Human interleukin-4 receptor alpha antibody glucocorticoid conjugates

Abstract

The present disclosure provides human interleukin-4 receptor alpha antibody glucocorticoid agonist conjugates and methods of using these conjugates to treat inflammatory diseases, such as type 2 inflammatory diseases.

Description

Technical Field

[0001] The present disclosure provides human interleukin-4 receptor alpha antibody glucocorticoid receptor agonist conjugates; methods of using these conjugates to treat inflammatory diseases, such as type 2 inflammatory diseases, such as atopic dermatitis, eosinophilic esophagitis, nasal polyposis, asthma, chronic sinusitis, allergic diseases, chronic obstructive pulmonary disease, or chronic spontaneous urticaria; methods for preparing these conjugates; and pharmaceutical compositions containing these conjugates. Background Art

[0002] Atopic dermatitis is a chronic, pruritic, relapsing and remitting inflammatory skin disease that occurs frequently in children but also affects many adults. Current treatments for atopic dermatitis include light therapy, topical creams containing corticosteroids or calcineurin inhibitors, or a subcutaneously injectable biologic called dupilumab. Despite advances in the treatment of atopic dermatitis, there remains a great need for new compounds that treat atopic dermatitis and other inflammatory and autoimmune diseases and that minimize or eliminate the shortcomings of currently approved treatments.

[0003] WO2017 / 210471 discloses certain glucocorticoid receptor agonists (GC) and immunoconjugates thereof that can be used to treat inflammatory diseases. WO2018 / 089373 discloses novel steroids, protein conjugates thereof, and methods for treating diseases, disorders, and conditions, the methods comprising administering steroids and conjugates. To date, there are no known human IL-4RαGC conjugates under development for the treatment of inflammatory diseases. Summary of the invention

[0004] The present disclosure provides certain novel human IL-4Rα antibody GC conjugates, wherein the antibody binds to human IL-4Rα. The present disclosure also provides compositions comprising novel anti-human IL-4Rα antibody GC conjugates and methods of using such anti-human IL-4Rα antibody GC conjugates and compositions thereof. The present disclosure also provides certain novel anti-human IL-4Rα GC conjugates, which can be used to treat inflammatory diseases, such as type 2 inflammatory diseases, such as atopic dermatitis, eosinophilic esophagitis, nasal polyposis, asthma, chronic sinusitis, allergic diseases, chronic obstructive pulmonary disease, or chronic spontaneous urticaria.

[0005] Certain anti-human IL-4Rα antibody GC conjugates provided herein have one or more of the following properties: 1) bind to a neo-epitope spanning the n-terminal fibronectin type III domains 1 and 2 of human and / or cynomolgus monkey IL-4Rα, 2) bind to human IL-4Rα with a desired binding affinity, 3) bind to cynomolgus monkey IL-4Rα with a desired binding affinity, 4) bind to human IL-4Rα on the cell surface and internalize into cells, 5) bind to human IL-4Rα and inhibit the binding of IL-4 and IL-13 to IL-4Rα, 6) bind to human IL-4Rα in vitro and inhibit IL-4R-mediated responses (pSTAT6 in B and T cells, B cell proliferation, CD23 expression, cytokine production (e.g., MDC, GM-CSF, etc.) F)), 7) binds to a unique functional epitope on human and / or cynomolgus monkey IL-4Rα, 8) modulates glucocorticoid receptor agonist-mediated responses in vitro (induces CD163 expression, inhibits B cell proliferation, inhibits cytokine production (e.g., IL-5, GM-CSF)), 9) modulates glucocorticoid receptor agonist-mediated gene expression (upregulation: Tsc22d3, Fkbp5, Zbtb16; downregulation: IL-5), 10) does not significantly induce ADCC or CDC activity, 11) retains Fcγ receptor binding to B cells and / or myeloid cells, 12) inhibits inflammatory responses in vivo, or 13) has good developmental characteristics, such as acceptable viscosity, solubility and aggregation stability, to facilitate development, manufacturing and / or formulation.

[0006] Thus, in one embodiment, the present disclosure provides a conjugate of Formula I:

[0007]

[0008] wherein Ab is an antibody that binds to human interleukin-4 receptor alpha ("anti-human IL-4Rα antibody"), and wherein

[0009] for:

[0010] or

[0011]

[0012] And n is 1 to 5.

