Anti-il-4r.alpha. antibody constructs and methods of use

Abstract

Antibody constructs that specifically bind to IL-4Ra 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.

Description

ANTI-IL-4Ra ANTIBODY CONSTRUCTS AND METHODS OF USEFIELD

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

[0002] IL-4Ra is a subunit of the interleukin-4 receptor (IL-4R). IL-4Ra can complex with the common cytokine receptor gamma chain (yc) to form a type I IL-4R or with the IL- 13 receptor subunit, IL- 13 Rai, to form a type II IL-4R. The IL-4Ra / IL-13Ral receptor complex can bind interleukin 4 (IL-4) as well as interleukin 13 (IL-13) to regulate IgE antibody production in B cells. IL-4 binding to IL-4R is further known to activate macrophages and to promote differentiation of type 2 helper T-cells (Th2 -cells), leading to Th2-driven inflammation.

[0003] IL-4R signalling has been shown to be associated with a variety of inflammatory and autoimmune diseases, including asthma, chronic obstructive pulmonary disease (COPD), atopic dermatitis and rhinitis.

[0004] A number of antibodies that bind IL-4Ra have been developed, including dupilumab (Dupixent®), which is approved for treatment of a number of indications including atopic dermatitis, asthma, chronic rhinosinusitis with nasal polyposis (CRSwNP) and prurigo nodularis.

[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.SUMMARY

[0006] Described herein are anti-IL-4Ra antibody constructs and methods of use. One aspect of the present disclosure relates to an antibody construct comprising one or more antigen-bindingdomains, wherein at least one of the antigen-binding domains is an IL-4Ra antigen-binding domain that specifically binds to human IL-4Ra, the IL-4Ra antigen-binding domain comprising the CDR sequences (HCDR1, HCDR2, HCDR3) of the VH domain as set forth in any one of SEQ ID NOs: 95, 97, 98, 101,102, 103, 104, 105, 106, 107, 108, 109, 110, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85 or 86, and the CDR sequences (LCDR1, LCDR2, LCDR3) of the VL domain as set forth in any one of SEQ ID NOs: 96, 99, 100, 111, 112, 87, 88, 89, 90, 91, 92, 93 or 94.

[0007] In certain embodiments, the IL-4Ra antigen-binding domain of the antibody construct comprises a VH domain comprising an HCDR1 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 26, an HCDR2 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 61, and an HCDR3 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 5, and a VL domain comprising an LCDR1 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 18, an LCDR2 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 55, and an LCDR3 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 20.

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

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

[0010] Another aspect of the present disclosure relates to a host cell comprising a polynucleotide or set of polynucleotides encoding an antibody construct as described herein or an expression vector or set of expression vectors comprising a polynucleotide or set of polynucleotides encoding an antibody construct as described herein.

[0011] 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 an antibody construct as described herein or an expression vector or set of expression vectors comprising a polynucleotide or set of polynucleotides encoding an antibodyconstruct as described herein, and culturing the host cell under conditions suitable for expression of the antibody construct.

[0012] Another aspect of the present disclosure relates to a multispecific antibody construct comprising an IL-4Ra antigen-binding domain that specifically binds to human IL-4Ra and one or more additional antigen-binding domains, wherein each additional antigen-binding domain binds to an antigen other than IL-4Ra, and wherein the IL-4Ra antigen-binding domain comprises the CDR sequences (HCDR1, HCDR2, HCDR3) of the VH domain as set forth in any one of SEQ ID NOs: 95, 97, 98, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85 or 86, and the CDR sequences (LCDR1, LCDR2, LCDR3) of the VL domain as set forth in any one of SEQ ID NOs: 96, 99, 100, 111, 112, 87, 88, 89, 90, 91, 92, 93 or 94.

[0013] Another aspect of the present disclosure relates to a multispecific antibody construct comprising an IL-4Ra antigen-binding domain that specifically binds to human IL-4Ra and one, two or three additional antigen-binding domains, wherein each additional antigen-binding domains binds to an antigen other than IL-4Ra, 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: 26, an HCDR2 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 61, and an HCDR3 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 5, and a VL domain comprising an LCDR1 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 18, an LCDR2 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 55, and an LCDR3 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 20, and wherein at least one of the additional antigen-binding domains binds to a cytokine or cytokine receptor.

[0014] Another aspect of the present disclosure relates to a polynucleotide or set of polynucleotides encoding a multispecific antibody construct as described herein.

[0015] 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 multispecific antibody construct as described herein.

[0016] Another aspect of the present disclosure relates to a host cell comprising a polynucleotide or set of polynucleotides encoding a multispecific antibody construct as described herein or an expression vector or set of expression vectors comprising a polynucleotide or set of polynucleotides encoding a multispecific antibody construct as described herein.

[0017] Another aspect of the present disclosure relates to a method of preparing a multispecific antibody construct as described herein comprising transfecting a host cell with a polynucleotide or set of polynucleotides encoding a multispecific antibody construct as described herein or an expression vector or set of expression vectors comprising a polynucleotide or set of polynucleotides encoding a multispecific antibody construct as described herein, and culturing the host cell under conditions suitable for expression of the multispecific antibody construct.

[0018] Another aspect of the present disclosure relates to a pharmaceutical composition comprising an antibody construct or a multispecific antibody construct as described herein.

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

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

[0022] Another aspect of the present disclosure relates to a use of an antibody construct or a multispecific antibody as described herein in the treatment of an inflammatory or autoimmune disease in a subject.BRIEF DESCRIPTION OF THE FIGURES

[0023] Figs. 1A-B show the inhibition of IL-4 / IL-13 mediated production of secreted embryonic alkaline phosphatase (SEAP) reporter in HEK-Blue™ IL-4 / IL-13 cells by monovalent (one- armed) anti-IL-4Ra antibody variants. Fig. 1A IL-4 stimulation, and Fig. IB IL- 13 stimulation.

[0024] Figs. 2A-C show binding of anti-IL-31 x anti-IL-4Ra and anti-IL-33 x anti-IL-4Ra bispecific IgG4 antibody variants on human peripheral blood mononuclear cells (PBMCs) by flow cytometry compared to dupilumab (anti-IL-4Ra), NM26-2198 (anti-IL-13 x 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.

[0025] Fig. 3 shows expression titers, post purification yield and % purity for a library of 190 anti-IL-31 x anti-IL-4Ra bispecific antibody variants each including a different engineered anti- IL-4Ra paratope.

[0026] Fig. 4 shows the results of non-specific ELISA (NS-ELISA) screening of a library of 190 anti-IL-31 x anti-IL-4Ra bispecific antibody variants each including a different engineered anti- IL-4Ra paratope.

[0027] Fig. 5 shows the results of AC-SINS screening of a library of 190 anti-IL-31 x anti-IL- 4Ra bispecific antibody variants each including a different engineered anti-IL-4Ra paratope.

[0028] Fig. 6 shows the results of thermal stability assessment by differential scanning fluorimetry of a library of 190 anti -IL-31 x anti-IL-4Ra bispecific antibody variants each including a different engineered anti-IL-4Ra paratope.

[0029] Figs. 7A-D 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-31 x anti-IL-4Ra bispecific IgG4 antibody variants comprising engineered anti-IL-4Ra paratopes. Figs. 7A and 7B show IL-4 stimulation and IL-13 stimulation, respectively, forv43190 and v43193 compared to v41790 comprising the parental anti-IL-4Ra paratope; Figs. 7C and 7D show IL-4 stimulation and IL-13 stimulation, respectively, for v43181, v43182 and v43188 compared to v41791 comprising the parental anti-IL-4Ra paratope.

[0030] Figs. 8A-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 engineered anti-IL-4Ra paratopes. Fig. 8A IL-4 stimulation, and Fig. 8B IL-13 stimulation.

[0031] Fig. 9 shows the serum pharmacokinetic (PK) profile from cynomolgus monkeys injected intravenously with 10 mg / kg of the anti -IL-31 x anti-IL-4Ra bispecific IgG4 antibody variant, v41791. (LLOQ: lower limit of quantification).

[0032] Fig. 10 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).

[0033] Fig. 11 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.

[0034] Figs. 12A-D 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-31 x anti -IL-4Ra bispecific IgG4 antibody variant, v41791. Fig. 12A serum IgE levels; Fig. 12B lung hIL-4 levels; Fig. 12C lung eosinophil levels, and Fig. 12D lung alveolar macrophage levels. Data are represented as mean + / - SEM.

[0035] Figs. 13A-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. 13A serum IgE levels; Fig. 13B lung hIL-4 levels; Fig. 13C lung eosinophil levels; Fig. 13D lung alveolar macrophage levels; Fig. 13E lung hIL-5 levels; Fig. 13F lung pathology evaluated by hemotoxylin and eosin staining of lung sections, and Fig. 13G total inflammation score. Data are represented as mean + / - SEM.

[0036] Fig. 14 presents the CDR sequences for the parental chimeric anti-IL-4Ra antibody variant (v36201) and humanized anti-IL-4Ra antibody variants (v36911 and v36912) (Table Al).

[0037] Fig. 15 presents the CDR sequences for affinity matured anti-IL-4Ra antibody variants (Table A2). Differences compared to parental chimeric (v36201) CDR sequences are marked in bold and underlined.

[0038] Fig. 16 presents the CDR sequences for engineered anti-IL-4Ra antigen-binding domains comprised by certain bispecific anti-IL-31 x anti-IL-4Ra and anti-IL-33 x anti-IL-4Ra antibody variants (Table A3). Differences compared to parental affinity matured anti-IL-4Ra (v38597) CDR sequences are marked in bold and underlined.

[0039] Fig. 17 presents the VH and VL sequences for engineered anti-IL-4Ra antigen-binding domains comprised by certain bispecific anti-IL-31 x anti-IL-4Ra and anti-IL-33 x anti-IL-4Ra antibody variants (Table B).

[0040] Fig. 18 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.

[0041] Figs. 19A-C 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-31 x anti-IL-4Ra bispecific antibody variants (v41791 and v43184). Fig. 19A shows inhibition of IL-4 stimulated SEAP production; Fig. 19B shows inhibition of IL- 13 stimulated SEAP production, and Fig. 19C shows inhibition of IL-31 stimulated SEAP production. v42104 is a negative control.

[0042] Fig. 20A-B show the results from a pharmacokinetic / pharmacodynamic study of the anti- IL-31 x anti-IL-4Ra bispecific antibody variant, v44927, in cynomolgus monkeys. Fig, 20A shows the serum pharmacokinetic profile, and Fig. 20B shows the serum IgE levels, from animals injected intravenously with 40 mg / kg of the bispecific antibody variant (LLOQ: lower limit of quantification).

[0043] Figs. 21A-C show the results from an oxazolone-induced atopic dermatitis efficacy study of the anti-IL-31 x anti-IL-4Ra bispecific IgG4 antibody variant, v44927, administered at 25 mg / kg. Fig. 21A shows body weight of the treated mice, Fig. 21B shows ear thickness by caliper measurement, and Fig. 21C shows hIL-4 serum concentrations.

[0044] Fig. 22A-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. 21A), or subcutaneously with 40 mg / kg (Fig. 21B), of the anti-IL-33 x anti-IL-4Ra bispecific antibody variant, v42101. (LLOQ: lower limit of quantification).

[0045] Fig. 23A-D shows the serum IgE levels from cynomolgus monkeys injected intravenously with 20 mg / kg (Fig. 23A), 40 mg / kg (Fig. 23B) or 100 mg / kg (Fig. 23C), orsubcutaneously with 40 mg / kg (Fig. 23D), of the anti-IL-33 x anti-IL-4Ra bispecific antibody variant, v42101.DETAILED DESCRIPTION

[0046] The present disclosure relates to antibody constructs that specifically bind to IL-4Ra. The antibody constructs may be monospecific antibody constructs comprising one or more antigenbinding domains that bind to the same epitope on IL-4Ra, or they may be biparatopic antibody constructs comprising two antigen-binding domains each of which binds to a different epitope on IL-4Ra, or they may be bispecific or multispecific antibody constructs that comprise at least one antigen-binding domain that binds to IL-4Ra and at least one antigen-binding domain that binds to a target antigen other than IL-4Ra. In certain embodiments, the bispecific or multispecific antibody constructs comprise at least one antigen-binding domain that binds to IL-4Ra and at least one antigen-binding construct that binds to another cytokine or cytokine receptor.

[0047] IL-4Ra 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-4Ra antibody constructs of the present disclosure in the treatment of such IL-4Ra related inflammatory and / or autoimmune diseases and disorders.Definitions

[0048] 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.

[0049] 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.

[0050] 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.”

[0051] 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.

[0052] 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.

[0053] 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 readilyapparent 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 I ICDR2. J ICDR3 and the light chain CDRs for all definitions except Contact, which uses Chothia numbering.2EICDR1 as shown is defined using Chothia numbering. The position in the Kabat numbering system that demarcates the end of the Chothia and IMGT CDR-EI1 loop varies depending on the length of the loop because Kabat numbering places insertions outside of those CDR definitions at positions 35A and 35B. Elowever, the IMGT and Chothia CORTI 1 loop can be unambiguously defined using Chothia numbering.

[0054] 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 sequencesare 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.

[0055] 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 the test 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).

[0056] 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.

[0057] It is contemplated that any embodiment described herein in relation to the IL-4Ra antibody constructs can be implemented with respect to any method, use or composition disclosed herein.

[0058] 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.

[0059] 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 of options 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.

[0060] 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-4Ra ANTIBODY CONSTRUCTS

[0061] The present disclosure relates to antibody constructs that specifically bind to human IL- 4Ra (anti- IL-4Ra 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.

[0062] In accordance with the present disclosure, the anti- IL-4Ra antibody constructs comprise at least one antigen-binding domain that specifically binds to human IL-4Ra (an “IL-4Ra antigenbinding domain”). By “specifically binds” to IL-4Ra, it is meant that the antigen-binding domain binds to human IL-4Ra and may bind to IL-4Ra 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, 2W2, Endocr Res, 28:217- 229).

[0063] In certain embodiments, the anti- IL-4Ra antibody constructs of the present disclosure may also be capable of binding to IL-4Ra from one or more non-human species. In certain embodiments, the anti- IL-4Ra antibody constructs of the present disclosure are capable of binding to cynomolgus monkey IL-4Ra.

[0064] The protein sequence for human IL-4Ra protein is known in the art and readily available from publicly accessible databases, such as GenBank or UniProtKB. For example, the amino acid sequence for human IL-4Ra is available under GenBank Accession No. P24394. The region between amino acids 26-232 of this sequence forms the extracellular domain (also provided in Table 2 as SEQ ID NO: 1). The IL-4Ra sequence forthe macaque (cynomolgus) monkey, Macaca fascicularis, is available under UniProt Accession No. G7Q0S7. The region between amino acids 26-232 of this sequence forms the extracellular domain (also provided in Table 2 as SEQ ID NO: 2).Table 2: Human and Macaque IL-4Ra Protein Sequences (Extracellular Domain)IL-4Ra Antigen-Binding Domains

[0065] The anti-IL-4Ra antibody constructs of the present disclosure comprise at least one antigen-binding domain that specifically binds to human IL-4Ra (an “IL-4Ra 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).

[0066] 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.

[0067] 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)).

