. DNA was transfected at an optimal DNAratio of the heavy chain A (HC-A), heavy chain B (HC-B), and light chain A (LC-A) and light chain B (LC-B) that allows for heterodimer formation (for example, HC-A:HC-B:LC-A:LC-B ratio of 15: 15:35:35). Transfected cells were harvested after 5-6 days and the culture medium collected after centrifugation at 4000 rpm and clarification using a 0.45 pm filter. The clarified culture medium was loaded onto a MabSelect™ SuRe™ (GE Healthcare) protein A column and washed with 10 column volumes of PBS buffer at pH 7.2 - 7.4. The antibody variant was eluted with 10 column volumes of 0. 1 M citrate buffer at pH 3.6 and the pooled fractions containing the antibody variant were neutralized with 1 M TRIS at pH 9. The amount of antibody variant was then quantified based on A280 nm (NanoDrop™ Spectrophotometer; Thermo Fisher Scientific).

[0379] The antibody variants were further purified by gel filtration chromatography using a Superdex™ 200 HiLoad™ 16 / 600 200pg column (GE Healthcare) via an AKTA Pure chromatography system at a flowrate of 1 mL / min with 20 mM histidine, 150 mM sodium chloride, pH 6.0 buffer. Fractions of eluted antibody variant were collected based on absorbance at A280 nm and the fractions were assessed by non-reducing and reducing CE-SDS or High Throughput Protein Express assay using Caliper LabChip™ GXII (Perkin Elmer, Waltham, MA) and UPLC- SEC using a Waters Acquity™ BEH200 SEC column (2.5 mL, 4.6x150 mm, stainless steel, 1.7 pm particles) (Waters Corporation, Mississauga, ON). Fractions corresponding to the purified antibody variants were collected, buffer exchanged into 20 mM histidine, pH 6.0 using a Zeba™ Spin desalting column (Thermo Fisher Scientific), concentrated to ~1 mg / mL and stored at -80°C.

[0380] Endotoxin levels were determined by the limulus amebocyte lysate (LAL) assay using the Endosafe™ Portable Test System (PTS) (Charles River Laboratories, Wilmington, MA). Antibody variants were quantified based on A280 nm absorbance ((NanoDrop™ Spectrophotometer) after protein A and SEC purification. UPLC-SEC was performed using a Waters Acquity™ BEH200 SEC column (2.5 mL, 4.6x150 mm, stainless steel, 1.7 pm particles) (Waters Corporation, Mississauga, ON) set to 30°C or 25°C and mounted on a Waters Acquity™ UPLC H-Class Bio system with a photodiode array (PDA) detector. Run times were 7 min with a total volume per injection of 5 pL and a running buffer of 200mM potassium phosphate pH 7.0 at 0.4 mL / min. Elution was monitored by UV absorbance in the range 210-500 nm, and chromatograms were extracted at 280 nm. Peak integration was performed using Agilent OpenLab software (Agilent Technologies, Inc., Santa Clara, CA).

[0381] The apparent purity and yield of the final antibody variant was estimated by UPLC- SEC and LC / MS generally as described in International Patent Publication No. WO 2015 / 109131. All antibody variants expressed and five of the variants were purified to >80% heterodimer purity without contaminating homodimers as shown in Table 16.3 below.Table 16.3: Post Purification Yield and Purity for Bispecific Anti-IL-33 x Anti-IL-4Ra Antibody Variants (20 mM Histidine pH 6.0 Buffer)* As determined by HPLC-SEC (size-exclusion chromatography HPLC)16.3 Functional Screening of the Engineered Anti-IL-4Ra Paratope in the Bispecific Anti-IL- 33 x Anti-IL-4Ra Antibody Variants

[0382] To determine the impact of the bispecific antibody variants on IL-4 / IL- 13 activation of the STAT6 pathway, selected variants (v42101 (parental), v43196, v43197, v43198 and v43199) were assessed for inhibition of IL-4 / IL-13 mediated production of STAT6 inducible secreted embryonic alkaline phosphatase (SEAP) reporter in HEK-Blue™ IL-4 / IL-13 cells as described below. An anti-IL-4Ra antibody (dupilumab), a tetravalent anti-IL-4Ra x IL-31 bispecific antibody (NM26-2198), an anti-RSV IgGl FcKO antibody (v39982) and an IgG4 isotype antibody (v42104) were used as controls.

[0383] Briefly, test articles were serially diluted starting at 20 000 pM in DMEM + 10% heat-inactivated fetal bovine serum (ThermoFisher Scientific, Waltham, MA) and plated into a 384-well black flat bottom assay plate. 125 pM IL-4 or 500-5000 pM IL- 13 (R&D Systems, Minneapolis, MN) was added, followed by 5000-12500 HEK-Blue™ IL-4 / IL-13 cells (InvivoGen, San Diego, CA). After 24 hr at 37°C, 5% CO2, SEAP production was assessed by incubatingsupernatant with QUANTI-Blue™ solution (InvivoGen, San Diego, CA) and measuring OD620nm on Synergy™ plate reader (BioTek Instruments, Winooski, VT).

[0384] The results are shown in Figs. 16A-B. Bispecific antibody variants including mutational variations of the IL-4Ra paratope showed a similar level of inhibition compared to the parental variant (v42101).EXAMPLE 17: ASSESSMENT OF HALF-LIFE OF BISPECIFIC ANTIBODY VARIANTS (IgG4 BACKBONE) IN A TG2 MOUSE MODEL #2

[0385] The pharmacokinetics (PK) of selected anti-IL-33 x anti-IL-4Ra bispecific antibody variants were evaluated in Tg32-SCID mice (Jackson Laboratories, Bar Harbor, ME) following the protocol described in Example 13. Serum samples were collected at 10 minutes, 24 hours, day 7, day 14, day 21 and day 28 post-injection.

[0386] The bispecific antibody variants tested were: v42101 and v42103 (see Table 4.1), and v43194, v43195, v43196, v43197, v43198, v43199 and v43200 (see Table 16.2). The anti-IL- 33 antibody itepekimab and the anti-IL-4Ra antibody dupilumab were used as controls.

[0387] The predicted half-life in human patients for each of the bispecific antibody variants was calculated from the data in the Tg32-SCID mice following the allometric scaling approach using scaling exponents published in Haraya K, et al., 2025, mAbs, 17(1).

[0388] The results are shown in Fig. 23A-C and Fig. 24 and in Table 17.1. Fig. 23A-C show the serum concentrations of the anti-IL-4Ra x anti-IL-33 bispecific antibody variants and benchmark controls. Table 17.1 provides a summary of the PK parameters (NCA calculation method: Linear log Trapezoidal, Half-life calculation method: Time range day 7-28). Fig. 24 shows the predicted half-life in humans by allometric scaling.Table 17.1: NCA of Total Serum Antibody Concentrations after 5 mg / kg Single IV Dose in Tg32-SCID MiceEXAMPLE 18: FUNCTIONAL CHARACTERIZATION OF ANTLIL-33 X ANTI-IL-4Ra BISPECIFIC ANTIBODY VARIANTS IN HEALTHY AND COPD PATIENT SETTINGS - IL-4 PBMC (CD23) ASSAY

[0389] To test the functional impact of blockade of IL-4Ra signalling by the bispecific antibodies, selected anti-IL-33 x anti-IL-4Ra bispecific antibody variants were assessed for inhibition of CD23 upregulation by PBMCs from healthy donors and from patients with chronic obstructive pulmonary disease (COPD) following IL-4 stimulation. Flow cytometry was used to assess CD23 expression following IL-4 stimulation as described below.