[0013] In some embodiments, the Ab in the conjugate of Formula I binds to an epitope of human IL-4Rα, wherein the epitope comprises one or more amino acid residues selected from the group consisting of: D12, M14, S15, I16, Y37, L39, F41, L42, L43, E45, H47, T48, C49, I50, E52, H62, L64, M65, D66, D67, V68, V69, D72, R99, P121, P123, P124, D125, P192 (amino acid residue positions corresponding to SEQ ID NO: 15). In some embodiments, the Ab in the conjugate of Formula I binds to an epitope of human IL-4Rα, wherein the epitope comprises at least one, at least two, at least three, at least four, or at least five or more amino acid residues selected from the following: D12, M14, S15, I16, Y37, L39, F41, L42, L43, E45, H47, T48, C49, I50, E52, H62, L64, M65, D66, D67, V68, V69, D72, R99, P121, P123, P124, D125, P192 (amino acid residue positions corresponding to SEQ ID NO: 15). In some embodiments, the Ab in the conjugate of Formula I binds to an epitope of human IL-4Rα, wherein the epitope comprises one or more amino acid residues selected from the group consisting of: D12, M14, S15, I16, L39, F41, L42, T48, C49, I50, E52, H62, L64, M65, D66, D67, V68, V69, D72, R99, P121, P123, P124, D125, P192 (amino acid residue positions corresponding to SEQ ID NO: 15). In some embodiments, the Ab in the conjugate of Formula I binds to an epitope of human IL-4Rα, wherein the epitope comprises at least one, at least two, at least three, at least four, at least five or more amino acid residues selected from the group consisting of D12, M14, S15, I16, L39, F41, L42, T48, C49, I50, E52, H62, L64, M65, D66, D67, V68, V69, D72, R99, P121, P123, P124, D125, P192 (amino acid residue positions corresponding to SEQ ID NO: 15). In some embodiments, the Ab in the conjugate of Formula I binds to an epitope of human IL-4Rα, wherein the epitope comprises one or more amino acid residues selected from the group consisting of D12, M14, S15, I16, Y37, L39, F41, L43, E45, H47, T48, C49, I50, H62, L64, M65, D66, D67, V69, D72, R99, P121, P123, P124, D125 (amino acid residue positions corresponding to SEQ ID NO: 15).In some embodiments, the Ab in the conjugate of Formula I binds to an epitope of human IL-4Rα, wherein the epitope comprises at least one, at least two, at least three, at least four, at least five or more amino acid residues selected from the group consisting of D12, M14, S15, I16, Y37, L39, F41, L43, E45, H47, T48, C49, I50, H62, L64, M65, D66, D67, V69, D72, R99, P121, P123, P124, D125 (amino acid residue positions corresponding to SEQ ID NO: 15). In some embodiments, the Ab in the conjugate of Formula I binds to an epitope of human IL-4Rα, wherein the epitope comprises one or more amino acid residues selected from the group consisting of: D12, M14, S15, I16, Y37, L39, T48, C49, I50, E52, H62, M65, R99, P121, P123, P124, D125, P192 (amino acid residue positions corresponding to SEQ ID NO: 15). In some embodiments, the Ab in the conjugate of Formula I binds to an epitope of human IL-4Rα, wherein the epitope comprises at least one, at least two, at least three, at least four, at least five or more amino acid residues selected from the group consisting of D12, M14, S15, I16, Y37, L39, T48, C49, I50, E52, H62, M65, R99, P121, P123, P124, D125, P192 (amino acid residue positions corresponding to SEQ ID NO: 15). In some embodiments, the Ab in the conjugate of Formula I binds to an epitope of human IL-4Rα, wherein the epitope comprises one or more amino acid residues selected from the group consisting of R99, P121, P123, P124, D125, P192 (amino acid residue positions corresponding to SEQ ID NO: 15), wherein these residues are located in domain 2 of the N-terminal fibronectin type III domain of IL-4Rα. In some embodiments, the Ab in the conjugate of Formula I binds to an epitope of human IL-4Rα, wherein the epitope comprises one or more amino acid residues selected from the group consisting of: D66, D67, and D125 (amino acid residue positions corresponding to SEQ ID NO: 15). In some embodiments, the Ab in the conjugate of Formula I binds to an epitope of human IL-4Rα, wherein the epitope comprises at least one amino acid residue selected from the group consisting of: D66 and D67 (amino acid residue positions corresponding to SEQ ID NO: 15). In some embodiments, the Ab in the conjugate of Formula I binds to an epitope of human IL-4Rα, wherein the epitope comprises at least one amino acid residue selected from the group consisting of: D66 and D125 (amino acid residue positions corresponding to SEQ ID NO: 15).In some embodiments, the Ab in the conjugate of Formula I binds to an epitope of human IL-4Rα, wherein the epitope comprises amino acid residue D66 (amino acid residue position corresponds to SEQ ID NO: 15). In yet other embodiments, the Ab in the conjugate of Formula I binds to a new structural and / or functional epitope of human IL-4Rα, wherein the epitope spans domain 1 and domain 2 of the n-terminal fibronectin type III domain of IL-4Rα. In specific embodiments, the Ab in the conjugate of Formula I binds to a new structural and / or functional epitope of human IL-4Rα, wherein the epitope overlaps with the IL-4 binding site of IL-4Rα. In such embodiments, the Ab in the conjugate of Formula I blocks the binding of IL-4 to human IL-4Rα. In specific embodiments, the Ab in the conjugate of Formula I binds to a new structural and / or functional epitope of human IL-4Rα, wherein the epitope overlaps with the IL-13 binding site of IL-4Rα. In such embodiments, the Ab in the conjugate of Formula I blocks the binding of IL-13 to human IL-4Rα. In a specific embodiment, the Ab in the conjugate of Formula I binds to a new structural and / or functional epitope of human IL-4Rα, wherein the epitope overlaps with the IL-4 and IL-13 binding sites of IL-4Rα. In such embodiments, the Ab in the conjugate of Formula I blocks the binding of IL-4 and IL-13 to human IL-4Rα. In some embodiments, the conjugate of Formula I, wherein the IL-4Rα epitope is determined by X-ray crystallography, alanine scanning mutagenesis, steric mutagenesis and / or HDX-MS. In yet other embodiments, the IL-4Rα epitope is determined by site-directed mutagenesis.

[0014] In further embodiments, the present disclosure provides a conjugate of Formula I:

[0015]

[0016] wherein Ab is an antibody that binds to human interleukin-4 receptor alpha ("anti-human IL-4Rα antibody"), wherein Ab comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein VH comprises heavy chain complementarity determining regions HCDR1, HCDR2, and HCDR3, and VL comprises light chain complementarity determining regions LCDR1, LCDR2, and LCDR3, wherein:

[0017] HCDR1 comprises SEQ ID NO: 1, 19 or 42;

[0018] HCDR2 comprises SEQ ID NO: 2 or 20;

[0019] HCDR3 comprises SEQ ID NO: 3;

[0020] LCDR1 comprises SEQ ID NO: 4 or 22;

[0021] LCDR2 comprises SEQ ID NO:5; and

[0022] LCDR3 comprises SEQ ID NO: 6 or 24;

[0023] And among them

[0024] for:

[0025] or

[0026]

[0027] And n is 1 to 5.