[0068] 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, Appt. Microbiol Biotechnol., Il W. 13-22).

[0069] In certain embodiments, the anti-IL-4Ra antibody constructs comprise an IL-4Ra antigen-binding domain that is a Fab or an scFv. In some embodiments, the anti-IL-4Ra antibody constructs comprise an IL-4Ra antigen-binding domain that is a Fab.

[0070] In those embodiments in which the anti- IL-4Ra 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-4Ra or they may bind to a different antigen. In certain embodiments, the anti-IL-4Ra antibody constructs comprise one or more additional antigen-binding domains that are immunoglobulin-based domains.

[0071] The present disclosure describes the identification of an antibody that specifically binds IL-4Ra (h054A03; see Example 1), as well as a chimeric version of this antibody (v36201), representative humanized versions of this antibody (variants v36911 and v36912) and representative affinity-matured versions of this antibody (for example, variants v38502, v38503, v38504, v38505, v38506, v38507, v38508, v38509, v38510, v38511, v38597 and v38598) (see Examples). The CDR sequences of the chimeric antibody v36201 and representative humanized and / or affinity-matured versions of this antibody are shown in Figs. 14 and 15 (Tables Al and A2). Also described herein are engineered versions of the affinity matured variant v38597. The CDR sequences of these engineered versions are shown in Fig. 16 (Table A3).

[0072] The VH and VL sequences of the parental chimeric antibody v36201 are the same as those for the original mouse antibody, h054A03, and are shown in Table 1.1 (see Example 1). The VH and VL sequences of the representative humanized and affinity-matured versions of this antibody are shown in Tables 1.2 and 1.4, respectively (see Example 1). The VH and VL sequences of the engineered versions of affinity-matured antibody variant, v38597, are shown in Fig. 17 (Table B).

[0073] In certain embodiments, the anti-IL-4Ra 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 (HCDR1, HCDR2, HCDR3) of the VH domain as set forth in any one of SEQ IDNOs: 95, 97, 98, 101-110 or 75-86. In some embodiments, the anti-IL-4Ra 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 (HCDR1, HCDR2, HCDR3) of the VH domain as set forth in SEQ ID NO: 103, 110, 75 or 78.

[0074] In certain embodiments, the anti-IL-4Ra 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 (LCDR1, LCDR2, LCDR3) of the VL domain as set forth in any one of SEQ ID NOs: 96, 99, 100, 111, 112 or 87-94. In some embodiments, the anti-IL-4Ra 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 (LCDR1, LCDR2, LCDR3) of the VL domain as set forth in SEQ ID NO: 112 or 91.

[0075] In certain embodiments, the anti-IL-4Ra 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 (HCDR1, HCDR2, HCDR3) of the VH domain as set forth in any one of SEQ ID NOs: 95, 97, 98, 101-110 or 75-86, and the CDR sequences (LCDR1, LCDR2, LCDR3) of the VL domain as set forth in any one of SEQ ID NOs: 96, 99, 100, 111, 112 or 87- 94. In some embodiments, the anti-IL-4Ra 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 (HCDR1, HCDR2, HCDR3) of the VH domain as set forth in SEQ ID NO:103, 110, 75 or 78, and the CDR sequences (LCDR1, LCDR2, LCDR3) of the VL domain as set forth in SEQ ID NO: 112 or 91.

[0076] In certain embodiments, the anti-IL-4Ra 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 heavy chain CDR amino acid sequences (HCDR1, HCDR2 and HCDR3) comprising the sequences as set forth in SEQ ID NOs: 124, 125 and 126, 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, 127 and 20 (see Table 3). SEQ ID NOs: 124, 125 and 126, and SEQ ID NOs: 21, 127 and 20 are consensus sequences based on the HCDR and LCDR sequences, respectively, of v36201 and representative humanized, affinity-matured and engineered versions of this antibody when defined by the Kabat system.Table 3: Consensus CDR Sequences by Kabat

[0077] In certain embodiments, the anti-IL-4Ra 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 heavy chain CDR amino acid sequences (HCDR1, HCDR2 and HCDR3) comprising the sequences as set forth in SEQ ID NOs: 124, 125 and 126, 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, 127 and 20, where X1is L, X2is K, X3is Y or A, X4is NQKFKG or NDKFKG, X5is GE, X6is D, X7is KRS and X8is Q. In some embodiments, the anti-IL-4Ra antibody constructs comprise at least one IL-4Ra antigen-binding domain, where the IL-4Ra antigen-binding domain comprises a VH domain comprising HCDR1, HCDR2 and HCDR3 amino acid sequences comprising the sequences as set forth in SEQ ID NOs: 124, 125 and 126, and a VL domain comprising LCDR1, LCDR2 and LCDR3 amino acid sequences comprising the sequences as set forth in SEQ ID NOs: 21, 127 and 20, where X1is L, X2is K, X3is A, X4is NQKFKG or NDKFKG, X5is GE, X6is D, X7is KRS and X8is Q.

[0078] In certain embodiments, the anti-IL-4Ra 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: 6, 27, 32, 37 or 43, an HCDR2 amino acid sequence comprising the sequence as set forth in any one of SEQ ID NOs: 7, 16, 62, 66, 67, 68, 69, 70, 71 or 72, and an HCDR3 amino acid sequence comprising the sequence as set forth in any one of SEQ ID NOs: 8, 44, 50 or 53.

[0079] In certain embodiments, the anti-IL-4Ra 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: 3, 26, 31 or 36, an HCDR2 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 4 or 61, and an HCDR3 amino acid sequence comprising the sequence as set forth in any one of SEQ ID NOs: 5, 42, 49 or 52. In some embodiments, the anti-IL-4Ra 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: 26, an HCDR2amino acid sequence comprising the sequence as set forth in SEQ ID NO: 4 or 61, and an HCDR3 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 5.

[0080] In certain embodiments, the anti-IL-4Ra 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: 21, an LCDR2 amino acid sequence comprising the sequence as set forth in any one of SEQ ID NOs: 22, 56, 59 or 73, and an LCDR3 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 20.

[0081] In certain embodiments, the anti-IL-4Ra 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: 18, an LCDR2 amino acid sequence comprising the sequence as set forth in any one of SEQ ID NOs: 19, 55 or 58, and an LCDR3 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 20. In some embodiments, the anti-IL-4Ra 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: 18, an LCDR2 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 55, and an LCDR3 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 20.

[0082] In certain embodiments, the anti-IL-4Ra 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: 6, 27, 32, 37 or 43, an HCDR2 amino acid sequence comprising the sequence as set forth in any one of SEQ ID NOs: 7, 16, 62, 66, 67, 68, 69, 70, 71 or 72, and an HCDR3 amino acid sequence comprising the sequence as set forth in any one of SEQ ID NOs: 8, 44, 50 or 53, and a VL domain comprising an LCDR1 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 21, an LCDR2 amino acid sequence comprising the sequence as set forth in any one of SEQ ID NOs: 22, 56, 59 or 73, and an LCDR3 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 20.

[0083] In certain embodiments, the anti-IL-4Ra 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: 3, 26, 31 or 36, an HCDR2 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 4 or 61, and an HCDR3 amino acid sequence comprising the sequence as set forth in any one of SEQ ID NOs: 5, 42, 49 or 52, and a VL domain comprising an LCDR1 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 18, an LCDR2 amino acid sequence comprising the sequence as set forth in any one of SEQ ID NOs: 19, 55 or 58, and an LCDR3 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 20.

[0084] In certain embodiments, the anti-IL-4Ra 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: 26, an HCDR2 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 4 or 61, and an HCDR3 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 5, and a VL domain comprising an LCDR1 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 18, an LCDR2 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 55, and an LCDR3 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 20.

[0085] In certain embodiments, the anti-IL-4Ra 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, an HCDR2 and an HCDR3, and a VL domain comprising an LCDR1, an LCDR2 and an LCDR3, where the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 have amino acid sequences as set forth for HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of IL-4Ra binding arm of any one of the variants set forth in Tables Al, A2 and A3 (see Figs. 14, 15 and 16).

[0086] In certain embodiments, the anti-IL-4Ra 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 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the VH sequence as set forth in any one of SEQ ID NOs: 95, 97, 98, 101-110or 75-86. In some embodiments, the anti-IL-4Ra 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 as set forth in any one of SEQ ID NOs: 95, 97, 98, 101-110 or 75-86. In some embodiments, the anti-IL-4Ra antibody constructs of the present disclosure comprise at least one IL-4Ra antigen-binding domain, where the IL-4Ra antigenbinding domain comprises a VH domain having a sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the VH sequence as set forth in any one of SEQ ID NOs: 103, 110, 75 or 78. In some embodiments, the anti-IL-4Ra antibody constructs of the present disclosure comprise at least one IL-4Ra antigen-binding domain, where the IL-4Ra antigenbinding domain comprises a VH domain having a sequence as set forth in any one of SEQ ID NOs: 103, 110, 75 or 78.

[0087] In certain embodiments, the anti-IL-4Ra 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 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the VL sequence as set forth in any one of SEQ ID NOs: 96, 99, 100, 111, 112 or 87-94. In some embodiments, the anti-IL-4Ra 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 the sequence as set forth in any one of SEQ ID NOs: 96, 99, 100, 111, 112 or 87-94. In some embodiments, the anti-IL-4Ra antibody constructs of the present disclosure comprise at least one IL-4Ra antigen-binding domain, where the IL-4Ra antigenbinding domain comprises a VL domain having a sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the VL sequence as set forth in SEQ ID NO: 112 or 91. In some embodiments, the anti-IL-4Ra 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 as set forth in SEQ ID NO: 112 or 91.

[0088] In certain embodiments, the anti-IL-4Ra 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 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the VH sequence as set forth in any one of SEQ ID NOs: 95, 97, 98, 101-110 or 75-86, and a VL domain having a sequence that is at least about 90%, 95%, 96%, 97%, 98%,99%, or 100% identical to the VL sequence as set forth in any one of SEQ ID NOs: 96, 99, 100, 111, 112 or 87-94. In some embodiments, the anti-IL-4Ra antibody constructs of the present disclosure comprise at least one IL-4Ra antigen-binding domain, where the IL-4Ra antigenbinding domain comprises a VH domain having a sequence as set forth in any one of SEQ ID NOs: 95, 97, 98, 101-110 or 75-86, and a VL domain having the sequence as set forth in any one of SEQ ID NOs: 96, 99, 100, 111, 112 or 87-94.

[0089] In certain embodiments, the anti-IL-4Ra 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 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the VH sequence as set forth in any one of SEQ ID NOs: 103, 110, 75 or 78, and a VL domain having a sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the VL sequence as set forth in SEQ ID NO: 112 or 91. In some embodiments, the anti-IL-4Ra 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: 103, 110, 75 or 78, and a VL domain having a sequence as set forth in SEQ ID NO: 112 or 91.

[0090] In certain embodiments, the anti-IL-4Ra antibody constructs of the present disclosure comprise at least one IL-4Ra antigen-binding domain, where the IL-4Ra antigen-binding domain comprises:(i) a VH domain having the sequence as set forth in SEQ ID NO: 95, and a VL domain having the sequence as set forth in SEQ ID NO: 96, or(ii) a VH domain having the sequence as set forth in SEQ ID NO: 97, and a VL domain having the sequence as set forth in SEQ ID NO: 99, or(iii) a VH domain having the sequence as set forth in any one of SEQ ID NOs: 98, 106, 107 or 108, and a VL domain having the sequence as set forth in SEQ ID NO: 100, or(iv) a VH domain having the sequence as set forth in any one of SEQ ID NOs: 101, 102, 104, 105, 107 or 109, and a VL domain having the sequence as set forth in SEQ ID NO: 111, or(v) a VH domain having the sequence as set forth in any one of SEQ ID NOs: 103, 105 or 110, and a VL domain having the sequence as set forth in SEQ ID NO: 112, or(vi) a VH domain having the sequence as set forth in SEQ ID NO: 75, and a VL domain having the sequence as set forth in SEQ ID NO: 87 or 88, or(vii) a VH domain having the sequence as set forth in SEQ ID NO: 78, and a VL domain having the sequence as set forth in SEQ ID NO: 91 or 92, or(viii) a VH domain having the sequence as set forth in SEQ ID NO: 76 or 82, and a VL domain having the sequence as set forth in SEQ ID NO: 89, or(ix) a VH domain having the sequence as set forth in SEQ ID NO: 77, and a VL domain having the sequence as set forth in SEQ ID NO: 90, or(x) a VH domain having the sequence as set forth in any one of SEQ ID NOs: 78, 81, 83 or 86, and a VL domain having the sequence as set forth in SEQ ID NO: 91, or(xi) a VH domain having the sequence as set forth in SEQ ID NO: 78 or 84, and a VL domain having the sequence as set forth in SEQ ID NO: 92, or(xii) a VH domain having the sequence as set forth in SEQ ID NO: 79 or 85, and a VL domain having the sequence as set forth in SEQ ID NO: 93, or(xiii) a VH domain having the sequence as set forth in SEQ ID NO: 80, and a VL domain having the sequence as set forth in SEQ ID NO: 94.

[0091] In certain embodiments, the anti-IL-4Ra antibody constructs of the present disclosure comprise at least one IL-4Ra antigen-binding domain, where the IL-4Ra antigen-binding domain comprises:(i) a VH domain having the sequence as set forth in SEQ ID NO: 75, and a VL domain having the sequence as set forth in SEQ ID NO: 87, or(ii) a VH domain having the sequence as set forth in SEQ ID NO: 78, and a VL domain having the sequence as set forth in SEQ ID NO: 91.Formats

[0092] The anti-IL-4Ra antibody constructs of the present disclosure may have various formats. The minimal component of the anti-IL-4Ra antibody construct is an antigen-binding domain that binds to human IL-4Ra. The anti-IL-4Ra 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-4Ra antibody construct comprises two or more antigen-binding domains, each additional antigen-binding domain may bind to the same epitope within IL-4Ra, may bind to a different epitope within IL-4Ra, or may bind to a different antigen. Thus, the anti-IL-4Ra antibody construct may be, for example, monospecific, biparatopic, bispecific or multispecific.

[0093] In certain embodiments, the anti-IL-4Ra antibody construct comprises at least one IL-4Ra antigen-binding domain and a scaffold, where the IL-4Ra 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.

[0094] In certain embodiments, the anti-IL-4Ra antibody construct comprises two antigen-binding domains optionally operably linked to a scaffold, where at least one of the antigen-binding domains is an IL-4Ra antigen-binding domain. In some embodiments, the anti-IL-4Ra 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-4Ra 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.

[0095] Anti-IL-4Ra antibody constructs that lack a scaffold may comprise a single IL-4Ra 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-4Ra antigen-binding domain. In such anti-IL-4Ra 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).

[0096] 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-4Ra 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.

[0097] 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.

[0098] In certain embodiments, the anti-IL-4Ra antibody construct may be in an antibody format that is based on an immunoglobulin (Ig). In certain embodiments, the anti-IL-4Ra 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-4Ra antibody construct may be based on an IgGl or IgG4 immunoglobulin. In the context of the present disclosure, when an anti-IL- 4Ra antibody construct is based on a specified immunoglobulin isotype, it is meant that the anti- IL-4Ra antibody construct comprises all or a portion of the constant region of the specified immunoglobulin isotype. For example, an anti-IL-4Ra antibody construct based on a given Ig isotype may comprise at least one IL-4Ra antigen-binding domain operably linked to an Ig scaffold, where the scaffold comprises an Fc region from the given isotype and optionally an Ighinge region from the same or a different isotype. It is to be understood that the anti-IL-4Ra 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.