[0390] The antibody variants tested were: v42101 (see Table 4.1) and v43196 (see Table 16.2). Dupilumab was used as a positive control for blocking Il-4Ra signalling and a hemagglutinin-specific IgG4 antibody (v42104) was used as a non-specific negative control.

[0391] Human PBMCs (Stemcell Technologies, Vancouver, Canada) were plated at 2xl05cells / well in 96 well round-bottom plates and incubated with serially diluted concentrations of test article for 30 minutes to allow binding. IL-4 (R&D Systems, Minneapolis, MN) was added to each well for a final concentration of 2ng / mL and cells were incubated at 37°C for 48 hours in 10% FBS RPMI (ThermoFisher Scientific, Waltham, MA). Following incubation cells were stained with Fixable Viability Dye eFluor™ 506 (Thermo Fisher Scientific, Waltham, MA) for 15 minutes in PBS, washed with PBS, washed with flow cytometry staining buffer (5% FBS, 2.5mM EDTA, PBS), and stained in flow cytometry staining buffer for 30 minutes with fluorescently-conjugated antibodies against human cell surface markers (CD3, CD14, CD16, CD19, CD23, CD27) to allow phenotyping of cells by flow cytometry. Cells were washed twice with flow cytometry staining buffer and fixed for 10 minutes in BD Cytofix™ Fixation Buffer (BD Biosciences, San Francisco, CA). Cells were washed and resuspended in flow cytometry staining buffer then analyzed by flow cytometry on an Attune™ NxT Acoustic Focusing Cytometer (ThermoFisher Scientific). Geometric mean fluorescent intensity (gMFI) of CD23 on various immune cell populations as defined by phenotyping antibodies (B-cells: CD19+CD3" CD27+ / ", T-cells: CD3+CD19", Monocytes: CD14+CD16+ / ) was used to assess inhibition of IL-4Ra signalling.

[0392] The results are shown in Fig. 25. The bispecific antibody variants were shown to limit changes to cellular phenotypes resulting from IL-4Ra stimulation by IL-4 as evidenced by decreasing CD23 expression as antibody concentration increases. Specifically, naive B-cells,which have been shown to increase CD23 expression following IL-4Ra stimulation, were both shown to have reduced CD23 expression which was dependent on antibody concentration. This effect was not seen in cells treated with isotype control showing that the effect was mediated by inhibition of IL-4Ra stimulation by the bispecific antibody variants.

[0393] In addition, PBMCs from COPD patients can be seen to show higher levels of CD23 expression than PBMCs from healthy donors when treated with highly diluted constructs suggesting COPD patient PBMCs are hyperresponsive to IL-4Ra stimulation (Fig. 25). Despite this increased level of CD23 expression, the bispecific antibody variants were able to reduce the level of CD23 expression in these PBMCs to levels comparable with healthy donor controls.

[0394] In summary, the anti -IL-33 x anti-IL-4Ra bispecific antibody variants were shown to decrease IL-4 stimulated CD23 production in both healthy and COPD patient derived PBMCs.EXAMPLE 19: FUNCTIONAL CHARACTERIZATION OF ANTI-IL-33 X ANTI-IL-4Ra BISPECIFIC ANTIBODY VARIANTS IN HEALTHY AND COPD PATIENT SETTINGS - IL-33 PBMC (INTRACELLULAR IFN-y) ASSAY

[0395] To test the functional impact of blockade of IL-33 signalling by the bispecific antibodies, selected anti-IL-33 x anti-IL-4Ra bispecific antibody variants were assessed for inhibition of IFN-y production by IL-12 stimulated PBMCs from healthy donors and from patients with chronic obstructive pulmonary disease (COPD). Flow cytometry was used to assess IFN-y production following PBMC stimulation as described below.

[0396] The antibody variants tested were: v42101 (see Table 14.1) and v43196 (see Table 16.2). Itepekemab was used as a positive control for blocking IL-33 signalling and dupilumab and a hemagglutinin-specific IgG4 antibody (v42104) were used as negative controls.

[0397] Test articles were serially diluted starting from 60000 pM in assay media, RPMI + 10% heat inactivated fetal bovine serum (ThermoFisher Scientific, Waltham, MA) in a 96-well round bottom plate (ThermoFisher Scientific, Waltham, MA). 50pM oxidation resistant human IL-33 (Adipogen Life Sciences, San Diego, CA) was added. Binding was allowed to proceed for 30 minutes followed by addition of 200 000 freshly thawed PBMCs (Stemcell Technologies, Vancouver Canada) from healthy donors or COPD patients in assay media containing 10 ng / mL IL-12 (R&D, Minneapolis USA). After 24 hr at 37°C, 5% CO2, eBioscience™ Brefeldin A Solution (1000X) (ThermoFisher Scientific, Waltham, MA) was added to cell media at 1 / 1000dilution from product vial to block cytokine secretion, followed by incubation for 6 hours. Cells were stained with Fixable Viability Dye eFluor™ 506 (Thermo Fisher Scientific, Waltham, MA) for 15 minutes in PBS, washed with PBS, washed with flow cytometry staining buffer (5% FBS, 2.5mM EDTA, PBS), and stained in flow cytometry staining buffer for 30 minutes with fluorescently-conjugated antibodies against human cell surface markers (CD3, CD4, CD8, CD16, CD 19, CD56) to allow phenotyping of cells by flow cytometry. Cells were washed twice with flow cytometry staining buffer and fixed for 20 minutes in eBioscience™ Foxp3 / Transcription Factor Fixation Buffer (ThermoFisher Scientific). Cells were washed twice in eBioscience™ Foxp3 / Transcription Factor Perm Buffer (ThermoFisher Scientific) and stained for 30 minutes with a fluorescently-conjugated antibody against human IFN-y in eBioscience™ Foxp3 / Transcription Factor Perm Buffer. Cells were washed twice in eBioscience™ Foxp3 / Transcription Factor Perm Buffer and resuspended in flow cytometry staining buffer then analyzed by flow cytometry on an Attune™ NxT Acoustic Focusing Cytometer (ThermoFisher Scientific). Percent of cells producing IFN-y in various immune cell populations as defined by phenotyping antibodies (CD4 T-cells: CD19’ CD3+, CD4+, CD8; CD8 T-cells: CD19’ CD3+, CD4' , CD8+, NK cells: CD19" CD3" CD16+ / " CD56+) was used to assess inhibition of IL-33 signalling.

[0398] The results are shown in Fig. 26. All anti-IL-33 x anti-IL-4Ra bispecific antibody variants demonstrated blockade of IL-33 mediated IFN-y production in IL-12 stimulated PBMCs from both healthy donors and from COPD patients. All bispecific antibody variants showed comparable inhibitory activity to the bivalent anti-IL33 itepekemab benchmark control.EXAMPLE 20: IN VIVO STUDY OF ANTI-IL-33 ANTIBODY VARIANTS IN A CHRONIC HOUSE DUST MITE MOUSE MODEL

[0399] The ability of anti-IL-33 antibody variants to diminish IL-33 protein levels in the lung was assessed in vivo in a chronic house dust mite mouse (HDM) model as described below. The variants tested were v40570 (anti-IL-33 OAA; see Table 1.5) and v41251 (anti-IL-33 x anti- IL-4Ra bispecific antibody; see Table 3.1). The anti-IL-33 monospecific antibody, itepekimab, was used as a positive control and an irrelevant antibody (palivizumab) or saline treatment was used as a negative control. Healthy mice not subjected to the HDM protocol were used as a reference for normal lung hIL-33 expression.