[0028] In further embodiments, the present disclosure provides a conjugate of Formula Ia:

[0029]

[0030] Wherein Ab is an antibody that binds to human IL-4Rα, wherein Ab comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein VH comprises heavy chain complementary determining regions HCDR1, HCDR2 and HCDR3, and VL comprises light chain complementary determining regions LCDR1, LCDR2 and LCDR3, wherein:

[0031] HCDR1 comprises SEQ ID NO: 1, 19 or 42;

[0032] HCDR2 comprises SEQ ID NO: 2 or 20;

[0033] HCDR3 comprises SEQ ID NO: 3;

[0034] LCDR1 comprises SEQ ID NO: 4 or 22;

[0035] LCDR2 comprises SEQ ID NO:5; and

[0036] LCDR3 comprises SEQ ID NO: 6 or 24;

[0037] And among them

[0038] for:

[0039] or

[0040]

[0041] And n is 1 to 5.

[0042] In further embodiments, the present disclosure provides a conjugate of Formula Ib:

[0043]

[0044] Wherein Ab is an antibody that binds to human IL-4Rα, wherein Ab comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein VH comprises heavy chain complementary determining regions HCDR1, HCDR2 and HCDR3, and VL comprises light chain complementary determining regions LCDR1, LCDR2 and LCDR3, wherein:

[0045] HCDR1 comprises SEQ ID NO: 1, 19 or 42;

[0046] HCDR2 comprises SEQ ID NO: 2 or 20;

[0047] HCDR3 comprises SEQ ID NO: 3;

[0048] LCDR1 comprises SEQ ID NO: 4 or 22;

[0049] LCDR2 comprises SEQ ID NO:5; and

[0050] LCDR3 comprises SEQ ID NO: 6 or 24;

[0051] And among them

[0052] for:

[0053] or

[0054]

[0055] And n is 1 to 5.

[0056] In further embodiments, the present disclosure provides a conjugate of Formula Ic:

[0057]

[0058] Wherein Ab is an antibody that binds to human IL-4Rα, wherein Ab comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein VH comprises heavy chain complementary determining regions HCDR1, HCDR2 and HCDR3, and VL comprises light chain complementary determining regions LCDR1, LCDR2 and LCDR3, wherein:

[0059] HCDR1 comprises SEQ ID NO: 1, 19 or 42;

[0060] HCDR2 comprises SEQ ID NO: 2 or 20;

[0061] HCDR3 comprises SEQ ID NO: 3;

[0062] LCDR1 comprises SEQ ID NO: 4 or 22;

[0063] LCDR2 comprises SEQ ID NO:5; and

[0064] LCDR3 comprises SEQ ID NO: 6 or 24;

[0065] And among them

[0066] for:

[0067] or

[0068]

[0069] And n is 1 to 5.

[0070] In further embodiments, the present disclosure provides a conjugate of Formula Id:

[0071]

[0072] Wherein Ab is an antibody that binds to human IL-4Rα, wherein Ab comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein VH comprises heavy chain complementary determining regions HCDR1, HCDR2 and HCDR3, and VL comprises light chain complementary determining regions LCDR1, LCDR2 and LCDR3, wherein:

[0073] HCDR1 comprises SEQ ID NO: 1, 19 or 42;

[0074] HCDR2 comprises SEQ ID NO: 2 or 20;

[0075] HCDR3 comprises SEQ ID NO: 3;

[0076] LCDR1 comprises SEQ ID NO: 4 or 22;

[0077] LCDR2 comprises SEQ ID NO:5; and

[0078] LCDR3 comprises SEQ ID NO: 6 or 24;

[0079] And among them

[0080] for:

[0081] or

[0082]

[0083] And n is 1 to 5.

[0084] In further embodiments, the present disclosure provides a conjugate of Formula Ie:

[0085]

[0086] Wherein Ab is an antibody that binds to human IL-4Rα, wherein Ab comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein VH comprises heavy chain complementary determining regions HCDR1, HCDR2 and HCDR3, and VL comprises light chain complementary determining regions LCDR1, LCDR2 and LCDR3, wherein:

[0087] HCDR1 comprises SEQ ID NO: 1, 19 or 42;

[0088] HCDR2 comprises SEQ ID NO: 2 or 20;

[0089] HCDR3 comprises SEQ ID NO: 3;

[0090] LCDR1 comprises SEQ ID NO: 4 or 22;

[0091] LCDR2 comprises SEQ ID NO:5; and

[0092] LCDR3 comprises SEQ ID NO: 6 or 24;

[0093] And among them

[0094] for:

[0095]

[0096] And n is 1 to 5.

[0097] In further embodiments, the present disclosure provides a conjugate of Formula If:

[0098]

[0099] Wherein Ab is an antibody that binds to human IL-4Rα, wherein Ab comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein VH comprises heavy chain complementary determining regions HCDR1, HCDR2 and HCDR3, and VL comprises light chain complementary determining regions LCDR1, LCDR2 and LCDR3, wherein:

[0100] HCDR1 comprises SEQ ID NO: 1, 19 or 42;

[0101] HCDR2 comprises SEQ ID NO: 2 or 20;

[0102] HCDR3 comprises SEQ ID NO: 3;

[0103] LCDR1 comprises SEQ ID NO: 4 or 22;

[0104] LCDR2 comprises SEQ ID NO:5; and

[0105] LCDR3 comprises SEQ ID NO: 6 or 24;

[0106] And among them

[0107] for:

[0108]

[0109] And n is 1 to 5.

[0110] In an embodiment, n is 2-5.

[0111] In an embodiment, n is 3-5.

[0112] In an embodiment, n is 3 to 4.

[0113] In an embodiment, n is about 4.

[0114] In an embodiment, n is about 3.

[0115] In an embodiment, n is about 2.