[0099] In some embodiments, the anti-IL-4Ra antibody constructs may be derived from two or more immunoglobulins that are from different species, for example, the anti-IL-4Ra 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.

[0100] 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.

[0101] 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.”

[0102] 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, et al., 1988, Nature, 332:323-329, and Presta, 1992, Curr.Op. Struct. Biol., 2:593-596, for example.

[0103] 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 limited subset of well-characterised human antibodies, irrespective of the sequence identity to the non- human 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).

[0104] 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 a , 2000, J Immunol, 164: 1432-1441; Gonzales, et a , 2004, Mol Immunol, 1:863-872, and Kashmiri, et al., 2005, Methods, 36:25-34).

[0105] In certain embodiments, the anti-IL-4Ra antibody construct of the present disclosure comprises humanized antibody sequences, for example, one or more humanized variable domains. In some embodiments, the anti-IL-4Ra antibody construct is a humanized antibody. Non-limiting examples of humanized antibodies based on the anti-IL-4Ra antibody h054A03 (see Example 1) are described herein (variants v36911 and v36912; see Examples and Table Al (Fig. 14)).

[0106] 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-4Ra construct of the present disclosure may comprise humanized, affinity matured antibody sequences, for example, one or more humanized, affinity matured variable domains. Non-limiting examples of anti-IL-4Ra affinity-matured antibodies are described herein (see Examples and Table A2 (Fig. 15)).Scaffolds

[0107] In certain embodiments, the anti-IL-4Ra antibody constructs of the present disclosure comprise one or more IL-4Ra antigen-binding domains operably linked to a scaffold. The antigen-binding 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).

[0108] 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-4Ra antibody construct may be linked to either the N- or C-terminus of the polypeptide scaffold. Anti- IL-4Ra antibody constructs comprising a polypeptide scaffold in which one or more of the antigenbinding 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.

[0109] In embodiments where the anti-IL-4Ra 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.

[0110] 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).

[0111] 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, 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).

[0112] 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-bindingmoieties (Muller et al., ibid.). Antigen-binding moieties may be fused at the N- and / or C-termini of albumin, optionally via a linker. 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.

[0113] In certain embodiments, the anti-IL-4Ra antibody construct may comprise a protein scaffold. In some embodiments, the anti-IL-4Ra 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-4Ra antibody construct may comprise a protein scaffold that is based on an immunoglobulin Fc region, for example, an IgG Fc region.Fc Regions

[0114] 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).

[0115] In certain embodiments, the anti-IL-4Ra 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-4Ra antibody constructs of the present disclosure comprise a scaffold that is a dimeric Fc region composed of two Fc polypeptides.

[0116] 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. Whenreferring 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.

[0117] An Fc region may comprise a CH3 domain or it may comprise both a CH3 and a CH2 domain. For example, in certain embodiments, an 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.

[0118] In some embodiments, the anti-IL-4Ra antibody construct may comprise a scaffold that is an IgG Fc region. In some embodiments, the anti-IL-4Ra antibody construct may comprise a scaffold that is a human IgG Fc region. In some embodiments, the anti-IL-4Ra antibody construct may comprise a scaffold that is an IgGl or IgG4 Fc region. In some embodiments, the anti-IL-4Ra antibody construct may comprise a scaffold that is a human IgGl or IgG4 Fc region.

[0119] In certain embodiments, the anti-IL-4Ra 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.

[0120] In certain embodiments, the anti-IL-4Ra antibody construct may comprise a scaffold based on an IgG 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-4Ra antibody construct comprises two or more different antigen-binding domains, for example, when the anti-IL-4Ra antibody construct is biparatopic, bispecific or multispecific.

[0121] In some embodiments, the anti-IL-4Ra antibody construct may comprise a scaffold based on an Fc region which comprises two CH3 sequences, at least one of which comprises one or more amino acid modifications, for example, amino acid substitutions. In some embodiments, the anti-IL-4Ra 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-4Ra antibody construct may comprise a scaffold based on an Fc region which comprises two CH3 sequences and two CH2 sequences, in which 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.

[0122] In some embodiments, the anti-IL-4Ra antibody construct may comprise a heterodimeric Fc region comprising a modified CH3 domain, where the modified CH3 domain is an 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.

[0123] In some embodiments, the anti-IL-4Ra 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.

[0124] 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 etal., 2010, J Biol Chem, 285, 19637-46 (“electrostatic steering”), Davis et al., 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-4Ra 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.

[0125] Table 4 provides the amino acid sequences of a human IgGl Fc region (SEQ ID NO: 128) and a human IgG4 Fc region (SEQ ID NO: 129). Table 5 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.

[0126] In certain embodiments, the anti-IL-4Ra antibody construct may comprise a heterodimeric Fc scaffold based on an IgGl or IgG4 Fc region having 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 5.Table 4: Amino Acid Sequence of the Human IgGl and IgG4 Fc Regions (CH2 and CH3 Domains)Table 5: CH3 Domain Amino Acid Substitutions Promoting Heterodimer Formation

[0127] In those embodiments in which the anti-IL-4Ra 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, for example, Handlogten, et al., 2020, MAbs, 12(1): 1779974; Namisaki, et al., 2020, PLoS ONE, 15(3): e0229027). In certain embodiments, the anti-IL-4Ra 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-4Ra 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-4Ra 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-4Ra antibody construct may comprise a heterodimeric IgG4 Fc region comprising the amino acid substitution R409K and amino acid substitutions of any one of Variant 1, Variant 2, Variant 3, Variant 4 or Variant 5, as shown in Table 5. In some embodiments, the anti-IL-4Ra antibody construct may comprise a heterodimeric IgG4 Fc region comprising theamino acid substitutions S228P and R409K and amino acid substitutions of any one of Variant 1, Variant 2, Variant 3, Variant 4 or Variant 5, as shown in Table 5.

[0128] In some embodiments, the anti-IL-4Ra 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-4Ra 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.

[0129] 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.

[0130] In certain embodiments, the anti-IL-4Ra 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 strategiesthat 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).

[0131] Examples of mutations that may be introduced into the hinge or CH2 domain of anIgGl 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-4Ra antibody construct comprises a scaffold based on an IgGl Fc having a modified CH2 domain that comprises the amino acid substitutions L234A / L235A / D265S in both chains of the CH2 domain.

[0132] In certain embodiments, the anti-IL-4Ra 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 all effector function (Bolt et al., 1993, Eur. J. Immunol., 23:403-411; Tao & Morrison, 1989, J. Immunol., 143:2595-2601).

[0133] 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-4Ra antibody constructs comprise YTE mutations.

[0134] In certain embodiments, the anti-IL-4Ra antibody constructs comprise an IgGl Fc region and comprise knock-out mutations and YTE mutations. In certain embodiments, the anti- IL-4Ra antibody constructs comprise an IgG4 Fc region and comprise YTE mutations.

[0135] In certain embodiments, the anti-IL-4Ra antibody constructs have the format of a full-size antibody (FSA). In some embodiments, the anti-IL-4Ra antibody constructs have the format of an IgG FSA, for example, an IgGl or IgG4 FSA. In some embodiments, the anti-IL-4Ra 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-4Ra 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-4Ra 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-4Ra 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.

[0136] In certain embodiments, the anti-IL-4Ra 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 E and F for any one of variants v36201, v36911, v36912, v38502, v38503, v38504, v38505, v38506, v38507, v38508, v38509, v38510, v38511, v38597, v38598 or v38597-Y54A. As is known in the art, expression of antibody heavy chain sequences in certain cell lines or from certain expression vectors 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- 4Ra 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 E and F for any one of variants v36201, v36911, v36912, v38502, v38503, v38504, v38505, v38506, v38507, v38508, v38509, v38510, v38511, v38597, v38598 or v38597-Y54A, in which one or both of the Hl and H2 sequences comprise a C-terminal lysine.MULTISPECIFIC ANTIBODY CONSTRUCTS

[0137] Certain embodiments of the present disclosure relate to multispecific antibody constructs that specifically bind to IL-4Ra and to one or more other target antigens, where the one or more other target antigens are not IL-4Ra. The multispecific antibody constructs of the present disclosure thus comprise an IL-4Ra antigen-binding domain and one or more additional antigenbinding domains, where the one or more additional antigen-binding domains each bind to an antigen other than IL-4Ra. In certain embodiments, the multispecific antibody constructs are bispecific, trispecific or tetraspecific. In some embodiments, the multispecific antibody constructs are bispecific or trispecific.

[0138] In certain embodiments, the multispecific antibody constructs comprise a first antigenbinding domain that binds to IL-4Ra (the IL-4Ra antigen-binding domain) and at least a second antigen-binding domain that binds to a target antigen other than IL-4Ra (a second target antigenbinding domain).

[0139] In some embodiments, the multispecific antibody construct is a bispecific antibody construct and comprises at least one IL-4Ra antigen-binding domain and at least one second target antigen-binding domain. 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 one IL-4Ra antigenbinding domain and one second target antigen-binding domain. In some embodiments, the bispecific antibody constructs are trivalent or tetravalent and comprise one or two IL-4Ra antigenbinding domains and one or two second target antigen-binding domains.

[0140] In certain embodiments, the multispecific antibody constructs of the present disclosure have higher specificities (for example, trispecific or tetraspecific) and include additional target antigen-binding domains, each binding to a different target antigen. For example, trispecific antibody constructs comprise at least one IL-4Ra antigen-binding domain, at least one second target antigen-binding domain that specifically binds to a second target antigen and at least one third target antigen-binding domain that specifically binds to a third target antigen, where the second and third target antigens are not IL-4Ra and are different from each other. A tetraspecific antibody construct comprises at least one IL-4Ra antigen-binding domain, at least one secondtarget antigen-binding domain that specifically binds to a second target antigen, at least one third target antigen-binding domain that specifically binds to a third target antigen and at least one fourth target antigen-binding domain that specifically binds to a fourth target antigen, where the second, third and fourth target antigens are not IL-4Ra and are different from each other.

[0141] In accordance with the present disclosure, the at least one IL-4Ra antigen-binding domain comprised by the multispecific antibody constructs may be the IL-4Ra antigen-binding domain of any one of the embodiments described above.

[0142] In certain embodiments, the multispecific antibody constructs comprise at least a second target antigen-binding domain that binds to a second target antigen, where 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-31 or IL-33.

[0143] The additional antigen-binding domains comprised by the multispecific antibody constructs may be derived from a known antibody that binds to the target antigen or it may be derived from an antibody against the target antigen generated by standard antibody generation techniques. For example, an additional antigen-binding domain may be derived from a known antibody that binds to IL- 13, IL-31 or IL-33. 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-31 include, but are not limited to, BMS-981164 and NM26-2198. Examples ofknown antibodies that bind to IL-33 include itepekimab, tozorakimab, etokimab and torudokimab. The VH and VL sequences for these known antibodies may be obtained from publicly accessible databases, such as the Therapeutic Antibody Database (Tabs).

[0144] In certain embodiments, the antigen-binding domains (for example, the IL-4Ra antigenbinding domain and the second, third and fourth target antigen-binding domains) comprised by the multispecific antibody constructs are immunoglobulin-based binding domains, such as antigenbinding antibody fragments. 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 singlechain Fv (scFv) and a single domain antibody (sdAb). In certain embodiments, the antigen-binding domains are each independently a Fab or an scFv.Multispecific Formats

[0145] The multispecific antibody constructs described herein comprise at least one IL-4Ra antigen-binding domain and at least one additional antigen-binding domain. In certain embodiments, the multispecific antibody constructs are bivalent (i.e. comprise two antigenbinding domains), trivalent (i.e. comprise three antigen-binding domains) or tetravalent (i.e. comprise four antigen-binding domains). In certain embodiments, the multispecific 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 other antigen-binding domains) to the scaffold.

[0146] In certain embodiments, the multispecific 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).

[0147] 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.

[0148] 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.

[0149] In certain embodiments, the multispecific 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 multispecific antibody constructs may be based on an IgGl, IgG2, IgG3 or IgG4 immunoglobulin. In some embodiments, the multispecific 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 multispecific 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 multispecific 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.

[0150] In certain embodiments, the multispecific antibody constructs comprise a scaffold based on an IgGl or IgG4 Fc region. Examples of such scaffolds are described in detail above. In some embodiments, the multispecific antibody constructs comprise two antigen-binding domains (i.e. are bivalent) and a scaffold based on an IgGl or IgG4 Fc region. In some embodiments, the multispecific antibody constructs comprise two antigen-binding domains and a scaffold based on an IgG4 Fc region. In some embodiments, the multispecific antibody constructs comprise three antigen-binding domains (i.e. are trivalent) and a scaffold based on an IgGl or IgG4 Fc region. In some embodiments, the multispecific antibody constructs comprise three antigen-binding domainsand a scaffold based on an IgG4 Fc region. In some embodiments, the multispecific antibody constructs comprise four antigen-binding domains (i.e. are tetravalent) and a scaffold based on an IgGl or IgG4 Fc region. In some embodiments, the multispecific antibody constructs comprise four antigen-binding domains and a scaffold based on an IgG4 Fc region. In those embodiments in which the multispecific antibody construct comprises an Fc region, the Fc region typically comprises a first Fc polypeptide and a second Fc polypeptide.

[0151] A bivalent multispecific antibody construct comprises one antigen-binding domain that binds to IL-4Ra and a second antigen-binding domain that binds to a second target antigen (i.e. is bispecific), whereas a trivalent multispecific antibody construct may be bispecific (comprising two antigen-binding domains that bind to one target antigen and a third antigenbinding domain that binds to a different target antigen) or it may be trispecific (comprising three antigen-binding domains that each bind to a different target antigen). Similarly, a tetravalent multispecific antibody construct may be bispecific (for example, comprising two antigen-binding domains that bind to one target antigen and two antigen-binding domains that binds to a different target antigen) or it may be trispecific (comprising two antigen-binding domains that bind to a first target antigen and two other antigen-binding domains that each bind to a different target antigen) or it may be tetraspecific (comprising four antigen-binding domains that each bind to a different target antigen).

[0152] In some embodiments, the multispecific antibody constructs are bivalent and bispecific and comprise a scaffold based on an IgGl or IgG4 Fc region. In some embodiments, the multispecific antibody constructs are trivalent and bispecific and comprise a scaffold based on an IgGl or IgG4 Fc region. In some embodiments, the multispecific antibody constructs are trivalent and trispecific and comprise a scaffold based on an IgGl or IgG4 Fc region. In some embodiments, the multispecific antibody constructs are tetravalent and bispecific and comprise a scaffold based on an IgGl or IgG4 Fc region. In some embodiments, the multispecific antibody constructs are tetravalent and trispecific and comprise a scaffold based on an IgGl or IgG4 Fc region. In some embodiments, the multispecific antibody constructs are tetravalent and tetraspecific and comprise a scaffold based on an IgGl or IgG4 Fc region.