[0400] Balb / c background, human IL-33 (hIL-33) knock-in mice were treated with 50 ug of house dust mite intranasally under isofluorane anesthesia three times weekly for 15 consecutive weeks. Four weeks after the initial HDM treatment, mice were administered with test articles at 0.1, 0.3, 1 or 3 mg / kg or with saline solution by subcutaneous injection twice weekly. Body weights and the general health and welfare of the mice were monitored three times a week. Mice were sacrificed 105 or 106 days post-HDM treatment initiation. Lung homogenates were analyzed for the abundance of hIL-33 -encoding mRNA transcripts and hIL-33 protein levels using quantitative polymerase chain reaction (qPCR) and ELISA, respectively.

[0401] The results are shown in Fig. 27A-B. Fig. 27A shows that the abundance of hlL- 33 -encoding mRNA transcripts was increased in animals subjected to the HDM protocol compared to untreated, healthy reference mice. The elevated mRNA transcript levels in animals subjected to the HDM protocol were similar across all treatment groups.

[0402] Fig.27B shows that the treatment with hIL-33 -specific antibodies reduced humanIL-33 protein levels and that the reduction was dose dependent for those test articles administered at multiple dose levels. Treatment with the anti -IL-33 OAA (v40570) significantly diminished hIL-33 protein levels at 0.3, 1 and 3 mg / kg doses, and treatment with the anti-IL-33 x anti-IL-4Ra bispecific antibody (v41251) at 1 mg / kg dose reduced hIL-33 protein to a similar level as itepekimab at the same dose. Thus, both the anti -IL-33 OAA and the bispecific antibody were capable of significantly reducing lung-derived hIL-33 protein levels in vivo in this chronic house dust mite mouse model.EXAMPLE 21: FUNCTIONAL CHARACTERIZATION OF ANTI-IL-33 x ANTI-IL-4Ra BISPECIFIC ANTIBODY VARIANTS - CONCURRENT BINDING TO IL-33 AND IL- 4Ra

[0403] The ability of the anti -IL-33 x anti-IL-4Ra bispecific antibody variant v42101 (see Table 4.1) to bind IL-4Ra and IL-33 concurrently was assessed using a Meso Scale Discovery assay (MSD). Dupilumab (v40883) and itepekimab (v32657) were used as positive controls. A hemagglutinin-specific IgG4 antibody (v42104) was used as a negative control.

[0404] MSD 384-well uncoated SECTOR™ plates (Meso Scale Diagnostics, LLC, Rockville, MD) were coated with 10 pl / well of 0.6pg / mL solutions of either IL-4Ra or IL-33 (ACROBiosystems, Newark, DE) in PBS pH 7.4. Plates were sealed with film and centrifuged toensure liquid was at the bottom of the wells, then incubated at 4°C overnight. The next day, plates were washed with PBST (PBS + 0.1% Tween-20) using a BioTek™ plate washer (Agilent Technologies, Inc., Santa Clara, CA). Plates were blocked with 40 pl of 5% MSD Blocker A (Meso Scale Diagnostics) for at least 1 hour at room temperature without shaking then washed with PBST. Antibodies were diluted in 1% MSD Blocker A and lOpL transferred to a coated MSD plate. Plates were sealed, centrifuged and incubated at room temperature on an orbital shaker for 1 hour then washed. At this time, lOpL of an anti -human Fc biotinylated antibody (Thermo Fisher Scientific, Waltham, MA), biotinylated IL-33 (ACROBiosystems) or biotinylated IL-4Ra (ACROBiosystems) were added to wells at 0.25pg / mL. Plates were spun down and incubated in an orbital shaker for 1 hour at room temperature. Plates were washed with PBST and lOpL of streptavidin SULFO-TAG™ labelled detection antibody (Meso Scale Diagnostics) were added to wells at 0.25pg / mL in 1% MSD Blocker A followed by a 30-minute incubation on an orbital shaker. Plates were washed and 35pL of MSD GOLD™ Read Buffer (Meso Scale Diagnostics) was added. Plates were read on a MESO SECTOR™ S600 analyzer (Meso Scale Diagnostics) within 15 minutes.

[0405] Results with biotinylated anti-human-Fc antibody (huFc) as the detection moiety are shown in Fig. 28A and Fig. 28B and demonstrate that there was specific binding by the bispecific antibody variant and positive controls to the target protein coated on the wells. Results with biotinylated IL-33 or biotinylated IL-4Ra as the detection moiety are shown in Fig. 28C and Fig. 28D and demonstrate that only the anti-IL-33 x anti-IL-4Ra bispecific antibody variant is capable of positive binding to these two proteins. Overall, these results show that the anti-IL-33 x anti-IL-4Ra bispecific antibody variant is capable of binding to IL-33 or IL-4Ra while also bound to the alternate target coated on the wells, thus confirming the ability of the bispecific antibody to bind to both targets at the same time.EXAMPLE 22: BLOCKADE OF IL-33 AND IL-4Ra SIGNALLING IN PBMC-DERIVED MONOCYTES

[0406] To demonstrate the bispecific effect of the anti -IL-33 x anti-IL-4Ra bispecific antibody variant v42101 (see Table 4.1), blockade of combined IL-33 and IL-4Ra signalling was examined in monocytes derived from PBMC cultures.

[0407] Antibodies were assessed for inhibition of expression of the immune activation marker HLA-DR on monocytes following IL-33 and IL-4Ra stimulation. Flow cytometry was used to assess cell surface markers. Itepekemab and dupilumab were used individually as positive controls for blocking IL-33 and IL-4Ra signalling, respectively, and in combination to examine advantages of a bispecific antibody over combination therapies. A hemagglutinin-specific IgG4 antibody (“isotype”) was used as a negative control.

[0408] PBMCs (STEMCELL Technologies, Vancouver, Canada) were thawed and resuspended in 10 mL PBS. Cells were counted and resuspended to 5x l06cells / mL in complete RPMI (RPMI 1640, 10% heat inactivated FBS, 100 U / mL penicillin / streptomycin, 2mM L- glutamine). 1 mL warm RPMI per well was added to 6 well plates, then 1 mL of cells was added and distributed by swirling the plate. Cells were incubated at 37°C, 5% CO2 for 1.5 hours to allow monocyte adherence, then gently washed twice with warm PBS. 2 mL of RPMI was added and cells were incubated overnight at 37°C, 5% CO2. Media was aspirated and 3 mL of fresh RPMI was added along with 0.5 mL of test antibody at 2000 pM and 0.5mL of IL-33 (AdipoGen Life Sciences, San Diego, CA), IL-4 (R&D Systems, Minneapolis, MN), or both cytokines, at 20 ng / mL. Plates were swirled to mix and incubated at 37°C, 5% CO2 for 72 hours. Media was aspirated and wells were gently washed with 2 mL room temperature PBS. Wells were then vigorously washed to detach cells which were transferred to 15 mL conical tubes and pelleted before being processed for flow cytometry. Cells were stained with Fixable Viability Dye eFluor™ 506 (Thermo Fisher Scientific, Waltham, MA) for 15 minutes in PBS, washed with PBS, washed with flow cytometry staining buffer (5% FBS, 2.5mM EDTA, PBS), and stained in flow cytometry staining buffer for 30 minutes with fluorescently-conjugated antibodies against human cell surface markers (CD11c, CD14, CD16, CD80, CD86, CD206, HLA-DR) to allow phenotyping of cells by flow cytometry. Cells were washed twice in flow cytometry staining buffer, resuspended in flow cytometry staining buffer, then analyzed by flow cytometry on an Attune™ NxT Acoustic Focusing Cytometer (Thermo Fisher Scientific). Monocytes were defined as CD14+CD16+and were measured for activation based on expression of HLA-DR.