[0116] In some embodiments, Ab comprises VH and VL, wherein VH comprises heavy chain complementary determining regions HCDR1, HCDR2, and HCDR3, and VL comprises light chain complementary determining regions LCDR1, LCDR2, and LCDR3, wherein HCDR1 comprises SEQ ID NO:1, HCDR2 comprises SEQ ID NO:2, HCDR3 comprises SEQ ID NO:3, LCDR1 comprises SEQ ID NO:4, LCDR2 comprises SEQ ID NO:5, and LCDR3 comprises SEQ ID NO:6. In some embodiments, Ab comprises VH comprising SEQ ID NO:7 and VL comprising SEQ ID NO:8. In some embodiments, Ab is Abl, wherein Abl comprises HC comprising SEQ ID NO:9 and LC comprising SEQ ID NO:10. In some embodiments, Ab is Ab2, wherein Ab2 comprises HC comprising SEQ ID NO:50 and LC comprising SEQ ID NO:10. In some embodiments, Ab is Ab3, wherein Ab3 comprises HC comprising SEQ ID NO:37 and LC comprising SEQ ID NO:10. In some embodiments, Ab is Ab4, wherein Ab4 comprises a HC comprising SEQ ID NO:31 and a LC comprising SEQ ID NO:10. In some embodiments, Ab is Ab5, wherein Ab5 comprises a HC comprising SEQ ID NO:35 and a LC comprising SEQ ID NO:10. In some embodiments, Ab is Ab6, wherein Ab6 comprises a HC comprising SEQ ID NO:33 and a LC comprising SEQ ID NO:10. In some embodiments, Ab is Ab7, wherein Ab7 comprises a HC comprising SEQ ID NO:13 and a LC comprising SEQ ID NO:10. In some embodiments, Ab is Ab8, wherein Ab8 comprises a HC comprising SEQ ID NO:52 and a LC comprising SEQ ID NO:10.

[0117] In some embodiments, Ab comprises VH and VL, wherein VH comprises heavy chain complementary determining regions HCDR1, HCDR2, and HCDR3, and VL comprises light chain complementary determining regions LCDR1, LCDR2, and LCDR3, wherein HCDR1 comprises SEQ ID NO: 42, HCDR2 comprises SEQ ID NO: 2, HCDR3 comprises SEQ ID NO: 3, LCDR1 comprises SEQ ID NO: 22, LCDR2 comprises SEQ ID NO: 5, and LCDR3 comprises SEQ ID NO: 6. In some embodiments, Ab comprises VH comprising SEQ ID NO: 44 and VL comprising SEQ ID NO: 45. In some embodiments, Ab is Ab9, wherein Ab9 comprises HC comprising SEQ ID NO: 46 and LC comprising SEQ ID NO: 47.

[0118] In some embodiments, Ab comprises VH and VL, wherein VH comprises heavy chain complementary determining regions HCDR1, HCDR2, and HCDR3, and VL comprises light chain complementary determining regions LCDR1, LCDR2, and LCDR3, wherein HCDR1 comprises SEQ ID NO: 19, HCDR2 comprises SEQ ID NO: 20, HCDR3 comprises SEQ ID NO: 3, LCDR1 comprises SEQ ID NO: 22, LCDR2 comprises SEQ ID NO: 5, and LCDR3 comprises SEQ ID NO: 24. In some embodiments, Ab comprises VH comprising SEQ ID NO: 25 and VL comprising SEQ ID NO: 26. In some embodiments, Ab is Ab10, wherein Ab10 comprises HC comprising SEQ ID NO: 27 and LC comprising SEQ ID NO: 28.

[0119] In some embodiments, the Ab in the conjugate of Formula I binds to human IL-4Rα and inhibits the binding of human IL-4 and human IL-13 to human IL-4Rα. In some embodiments, the Ab in the conjugate of Formula I binds to human IL-4Rα and inhibits IL-4R receptor-mediated responses (e.g., inhibiting pSTAT6 in B and T cells, inhibiting B cell proliferation, inhibiting CD23 expression, inhibiting cytokine secretion (e.g., MDC, GM-CSF). In some embodiments, the Ab in the conjugate of Formula I binds to human IL-4Rα and does not significantly induce ADCC or CDC activity. In some embodiments, the Ab in the conjugate of Formula I binds to human IL-4Rα on the cell surface and internalizes the conjugate of Formula I into the cell.

[0120] In some embodiments of the present disclosure, the Ab in the conjugate of Formula I is a fully human antibody. In some embodiments of the present disclosure, the Ab in the conjugate of Formula I has a human IgG4 or human IgG1 isotype.

[0121] In some embodiments of the present disclosure, the Ab in the conjugate of Formula I has a modified human IgG4 hinge region comprising an S228P substitution (EU numbering), also referred to as IgG4P, which reduces in vivo IgG4 Fab-arm exchange (see Labrijn et al., Nat. Biotechnol. 2009, 27(8):767). In some embodiments, when compared to human IgG4 and / or human IgG4P IL-4Rα antibodies with an effector empty backbone, the Ab with a human IgG4P backbone has improved binding to B cells and / or myeloid cells.

[0122] In some embodiments of the present disclosure, the Ab in the conjugate of Formula I has a modified human IgG1 Fc region comprising alanine (K322A substitution) at amino acid residue 322 (EU numbering) (also referred to as IgG1A). In such embodiments, the Ab has reduced or eliminated complement activity. In some embodiments, the Ab has a modified human IgG1 Fc region comprising L234A, L235A and / or P329A (also referred to as IgG1AA or IgGIAAA, respectively), which has reduced or eliminated binding to Fcγ and C1q receptors (all residues are numbered according to EU numbering). In some embodiments, when compared to an anti-human IL-4Rα antibody with a human IgG1AAA effector empty main chain, the Ab with a human IgG1A main chain shows improved binding to B cells and / or myeloid cells.

[0123] In some embodiments of the present disclosure, the Ab in the conjugate of Formula I has a modified human IgG4 HC constant region, which reduces the viscosity of the antibody compared to the same antibody with a wild-type human IgG4 HC constant region. In such embodiments, the Ab has a modified human IgG4 HC constant region, which includes amino acid substitutions at any one or more of the following amino acid residues: E137G, D203N, Q274K, Q355R, E419Q (all positions are numbered according to EU numbering). In some embodiments, the Ab has a modified human IgG4 HC constant region, which includes amino acid substitutions at any one or more of the following amino acid residues: E137G, D203N, Q274K, Q355R, E419Q (all positions are numbered according to EU numbering). In some embodiments, the Ab has a modified human IgG4 HC constant region, which includes amino acid substitutions at any one or more of the following amino acid residues: Q274K, Q355R, E419Q (all positions are numbered according to EU numbering). In some embodiments, the Ab has a modified human IgG4 HC constant region comprising amino acid substitutions at the following amino acid residues: Q274K, Q355R, E419Q (all positions are numbered according to EU numbering).