[0153] As would be appreciated by one of skill in the art, the multispecific antibody constructs may be constructed in various formats. For example, each of the antigen-binding domains comprised by the multispecific antibody may be in a different format (for example, Fab, scFv), or they may all be in the same format. Where the multispecific 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.

[0154] In certain embodiments, the antigen-binding domains comprised by the multispecific antibody construct are all in Fab format. In some embodiments, at least one of the antigen-binding domains comprised by the multispecific antibody construct is in Fab format and the other antigen-binding domains are in Fab or scFv format. In some embodiments, at least one of the antigen-binding domains comprised by the multispecific antibody construct is in scFv format and the other antigen-binding domains are in Fab or scFv format. In some embodiments, the antigen-binding domains comprised by the multispecific antibody construct are all in scFv format.

[0155] In certain embodiments, the multispecific antibody constructs are bivalent and comprise one IL-4Ra antigen-binding domain and one second target antigen-binding domain and an IgG Fc region, where (a) both antigen-binding domains are in Fab format, or (b) both antigenbinding 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.

[0156] In those embodiments in which the multispecific 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 a heterodimeric Fc over a homodimeric Fc, as described above. In certain embodiments in which the multispecific antibody constructs comprise a scaffold based on an IgGl or IgG4 Fc region, theFc region may comprise 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 5.

[0157] When the multispecific antibody constructs comprise two or more antigen-binding domains in Fab format, the CHI and CL domains of each Fab may comprise sets of mutations to drive the correct pairing between the heavy and light chains of each 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 some embodiments, the multispecific antibody constructs comprise at least two antigen-binding domains in Fab format and each antigen-binding domain comprises a set of mutations to drive the correct pairing between the heavy and light chains of the antigen-binding domain, where the set of mutations is Set 1 or Set 2 as shown in Table 6.Table 6: Sets of CH1 / CL Mutations to Promote Correct Pairing* Numbering of amino acid positions according to EU

[0158] In certain embodiments, the multispecific antibody construct comprises one or more IL-4Ra antigen-binding domains where each IL-4Ra antigen-binding domain comprises the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 sequences of the IL-4Ra antigenbinding domain as set forth in Tables E-G for any one of variants v43181, v43182, v43183,V43184, v43185, v43186, v43187, v43188, v43189, v43190, v43191, v43192, v43193, v43194, v43195, v43196, v43197, v43198, v43199 or v43200.

[0159] In certain embodiments, the multispecific antibody construct comprises one or more IL-4Ra antigen-binding domains where each IL-4Ra antigen-binding domain comprises the VH and VL sequences of the IL-4Ra antigen-binding domain as set forth in Tables E-G for any one of variants v43181, v43182, v43183, v43184, v43185, v43186, v43187, v43188, v43189, v43190, v43191, v43192, v43193, v43194, v43195, v43196, v43197, v43198, v43199 or v43200.

[0160] In certain embodiments, the multispecific antibody construct comprises one or more IL-4Ra antigen-binding domains where each IL-4Ra antigen-binding domain comprises a VH domain having a sequence as set forth in any one of SEQ ID NOs: 95, 97, 98, 101-110 or 75- 86, and a VL domain having the sequence as set forth in any one of SEQ ID NOs: 96, 99, 100, 111, 112 or 87-94.

[0161] In certain embodiments, the multispecific antibody construct comprises one or more IL-4Ra antigen-binding domains where each IL-4Ra antigen-binding domain comprises a VH domain having a sequence as set forth in any one of SEQ ID NOs: 103, 110, 75 or 78, and a VL domain having a sequence as set forth in SEQ ID NO: 112 or 91.

[0162] In certain embodiments, the multispecific antibody constructs have the format of a full-size antibody (FSA), for example, an IgGl or IgG4 FSA. In some embodiments, the multispecific antibody construct is an 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.METHODS OF PREPARING ANTIBODY CONSTRUCTS

[0163] The anti- IL-4Ra antibody constructs and multispecific 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).

[0164] 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 theantibody 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 antigenbinding 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 multispecific antibody construct in a full-size antibody format with two Fab antigen-binding domains, each binding a different antigen, two polynucleotides each encoding a different heavy chain and two polynucleotides each encoding a different light chain will be required. When multiple polynucleotides are required, they may be incorporated into one vector or into more than one vector.

[0165] 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 antibody construct 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.

[0166] 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 orPichia cells). Prokaryotic host cells include, for example, E. colt, A. salmonicida or B. subtilis cells.

[0167] 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.

[0168] 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).

[0169] 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), human lung 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, 77:4216), and myeloma cell lines (such as Y0, NS0 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).

[0170] 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.

[0171] 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.

[0172] 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.

[0173] In certain embodiments, the antibody constructs are substantially pure. The term “substantially pure” (or “substantially purified”) when used in reference to an antibody constructdescribed 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.

[0174] Certain embodiments of the present disclosure relate to a method of making an anti-IL-4Ra antibody construct or a multispecific 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

[0175] 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.

[0176] 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.

[0177] 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.

[0178] 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.

[0179] 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.

[0180] 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

[0181] Certain embodiments of the present disclosure relate to an isolated polynucleotide or a set of polynucleotides encoding an anti-IL-4Ra antibody construct or multispecific antibody construct described herein.

[0182] 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.

[0183] 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.

[0184] Certain embodiments of the present disclosure relate to vectors (such as expression vectors) comprising one or more polynucleotides encoding an anti-IL-4Ra antibody construct or a multispecific 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.

[0185] Certain embodiments of the present disclosure relate to host cells comprising polynucleotide(s) encoding an anti-IL-4Ra antibody construct or a multispecific 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

[0186] For therapeutic use, the anti-IL-4Ra antibody construct or multispecific antibody construct may be provided in the form of pharmaceutical compositions comprising the antibody construct and a pharmaceutically acceptable carrier or diluent. The compositions may be prepared by known procedures using well-known and readily available ingredients.

[0187] Pharmaceutical compositions may be formulated for administration to a subject by, for example, parenteral, oral (including, for example, buccal or sublingual), topical, rectal orvaginal 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.

[0188] 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.

[0189] 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).

[0190] 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 the antibody 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 orisotonic 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.

[0191] 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.

[0192] 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

[0193] Certain aspects of the present disclosure relate to the therapeutic use of the anti-IL- 4Ra antibody constructs and multispecific antibody constructs described herein.

[0194] Certain embodiments relate to use of the anti-IL-4Ra antibody constructs and multispecific 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-4Ra antibody construct or multispecific antibody construct to the subject.

[0195] Examples of inflammatory or autoimmune diseases that may be treated with the anti-IL-4Ra antibody constructs or multispecific 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.

[0196] 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.

[0197] The dosage of the anti-IL-4Ra antibody construct or multispecific 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

[0198] Certain embodiments relate to pharmaceutical kits (or articles of manufacture) comprising an anti-IL-4Ra antibody construct or a multispecific antibody construct as described herein.

[0199] 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, thecontainer may have a sterile access port. For example, the container may be an intravenous solution bag or a vial having a stopper that may be pierced by a hypodermic injection needle.

[0200] 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. 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).

[0201] The kit may further include other materials desirable from a commercial or user standpoint, such as filters, needles, and syringes.

[0202] The following Examples are provided for illustrative purposes and are not intended to limit the scope of the claimed invention in any way.EXAMPLES

[0203] 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 OF HUMANIZED AND AFFINITY-MATURED ANTLIL-4Ra ANTIBODIES1.1 Antibody Generation

[0204] Antibodies that specifically bind IL-4Ra were generated by immunizing mice with human IL-4Ra as described below.

[0205] Ten B6X129 mice were subcutaneously immunized with recombinant human IL-4Ra (R&D Systems, Minneapolis, MN; Cat. No. 7700-4R) over 63 days, after which blood was drawn. Anti -human IL-4Ra antibody titers were determined by flow cytometry using HEK293-6E cells (National Research Council of Canada) that were transiently transfected with a pTT5 -based expression plasmid (National Research Council of Canada) encoding human IL-4Ra according to manufacturer’s instructions for Lipofectamine™ 2000 (Thermo Fisher Scientific, Waltham, MA). Test bleed sera mounted a significant response against human IL-4Ra.

[0206] Immunized mice were sacrificed and spleens harvested. Splenocytes for each mouse were pooled and used for B cell enrichment and sorted on a FACSAria™ system (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).

[0207] 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 et al., 1996, Proc Natl Acad Sci USA, 93(15):7843-7848 and von Boehmer et al., 2016, Nat Protoc., 11(10): 1908, 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 familyspecific 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 ExpiCHO™ cells (Thermo Fisher Scientific, Waltham, MA).

[0208] Cell supernatants containing secreted antibodies were assessed for blocking human IL- 4Ra in a reporter gene assay. The wells corresponding to human IL-4Ra antibody containing supernatant were selected for sequencing.

[0209] Heavy and light chain PCR amplicons were sequenced using NGS-based Amplicon-EZ and analyzed for unique antibody-coding sequences. The following mouse anti -human IL-4Ra antibody VH and VL sequences were identified for the antibody h054A03.Table 1.1: Mouse VH and VL Sequences for Anti-Human IL-4Rot Antibody (h054A03)

[0210] The mouse VH and VL sequences were used to prepare a mouse-human chimeric IgGl / kappa antibody variant, v36201, 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 for human and cynomolgus IL-4Ra.1.2 Humanization

[0211] The mouse VH and VL sequences from the chimeric antibody variant v36201 were aligned against human immunoglobulin germline sequences to select basis germline sequences for humanization. Human germline IGHV1-169*O4 with IGHJ6*01 was selected for VH humanization. Human germline IGKV1-139*O1 with IGKJ2*01 was selected for VL humanization. The CDR sequences by AbM definition for CDRH1, CDRH2 and CDRH3 from v36201 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 theoriginal mouse parental sequence to minimize potential disruption to antigen binding. Humanization was performed with five new candidate humanized VH sequences, and three new candidate humanized VL sequences created. Recombinant human IgGl-based monoclonal antibody variants containing each combination of the candidate VH and VL humanized sequences were expressed in Expi293F™ cells and the supernatants were screened for activity using IL-4 / IL- 13 reporter gene assay (RGA) as described in Example 3. Two antibody variants, v36911 and v36912, represented by 2 humanized VH (Hl and H4) and 2 humanized VL sequences (LI and L2) (see Tables 1.2 and 1.3) demonstrated similar activity to the parental chimeric antibody variant v36201 (see Example 3). Table 1.2: Humanized Anti-IL-4Ra Antibody VH and VL Domain SequencesTable 1.3: VH and VL Composition of Selected Humanized Antibody Variants1.3 Affinity maturation

[0212] One VH sequence (H4) and one VL sequence (L2) from humanization (v36912) were affinity matured using the HuTarg™ system (Innovative Targeting Solutions, Vancouver, BC, Canada) and the resulting affinity matured anti-IL-4Ra antibody variants were expressed in Expi293™ cells and purified by affinity chromatography using by MabSelect™ SuRe™ ProteinA resin (GE Healthcare). The purified antibody variants were screened for activity by reporter gene assay (RGA) using human IL-4 and IL- 13 as described in Example 3 and three affinity matured antibody variants v38597, v38504 and v38598 represented by 3 affinity matured VH and 2 affinity matured VL sequences (see Table 1.4 and 1.5) were selected for further characterization. Table 1.4: Affinity Matured Anti-IL-4Ra Antibody VH and VL Sequences* SEQ ID NO: 112 is identical to SEQ ID NO: 87Table 1.5: VH and VL Composition of Affinity Matured Anti-IL-4Ra Antibody VariantsEXAMPLE 2: PRODUCTION AND CHARACTERIZATION OF AFFINITY MATUREDANTI-IL-4Ra ANTIBODY VARIANTS2.1 Production of Affinity Matured Anti-IL-31 Antibody Constructs

[0213] Each of the three humanized, affinity matured antibody variants from Example 1 (v38597, v38504 and v38598) was produced in full-size antibody (FSA) IgGl format and one- armed antibody (OAA) format (see Table 2.1).

[0214] The FSA variants contained two identical full length heavy chains and two identical kappa light chains. Each of the humanized VH domain sequences was appended to a human CH1- hinge-CH2-CH3 domain sequence of IGHGl*01 (SEQ ID NO: 113; see Table 2.2) and each of the humanized VL domain sequences was appended to the human kappa CL domain sequence of IGKC*01 (SEQ ID NO: 114; see Table 2.2).

[0215] In addition to the FSA variants, the two humanized, affinity matured antibody variants were produced in one-armed antibody (OAA) format containing one full length heavy chain (Heavy Chain A), one truncated heavy chain (Heavy Chain B) and one kappa light chain (Light Chain). The full-length heavy chain (Heavy Chain A) contained the human CHl-hinge-CH2-CH3 domain sequence of IGHGl*01 with the mutations: T350V_L351Y_F405A_Y407V. Each of the humanized, affinity matured VH domain sequences was appended to the CHl-hinge-CH2-CH3 domain sequence of Heavy Chain A. The truncated heavy chain (Heavy Chain B) contained hinge- CH2-CH3 domain sequence of IGHGl*01 with the mutations: T350V_T366L_K392L_T394W. 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: 114; see Table 2.2). 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 2.1: VH, VL and Fc Composition of Affinity Matured Anti-IL-4Ra Antibody VariantsTable 2.2: Sequences of Wild-Type IgGl Heavy Chain and Kappa Light Chain

[0216] All sequences were reverse translated to DNA, codon optimized for mammalian expression and gene synthesized.

[0217] Heavy chain vector inserts comprising a signal peptide (artificially designed sequence: MRPTWAWWLFLVLLLALWAPARG (SEQ ID NO: 115) (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 pTT5vector 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.2.1.1 Cloning and Transfection Conditions

[0218] Gene products were cloned (by GenScript BioTech Corporation, Piscataway, NJ) into the pTT5 mammalian expression vector and transiently expressed in Expi293™ cells (ThermoFisher Scientific, Waltham, MA). Transfected cells were cultured at 37°C in Expi293™ expression medium (Thermo Fisher Scientific) on an orbital shaker rotating at 120 rpm in a humidified atmosphere of 8% CO2. Each 1 mb of cells at a density of 3 x 106cells / mL was transfected with a total of 1 pg DNA. Prior to transfection the DNA was diluted in 60 pL Opti-MEM™ I Reduced Serum Medium (Thermo Fisher Scientific). In a volume of 56.8 pL Opti-MEM™ I Reduced Serum Medium, 3.2 pL of ExpiFectamine™ 293 Reagent (Thermo Fisher Scientific) was diluted and, after incubation for 3 - 5 minutes, combined with the diluted DNA to a total volume of 120 pL. After incubation for 10-20 minutes, the DNA-ExpiFectamine™ 293 Reagent complex was added to the cell culture (120 pL complex per 1 mb of cell culture) and incubated in a 120 rpm shaking incubator at 37°C and 8% CO2. 18-22 hours post transfection, 6 pL of ExpiFectamine 293 Transfection Enhancer 1 (Thermo Fisher Scientific) and 60 pL of ExpiFectamine 293 Transfection Enhancer 2 (Thermo Fisher Scientific) was added per ImL of cell culture. Cells were maintained in culture at 37°C for a total of 5 - 7 days. Supernatants were harvested by transferring each culture into appropriately sized Falcon tubes and centrifuging at 3500 rpm for 15 minutes. Following centrifugation, supernatants were filtered using a 0.2mm polyethersulfone membrane (Thermo Fisher Scientific), then analyzed by non-reducing SDS-PAGE and Octet™ biolayer interferometry (ForteBio, Fremont, CA).2.1.2 Purification by Protein A Affinity Chromatography

[0219] The clarified culture medium was loaded onto 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. The antibody variant was eluted with 10 column volume s 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).