[0409] The results are shown in Fig. 29 and show that the anti -IL-33 x anti-IL-4Ra bispecific antibody variant was able to influence IL-4 and IL-33 stimulation-induced differential expression of the activation marker HLA-DR when measured by flow cytometry. Overall, the results indicate a trend towards reduced HLA-DR expression in monocytes as measured bygeometric mean fluorescent intensity (gMFI) when monocytes were cultured with the anti -IL-33 x anti-IL-4Ra bispecific antibody variant compared to the combination of itepekimab and dupilumab, and greater reduction compared to each monospecific antibody individually. The decreased gMFI at a whole population level suggests that individual cells received less stimulation when treated with the bispecific antibody variant compared to the monospecific antibodies.EXAMPLE 23: BINDING AFFINITY CHARACTERIZATION OF ANTI-IL-33 x ANTI- IL-4Ra BISPECIFIC ANTIBODY VARIANT

[0410] The equilibrium binding affinity of the anti-IL-33 x anti-IL-4Ra bispecific antibody variant, v42101 (see Table 4.1) to human and cynomolgus IL-33, and to human and cynomolgus IL-4Ra was determined using a KinExA™ instrument (Sapidyne Inc., Boise, ID) as described below. The anti-IL-33 antibody, itepekimab, or the anti-IL-4Ra antibody, dupilumab, was included for comparison.Binding to Human IL-33 and Cynomolgus IL-33

[0411] Briefly, histidine tagged human and cynomolgus IL-33 (hu IL-33-His (Sino Biological, Inc.; Cat # 10368-HNAE) and cyno IL-33-His (Sino Biological, Inc.; Cat # 90912- CNAE)) were immobilized on azlactone beads (Sapidyne Inc.; Part # 444110). Antibodies were used as the constant binding partner (CBP), and antigens (hu IL-33 -his or cyno IL-33 -his) were used as titrants. A series of equilibrium mixtures were prepared in KinExA™ sample buffer (IX PBS, pH7.4 + Img / mL BSA + 0.02% sodium azide) by 2-fold serial dilution of titrants (antigen) with two different antibody concentrations (z. e. high CBP and low CBP) and incubated at room temperature for 6 days. The antibodies were tested at 50 pM and 5 pM or 20 pM and 2 pM. The titrants, hu IL-33 -His and cyno IL-33 -His were titrated by 2-fold serial dilutions of 500 pM or 300 pM, and 1000 pM, respectively. For KD measurements, the equilibrium mixtures were loaded onto the instrument into the flow cell filled with the respective titrant coated solid phase to measure the free CBP concentration using 0.5 pg / mL fluorescent labelled detection antibody, Alexa Fluor® 647 AffmiPure Goat Anti-Human IgG (H+L) (Jackson ImmunoResearch; Cat # 109-605-003). The voltage signal generated corresponded to the free CBP in the equilibrium mixture. For each antibody, a global analysis (n-curve analysis) of two binding curves, at different antibody concentrations, was performed on KinExA™ Pro software (version 4.7.6) to obtain the best-fit KDvalue. Final KD values are reported as the mean ± standard deviation (SD) of n=3 measurements and are listed in Table 23.1.Table 23.1: Binding Affinity (KD) of v42101 to Human IL-33 and Cynomolgus IL-33Binding to Human IL-4Ra and Cynomolgus IL-4Ra

[0412] Briefly, histidine tagged human and cynomolgus IL-4Ra (hu IL-4Ra-His (ACROBiosystems, Newark, DE; Cat # ILR-H5221) and cyno IL-4Ra-His (ACROBiosystems; Cat # ILR-C52H8)) were immobilized on azlactone beads (Sapidyne Inc.; Part # 444110). Antibodies were used as the constant binding partner (CBP) and antigens (hu IL-4Ra-His or cyno IL-4Ra-His) were used as the titrants. A series of equilibrium mixtures were prepared in KinExA™ sample buffer (IX PBS, pH7.4 + Img / mL BSA + 0.02% sodium azide) by 2-fold serial dilution of titrants (antigen) with two different antibody concentrations (i.e. high CBP and low CBP) and incubated at room temperature for 5 days. The antibodies were tested at constant concentrations of 50 pM and 5 pM. The titrant, hu IL-4Ra-His and cyno IL-4Ra-His were each titrated by 2-fold serial dilutions of 500 pM. For KD measurements, the equilibrium mixtures were loaded onto the instrument into the flow cell filled with the respective titrant coated solid phase to measure the free CBP concentration using 0.5 pg / mL fluorescent labelled detection antibody, Alexa Fluor® 647 AffiniPure Goat Anti-Human IgG (H+L) (Jackson ImmunoResearch; Cat # 109-605-003). The voltage signal generated corresponded to the free CBP in the equilibrium mixture . For each antibody, a global analysis (n-curve analysis) of two binding curves, at different antibody concentrations, was performed on KinExA™ Pro software (version 4.7.6) to obtain the best-fit KD value. Final KD values are reported as the mean ± standard deviation (SD) of n=3 measurements and are listed in Table 23.2.Table 23.2: Binding Affinity (KD) of v42101 to Human IL-4Ra and Cynomolgus IL-4RaEXAMPLE 24: PHARMACOKINETIC / PHARMACODYNAMIC STUDY OF A BISPECIFIC ANTIBODY VARIANT (IgG4 BACKBONE) IN CYNOMOLGUS MONKEYS #2

[0413] Pharmacokinetics (PK), pharmacodynamics (PD) and toxicity parameters for the anti-IL-33 x anti-IL-4Ra bispecific antibody variant v42101 (see Table 4.1) was assessed in vivo in naive cynomolgus monkeys as described below.

[0414] Three groups, each consisting of two cynomolgus monkeys (one female and one male) per dose level, were injected intravenously with 20 mg / kg, 40 mg / kg or 100 mg / kg of the anti -IL-33 x anti-IL-4Ra bispecific antibody variant v42101. A fourth group of two cynomolgus monkeys was injected subcutaneously with 40 mg / kg of v42101. A control group of two cynomolgus monkeys was injected intravenously and subcutaneously with equal volumes of vehicle (histidine buffer). All monkeys were injected once a week for four weeks (QWx4). Accordingly, injections were performed on days 1, 8, 15, and 22. All monkeys were euthanized for necropsy on day 29.

[0415] Blood was collected at each pre-dose and 5 min after each injection, as well as 3h, 8h, 24h, 48h, 96h, 507h, 512h, 528h, 552h, 600h and 672h post first injection and analyzed for serum PK by an anti-human IgG ELISA assay. In addition, serum IgE levels were monitored by ELISA assay at each pre-dose and at 672h post first dose (study termination). Blood samples were collected throughout the study to monitor clinical chemistry, hematology and coagulation parameters.