[0124] In another embodiment, the Ab in the conjugate of Formula I has a modified human IgG1 or human IgG4 constant domain, which includes one or more engineered cysteine ​​residues (see WO 2018 / 232088A1). In such embodiments, Ab includes a cysteine ​​at amino acid residue 124 (EU numbering) in the heavy chain constant domain 1 (CH1), or a cysteine ​​at amino acid residue 378 (EU numbering) in the heavy chain constant domain 2 (CH2). In other embodiments, Ab includes a cysteine ​​at amino acid residue 124 (EU numbering) in the CH1 domain and a cysteine ​​at amino acid residue 378 (EU numbering) in the CH2 domain.

[0125] In some embodiments, Ab in the conjugate of Formula I is selected from Abl, Ab2, Ab3, Ab4, Ab5, Ab6, Ab7, Ab8, Ab9 and AblO. In some embodiments, Abl, Ab2, Ab3, Ab4, Ab5, Ab6, Ab7 and Ab8 have the same HCDR and LCDR amino acid sequences. Thus, as shown below, Abl, Ab2, Ab3, Ab4, Ab5, Ab6, Ab7 and Ab8 bind to an epitope comprising amino acid residues selected from the group consisting of D12, M14, S15, I16, L39, F41, L42, T48, C49, I50, E52, H62, L64, M65, D66, D67, V68, V69, D72, R99, P121, P123, P124, D125, P192 (amino acid residue positions corresponding to SEQ ID NO: 15). In some embodiments shown below, Ab9 and Ab10 bind to an epitope of human IL-4Rα, wherein the epitope comprises one or more amino acid residues selected from the group consisting of: D12, M14, S15, I16, Y37, L39, F41, L43, E45, H47, T48, C49, I50, H62, L64, M65, D66, D67, V69, D72, R99, P121, P123, P124, D125 (amino acid residue positions corresponding to SEQ ID NO: 15). In some embodiments, as shown below, Abl, Ab2, Ab3, Ab4, Ab5, Ab6, Ab7, Ab8, Ab9 and AblO bind to an epitope of human IL-4Rα, wherein the epitope comprises one or more amino acid residues selected from the group consisting of: D12, M14, S15, I16, Y37, L39, T48, C49, I50, E52, H62, M65, R99, P121, P123, P124, D125, P192 (amino acid residue positions corresponding to SEQ ID NO: 15). In some embodiments, as shown below, Abl, Ab2, Ab3, Ab4, Ab5, Ab6, Ab7, Ab8, Ab9 and AblO bind to an epitope of human IL-4Rα, wherein the epitope comprises one or more amino acid residues selected from the group consisting of R99, P121, P123, P124, D125, P192 (amino acid residue positions corresponding to SEQ ID NO: 15), wherein these residues are located in domain 2 of the N-terminal fibronectin type III domain of IL-4Rα. In some embodiments, as shown below, Abl, Ab2, Ab3, Ab4, Ab5, Ab6, Ab7, Ab8, Ab9 and AblO bind to an epitope of human IL-4Rα, wherein the epitope comprises one or more amino acid residues selected from the group consisting of D66, D67 and D125 (amino acid residue positions corresponding to SEQ ID NO: 15).In additional embodiments as shown below, Abl, Ab2, Ab3, Ab4, Ab5, Ab6, Ab7, Ab8, Ab9 and Ab10 bind to an epitope of human IL-4Rα, wherein the epitope comprises at least one amino acid residue selected from the group consisting of D66 and D67 (amino acid residue positions corresponding to SEQ ID NO: 15). In yet another embodiment as shown below, Abl, Ab2, Ab3, Ab4, Ab5, Ab6, Ab7, Ab8, Ab9 and Ab10 bind to an epitope of human IL-4Rα, wherein the epitope comprises at least one amino acid residue selected from the group consisting of D66 and D125 (amino acid residue positions corresponding to SEQ ID NO: 15). In yet another embodiment as shown below, Abl, Ab2, Ab3, Ab4, Ab5, Ab6, Ab7, Ab8, Ab9 and Ab10 bind to an epitope of human IL-4Rα, wherein the epitope comprises amino acid residue D66 (amino acid residue positions corresponding to SEQ ID NO: 15). In such embodiments, Abl, Ab2, Ab3, Ab4, Ab5, Ab6, Ab7, Ab8, Ab9 and Ab10 bind to a novel structural and / or functional epitope of human IL-4Rα, wherein the epitope spans domain 1 and domain 2 of the n-terminal fibronectin type III domain of IL-4Rα. In specific embodiments, Abl, Ab2, Ab3, Ab4, Ab5, Ab6, Ab7, Ab8, Ab9 and Ab10 bind to a novel structural and / or functional epitope of human IL-4Rα, wherein the epitope overlaps with the IL-4 binding site of IL-4Rα. In such embodiments, Abl, Ab2, Ab3, Ab4, Ab5, Ab6, Ab7, Ab8, Ab9 and Ab10 inhibit the binding of IL-4 to human IL-4Rα. In a specific embodiment, Ab1, Ab2, Ab3, Ab4, Ab5, Ab6, Ab7, Ab8, Ab9 and Ab10 bind to a new structural and / or functional epitope of human IL-4Rα, wherein the epitope overlaps with the IL-13 binding site of IL-4Rα. In such embodiments, Ab1, Ab2, Ab3, Ab4, Ab5, Ab6, Ab7, Ab8, Ab9 and Ab10 inhibit the binding of IL-13 to human IL-4Rα. In some embodiments, Ab1, Ab2, Ab3, Ab4, Ab5, Ab6, Ab7, Ab8, Ab9 and Ab10 bind to a new structural and / or functional epitope of human IL-4Rα, wherein the epitope overlaps with the IL-4 and IL-13 binding sites of IL-4Rα. In such embodiments, Ab1, Ab2, Ab3, Ab4, Ab5, Ab6, Ab7, Ab8, Ab9 and Ab10 inhibit the binding of IL-4 and IL-13 to human IL-4Rα.In some embodiments, the IL-4Rα epitope is determined by X-ray crystallography, alanine scanning mutagenesis, steric mutagenesis, and / or HDX-MS. In yet other embodiments, the IL-4Rα epitope is determined by site-directed mutagenesis.