[0220] Purity of samples was assessed by electrophoresis under non-reducing 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 2 pl or 5 pl (concentration range 5-2000 ng / pl), were added to separate wells in 96 well plates along with 7pl of HT Protein Express Sample Buffer (Perkin Elmer, Cat # 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.

[0221] Species homogeneity of the humanized, affinity matured antibody variants and parental chimeric antibody variant samples was assessed by UPLC-SEC. UPLC-SEC was performed on Agilent Technologies 1290 Infinity system with a diode array detector (DAD) using the Agilent Technologies AdvanceBio SEC300A column (7.8 x 150 mm, 2.7 um) at 25 °C. The mobile phase was 200 mM KPOr + 200 mM KC1, pH 7.4 or PBS pH 7.4 and the flow rate was 1 mL / min. 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, Santa Clara, CA).

[0222] The antibody variants were further purified by gel filtration chromatography using a Superdex™ 200 Increase column (GE Healthcare) and an AKTA Pure™ chromatography system (Cytiva, Marlborough, MA) at a flowrate of 1 mL / min. PBS buffer at pH 7.4 was used at a flowrate of 1 mL / min. Fractions of eluted antibody variant were collected based on absorbance at A280 nm and the fractions were assessed by non-reducing and reducing High Throughput Protein Express assay using Caliper LabChip® GXII and UPLC-SEC using Agilent Technologies 1290 Infinity system with a DAD detector and the Agilent Technologies AdvanceBio SEC300A column (7.8 x 150 mm, 2.7 um) at 25 °C. Fractions with high purity were pooled and endotoxin levels were determined by the limulus amebocyte lysate (LAL) assay using the Endosafe™ Portable Test System (PTS, Charles River, Wilmington, MA).

[0223] The yields and purity for the humanized, affinity matured antibody are shown in Table 2.3.Table 2.3: Post Purification Yield and Final Purity (%) for Affinity Matured Anti-IL-4Ra Antibody Variants2.2 Characterization of Affinity Matured Anti-IL-4Ra Antibody Variants2.2.1 Binding to IL-4Ra

[0224] The equilibrium binding affinity of the humanized antibody variant v36912 and the two affinity matured OAA variants, v38606, v38607 and v38535, to human IL-4Ra was determined by KinExA™ (Sapidyne Inc., Boise, ID). The anti-IL-4Ra antibody, dupilumab, was included for comparison.

[0225] Briefly, human IL-4Ra (R&D Systems; Cat. No. 230-4RB) and cynomolgus IL-4Ra (ACRO BioSystems; Cat. No. ILR-C52H8) were each immobilized on azlactone beads. Antibody variants were used as the constant binding partner (CBP) and antigen (hu IL-4Ra) was used as the titrant. The OAA antibody variants were tested at constant concentrations of 2 pM and 20 pM or 5 pM and 50 pM. The titrants, human IL-4Ra and cynomolgus IL-4Ra, were each titrated by 2- fold serial dilutions of 0.5 nM. The antibody / antigen mixture was incubated at 25°C for 5 days and assessed using a goat anti-human IgG (H+L) conjugated to Alexa Fluor™ 647 with data analyzed using the KinExA™ software. The measured binding affinities (KD) are listed in Table 2.4.Table 2.4: Binding Affinity of Humanized and Affinity Matured Anti-IL-4Ra AntibodyVariants to Human and Cynomolgus IL-4Rot2.2.2 Assessment of Non-specific Interactions and Self-interaction Propensity

[0226] The potential propensity of the affinity matured antibody variants for non-specific interactions and self-interaction were assessed by non-specific ELISA (NS-ELISA) and affinitycapture self-interaction nanoparticle spectroscopy (AC-SINS), respectively, as described below. The affinity matured antibody variants were tested in OAA format. Trastuzumab (anti-HER2) was used as a negative control (low propensity) and lenzilumab (anti-GM-CSF) was used as a positive control (high propensity).

[0227] ELISA-based non-specific binding to several coating materials was adapted as previously described (Jain, ef a / ., 2017, Proc Natl Acad Sci 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 incubatedfor 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.

[0228] 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 2.5. All the antibody variants tested showed high non-specific binding scores.Table 2.5: Assessment of Non-specific Interactions of the Anti-IL-4Ra Antibody Variants

[0229] 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.

[0230] Briefly, 20 nm gold nanoparticles (Ted Pella, Inc., #15705) washed with 0.22 pm filteredMilli-Q™ water were coated with a mixture of 80% AffiniPure™ 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 nanoparticleswere 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 read from 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 2.6. All the antibody variants tested showed similar self-interaction propensity to the positive control.Table 2.6: Assessment of Self-interaction Propensity of the Anti-IL-4Ra Antibody VariantsEXAMPLE 3: FUNCTIONAL CHARACTERIZATION OF ANTI-IL-4Rot ANTIBODY VARIANTS - IL-4 / 13 REPORTER GENE ASSAY

[0231] To determine the impact of the anti-IL-4Ra antibody variants on IL-4 / IL-13 activation of the STAT6 signalling pathway, selected variants were assessed for 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 as described below. The anti-IL-4Ra antibody variants were tested in OAA format. The variants tested were: v38533, v38535, v38536, v38538, v38606 and v38607, which are OAA versions of v38502, v38504, v38505, v38507, v38597 and v38598, respectively (see Table 1.5). The anti-IL-4Ra antibodies dupilumab and Antibody 37GL (see US2015 / 0079092) (referred to herein as “Medi-37GL) in monovalent format were used as positive controls, and the anti-RSV antibody palivizumab (v22277) was used as a negative control.

[0232] 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 (both from 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 OD620nmon a Synergy™ plate reader (BioTek Instruments, Inc., Winooski, VT).

[0233] The results are shown in Fig. 1A-B. All the anti-IL-4Ra antibody variants in monovalent (OAA) format showed similar inhibition of IL-4 / IL-13 mediated production of STAT6 inducible SEAP reporter in HEK-Blue™ IL-4 / 13 cells. The inhibitory activity of the antibody variants was similar to that shown by the benchmark anti-IL-4Ra antibody controls (Dupilumab and Medi- 37GL).EXAMPLE 4: ENGINEERING AN ANTI-IL-4Ra ANTIBODY VARIANT TO LOWER HYDROPHOBICITY

[0234] To potentially decrease the non-specific and self-interactions observed for the anti-IL- 4Ra antibody variants described in Examples 1 and 2, a hydrophobicity reducing mutation was introduced into the affinity matured anti-IL-4Ra antibody variant v38597 (see Table 2. 1).

[0235] Positions were identified for potential introduction of hydrophobicity reducing mutations by assessing the affinity matured parental VH and VL sequences for hydrophobic residues in the CDR regions. Residues that had been mutated in the affinity maturation process were excluded to avoid loss in antigen binding affinity. Hydrophobic residues with high surface exposure and minimal involvement in intra-molecular interactions were selected based on a 3D structure model. A residue in CDRH2, Y54 was selected for mutation. The residue was mutated to alanine (Y 54A) to potentially lower hydrophobicity. The sequence of the mutated VH region of the v38597-Y54A variant is provided in Table 4.1.Table 4.1: VH Sequence of Anti-IL-4Ra Antibody Variant v38597-Y54A

[0236] This mutation was assessed in bispecific antibody format (anti-IL-31 x anti-IL-4Ra and anti-IL-31 x anti-IL-4Ra) as described in Examples 5 and 6. Briefly, no significant differences in protein expression, purification and functional characterization were observed.EXAMPLE 5: PREPARATION AND CHARACTERIZATION OF IgG4 BISPECIFIC ANTIBODIES5.1 Anti-IL-31 x Anti-IL-4Ra Bispecific Antibody Variants

[0237] Bivalent, bispecific anti-IL-31 x anti-IL-4Ra antibody variants were produced in an IgG4 background using the anti-IL-4Ra paratope from the antibody variant v38597 (see Example 2) or v38597-Y54A (see Example 4), and an anti-IL-31 paratope as described below.

[0238] Bispecific antibody variants that are monovalent for each antigen were prepared in a format in which both the IL-31 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 5.1.

[0239] The heavy chains comprised by the bispecific antibody variants described in Table 5.1 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.

[0240] The CHI domain amino acid substitutions promoting correct heavy and light chain pairing are:

[0241] Chain A: Q175R (HetFab HCA) and Chain B: A141W / L145E / K147T / Q175E (HetFab HCB)

[0242] The CH3 domain amino acid substitutions promoting heterodimeric Fc formation are:

[0243] Chain A: T350V / L351Y / F405A / Y407V / R409K (HetFcA) and Chain B: T350V / T366L / K392L / T394W / R409K (HetFcB) .

[0244] 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.

[0245] In addition, 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.

[0246] The light chains comprised by the bispecific antibody variants described in Table 5. 1 are human kappa light chains which comprise amino acid substitutions in the CL domain that promote formation of correct heavy and light chain pairs. The CL domain amino acid substitutions are:

[0247] Chain A: Q124E / L135W / A144I / T178E / T180E (HetFab LCA) and Chain B: Fl 16A / Q124R / L135V / T178R (HetFab LCB)

[0248] Numbering of amino acids in the CL domain is according to the EU index.

[0249] In the bispecific antibody variants, the VH and VL sequences of each of the anti-IL- 31 Fab domain and the anti-IL-4Ra Fab domain were coupled to human IgG4 CHI and CL sequences, respectively.

[0250] The anti-IL-31 Fab domain sequences were generated from the VH and VL sequences of a humanized, affinity matured anti-IL-31 antibody variant v36542. The Vn and VL sequences of the anti-IL-31 antibody variant v36542 are provided in Table 5.2.Table 5.1: Description of Anti-IL-31 x Anti-IL-4Ra Bispecific Antibody Variants (IgG4)Table 5.2: VH and VL Sequences of Anti-IL-31 Antibody Variant v36542

[0251] The bispecific antibody variants were expressed and characterized following the protocols described in International Patent Publication No. WO 2015 / 109131.

[0252] 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 KI cells. The CHO 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 wastransfected 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 buffer or 0.1M acetate buffer at pH 3.0 with the pooled fractions containing 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).

[0253] The antibody variants were further purified by cation exchange chromatography using a POROS™ XS column with a salt gradient of 20 mM sodium acetate, IM NaCl buffer, pH 5.5. 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 variants were collected, buffer exchanged into 20 mM histidine, pH 6.0, concentrated to ~20 mg / mL, and stored at -80 °C.

[0254] The apparent purity and yield of the final antibody variants was estimated by HPLC-SEC and LC / MS following generally the protocols as described in International Patent Publication No. WO 2015 / 109131. Both antibody variants were expressed and purified to >95% heterodimer purity without contaminating homodimers (see Table 5.3).Table 5.3: Post Purification Yield and Purity for Anti-IL-31 x Anti-IL-4Ra Bispecific Antibody Variants (IgG4)1As determined by size-exclusion chromatography HPLC (HPLC-SEC)2As determined by LCMS intensity5.2 Anti- IL-33 x Anti-IL-4Ra Bispecific Antibody Variants

[0255] Bivalent, bispecific anti -IL-33 x anti-IL-4Ra antibody variants were produced in an IgG4 background (including the same HetFc, S228P and YTE mutations as described in Section 5.1) using the anti -IL-4Ra paratope from antibody variants v38597 (see Example 2) and v38597-Y54A (see Example 4) and the anti-IL-33 paratopes from variants v33096-N31A and v33101-N31A (described below) following the protocols described in Section 5.1. The following amino acid substitutions in the CHI domain and CL domain that promote correct heavy and light chain pairing (HetFab) were used in place of those described in Section 5.1.

[0256] CHI domain amino acid substitutions:

[0257] Chain A: L145E / K147T / Q175E (HetFab HCA) and Chain B: L145R (HetFab HCB)

[0258] CL domain amino acid substitutions:

[0259] Chain A: Q124R / T178R (HetFab LCA) and Chain B: Q124E / V133E (HetFab LCB)

[0260] The anti-IL-33 x anti-IL-4Ra bispecific antibodies are described in Table 5.4 and the sequences of the VH and VL domains of the anti-IL-33 antibody variants v33096-N31A and v33101-N31A are provided in Table 5.5. Table 5.4: Description of Anti-IL-33 x Anti-IL-4Ra Bispecific Antibody Variants (IgG4)Table 5.5: VH and VL Sequences of Anti-IL-33 Antibody Variants v33096-N31A and v33101-N31AEXAMPLE 6: CELLULAR BINDING OF BISPECIFIC ANTIBODIES COMPRISING ANTI-IL-4Ra PARATOPE

[0261] Bispecific antibody variants comprising an anti-IL4Ra paratope based on the anti-IL4Ra antibody variants described in Example 2 were tested for their ability to bind to bind human IL- 4Ra as described below.

[0262] The bispecific antibody variants tested were the anti -IL-31 x anti-IL-4Ra and anti-IL-33 x anti-IL-4Ra bispecific antibody variants described in Example 5 (see Tables 5.1 and 5.4). The anti-IL-4Ra antibody dupilimab used as a positive control for IL-4Ra binding and a human-IgGisotype (v36992) was used as a non-specific negative control. A tetravalent, bispecific anti-IL-31 x anti-IL-4Ra antibody (NM26-2198) was also included as a positive control.

[0263] Flow cytometry was performed on human PBMCs (StemCell Technologies, Vancouver Canada). Briefly, PBMCs were thawed and plated in 96 well v-bottom plates at IxlO5cells / well. Bispecific 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 cell surface markers (CD3, CD4, CD 14, CD15, CD16, CD19) 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+CD19" CD4+), B cell (CDS’ CD19+) and monocyte (CD-CD14+CD15+CD16+ / ) populations as defined by phenotyping antibody panel staining.

[0264] 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 of anti-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 7: ENGINEERING AN ANTI-IL-4Ra ANTIBODY PARATOPE TO REDUCE CHARGE ASYMMETRY

[0265] To potentially decrease the non-specific and self-interactions observed for the anti-IL- 4Ra antibody variants described in Examples 1 and 2, mutations were introduced into the affinitymatured anti-IL-4Ra antibody variant v38597 (see Table 2.1) to reduce the charge asymmetry in the VH and VL domains as described below.

[0266] A library of antibody variants was constructed by mutating residues in the framework regions of VH and VL domains of the anti-IL-4Ra antibody variant v38597, specifically avoiding the residues within the CDR domains (as defined by IMGT). Positions were selected for charge lowering mutations by comparing the parental VH and VL sequences with the germline sequences and the most frequent human germlines with lower framework charges. The positions conserved in the most frequent human germlines with lower framework charge were selected to be mutated by either charge neutralization or charge reversal, for example, by changing a positively charged residue to a neutral or negatively charged residue. Additional mutation sites within framework regions based on 3D structure modeling to identify charged residues that are likely to be solvent exposed, not participating in intra-domain interactions and positioned to hypothetically not contribute to antigen binding interactions were also considered for mutations to modulate total charge on the antibody. A panel of anti-IL-4Ra paratopes were thus designed with a wide range of mutations to screen charge reduction by 3, 4, 5 or 6 charges.