[0416] Treatment with the anti -IL-33 x anti-IL-4Ra bispecific antibody variant v42101 was well tolerated in monkeys at all tested dose levels. No adverse responses were observed throughout the study. Test article-related histopathological changes were limited to the two monkeys administered 40 mg / kg v42101 subcutaneously, and the affected tissues were injection site (both animals), kidney (male), and bone marrow (male). Analysis of serum PK for all intravenously injected monkeys showed that v42101 had antibody-like PK with dose accumulationin AUC between dose 1 and 4 (see Fig. 30). Similar PK was observed for one of the subcutaneously dosed monkeys, while the second animal in this group showed impacted PK from the third dose until termination. As expected, subcutaneous administration resulted in lower Cmax and AUClast post doses 1 and 4 compared to the monkeys intravenously injected with the same dose of v42101 (40 mg / kg).

[0417] To determine the activity of the IL-33 x IL-4Ra bispecific antibody variant, serum IgE levels were monitored. IL-4 signalling triggers class switch recombination (CSR) in B cells to produce IgE, a critical mediator that induces mast cell degranulation and the release of histamine during allergic reactions. Blocking IL-4 signalling should result in a decrease in serum IgE levels. As the monkeys used in this study are naive (i.e. are not showing allergic inflammation symptoms), the initial IgE serum levels will be low. Thus, changes in serum IgE levels are expected to be modest after treatment with the bispecific antibody blocking IL-4Ra.

[0418] Compared to vehicle-dosed monkeys in the control group, treatment with v42101 once a week for four weeks (QWx4) transiently decreased serum IgE levels between days 8 and 15 (see Fig. 31). A reduction of serum IgE levels has been similarly observed in patients treated with IL-4Ra targeting monoclonal antibody therapies suggesting that v42101 can function in a similar manner to clinically approved IL-4Ra targeting monoclonal antibodies.

[0419] The disclosures of all patents, patent applications, publications and database entries referenced in this specification are hereby specifically incorporated by reference in their entirety to the same extent as if each such individual patent, patent application, publication and database entry were specifically and individually indicated to be incorporated by reference

[0420] Modifications of the specific embodiments described herein that would be apparent to those skilled in the art are intended to be included within the scope of the following claims.SEQUENCE TABLESTable F: Clone Numbers for VariantsTable G: Clone Sequences

Claims

WE CLAIM:

1. An antibody construct comprising one or more antigen-binding domains, wherein at least one of the antigen-binding domains is an IL-33 antigen-binding domain that specifically binds to human IL-33, the IL-33 antigen-binding domain comprising the CDR sequences (HCDR1, HCDR2, HCDR3) of the VH domain as set forth in any one of SEQ ID NOs: 30, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 or 42, and the CDR sequences (LCDR1, LCDR2, LCDR3) ofthe VL domain as set forth in any one of SEQ ID NOs: 31, 43, 44, 45 or 46.

2. The antibody construct according to claim 1, wherein the IL-33 antigen-binding domain comprises the CDR sequences (HCDR1, HCDR2, HCDR3) of the VH domain as set forth in SEQ ID NO: 32 or 38, and the CDR sequences (LCDR1, LCDR2, LCDR3) of the VL domain as set forth in SEQ ID NO: 45 or 46.

3. The antibody construct according to claim 1, wherein the IL-33 antigen-binding domain comprises a VH domain comprising heavy chain CDR amino acid sequences (HCDR1, HCDR2 and HCDR3) comprising the sequences as set forth in SEQ ID NOs: 6, 47 and 8, and a VL domain comprising light chain CDR amino acid sequences (LCDR1, LCDR2 and LCDR3) comprising the sequences as set forth in SEQ ID NOs: 48, 20 and 18.

4. The antibody construct according to claim 1, wherein the IL-33 antigen-binding domain comprises a VH domain comprising heavy chain CDR amino acid sequences (HCDR1, HCDR2 and HCDR3) comprising the sequences as set forth in SEQ ID NOs: 13, 49 and 15, respectively, and a VL domain comprising light chain CDR amino acid sequences (LCDR1, LCDR2 and LCDR3) comprising the sequences as set forth in SEQ ID NOs: 21, 22 and 23.

5. The antibody construct according to claim 3, wherein X1is DSV or DWA, and X2is R.

6. The antibody construct according to claim 4, wherein X3is VA or VS.

7. The antibody construct according to claim 1, wherein the IL-33 antigen-binding domain comprises a VH domain comprising an HCDR1 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 3 or 6, an HCDR2 amino acid sequence comprising the sequence as set forth in any one of SEQ ID NOs: 4, 7, 24, 26 or 28, and an HCDR3 amino acid sequencecomprising the sequence as set forth in SEQ ID NO: 5 or 8, and a VL domain comprising an LCDR1 amino acid sequence comprising the sequence as set forth in any one of SEQ ID NOs: 16, 19 or 29, an LCDR2 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 17 or 20, and an LCDR3 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 18.

8. The antibody construct according to claim 7, wherein the HCDR2 amino acid sequence comprises the sequence as set forth in any one of SEQ ID NOs: 4, 24 or 26.

9. The antibody construct according to claim 1, wherein the IL-33 antigen-binding domain comprises:(a) a VH domain comprising either(i) an HCDR1 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 3 or 6, an HCDR2 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 4 or 24, and an HCDR3 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 5 or 8, or(ii) an HCDR1 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 3 or 6, an HCDR2 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 4 or 26, and an HCDR3 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 5 or 8, and(b) a VL domain comprising an LCDR1 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 16 or 29, an LCDR2 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 17 or 20, and an LCDR3 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 18.

10. The antibody construct according to claim 1, wherein the IL-33 antigen-binding domain comprises a VH domain comprising an HCDR1 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 3 or 6, an HCDR2 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 4 or 26, and an HCDR3 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 5 or 8, and a VL domain comprising an LCDR1 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 16 or 29, an LCDR2 amino acid sequencecomprising the sequence as set forth in SEQ ID NO: 17 or 20, and an LCDR3 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 18.

11. The antibody construct according to any one of claims 1 to 10, wherein the IL-33 antigen binding domain comprises a VH domain comprising a sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the VH sequence as set forth in any one of SEQ ID NOs: 30, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 or 42.

12. The antibody construct according to any one of claims 1 to 11, wherein the IL-33 antigen binding domain comprises a VL domain comprising a sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the VL sequence as set forth in any one of SEQ ID NOs: 31, 43, 44, 45 or 46.

13. The antibody construct according to any one of claims 1 to 10, wherein the IL-33 antigen binding domain comprises a VH domain comprising a sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the VH sequence as set forth in SEQ ID NO: 32 or 38.

14. The antibody construct according to any one of claims 1 to 13, wherein the IL-33 antigen binding domain comprises a VL domain comprising a sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the VL sequence as set forth in SEQ ID NO: 45 or 46.

15. The antibody construct according to any one of claims 1 to 13, wherein the IL-33 antigen binding domain comprises a VH domain comprising a sequence as set forth in SEQ ID NO: 32 or 38, and a VL domain comprising a sequence as set forth in SEQ ID NO: 45 or 46.

16. The antibody construct according to any one of claims 1 to 13, wherein the IL-33 antigen binding domain comprises a VH domain comprising a sequence as set forth in SEQ ID NO: 32, and a VL domain comprising a sequence as set forth in SEQ ID NO: 46.