[0126] In some embodiments, the present disclosure provides a nucleic acid encoding the HC or LC, or VH or VL of an antibody that binds to human IL-4Rα, or a vector comprising such a nucleic acid.

[0127] In some embodiments, the present disclosure provides a nucleic acid comprising the sequence of SEQ ID NO: 11, 12, 14, 29, 30, 32, 34, 36, 38, 48, 49, 51 or 53.

[0128] In some embodiments, nucleic acids encoding heavy or light chains of antibodies that bind to human IL-4Rα are provided. In some embodiments, nucleic acids comprising sequences encoding SEQ ID NO:9, 10, 13, 27, 28, 31, 33, 35, 37, 46, 47, 50, or 52 are provided. In some embodiments, nucleic acids comprising sequences encoding heavy chains of antibodies comprising SEQ ID NO:9, 13, 27, 31, 33, 35, 37, 46, 50, or 52 are provided. For example, the nucleic acid may comprise a sequence of SEQ ID NO:11, 14, 29, 32, 34, 36, 38, 48, 51, or 53. In some embodiments, nucleic acids comprising sequences encoding light chains of antibodies comprising SEQ ID NO:10, 28, or 47 are provided. For example, the nucleic acid may comprise a sequence of SEQ ID NO:12, 30, or 49.

[0129] In some embodiments of the present disclosure, nucleic acids encoding VH or VL of antibodies that specifically bind to human IL-4Rα are provided. In some embodiments, nucleic acids comprising sequences encoding SEQ ID NO: 7, 8, 25, 26, 44 or 45 are provided. In some embodiments, nucleic acids comprising sequences encoding antibody VH comprising SEQ ID NO: 7, 25 or 44 are provided. In some embodiments, nucleic acids comprising sequences encoding antibody VL comprising SEQ ID NO: 8, 26 or 45 are provided.

[0130] Some embodiments of the present disclosure provide vectors comprising a nucleic acid sequence encoding an antibody heavy chain or light chain. For example, such vectors may comprise a nucleic acid sequence encoding SEQ ID NO: 9, 13, 27, 31, 33, 35, 37, 46, 50 or 52. In some embodiments, the vector comprises a nucleic acid sequence encoding SEQ ID NO: 10, 28 or 47.

[0131] Also provided herein are vectors comprising a nucleic acid sequence encoding an antibody VH or VL. For example, such vectors may comprise a nucleic acid sequence encoding SEQ ID NO: 7, 25, or 44. In some embodiments, the vector comprises a nucleic acid sequence encoding SEQ ID NO: 8, 26, or 45.

[0132] Also provided herein is a vector comprising a first nucleic acid sequence encoding an antibody heavy chain and a second nucleic acid sequence encoding an antibody light chain. In some embodiments, the vector comprises a first nucleic acid sequence encoding SEQ ID NO:9, 13, 27, 31, 33, 35, 37, 46, 50 or 52 and a second nucleic acid sequence encoding SEQ ID NO:10, 28 or 47. In some embodiments, the vector comprises a first nucleic acid sequence encoding SEQ ID NO:9 and a second nucleic acid sequence encoding SEQ ID NO:10. In some embodiments, the vector comprises a first nucleic acid sequence encoding SEQ ID NO:13 and a second nucleic acid sequence encoding SEQ ID NO:10. In some embodiments, the vector comprises a first nucleic acid sequence encoding SEQ ID NO:27 and a second nucleic acid sequence encoding SEQ ID NO:28. In some embodiments, the vector comprises a first nucleic acid sequence encoding SEQ ID NO:31 and a second nucleic acid sequence encoding SEQ ID NO:10. In some embodiments, the vector comprises a first nucleic acid sequence encoding SEQ ID NO:33 and a second nucleic acid sequence encoding SEQ ID NO:10. In some embodiments, the vector comprises a first nucleic acid sequence encoding SEQ ID NO:35 and a second nucleic acid sequence encoding SEQ ID NO:10. In some embodiments, the vector comprises a first nucleic acid sequence encoding SEQ ID NO:37 and a second nucleic acid sequence encoding SEQ ID NO:10. In some embodiments, the vector comprises a first nucleic acid sequence encoding SEQ ID NO:50 and a second nucleic acid sequence encoding SEQ ID NO:10. In some embodiments, the vector comprises a first nucleic acid sequence encoding SEQ ID NO:52 and a second nucleic acid sequence encoding SEQ ID NO:10. In some embodiments, the vector comprises a first nucleic acid sequence encoding SEQ ID NO:46 and a second nucleic acid sequence encoding SEQ ID NO:47.