[0267] To test these mutations, a library of 190 anti -IL-31 x anti-IL-4Ra bivalent, bispecific antibody constructs were created using the anti -IL-31 paratope from the anti -IL-31 antibody variant, v36542 (see Example 5 and Table 5.2), and 190 different engineered anti-IL-4Ra paratopes. These bispecific antibody constructs were produced in an IgG4 background including the same mutations (HetFab, HetFc, S228P, YTE) as described in Section 5.1 and following the protocols described in Section 5.1.7.1 Production of the Mutational Library in the Anti-IL-31 x Anti-IL-4Ra Bispecific Antibody Format (190 mutants)

[0268] Generally, the final gene products were sub-cloned into the mammalian expression vector pTT5 (NRC-BRI, Canada) and expressed in ExpiCHO™ cells. ExpiCHO™ cells were cultured at 37°C in ExpiCHO™ expression medium (Thermo Fisher, Waltham, MA) on an orbital shaker rotating at 120 rpm in a humidified atmosphere of 8% CO2. Each 1 ml of cells at a density of 6 x 106cells / ml was transfected with a total of 0.8 pg DNA. The DNA was transfected in optimal DNA ratios of the heavy chain 1 (HC-1), heavy chain 2 (HC-2), and light chain 1 (LC-1) and lightchain 2 (LC-2) that allow for heterodimer formation (for example, HC-l:HC-2:LC-l:LC-2 ratios of 15: 15:35:35). Prior to transfection the DNA was diluted in 76.8 pL OptiPRO™ SFM (Thermo Fisher, Waltham, MA), after which 3.2 pL of ExpiFectamine™ CHO reagent (Thermo Fisher, Waltham, MA) was directly added to make a total volume of 80 pL. After incubation for 1 - 5 minutes, the DNA-ExpiFectamine™ CHO Reagent complex was added to the cell culture (80 pL complex per 1 mL of cell culture) then incubated in a 120 rpm shaking incubator at 37 °C and 8% CO2. Following incubation at 37°C for 18-22 hours, 6 pL of ExpiCHO™ Enhancer and 240 pL of ExpiCHO™ Feed (Thermo Fisher, Waltham, MA) were added per ImL of culture. Cells were maintained in culture at 37°C for a total of 8 - 10 days, after which each culture was harvested by transferring into appropriately sized falcon tubes and centrifuging at 3500 rpm for 15 minutes. Supernatants were filtered using a 0.2 mm polyethersulfone membrane (Thermo Fisher, Waltham, MA), then analyzed by non-reducing SDS-PAGE and Octet™ biolayer interferometry (ForteBio, Fremont, CA).

[0269] The supernatants were used to perform high throughput Protein A purification using the KingFisher™ instrument (Thermo Fisher Scientific) with Mag Sepharose™ PrismA magnetic beads. Briefly, all supernatants were transferred to KingFisher™ 96 deep well sample plate with the magnetic beads and washed with PBS, pH 7.4. The bispecific antibody variants were eluted with 0.1 M sodium citrate, pH 3.5 and further neutralized with IM Tris, pH 9.0. The purified bispecific constructs were quantified based on A280 nm (NanoDrop™ Spectrophotometer; Thermo Fisher Scientific) and assessed for purity by non-reducing and reducing 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 Ltd., Mississauga, Canada). The bispecific antibody variants were expressed with varying titers and a majority of the antibody variants were purified to >70% purity without contaminating homodimers (see Fig. 3).7.2 Library Screening

[0270] To screen the library of 190 bispecific antibody variants to identify those comprising an engineered anti-IL-4Ra paratope that has improved properties, three assays were performed. The first assay assessed the propensity of the bispecific antibodies for non-specific interactions usingnon-specific ELISA (NS-ELISA) and the second assessed the propensity of the bispecific antibody variants for self-interaction by self-interaction nanoparticle spectroscopy (AC-SINS). The third assay evaluated the thermal stability of the bispecific antibody variants by differential scanning fluorimetry (DSF). In each case, the values obtained were compared to those of the corresponding anti -IL-31 x anti-IL-4Ra antibody variant comprising the parental anti-IL-4Ra paratope.7.2.1 Assessment of Non-Specific Interactions by NS-ELISA

[0271] The library of 190 bispecific antibody variants was assessed by NS-ELISA following the protocol described in Example 2 (Section 2.2.2) using insulin and ssDNA. The results are shown in Fig. 4. A large majority of the bispecific antibody variants comprising engineered anti-IL-4Ra paratopes (“mutants”) showed significant improvement in the NS-ELISA total binding score as compared to the bispecific antibody variant comprising the parental anti-IL-4Ra paratope (“WT parental”).7.2.2 Assessment of Self Interaction by AC-SINS

[0272] The library of 190 bispecific antibody variants was assessed by AC-SINS following the protocol described in Example 2 (Section 2.2.2). The results are shown in Fig. 5. All bispecific antibody variants comprising engineered anti-IL-4Ra paratopes (“mutants”) showed significant improvement in the AC-SINS score as compared to the bispecific antibody variant comprising the parental anti -IL-4Ra paratope (“WT parental”).7.2.3 Assessment of Thermal Stability

[0273] All DSF experiments were carried out using a CFX96 Touch™ Real-Time PCR instrument (Bio-Rad Laboratories, Inc., Hercules, CA). The antibodies 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 buffer background subtracted to determine the maximum melting temperatures (Tm) for each of the peaks in the thermograms.

[0274] The results are shown in Fig. 6, which shows the Tml for each of the bispecific antibody variants comprising engineered anti-IL-4Ra paratopes (“mutants”). All bispecific antibodyvariants comprising engineered anti-IL-4Ra arms displayed higher melting temperatures (Tml) corresponding to greater thermal stability as compared to the bispecific antibody variant comprising the parental anti-IL-4Ra paratope (“WT parental”).EXAMPLE 8: PRODUCTION AND CHARACTERIZATION OF ANTI-IL-31 x ANTLIL- 4Ra BISPECIFIC ANTIBODY VARIANTS COMPRISING ENGINEERED ANTI-IL-4Ra ANTIBODY PARATOPES #1

[0275] Eight engineered IL-4Ra paratopes from the library described in Example 7 were selected for additional assessment. The engineered IL-4Ra paratopes are described in Table 8.1. The CDR sequences of the engineered IL-4Ra paratopes are shown in Fig. 16 (Table A3) and the VH and VL sequences are shown in Fig. 17 (Table B). The anti -IL-31 paratope used was from the anti -IL-31 antibody variant, v36542 (see Example 5 and Table 5.2).

[0276] Bispecific antibody variants that are monovalent for each antigen were prepared in a format in which both the IL-31 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 5. 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 8.2. All bispecific antibody variants comprised the HetFab, HetFc and S228P mutations described in Example 5, Section 5.1. In addition, certain of the bispecific antibody variants further comprise the YTE mutations (M252Y / S254T / T256E) as noted in Table 8.2.Table 8.1: IL-4Ra Paratope Mutational Designs** All amino acid positions are numbered using AbM numbering.Table 8.2: Description of Anti-IL-31 x Anti-IL-4Ra Bispecific Antibody Variants (IgG4)8.1 Preparation of Bispecific Anti-IL-31 x Anti-IL-4Ra Antibody Variants

[0277] The antibody variants shown in Table 8.2 were expressed in ExpiCHO™ cells at a 200 mL 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 a PEI: DNA ratio of 2.5: 1. DNA was transfected at an optimal DNA ratio 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-Bratio 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 gm 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).

[0278] 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.

[0279] 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).

[0280] The apparent purity and yield of each final antibody variant was estimated by UPLC-SEC and LC / MS 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 8.3 below. Table 8.3: Post Purification Yield and Purity for Bispecific Anti-IL-31 x Anti-IL-4RaAntibody Variants (20 mM Histidine pH 6.0 Buffer)* As determined by HPLC-SEC (size-exclusion chromatography HPLC)8.2 Functional Screening of the Engineered Anti-IL-4Ra Paratope in the Bispecific Anti-IL- 31 x Anti-IL-4Ra Antibody Variants

[0281] To determine the impact of the engineered IL-4Ra paratope comprised by the bispecific antibody variants on IL-4 / IL-13 activation of the STAT6 pathway, selected variants (v43181, v43182, v43188, v43190 and v43193) 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. Bispecific antibody variants comprising the parental anti-IL-4Ra arm (v41790 and v41791; see Table 5.1) were also included. 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.

[0282] 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 incubating supernatant with QUANTI-Blue™ solution (InvivoGen, San Diego, CA) and measuring OD620nm on Synergy™ plate reader (BioTek Instruments, Winooski, VT).

[0283] The results are shown in Figs. 7A-D. Bispecific antibody variants including engineered IL-4Ra paratopes showed a deceased level of inhibition compared to the parental variant (v41790 or v41791; see Table 5.1). However, all tested bispecific antibody variants blocked IL-4 and IL- 13 mediated production of STAT6 inducible SEAP reporter in HEK-Blue™ IL-4 / IL-13 cells.EXAMPLE 9: PRODUCTION AND CHARACTERIZATION OF ANTI-IL-33 x ANTI-IL- 4Ra BISPECIFIC ANTIBODY VARIANTS COMPRISING ENGINEERED ANTI-IL-4Ra ANTIBODY PARATOPES

[0284] Anti-IL-33 x anti-IL-4Ra bispecific antibody variants were prepared using engineered versions of the IL-4Ra paratope v38597 described in Example 7. The engineered IL-4Ra paratopes are described in Example 8, Table 8.1. The CDR sequences of the engineered IL-4Ra paratopes are shown in Fig. 16 (Table A3) and the VH and VL sequences are shown in Fig. 17 (Table B). The anti -IL-33 paratope used was from the anti -IL-31 antibody variant, v33101. The VH and VL sequences for v33101 are provided in Table 9.2.

[0285] 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 5. 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 9.1. All bispecific antibody variants comprised the HetFab, HetFc and S228P mutations described in Example 5, Section 5.2. Inaddition, the bispecific antibody variants further comprise the YTE mutations (M252Y / S254T / T256E) as noted in Table 9.1.Table 9.1: Description of Anti-IL-33 x Anti-IL-4Ra Bispecific Antibody Variants (IgG4)Table 9.2: VH and VL Sequences of Anti-IL-33 Antibody Variant v331019.1 Preparation of Bispecific Anti-IL-33 x Anti-IL-4Ra Antibody Variants

[0286] The antibody variants shown in Table 9.1 were prepared and purified following the protocols described in Example 8 (Section 8.1).

[0287] The apparent purity and yield of each final antibody variant was estimated by UPLC-SEC and LC / MS as described in International Patent Publication No. WO 2015 / 109131. All antibody variants expressed and four of the variants were purified to >80% heterodimer purity without contaminating homodimers as shown in Table 9.3.Table 9.3: Post Purification Yield and Purity for Bispecific Anti-IL-33 x Anti-IL-4RaAntibody Variants (20 mM Histidine pH 6.0 Buffer)* As determined by HPLC-SEC (size-exclusion chromatography HPLC)9.2 Functional Screening of the Engineered Anti-IL-4Ra Paratope in the Bispecific Anti-IL-33 x Anti-IL-4Ra Antibody Variants

[0288] To determine the impact of the engineered IL-4Ra paratopes comprised by the bispecific antibody variants on IL-4 / IL-13 activation of the STAT6 pathway, selected variants (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 in Example 8 (Section 8.2). A bispecific antibody variant comprising the same anti-IL-33 arm and the parental anti-IL-4Ra arm (v42101) was also included (see Table 9.4). 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.

[0289] The results are shown in Figs. 8A-B. Bispecific antibody variants including engineered IL-4Ra paratopes showed a similar level of inhibition compared to the parental variant (v42101).Table 9.4: Description of Anti-IL-33 x Anti-IL-4Ra Bispecific Antibody Variant v42101EXAMPLE 10: PHARMACOKINETIC STUDY OF AN ANTI-IL-31 x ANTI-IL-4RaBISPECIFIC ANTIBODY VARIANT IN CYNOMOLGUS MONKEYS

[0290] Pharmacokinetics (PK) of the anti -IL-31 x anti-IL-4Ra antibody variant v41791 (see Table 5.1) were assessed in vivo in cynomolgus monkeys as described below. The tetravalent, bispecific anti-IL-31 x anti-IL-4Ra antibody NM26-2198 was used as a control antibody.

[0291] 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 infusion), 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. Blood samples were collected throughout the study to monitor clinical chemistry, hematology and coagulation parameters.

[0292] Overall, the study showed that treatment with v41791 was well tolerated in monkeys. No adverse responses were observed throughout the study. Analysis for serum PK showed that v41791 had antibody-like PK similar to that observed with NM26-2198 treatment (see Fig. 9).EXAMPLE 11: PHARMACOKINETIC STUDY OF AN ANTI-IL-33 x ANTI-IL-4Ra BISPECIFIC ANTIBODY VARIANT IN CYNOMOLGUS MONKEYS

[0293] Activity and pharmacokinetics (PK) of the anti-IL-33 x anti-IL-4Ra bispecific antibody variant v42101 (see Table 9.4) was assessed in vivo in cynomolgus monkeys as described below.

[0294] 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 168h, 336h, 504h, and 672h post dose. Blood samples were collected throughout the study to monitor clinical chemistry, hematology and coagulation parameters.

[0295] Treatment with v42101 was well tolerated in monkeys. No adverse responses were observed throughout the study. Analysis for serum PK showed that v42101 had antibody-like PK (see Fig. 10).

[0296] To determine the activity of v42101, 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. Treatment with a single dose of v42101 was sufficient to decrease serum IgE levels up to 28 days post dosing (see Fig. 11).EXAMPLE 12: IN VIVO EFFICACY STUDY OF ANTI-IL-31 x ANTI-IL-4Ra BISPECIFIC ANTIBODY VARIANT IN AN ACUTE HOUSE DUST MITE MOUSE MODEL

[0297] The anti-inflammatory activity of the anti -IL-31 x anti-IL-4Ra antibody variant v41791 (see Table 5.1) 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).

[0298] Briefly, 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 intranasally under 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 by ELISA. 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 wereidentified by flow cytometry using the cellular surface markers: CD45, CD 1 lb, Ly6G, Ly6C, CD3, CD4, CD8, ST2, CD 19, MERTK and Siglec-F.

[0299] The results are shown in Figs. 12A-D. As shown in Fig. 12A, treatment with 25 mg / kg v41791 decreased serum levels of IgE to levels similar to those observed in the no HDM control group. The IgE level reduction was similar to that observed with 25 mg / kg and 10 mg / kg dupilumab treatment.

[0300] To specifically monitor the immune response in the lung, hIL-4 levels were quantified at endpoint. As shown in Fig. 12B, treatment with 25 mg / kg v41791 decreased levels of hIL-4 to levels similar to those observed in the no HDM control group. This decrease was also observed in mice treated with 25 mg / kg and 10 mg / kg dupilumab.