17. The antibody construct according to any one of claims 1 to 16, wherein the IL-33 antigen binding domain also binds to cynomolgus monkey IL-33.

18. The antibody construct according to any one of claim 1 to 17, wherein the antibody construct comprises two, three or four antigen-binding domains.

19. The antibody construct according to any one of claims 1 to 18, wherein the antibody construct comprises two IL-33 antigen-binding domains.

20. The antibody construct according to any one of claims 1 to 19, further comprising a scaffold, wherein the IL-33 antigen-binding domain is operably linked to the scaffold.

21. The antibody construct according to claim 20, wherein the scaffold is an IgG Fc region.

22. The antibody construct according to claim 21, wherein the scaffold is a human IgG Fc region.

23. The antibody construct according to claim 21 or claim 22, wherein the IgG Fc region is an IgGl or IgG4 Fc region.

24. The antibody construct according to any one of claims 1 to 18, wherein the antibody construct comprises a second target antigen-binding domain that specifically binds to a second target antigen, wherein the second target antigen is other than IL-33.

25. The antibody construct according to claim 24, wherein the second target antigen is a cytokine or cytokine receptor.

26. The antibody construct according to claim 24 or claim 25, wherein the second target antigen is human IL-4Ra.

27. The antibody construct according to any one of claims 24 to 26, further comprising a scaffold, wherein the IL-33 antigen-binding domain and the second target antigen-binding domain are both operably linked to the scaffold.

28. The antibody construct according to claim 27, wherein the scaffold is an IgG Fc region.

29. The antibody construct according to claim 28, wherein the scaffold is a human IgG Fc region.

30. The antibody construct according to claim 28 or claim 29, wherein the IgG Fc region is an IgGl or IgG4 Fc region.

31. The antibody construct according to any one of claims 28 to 30, wherein the IgG Fc region is a heterodimeric Fc region comprising a first Fc polypeptide and a second Fc polypeptide.

32. The antibody construct according to claim 31, wherein the heterodimeric Fc region comprises a modified CH3 domain, and wherein the modified CH3 domain comprises one or more amino acid modifications that promote formation of the heterodimeric Fc over formation of a homodimeric Fc.

33. The antibody construct according to claim 32, wherein:(a) the first Fc polypeptide comprises the amino acid substitutions L351Y, F405A and Y407V, and the second Fc polypeptide comprises the amino acid substitutions T366L, K392M and T394W; or(b) the first Fc polypeptide comprises the amino acid substitutions L351Y, F405A and Y407V, and the second Fc polypeptide comprises the amino acid substitutions T366L, K392L and T394W; or(c) the first Fc polypeptide comprises the amino acid substitutions T350V, L351Y, F405A and Y407V, and the second Fc polypeptide comprises the amino acid substitutions T350V, T366L, K392M and T394W; or(d) the first Fc polypeptide comprises the amino acid substitutions T350V, L351Y, F405A and Y407V, and the second Fc polypeptide comprises the amino acid substitutions T350V, T366L, K392L and T394W; or(e) the first Fc polypeptide comprises the amino acid substitutions T350V, L351Y, S400E, F405A and Y407V, and the second Fc polypeptide comprises the amino acid substitutions T350V, T366L, N390R, K392M and T394W, and wherein the numbering of amino acids is EU numbering.

34. The antibody construct according to any one of claims 21 to 23 and 28 to 33, wherein the IgG Fc region comprises the amino acid substitutions M252Y, S254T and T256E, and wherein the numbering of amino acids is EU numbering.

35. The antibody construct according to any one of claims 21 to 23 and 28 to 34, wherein the IgG Fc region is an IgGl Fc region.

36. The antibody construct according to claim 35, wherein the IgGl Fc region comprises the amino acid substitutions L234A, L235A and D265S, and wherein the numbering of amino acids is EU numbering.

37. The antibody construct according to any one of claims 21 to 23 and 28 to 34, wherein the IgG Fc region is an IgG4 Fc region.

38. The antibody construct according to claim 37, wherein the IgG4 Fc region comprises the amino acid substitution S228P and / or the amino acid substitution R409K.

39. A polynucleotide or set of polynucleotides encoding the antibody construct according to any one of claims 1 to 38.

40. An expression vector or set of expression vectors comprising the polynucleotide or set of polynucleotides according to claim 39.

41. A host cell comprising the polynucleotide or set of polynucleotides according to claim 39 or the expression vector or set of expression vectors according to claim 40.

42. A method of preparing the antibody construct according to any one of claims 1 to 38 comprising transfecting a host cell with the polynucleotide or set of polynucleotides according to claim 39 or the expression vector or set of expression vectors according to claim 40, and culturing the host cell under conditions suitable for expression of the antibody construct.

43. A bispecific antibody construct comprising an IL-33 antigen-binding domain that specifically binds to human IL-33, and a second target antigen-binding domain, that specifically binds to a second target antigen, wherein the second target antigen is other than IL-33, and wherein the IL-33 antigen-binding domain comprising the CDR sequences (HCDR1, HCDR2, HCDR3) ofthe VH domain as set forth in any one of SEQ ID NOs: 30, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 or 42, and the CDR sequences (LCDR1, LCDR2, LCDR3) of the VL domain as set forth in any one of SEQ ID NOs: 31, 43, 44, 45 or 46.

44. The bispecific antibody construct according to claim 43, wherein the second target antigen is a cytokine or cytokine receptor.

45. The bispecific antibody construct according to claim 43 or claim 44, wherein the second target antigen is human IL-4Ra.

46. The bispecific antibody construct according to any one of claims 43 to 45, wherein the IL- 33 antigen-binding domain comprises the CDR sequences (HCDR1, HCDR2, HCDR3) of the VH domain as set forth in SEQ ID NO: 32 or 38, and the CDR sequences (LCDR1, LCDR2, LCDR3) of the VL domain as set forth in SEQ ID NO: 45 or 46.

47. The bispecific antibody construct according to any one of claims 43 to 45, wherein the IL- 33 antigen-binding domain comprises a VH domain comprising heavy chain CDR amino acid sequences (HCDR1, HCDR2 and HCDR3) comprising the sequences as set forth in SEQ ID NOs: 6, 47 and 8, and a VL domain comprising light chain CDR amino acid sequences (LCDR1, LCDR2 and LCDR3) comprising the sequences as set forth in SEQ ID NOs: 48, 20 and 18.

48. The bispecific antibody construct according to any one of claims 43 to 45, wherein the IL- 33 antigen-binding domain comprises a VH domain comprising heavy chain CDR amino acid sequences (HCDR1, HCDR2 and HCDR3) comprising the sequences as set forth in SEQ ID NOs: 13, 49 and 15, respectively, and a VL domain comprising light chain CDR amino acid sequences (LCDR1, LCDR2 and LCDR3) comprising the sequences as set forth in SEQ ID NOs: 21, 22 and 23.

49. The bispecific antibody construct according to claim 47, wherein X1is DSV or DWA, and X2is R.

50. The bispecific antibody construct according to claim 48, wherein X3is VA or VS.

51. The bispecific antibody construct according to any one of claims 43 to 45, wherein the IL- 33 antigen-binding domain comprises a VH domain comprising an HCDR1 amino acid sequencecomprising the sequence as set forth in SEQ ID NO: 3 or 6, an HCDR2 amino acid sequence comprising the sequence as set forth in any one of SEQ ID NOs: 4, 7, 24, 26 or 28, and an HCDR3 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 5 or 8, and a VL domain comprising an LCDR1 amino acid sequence comprising the sequence as set forth in any one of SEQ ID NOs: 16, 19 or 29, an LCDR2 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 17 or 20, and an LCDR3 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 18.