[0133] Also provided herein is a composition comprising a first vector containing a nucleic acid sequence encoding an antibody heavy chain and a second vector containing a nucleic acid sequence encoding an antibody light chain. In some embodiments, the composition comprises a first vector containing a nucleic acid sequence encoding SEQ ID NO:9, 13, 27, 31, 33, 35, 37, 46, 50 or 52 and a second vector containing a nucleic acid sequence encoding SEQ ID NO:10, 28 or 47. In some embodiments, the composition comprises a first vector containing a nucleic acid sequence encoding SEQ ID NO:9 and a second vector containing a nucleic acid sequence encoding SEQ ID NO:10. In some embodiments, the composition comprises a first vector containing a nucleic acid sequence encoding SEQ ID NO:13 and a second vector containing a nucleic acid sequence encoding SEQ ID NO:10. In some embodiments, the composition comprises a first vector containing a nucleic acid sequence encoding SEQ ID NO:27 and a second vector containing a nucleic acid sequence encoding SEQ ID NO:28. In some embodiments, the composition comprises a first vector containing a nucleic acid sequence encoding SEQ ID NO:31 and a second vector containing a nucleic acid sequence encoding SEQ ID NO:10. In some embodiments, the composition comprises a first vector containing a nucleic acid sequence encoding SEQ ID NO:33 and a second vector containing a nucleic acid sequence encoding SEQ ID NO:10. In some embodiments, the composition comprises a first vector containing a nucleic acid sequence encoding SEQ ID NO:35 and a second vector containing a nucleic acid sequence encoding SEQ ID NO:10. In some embodiments, the composition comprises a first vector containing a nucleic acid sequence encoding SEQ ID NO:37 and a second vector containing a nucleic acid sequence encoding SEQ ID NO:10. In some embodiments, the composition comprises a first vector containing a nucleic acid sequence encoding SEQ ID NO:50 and a second vector containing a nucleic acid sequence encoding SEQ ID NO:10. In some embodiments, the composition comprises a first vector containing a nucleic acid sequence encoding SEQ ID NO:52 and a second vector containing a nucleic acid sequence encoding SEQ ID NO:10. In some embodiments, the composition comprises a first vector containing a nucleic acid sequence encoding SEQ ID NO:46 and a second vector containing a nucleic acid sequence encoding SEQ ID NO:47.

[0134] Also provided herein is a composition comprising a vector comprising a nucleic acid sequence encoding an antibody heavy chain and a nucleic acid sequence encoding an antibody light chain. In some embodiments, the composition comprises a vector comprising a nucleic acid sequence encoding SEQ ID NO: 9, 13, 27, 31, 33, 35, 37, 46, 50 or 52 and a second nucleic acid sequence encoding SEQ ID NO: 10, 28 or 47.

[0135] As used herein, "GC" in the formula:

[0136]

[0137] refers to a suitable glucocorticoid receptor agonist payload and includes the following Formula IIa, IIb or IIc:

[0138]

[0139] and

[0140]

[0141] As used herein, the "L" in the formula

[0142]

[0143] Refers to a suitable linker group that connects Ab to GC. Suitable linkers known to those of ordinary skill in the art include, for example, cleavable and non-cleavable linkers. More specifically, suitable linkers "L" include the following formulas IIIa to IIIf:

[0144]

[0145] and

[0146]

[0147] In an embodiment, the present disclosure provides a glucocorticoid receptor agonist payload-linker of Formula IV:

[0148]

[0149] In an embodiment, the present disclosure provides a glucocorticoid receptor agonist payload-linker of Formula IVa:

[0150]

[0151] In an embodiment, the disclosure provides a glucocorticoid receptor agonist payload-linker of Formula IVb:

[0152]

[0153] In an embodiment, the present disclosure provides a glucocorticoid receptor agonist payload-linker of Formula IVc:

[0154]

[0155] In an embodiment, the present disclosure provides a glucocorticoid receptor agonist payload-linker of Formula IVd:

[0156]

[0157] In an embodiment, the present disclosure provides a compound of Formula V:

[0158]

[0159] In further embodiments, the present disclosure provides compounds of Formula Va:

[0160]

[0161] In an embodiment, the disclosure also provides a method for treating an inflammatory disease in a subject in need thereof, the method comprising administering an effective amount of a conjugate of Formula I or a pharmaceutically acceptable salt thereof to the subject. In an embodiment, the disclosure also provides a method for treating an inflammatory disease, wherein the inflammatory disease is a type 2 inflammatory disease in a subject in need thereof, the method comprising administering an effective amount of a conjugate of Formula I or a drug or salt thereof to the subject. In certain embodiments, type 2 inflammatory diseases are, for example, atopic dermatitis, eosinophilic esophagitis, nasal polyposis, asthma, chronic sinusitis, allergic diseases, chronic obstructive pulmonary disease, or chronic spontaneous urticaria. In an embodiment, the disclosure also provides a method for treating atopic dermatitis in a subject in need thereof, the method comprising administering an effective amount of a conjugate of Formula I or a pharmaceutically acceptable salt thereof to the subject. In an embodiment, the disclosure also provides a method for treating eosinophilic esophagitis in a subject in need thereof, the method comprising administering an effective amount of a conjugate of Formula I or a pharmaceutically acceptable salt thereof to the subject. In an embodiment, the present disclosure also provides a method for treating nasal polyposis in a subject in need thereof, the method comprising administering an effective amount of a conjugate of Formula I or a pharmaceutically acceptable salt thereof to the subject. In an embodiment, the present disclosure also provides a method for treating asthma in a subject in need thereof, the method comprising administering an effective amount of a conjugate of Formula I or a pharmaceutically acceptable salt thereof to the subject. In an embodiment, the present disclosure also provides a method for treating chronic sinusitis in a subject in need thereof, the method comprising administering an effective amount of a conjugate of Formula I or a pharmaceutically acceptable salt thereof to the subject. In an embodiment, the present disclosure also provides a method for treating allergic diseases in a subject in need thereof, the method comprising administering an effective amount of a conjugate of Formula I or a pharmaceutically acceptable salt thereof to the subject. In an embodiment, the present disclosure also provides a method for treating chronic obstructive pulmonary disease in a subject in need thereof, the method comprising administering an effective amount of a conjugate of Formula I or a pharmaceutically acceptable salt thereof to the subject. In an embodiment, the present disclosure also provides a method for treating chronic spontaneous urticaria in a subject in need thereof, the method comprising administering an effective amount of a conjugate of Formula I or a pharmaceutically acceptable salt thereof to the subject.