[0301] In addition to monitoring hIL-4 levels in the lung, immune infiltrate was characterized following treatment. As shown in Fig. 12C, tissue resident eosinophils were decreased in number in mice treated with 25 mg / kg v41791 compared to the negative control antibody group. Furthermore, as shown in Fig. 12D, mice treated with 25 mg / kg v41791 had an increased number of alveolar macrophages in the lung compared to the negative control antibody group. Both the changes in eosinophil number and alveolar macrophage number were also observed in mice treated with 25 mg / kg or 10 mg / kg dupilumab.

[0302] The dose response difference observed between v41791 and dupilumab in the levels of serum IgE, lung hIL-4 and lung infiltrate may be attributed to the bivalency of dupilumab compared to the monovalency of the anti-IL-4Ra arm of the v41791 bispecific antibody. Overall, the data shown in Figs. 12A-D indicate that the anti-IL-31 x anti-IL-4Ra bispecific antibody, v41791, is capable of regulating IL-4 and IL- 13 linked immune processes associated with inflammatory disease.EXAMPLE 13: IN VIVO EFFICACY STUDY OF ANTI-IL-33 x ANTI-IL-4Ra BISPECIFIC ANTIBODY VARIANT IN AN ACUTE HOUSE DUST MITE MOUSE MODEL

[0303] The anti-inflammatory activity of the anti-IL-33 x anti-IL-4Ra antibody variant v42101 (see Table 9.4) was assessed in vivo in an acute house dust mite mouse (HDM) model asdescribed in Example 12. Variant v42101 comprises the same IL-4Ra paratope as the anti-IL-31 x anti-IL-4Ra bispecific antibody v41791 that was tested in Example 12. This Example and Example 12 show that the IL-4Ra paratope functions equally well in different bispecific antibody contexts.

[0304] The results are shown in Figs. 13A-G. To determine if v42101 can reduce systemic allergy-associated responses, serum IgE concentrations were quantified following treatment. The results are shown in Fig. 13A. 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.

[0305] 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. 13B (hIL-4) and Fig. 13E (hIL-5). Treatment with 25 mg / kg and 10 mg / kg v42101 decreased levels ofhIL-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.

[0306] 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. 13C and Fig. 13D. Treatment with 25 mg / kg and 10 mg / kg v42101 was sufficient to decrease eosinophil numbers (see Fig. 13C) and increase alveolar macrophage numbers (see Fig. 13D) 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.

[0307] Lung pathology following treatment was evaluated by hemotoxylin and eosin staining. The results are shown in Fig. 13F. Lung sections from mice receiving irrelevant antibody along with HDM showed severe inflammation in the lungs compared to no HDM controls 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 beseen, 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 or v42101 showed much less severe physiological changes to lung architecture as compared to those seen in mice receiving irrelevant antibody controls.

[0308] 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. 13G and show that treatment with 25 mg / kg and 10 mg / kg v42101 decreased the total inflammation score in mice.

[0309] 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 anti-IL-4Ra monoclonal antibody benchmark, dupilumab.EXAMPLE 14: FUNCTIONAL CHARACTERIZATION OF ANTI-IL-33 X ANTI-IL-4Ra BISPECIFIC ANTIBODY VARIANTS IN HEALTHY AND COPD PATIENT SETTINGS - IL-4 PBMC (CD23) ASSAY

[0310] To test the functional impact of blockade of IL-4Ra signalling, 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.

[0311] The antibody variants tested were: v42101 and v43196 (see Table 9.4 and Table 9.1). Dupilumab was used as a positive control for blocking Il-4Ra signalling and a hemagglutininspecific IgG4 antibody (v42104) was used as a non-specific negative control.

[0312] 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 antibody 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 werestained 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.

[0313] The results are shown in Fig. 18. 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.

[0314] 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. 18). 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.

[0315] 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 15: PRODUCTION AND CHARACTERIZATION OF ANTI-IL-31 x ANTLIL- 4Ra BISPECIFIC ANTIBODY VARIANTS COMPRISING ENGINEERED ANTI-IL-4Ra ANTIBODY PARATOPES #2

[0316] Selected antibody variants from Example 8 were re-expressed in CHO3E7 cells at a 500 mL culture volume. In brief, cells were transfected in exponential growth phase (1.5 to 2 million cells / mL) with aqueous 1 mg / mL polyethyleneimine (PEI) using a PEEDNA ratio of 2.5: 1. DNA was transfected at an optimal DNA ratio 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: 15:53: 17 or 15: 15:35:35). Transfected cells were harvested after 5-6 days and the antibody variant recovered from the culture medium as described in Example 8 and quantified based on A280 nm.

[0317] The antibody variants were further purified by gel filtration chromatography using one or two Superdex™ 200 HiLoad™ 26 / 600 200pg columns (Cytiva) in tandem via an AKTA Pure chromatography system following the procedure described in Example 15. 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.

[0318] The apparent purity and yield of the final antibody variant was estimated by UPLC-SEC and LC / MS as generally described in International Patent Publication No. WO 2015 / 109131. All antibody variants expressed and were purified to >93% heterodimer purity without contaminating homodimers as shown in Table 15.1.Table 15.1: Post Purification Yield and Purity for Bispecific Anti-IL-31 x Anti-IL-4RaAntibody Variants (20 mM Histidine pH 5.5 Buffer)1As determined by UPLC-SEC2As determined by LC / MS intensity15.1 Thermal Stability of Bispecific Antibody Variants by Differential Scanning Fluorimetry (DSF)

[0319] The thermal stability of the bispecific anti-IL-31 x anti-IL-4Ra antibody variants was assessed by Differential Scanning Fluorimetry (DSF). All DSF experiments were carried out using a CFX96 Touch™ Real-Time PCR instrument (BioRad Laboratories, Inc., Hercules, CA) as described in Example 3 (Section 3.2.1).

[0320] The results are shown in Table 15.2, which shows the maximum melting temperatures (Tm) for each of the peaks in the thermograms of the tested bispecific antibody variants.Table 15.2: Thermal Stability of Bispecific Antibody Variants15.2 Stability of Bispecific Antibody Variants under Accelerated Stress

[0321] Stability of the bispecific antibody variants v43184, v43185 and v43187 was tested using an accelerated stress test as described below. Purity of the bispecific antibody variants pre- and post-treatment was assessed by UPLC-SEC.

[0322] Bispecific antibody variants were incubated at 40 °C for 14 and 28 days using a protein concentration of 5 mg / mL in 20 mM histidine, pH 5.5 buffer. The results are shown in Table 15.3. All bispecific antibody variants tested showed only a minimal change in purity after the 28-day incubation.Table 15.3: Purity (%) of Bispecific Antibody Variants After 28 Days at 40 °C15.3 Functional Characterization

[0323] The impact of the bispecific antibody variant v43184 on IL-4 / IL-13 activation of the STAT6 signalling pathway was assessed by measuring 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 following the protocol described in Example 8 (Section 8.2). The impact of the bispecific antibody variant on IL-31 activation of the STAT5 signalling pathway was assessed by measuring inhibition of IL-31 mediated production of the STAT5 inducible SEAP reporter in HEK-Blue™ IL-31 cells as described below. The anti-IL-31 x anti-IL-4Ra bispecific antibody variant v41791 (see Table 5.1), atetravalent, bispecific anti -IL-31 x anti -IL-4Ra antibody based on NM26-2198 (Numab Therapeutics), a bivalent anti-IL-31 antibody (BMS-981164), abivalent anti-IL-4Ra antibody (dupilumab) and an IgG4 isotype antibody (v42104) were also included in the assay.

[0324] Briefly, test articles were serially diluted starting at a concentration of 20 nM in DMEM + 10% heat-inactivated fetal bovine serum (ThermoFisher Scientific, Waltham, MA) and plated into a 384-well black flat bottom assay plate. 3 pM IL-31 (R&D Systems, Minneapolis, MN) was added, followed by 12500 HEK-Blue™ IL-31 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).

[0325] The results for IL-4 and IL- 13 mediated production of STAT6 inducible SEAP reporter in HEK-Blue™ IL-4 / IL-13 cells are shown in Figs. 19A & 19B, respectively. Both bispecific antibody variants showed similar inhibitory activity to each other and to both the bivalent anti-IL-4Ra antibody control (dupilumab) and the tetravalent, bispecific anti-IL-31 x anti- IL-4Ra antibody NM26-2198. The anti -IL-31 antibody control (BMS-981164) and the IgG4 isotype control showed no activity as expected.

[0326] The results for IL-31 mediated production of STAT6 inducible SEAP reporter in HEK-Blue™ IL-31 cells are shown in Fig. 19C. Both bispecific antibody variants showed similar inhibitory activity to each other and similar or better inhibitory activity compared to the bivalent anti-IL-31 antibody control (BMS-981164) and the tetravalent, bispecific anti-IL-31 x anti-IL-4Ra antibody NM26-2198. The anti-IL-4Ra antibody control (dupilumab) and the IgG4 isotype control showed no activity as expected.EXAMPLE 16: PHARMACOKINETIC / PHARMACODYNAMIC STUDY OF AN ANTI- IL-31 x ANTI-IL-4Ra BISPECIFIC ANTIBODY VARIANT IN CYNOMOLGUS MONKEYS

[0327] Pharmacokinetic (PK) and pharmacodynamic (PD) parameters for the anti-IL-31 x anti-IL-4Ra antibody variant v44927 were assessed in vivo in naive cynomolgus monkeys as described below. v44927 is identical to v43184 (see Table 8.2) except that v44927 includes a lysine residue at the C-terminus of the heavy chains.

[0328] The test group, consisting of two cynomolgus monkeys (one female and one male), was injected intravenously with 40 mg / kg of v44927. A control group of two cynomolgus monkeys was injected intravenously and subcutaneously with equal volumes of saline. All monkeys were injected once a week for four weeks (QWx4). Injections were performed on days 1, 8, 15, and 22.

[0329] Blood was collected at each pre-dose and 5min after each infusion, 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. 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.

[0330] Treatment with v44927 was well tolerated in monkeys. No adverse responses were observed throughout the study. Analysis for serum PK of the test article -injected monkeys showed that v44927 had antibody-like PK, with a modest decrease in AUC after the second, third, and fourth administration and differing outcomes per test animal (see Fig. 20A).

[0331] To determine the activity of the IL-31 x IL-4Ra bispecific antibody, serum IgE levels were monitored. IL-4 signalling triggers class switch recombination (CSR) in B cells to produce IgE and blocking IL-4 signalling should, therefore, 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 and changes in serum IgE levels are expected to be modest after treatment with the bispecific antibody blocking IL-4Ra.

[0332] Compared to saline-administered monkeys in the control group, treatment with v44927 once a week for four weeks (QWx4) transiently decreased serum IgE levels between days 8 and 15 (see Fig. 20B). A reduction of serum IgE levels has been similarly observed in patients treated with IL-4Ra targeting monoclonal antibody therapies indicating that v44927 can function in a similar manner to clinically approved IL-4Ra targeting monoclonal antibodies.EXAMPLE 17: OXAZOLONE-INDUCED ATOPIC DERMATITIS EFFICACY STUDYOF AN ANTI-IL-31 x ANTI-IL-4Ra BISPECIFIC ANTIBODY VARIANT

[0333] The anti -IL-31 x anti-IL-4Ra antibody variant v44927 (see Example 16) was assessed in vivo in an oxazolone-induced atopic dermatitis mouse model using hIL3 l / hIL3 lRA / hOSMR / hIL4 / hIL4RA C57BL / 6 mice. Body weight, ear thickness and human IL-4 (hIL-4) serum cytokine levels for the mice were assessed as described below.

[0334] Three groups of hIL3 l / hIL3 lRA / hOSMR / hIL4 / hIL4RA C57BL / 6 mice were used with six mice allocated to each study group. The first group was treated subcutaneously with 25 mg / kg v44927. The second group was treated with 25 mg / kg isotype control antibody. The third group of animals was left untreated. The test article and isotype control administrations were performed twice a week for a total of eight doses, on days -1, 2, 6, 9, 13, 16, 20 and 23. Induction of atopic dermatitis was initiated by application of 0.8% oxazolone solution (in a 4: 1 mixture of acetone and olive oil) onto the right ear of each antibody-treated animal on day 0, followed by application of 0.4% oxazolone solution on days 7, 9, 11, 13, 16, 20 and 23. The third group of untreated animals received a vehicle solution only (4: 1 mixture of acetone and olive oil).

[0335] Body weight and ear thickness (measured by caliper) were recorded on days -2, 0, 7, 9, 11, 14, 16, 18, 21, 23, 25 and 26. Blood was collected on days 9 and 26 to determine hIL-4 serum cytokine concentrations using the Mesoscale Discovery (MSD) method.

[0336] Body weight of the treated mice did not change over the course of the experiment (see Fig. 21A) indicating that the oxazolone-mediated interventions, as well as test article (v44927) and isotype administrations, were well tolerated with no adverse events observed throughout the study. Compared to isotype-treated animals, treatment with v44927 significantly reduced ear thickness on days 14, 16, and 18 after initial oxazolone sensitization on day 0 (see Fig. 261B). In addition, compared to isotype-treated mice, treatment with v44927 significantly diminished hIL-4 serum cytokine concentrations on days 9 and 26 after initial oxazolone sensitization on day 0 (see Fig. 21C). The hIL-4 serum cytokine concentrations in animals treated with v44927 were similar to those detected in vehicle-treated control mice.EXAMPLE 18: BINDING AFFINITY OF AN ANTLIL-33 x ANTI-IL-4Ra BISPECIFIC ANTIBODY VARIANT TO IL-4Ra

[0337] The equilibrium binding affinity of the IL-4Ra paratope comprised by the anti-IL-33 x anti-IL-4Ra bispecific antibody variant, v42101 (see Table 9.4), to human and cynomolgus IL-4Ra was determined using a KinExA™ instrument (Sapidyne Inc., Boise, ID) as described below. The anti-IL-4Ra antibody, dupilumab, was included for comparison.

[0338] 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 18.1.Table 18.1: Binding Affinity (KD) of v42101 to Human IL-4Ra and Cynomolgus IL-4RaEXAMPLE 19: PHARMACOKINETIC / PHARMACODYNAMIC STUDY OF AN ANTI- IL-33 x ANTI-IL-4Ra BISPECIFIC ANTIBODY VARIANT IN CYNOMOLGUS MONKEYS

[0339] Pharmacokinetics (PK), pharmacodynamics (PD) and toxicity parameters for the anti-IL-33 x anti-IL-4Ra bispecific antibody variant v42101 (see Table 9.4) were assessed in vivo in naive cynomolgus monkeys as described below.

[0001] 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.

[0002] 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.

[0003] 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. 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 accumulation in AUC between dose 1 and 4 (see Fig. 22). 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 postdoses 1 and 4 compared to the monkeys intravenously injected with the same dose of v42101 (40 mg / kg).

[0004] 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 and blocking IL-4 signalling should, therefore, 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 and changes in serum IgE levels are expected to be modest after treatment with the bispecific antibody blocking IL-4Ra.

[0005] 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. 23). 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.

[0340] 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.

[0341] 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 C: Clone Numbers for Monospecific VariantsTable D: Clone SequencesTable E: Clone Numbers for Bispecific VariantsTable F: Clone SequencesTable G: Clone Sequences

Claims

1. 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-4Ra antigen-binding domain that specifically binds to human IL-4Ra, the IL-4Ra antigen-binding domain comprising the CDR sequences (HCDR1, HCDR2, HCDR3) of the VH domain as set forth in any one of SEQ ID NOs: 95, 97, 98, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85 or 86, and the CDR sequences (LCDR1, LCDR2, LCDR3) of the VL domain as set forth in any one of SEQ ID NOs: 96, 99, 100, 111, 112, 87, 88, 89, 90, 91, 92, 93 or 94.