52. The bispecific antibody construct according to claim 51, wherein the HCDR2 amino acid sequence comprises the sequence as set forth in any one of SEQ ID NOs: 4, 24 or 26.

53. The bispecific antibody construct according to any one of claims 43 to 45, wherein the IL- 33 antigen-binding domain comprises:(a) a VH domain comprising either(i) an HCDR1 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 3 or 6, an HCDR2 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 4 or 24, and an HCDR3 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 5 or 8, or(ii) an HCDR1 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 3 or 6, an HCDR2 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 4 or 26, and an HCDR3 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 5 or 8, and(b) a VL domain comprising an LCDR1 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 16 or 29, an LCDR2 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 17 or 20, and an LCDR3 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 18.

54. The bispecific antibody construct according to any one of claims 43 to 45, wherein the IL- 33 antigen-binding domain comprises a VH domain comprising an HCDR1 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 3 or 6, an HCDR2 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 4 or 26, and an HCDR3 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 5 or 8, and a VL domain comprising anLCDR1 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 16 or 29, an LCDR2 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 17 or 20, and an LCDR3 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 18.

55. The bispecific antibody construct according to any one of claims 42 to 53, wherein the IL- 33 antigen binding domain comprises a VH domain comprising a sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the VH sequence as set forth in any one of SEQ ID NOs: 30, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 or 42.

56. The bispecific antibody construct according to any one of claims 42 to 54, wherein the IL- 33 antigen binding domain comprises a VL domain comprising a sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the VL sequence as set forth in any one of SEQ ID NOs: 31, 43, 44, 45 or 46.

57. The bispecific antibody construct according to any one of claims 42 to 56, wherein the IL- 33 antigen binding domain comprises a VH domain comprising a sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the VH sequence as set forth in SEQ ID NO: 32 or 38.

58. The bispecific antibody construct according to any one of claims 42 to 57, wherein the IL- 33 antigen binding domain comprises a VL domain comprising a sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the VL sequence as set forth in SEQ ID NO: 45 or 46.

59. The bispecific antibody construct according to any one of claims 42 to 58, wherein the IL- 33 antigen binding domain comprises a VH domain comprising a sequence as set forth in SEQ ID NO: 32 or 38, and a VL domain comprising a sequence as set forth in SEQ ID NO: 45 or 46.

60. The bispecific antibody construct according to any one of claims 42 to 58, wherein the IL- 33 antigen binding domain comprises a VH domain comprising a sequence as set forth in SEQ ID NO: 32, and a VL domain comprising a sequence as set forth in SEQ ID NO: 46.

61. The bispecific antibody construct according to any one of claims 43 to 60, wherein the IL- 33 antigen binding domain also binds to cynomolgus monkey IL-31.

62. The bispecific antibody construct according to any one of claims 43 to 61, wherein the second target antigen-binding domain specifically binds to IL-4Ra and comprises the CDR sequences of the VH domain as set forth in any one of SEQ ID NOs: 90, 91, 93, 94, 95, 96, 97, 98 or 99, and the CDR sequences of the VL domain as set forth in any one of SEQ ID NOs: 92, 100, 101, 102, 103, 104, 105 or 106.

63. The bispecific antibody construct according to any one of claims 43 to 61, wherein the second target antigen-binding domain specifically binds to IL-4Ra and comprises a VH domain comprising an HCDR1 amino acid sequence comprising the sequence as set forth in any one of SEQ ID NOs: 53, 56, 66 or 67, an HCDR2 amino acid sequence comprising the sequence as set forth in any one of SEQ ID NOs: 54, 57, 79, 80, 84, 85, 86 or 87, and an HCDR3 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 55 or 58, and a VL domain comprising an LCDR1 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 71 or 74, an LCDR2 amino acid sequence comprising the sequence as set forth in any one of SEQ ID NOs: 72, 75 or 88, and an LCDR3 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 73.

64. The bispecific antibody construct according to claim 63 , wherein the VH domain comprises an HCDR1 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 53 or 56, an HCDR2 amino acid sequence comprising the sequence as set forth in any one of SEQ ID NOs: 54, 57, 79, 80, 84, 85, 86 or 87, and an HCDR3 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 55 or 58.

65. The bispecific antibody construct according to any one of claims 43 to 64, wherein the second target antigen-binding domain specifically binds to IL-4Ra and comprises(a) a VL domain comprising an LCDR1 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 71 or 74, an LCDR2 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 72 or 75, and an LCDR3 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 73, and a VH domain comprising:(i) an HCDR1 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 53 or 56, an HCDR2 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 54 or 57, and an HCDR3 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 55 or 58, or(ii) an HCDR1 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 66 or 67, an HCDR2 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 54 or 57, and an HCDR3 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 55 or 58, or(iii) an HCDR1 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 53 or 56, an HCDR2 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 79 or 80, and an HCDR3 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 55 or 58, or(iv) an HCDR1 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 53 or 56, an HCDR2 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 79 or 84, and an HCDR3 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 55 or 58, or(v) an HCDR1 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 53 or 56, an HCDR2 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 79 or 85, and an HCDR3 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 55 or 58, or(vi) an HCDR1 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 53 or 56, an HCDR2 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 79 or 86, and an HCDR3 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 55 or 58, or(vii) an HCDR1 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 53 or 56, an HCDR2 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 79 or 87, and an HCDR3 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 55 or 58, or(b) a VL domain comprising an LCDR1 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 71 or 74, an LCDR2 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 72 or 88, and an LCDR3 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 73, and a VH domain comprising an HCDR1 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 53 or 56, an HCDR2 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 79 or 80, and an HCDR3 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 55 or 58.

66. The bispecific antibody construct according to any one of claims 43 to 64, wherein the second target antigen-binding domain specifically binds to IL-4Ra and comprises a VL domain comprising an LCDR1 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 71 or 74, an LCDR2 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 72 or 75, and an LCDR3 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 73, and a VH domain comprising an HCDR1 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 53 or 56, an HCDR2 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 79 or 80, and an HCDR3 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 55 or 58.

67. The bispecific antibody construct according to any one of claims 43 to 66, wherein the second target antigen-binding domain specifically binds to IL-4Ra and comprises a VH domain having a sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the VH sequence as set forth in any one of SEQ ID NOs: 90, 91, 93, 94, 95, 96, 97, 98 or 99, and a VL domain having a sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the VL sequence as set forth in any one of SEQ ID NOs: 92, 100, 101, 102, 103, 104, 105 or 106.

68. The bispecific antibody construct according to any one of claims 43 to 66, wherein the second target antigen-binding domain specifically binds to IL-4Ra and comprises a VH domain having a sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the VH sequence as set forth in SEQ ID NO: 93, and a VL domain having a sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the VL sequence as set forth in SEQ ID NO: 92.

69. The bispecific antibody construct according to any one of claims 43 to 68, further comprising a scaffold, wherein the IL-33 antigen-binding domain and the second target antigenbinding domain are both operably linked to the scaffold.

70. The bispecific antibody construct according to claim 69, wherein the IL-33 antigen-binding domain and the second target antigen-binding domain are each a Fab.