2. The antibody construct according to claim 1, wherein the IL-4Ra antigen-binding domain comprises the CDR sequences (HCDR1, HCDR2, HCDR3) of the VH domain as set forth in any one of SEQ ID NOs: 103, 110, 75 or 78, and the CDR sequences (LCDR1, LCDR2, LCDR3) of the VL domain as set forth in SEQ ID NO: 112 or 91.

3. The antibody construct according to claim 1, wherein 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: 6, 27, 32, 37 or 43, an HCDR2 amino acid sequence comprising the sequence as set forth in any one of SEQ ID NOs: 7, 16, 62, 66, 67, 68, 69, 70, 71 or 72, and an HCDR3 amino acid sequence comprising the sequence as set forth in any one of SEQ ID NOs: 8, 44, 50 or 53, and a VL domain comprising an LCDR1 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 21, an LCDR2 amino acid sequence comprising the sequence as set forth in any one of SEQ ID NOs: 22, 56, 59 or 73, and an LCDR3 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 20.

4. The antibody construct according to claim 1, 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: 3, 26, 31 or 36, an HCDR2 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 4 or 61, and an HCDR3 amino acid sequence comprising the sequence as set forth in any one of SEQ ID NOs: 5, 42, 49 or 52, and a VL domain comprising an LCDR1 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 18, an LCDR2 amino acid sequence comprising the sequence as set forth in any one of SEQ ID NOs: 19, 55 or 58, and an LCDR3 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 20.

5. The antibody construct according to claim 1, 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: 26, an HCDR2 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 61, and an HCDR3 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 5, and a VL domain comprising an LCDR1 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 18, an LCDR2 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 55, and an LCDR3 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 20.

6. The antibody construct according to any one of claims 1 to 5, wherein the IL-4Ra antigen binding domain comprises a VH domain comprising a sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the VH sequence as set forth in any one of SEQ ID NOs: 95, 97, 98, 101,102, 103, 104, 105, 106, 107, 108, 109, 110, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85 or 86.

7. The antibody construct according to any one of claims 1 to 6, wherein the IL-4Ra antigen binding domain comprises a VL domain comprising a sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the VL sequence as set forth in any one of SEQ ID NOs: 96, 99, 100, 111, 112, 87, 88, 89, 90, 91, 92, 93 or 94.

8. The antibody construct according to any one of claims 1 to 5, wherein the IL-4Ra antigen binding domain comprises a VH domain comprising a sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the VH sequence as set forth in SEQ ID NO: 103, 110, 75 or 78.

9. The antibody construct according to any one of claims 1 to 5 and 8, wherein the IL-4Ra antigen binding domain comprises a VL domain comprising a sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the VL sequence as set forth in SEQ ID NO: 112 or 91.

10. The antibody construct according to claim 1, wherein the IL-4Ra antigen binding domain comprises a VH domain comprising a sequence as set forth in SEQ ID NO: 103, 110, 75 or 78, and a VL domain comprising a sequence as set forth in SEQ ID NO: 112 or 91.

11. The antibody construct according to claim 1, wherein the IL-4Ra antigen binding domain comprises:(i) a VH domain comprising a sequence as set forth in SEQ ID NO: 75 and a VL domain comprising a sequence as set forth in SEQ ID NO: 112, or(ii) a VH domain comprising a sequence as set forth in SEQ ID NO: 78 and a VL domain comprising a sequence as set forth in SEQ ID NO: 91.

12. The antibody construct according to any one of claims 1 to 11, wherein the IL-4Ra antigen binding domain is a Fab or an scFv.

13. The antibody construct according to any one of claim 1 to 12, wherein the antibody construct comprises two, three or four antigen-binding domains.

14. The antibody construct according to any one of claims 1 to 13, wherein the antibody construct comprises two IL-4Ra antigen-binding domains.

15. The antibody construct according to claim 14, wherein the two IL-4Ra antigen-binding domains are the same.

16. The antibody construct according to any one of claims 1 to 15, further comprising a scaffold, wherein the IL-4Ra antigen-binding domain is operably linked to the scaffold.

17. The antibody construct according to any one of claims 1 to 12, wherein the antibody construct comprises a second target antigen-binding domain that specifically binds to a second target antigen, and wherein the second target antigen is other than IL-4Ra.

18. The antibody construct according to claim 17, wherein the second target antigen is a cytokine or cytokine receptor.

19. The antibody construct according to claim 17 or claim 18, further comprising a scaffold, wherein the IL-4Ra antigen-binding domain and the second target antigen-binding domain are both operably linked to the scaffold.

20. The antibody construct according to claim 16 or claim 19, wherein the scaffold is an IgG Fc region.

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

22. The antibody construct according to claim 20 or claim 21, wherein the IgG Fc region is an IgGl or IgG4 Fc region.

23. The antibody construct according to any one of claims 20 to 22, wherein the IgG Fc region is a heterodimeric Fc region comprising a first Fc polypeptide and a second Fc polypeptide.

24. The antibody construct according to claim 23, 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.

25. The antibody construct according to claim 24, 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.

26. The antibody construct according to any one of claims 20 to 25, wherein the IgG Fc region comprises the amino acid substitutions M252Y, S254T and T256E, and wherein the numbering of amino acids is EU numbering.

27. The antibody construct according to any one of claims 20 to 26, wherein the IgG Fc region is an IgGl Fc region.

28. The antibody construct according to claim 27, wherein the IgGl Fc region comprises the amino acid substitutions L234A, L235A and D265S, and wherein the numbering of amino acids is EU numbering.

29. The antibody construct according to any one of claims 20 to 26, wherein the IgG Fc region is an IgG4 Fc region.

30. The antibody construct according to claim 29, wherein the IgG4 Fc region comprises the amino acid substitution S228P and / or the amino acid substitution R409K.

31. A polynucleotide or set of polynucleotides encoding the antibody construct according to any one of claims 1 to 30.

32. An expression vector or set of expression vectors comprising the polynucleotide or set of polynucleotides according to claim 31.

33. A host cell comprising the polynucleotide or set of polynucleotides according to claim 31 or the expression vector or set of expression vectors according to claim 32.

34. A method of preparing the antibody construct according to any one of claims 1 to 30 comprising transfecting a host cell with the polynucleotide or set of polynucleotides according to claim 31 or the expression vector or set of expression vectors according to claim 32, and culturing the host cell under conditions suitable for expression of the antibody construct.

35. A multispecific antibody construct comprising an IL-4Ra antigen-binding domain that specifically binds to human IL-4Ra and one or more additional antigen-binding domains, wherein each additional antigen-binding domain binds to an antigen other than IL-4Ra, and wherein the IL-4Ra antigen-binding domain comprises the CDR sequences (HCDR1, HCDR2, HCDR3) of the VH domain as set forth in any one of SEQ ID NOs: 95, 97, 98, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85 or 86, and the CDR sequences (LCDR1, LCDR2, LCDR3) of the VL domain as set forth in any one of SEQ ID NOs: 96, 99, 100, 111, 112, 87, 88, 89, 90, 91, 92, 93 or 94.

36. The multispecific antibody construct according to claim 35, wherein at least one of the additional antigen-binding domains binds to a cytokine or cytokine receptor.

37. The multispecific antibody construct according to claim 35 or claim 36, wherein the IL- 4Ra antigen-binding domain comprises the CDR sequences (HCDR1, HCDR2, HCDR3) of the VH domain as set forth in any one of SEQ ID NOs: 103, 110, 75 or 78, and the CDR sequences (LCDR1, LCDR2, LCDR3) of the VL domain as set forth in SEQ ID NO: 112 or 91.

38. The multispecific antibody construct according to claim 35 or claim 36, wherein 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: 6, 27, 32, 37 or 43, an HCDR2 amino acid sequence comprising the sequence as set forth in any one of SEQ ID NOs: 7, 16, 62, 66, 67, 68, 69, 70, 71 or 72, and an HCDR3 amino acid sequence comprising the sequence as set forth in any one of SEQ ID NOs: 8, 44, 50 or 53, and a VL domain comprising an LCDR1 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 21, an LCDR2 amino acid sequence comprising the sequence as set forth in any one of SEQ ID NOs: 22, 56, 59 or 73, and an LCDR3 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 20.

39. The multispecific antibody construct according to claim 35 or claim 36, 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: 3, 26, 31 or 36, an HCDR2 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 4 or 61, and an HCDR3 amino acid sequence comprising the sequence as set forth in any one of SEQ ID NOs: 5, 42, 49 or 52, and a VL domain comprising an LCDR1 amino acid sequence comprising the sequence as set forth inSEQ ID NO: 18, an LCDR2 amino acid sequence comprising the sequence as set forth in any one of SEQ ID NOs: 19, 55 or 58, and an LCDR3 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 20.

40. The multispecific antibody construct according to claim 35 or claim 36, 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: 26, an HCDR2 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 61, and an HCDR3 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 5, and a VL domain comprising an LCDR1 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 18, an LCDR2 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 55, and an LCDR3 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 20.

41. The multispecific antibody construct according to any one of claims 35 to 40, wherein the IL-4Ra antigen binding domain comprises a VH domain comprising a sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the VH sequence as set forth in any one of SEQ ID NOs: 95, 97, 98, 101,102, 103, 104, 105, 106, 107, 108, 109, 110, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85 or 86.

42. The multispecific antibody construct according to any one of claims 35 to 41, wherein the IL-4Ra antigen binding domain comprises a VL domain comprising a sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the VL sequence as set forth in any one of SEQ ID NOs: 96, 99, 100, 111, 112, 87, 88, 89, 90, 91, 92, 93 or 94.

43. The multispecific antibody construct according to any one of claims 35 to 40, wherein the IL-4Ra antigen binding domain comprises a VH domain comprising a sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the VH sequence as set forth in any one of SEQ ID NOs: 103, 110, 75 or 78.

44. The multispecific antibody construct according to any one of claims 35 to 40 and 43, wherein the IL-4Ra antigen binding domain comprises a VL domain comprising a sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the VL sequence as set forth in SEQ ID NO: 112 or 91.

45. The multispecific antibody construct according to claim 35 or claim 36, wherein the IL- 4Ra antigen binding domain comprises a VH domain comprising a sequence as set forth in any one of SEQ ID NO: 103, 110, 75 or 78, and a VL domain comprising a sequence as set forth in SEQ ID NO: 112 or 91.

46. The multispecific antibody construct according to claim 35 or claim 36, wherein the IL- 4Ra antigen binding domain comprises:(i) a VH domain comprising a sequence as set forth in SEQ ID NO: 75 and a VL domain comprising a sequence as set forth in SEQ ID NO: 112, or(ii) a VH domain comprising a sequence as set forth in SEQ ID NO: 78 and a VL domain comprising a sequence as set forth in SEQ ID NO: 91.

47. The multispecific antibody construct according to any one of claims 35 to 46, wherein the one or more additional antigen-binding domains are one, two or three additional antigen-binding domains.

48. A multispecific antibody construct comprising an IL-4Ra antigen-binding domain that specifically binds to human IL-4Ra and one, two or three additional antigen-binding domains, wherein each additional antigen-binding domains binds to an antigen other than IL-4Ra, 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: 26, an HCDR2 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 61, and an HCDR3 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 5, and a VL domain comprising an LCDR1 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 18, an LCDR2 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 55, and an LCDR3 amino acid sequence comprising the sequence as set forth in SEQ ID NO: 20, and wherein at least one of the additional antigen-binding domains binds to a cytokine or cytokine receptor.

49. The multispecific antibody construct according to any one of claims 35 to 48, wherein the IL-4Ra antigen-binding domain is a Fab or an scFv.

50. The multispecific antibody construct according to any one of claims 35 to 49, wherein each additional antigen-binding domain is independently a Fab or an scFv.

51. The multispecific antibody construct according to any one of claims 35 to 50 further comprising a scaffold, wherein the IL-4Ra antigen-binding domain and at least one additional antigen-binding domain are operably linked to the scaffold.

52. The multispecific antibody construct according to claim 51, wherein the scaffold is an IgG Fc region.

53. The multispecific antibody construct according to claim 51 or claim 52, wherein the scaffold is a human IgG Fc region.

54. The multispecific antibody construct according to claim 52 or claim 53, wherein the IgG Fc region is an IgGl or IgG4 Fc region.

55. The multispecific antibody construct according to any one of claims 52 to 54, wherein the IgG Fc region is a heterodimeric Fc region comprising a first Fc polypeptide and a second Fc polypeptide.

56. The multispecific antibody construct according to claim 55, 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.

57. The multispecific antibody construct according to claim 56, 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.

58. The multispecific antibody construct according to any one of claims 52 to 57, wherein the IgG Fc region comprises the amino acid substitutions M252Y, S254T and T256E, and wherein the numbering of amino acids is EU numbering.

59. The multispecific antibody construct according to any one of claims 52 to 58, wherein the IgG Fc region is an IgGl Fc region.

60. The multispecific antibody construct according to claim 59, wherein the IgGl Fc region comprises the amino acid substitutions L234A, L235A and D265S, and wherein the numbering of amino acids is EU numbering.

61. The multispecific antibody construct according to any one of claims 52 to 58, wherein the IgG Fc region is an IgG4 Fc region.

62. The multispecific antibody construct according to claim 61, wherein the IgG4 Fc region comprises the amino acid substitution S228P and / or the amino acid substitution R409K.

63. A polynucleotide or set of polynucleotides encoding the multispecific antibody construct according to any one of claims 35 to 62.

64. An expression vector or set of expression vectors comprising the polynucleotide or set of polynucleotides according to claim 63.

65. A host cell comprising the polynucleotide or set of polynucleotides according to claim 63 or the expression vector or set of expression vectors according to claim 64.

66. A method of preparing the multispecific antibody construct according to any one of claims 35 to 62 comprising transfecting a host cell with the polynucleotide or set of polynucleotides according to claim 63 or the expression vector or set of expression vectors according to claim 64, and culturing the host cell under conditions suitable for expression of the multispecific antibody construct.

67. A pharmaceutical composition comprising the antibody construct according to any one of claims 1 to 30 or the multispecific antibody construct according to any one of claims 35 to 62.

68. An antibody construct according to any one of claims 1 to 30 or a multispecific antibody construct according to any one of claims 35 to 62 for use in therapy.

69. The antibody construct or multispecific antibody construct for use according to claim 68, wherein the therapy comprises treatment of an inflammatory or autoimmune disease in a subject.

70. Use of an antibody construct according to any one of claims 1 to 30 or a multispecific antibody construct according to any one of claims 35 to 62 in the manufacture of a medicament.

71. The use according to claim 70, wherein the medicament is for treatment of an inflammatory or autoimmune disease in a subject.

72. 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 30 or a multispecific antibody construct according to any one of claims 35 to 62.

73. Use of an antibody construct according to any one of claims 1 to 30 or a multispecific antibody construct according to any one of claims 35 to 62 in the treatment of an inflammatory or autoimmune disease in a subject.

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