71. The bispecific antibody construct according to claim 70, wherein the CHI and CL domains of the IL-33 antigen-binding domain and the CHI and CL domains of the second target antigen-binding domain comprise sets of mutations to drive correct pairing between the heavy and light chains of the IL-33 antigen-binding domain and between the heavy and light chains of the second target antigen-binding domain.

72. The bispecific antibody construct according to any one of claims 69 to 71, wherein the scaffold is an IgG Fc region.

73. The bispecific antibody construct according to any one of claims 69 to 72, wherein the scaffold is a human IgG Fc region.

74. The bispecific antibody construct according to claim 72 or claim 73, wherein the IgG Fc region is an IgGl or IgG4 Fc region.

75. The bispecific antibody construct according to any one of claims 72 to 74, wherein the IgG Fc region is a heterodimeric Fc region comprising a first Fc polypeptide and a second Fc polypeptide.

76. The bispecific antibody construct according to claim 75, wherein the heterodimeric Fc region comprises a modified CH3 domain, and wherein the modified CH3 domain comprises one or more amino acid modifications that promote formation of the heterodimeric Fc over formation of a homodimeric Fc.

77. The bispecific antibody construct according to claim 76, wherein:(a) the first Fc polypeptide comprises the amino acid substitutions L351Y, F405A and Y407V, and the second Fc polypeptide comprises the amino acid substitutions T366L, K392M and T394W; or(b) the first Fc polypeptide comprises the amino acid substitutions L351Y, F405A and Y407V, and the second Fc polypeptide comprises the amino acid substitutions T366L, K392L and T394W; or(c) the first Fc polypeptide comprises the amino acid substitutions T350V, L351Y, F405A and Y407V, and the second Fc polypeptide comprises the amino acid substitutions T350V, T366L, K392M and T394W; or(d) the first Fc polypeptide comprises the amino acid substitutions T350V, L351Y, F405A and Y407V, and the second Fc polypeptide comprises the amino acid substitutions T350V, T366L, K392L and T394W; or(e) the first Fc polypeptide comprises the amino acid substitutions T350V, L351Y, S400E, F405A and Y407V, and the second Fc polypeptide comprises the amino acid substitutions T350V, T366L, N390R, K392M and T394W, and wherein the numbering of amino acids is EU numbering.

78. The bispecific antibody construct according to any one of claims 72 to 77, wherein the IgG Fc region comprises the amino acid substitutions M252Y, S254T and T256E, and wherein the numbering of amino acids is EU numbering.

79. The bispecific antibody construct according to any one of claims 72 to 78, wherein the IgG Fc region is an IgGl Fc region.

80. The bispecific antibody construct according to claim 79, wherein the IgGl Fc region comprises the amino acid substitutions L234A, L235A and D265S, and wherein the numbering of amino acids is EU numbering.

81. The bispecific antibody construct according to any one of claims 72 to 78, wherein the IgG Fc region is an IgG4 Fc region.

82. The bispecific antibody construct according to claim 81, wherein the IgG4 Fc region comprises the amino acid substitution S228P and / or the amino acid substitution R409K.

83. A bispecific antibody construct comprising an IL-33 antigen-binding domain that specifically binds to human IL-33 and an IL-4Ra antigen-binding domain that specifically binds to human IL-4Ra, wherein the IL-33 antigen-binding domain comprises a VH domain comprising an HCDR1 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 3 or 6, an HCDR2 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 4 or 26, and an HCDR3 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 5 or 8, and a VL domaincomprising an LCDR1 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 16 or 29, an LCDR2 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 17 or 20, and an LCDR3 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 18, wherein the IL-4Ra antigen-binding domain comprises a VH domain comprising an HCDR1 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 53 or 56, an HCDR2 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 79 or 80, and an HCDR3 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 55 or 58, and a VL domain comprising an LCDR1 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 71 or 74, an LCDR2 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 72 or 75, and an LCDR3 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 73, and wherein the IL-33 antigen-binding domain and the IL-4Ra antigen-binding domain are both operably linked to a human IgG Fc region.

84. The bispecific antibody construct according to claim 83, wherein the IgG Fc region is an IgG4 Fc region.

85. The bispecific antibody construct according to claim 83 or claim 84 comprising a first heavy chain comprising the sequence as set forth in SEQ ID NO: 166, a first light chain comprising the sequence as set forth in SEQ ID NO: 149, a second heavy chain comprising the sequence as set forth in SEQ ID NO: 164, and a second light chain comprising the sequence as set forth in SEQ ID NO: 155.

86. A polynucleotide or set of polynucleotides encoding the bispecific antibody construct according to any one of claims 43 to 85.

87. An expression vector or set of expression vectors comprising the polynucleotide or set of polynucleotides according to claim 86.

88. A host cell comprising the polynucleotide or set of polynucleotides according to claim 86 or the expression vector or set of expression vectors according to claim 87.

89. A method of preparing the bispecific antibody construct according to any one of claims 43 to 85 comprising transfecting a host cell with the polynucleotide or set of polynucleotides according to claim 86 or the expression vector or set of expression vectors according to claim 87, and culturing the host cell under conditions suitable for expression of the bispecific antibody construct.

90. A pharmaceutical composition comprising the antibody construct according to any one of claims 1 to 38 or the bispecific antibody construct according to any one of claims 43 to 85.

91. An antibody construct according to any one of claims 1 to 38 or a bispecific antibody construct according to any one of claims 43 to 85 for use in therapy.

92. Use of an antibody construct according to any one of claims 1 to 38 or a bispecific antibody construct according to any one of claims 43 to 85 in therapy.

93. Use of an antibody construct according to any one of claims 1 to 38 or a bispecific antibody construct according to any one of claims 43 to 85 in the manufacture of a medicament.

94. A method of treating an inflammatory or autoimmune disease in a subject comprising administering to the subject an effective amount of the antibody construct according to any one of claims 1 to 38 or the bispecific antibody construct according to any one of claims 43 to 85.

95. The method according to claim 94, wherein the inflammatory or autoimmune disease is atopic dermatitis, psoriasis, asthma, rhinosinusitis, chronic obstructive pulmonary disease (COPD), inflammatory bowel disease (IBD) or a rheumatological disease.

96. An antibody construct according to any one of claims 1 to 38 or a bispecific antibody construct according to any one of claims 43 to 85 for use in the treatment of an inflammatory or autoimmune disease in a subject.

97. The antibody construct or bispecific antibody construct for use according to claim 96, wherein the inflammatory or autoimmune disease is atopic dermatitis, psoriasis, asthma, rhinosinusitis, chronic obstructive pulmonary disease (COPD), inflammatory bowel disease (IBD) or a rheumatological disease.

98. Use of an antibody construct according to any one of claims 1 to 38 or a bispecific antibody construct according to any one of claims 43 to 85 in the treatment of an inflammatory or autoimmune disease in a subject.

99. Use of an antibody construct according to any one of claims 1 to 38 or a bispecific antibody construct according to any one of claims 43 to 85 in the manufacture of a medicament for the treatment of an inflammatory or autoimmune disease.

100. The use according to claim 98 or claim 99, wherein the inflammatory or autoimmune disease is atopic dermatitis, psoriasis, asthma, rhinosinusitis, chronic obstructive pulmonary disease (COPD), inflammatory bowel disease (IBD) or a rheumatological disease.

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