[0162] In an embodiment, the present disclosure also provides a conjugate of Formula I or a pharmaceutically acceptable salt thereof for use in therapy. In an embodiment, the present disclosure provides a conjugate of Formula I or a pharmaceutically acceptable salt thereof for use in the treatment of inflammatory diseases. In certain embodiments, the inflammatory disease is a type 2 inflammatory disease. In certain embodiments, a type 2 inflammatory disease is, for example, atopic dermatitis, eosinophilic esophagitis, nasal polyposis, asthma, chronic sinusitis, allergic diseases, chronic obstructive pulmonary disease, or chronic spontaneous urticaria. In an embodiment, the present disclosure provides a conjugate of Formula I or a pharmaceutically acceptable salt thereof for use in the treatment of atopic dermatitis. In an embodiment, the present disclosure provides a conjugate of Formula I or a pharmaceutically acceptable salt thereof for use in the treatment of eosinophilic esophagitis. In an embodiment, the present disclosure provides a conjugate of Formula I or a pharmaceutically acceptable salt thereof for use in the treatment of nasal polyposis. In an embodiment, the present disclosure provides a conjugate of Formula I or a pharmaceutically acceptable salt thereof for use in the treatment of asthma. In an embodiment, the present disclosure provides a conjugate of Formula I or a pharmaceutically acceptable salt thereof for use in the treatment of chronic sinusitis. In an embodiment, the present disclosure provides a conjugate of Formula I or a pharmaceutically acceptable salt thereof for use in treating allergic diseases. In an embodiment, the present disclosure provides a conjugate of Formula I or a pharmaceutically acceptable salt thereof for use in treating chronic obstructive pulmonary disease. In an embodiment, the present disclosure provides a conjugate of Formula I or a pharmaceutically acceptable salt thereof for use in treating chronic spontaneous urticaria.

[0163] In an embodiment, the present disclosure also provides the use of a conjugate of Formula I or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for the treatment of an inflammatory disease. In certain embodiments, the inflammatory disease is a type 2 inflammatory disease. In certain embodiments, a type 2 inflammatory disease is, for example, atopic dermatitis, eosinophilic esophagitis, nasal polyposis, asthma, chronic sinusitis, allergic diseases, chronic obstructive pulmonary disease, or chronic spontaneous urticaria. In an embodiment, the present disclosure provides the use of a conjugate of Formula I or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for the treatment of atopic dermatitis. In an embodiment, the present disclosure provides the use of a conjugate of Formula I or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for the treatment of eosinophilic esophagitis. In an embodiment, the present disclosure provides the use of a conjugate of Formula I or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for the treatment of nasal polyposis. In an embodiment, the present disclosure provides the use of a conjugate of Formula I or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for the treatment of asthma. In an embodiment, the present disclosure provides the use of a conjugate of Formula I or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for the treatment of chronic sinusitis. In an embodiment, the present disclosure provides the use of a conjugate of Formula I or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for treating an allergic disease. In an embodiment, the present disclosure provides the use of a conjugate of Formula I or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for treating chronic obstructive pulmonary disease. In an embodiment, the present disclosure provides the use of a conjugate of Formula I or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for treating chronic spontaneous urticaria.

[0164] Nucleic acids of the present disclosure can be expressed in host cells, for example, after the nucleic acids have been operably connected to expression control sequences. Expression control sequences capable of expressing nucleic acids operably connected thereto are well known in the art. Expression vectors may include sequences encoding one or more signal peptides that promote secretion of polypeptides from host cells. Expression vectors containing nucleic acids of interest (for example, nucleic acids encoding heavy or light chains of antibodies) can be transferred to host cells by well-known methods (for example, stable or transient transfection, transformation, transduction or infection). In addition, the expression vector may include one or more selection markers, such as tetracycline, neomycin and dihydrofolate reductase, to help detect host cells transformed with the desired nucleic acid sequence.

[0165] In another aspect, provided herein are cells, such as host cells, comprising nucleic acids, vectors, or nucleic acid compositions described herein. The host cell may be a cell stably or transiently transfected, transformed, transduced, or infected with one or more expression vectors expressing all or a portion of an antibody described herein. In some embodiments, the host cell may be stably or transiently transfected, transformed, transduced, or infected with expression vectors expressing the HC and LC polypeptides of the antibodies disclosed herein. In some embodiments, the host cell may be stably or transiently transfected, transformed, transduced, or infected with a first vector expressing the HC polypeptide of an antibody described herein and a second vector expressing the LC polypeptide of an antibody described herein. Such host cells (e.g., mammalian host cells) may express antibodies that bind to human IL-4Rα as described herein. Mammalian host cells known to be capable of expressing antibodies include CHO cells, HEK293 cells, COS cells, and NS0 cells.

[0166] In some embodiments, the cell (e.g., a host cell) comprises a vector comprising a first nucleic acid sequence encoding SEQ ID NO: 9, 13, 27, 31, 33, 35, 37, 46, 50, or 52 and a second nucleic acid sequence encoding SEQ ID NO: 10, 28, or 47.

[0167] In some embodiments, the cell (e.g., a host cell) comprises a first vector comprising a nucleic acid sequence encoding SEQ ID NO: 9, 13, 27, 31, 33, 35, 37, 46, 50, or 52 and a second vector comprising a nucleic acid sequence encoding SEQ ID NO: 10, 28, or 47.

[0168] The present disclosure also provides a method for producing an antibody that binds to human IL-4Rα as described herein by culturing the above-mentioned host cells, such as mammalian host cells, under conditions such that the antibody is expressed, and recovering the expressed antibody from the culture medium. The culture medium in which the antibody has been secreted can be purified by conventional techniques. Various protein purification methods can be used, and such methods are known in the art and described, for example, in Deutscher, Methods in Enzymology 182: 83-89 (1990) and Scopes, Protein Purification: Principles and Practice, 3rd Edition, Springer, NY (1994).

[0169] The present disclosure provides a method of producing a conjugate, the method comprising conjugating a compound of the present disclosure to an anti-human IL-4Rα antibody.

[0170] The present disclosure provides a method for producing a conjugate, the method comprising conjugating a compound of formula IV with an anti-human IL-4Rα antibody. The present disclosure provides a method for producing a conjugate, the method comprising conjugating a compound of formula IVa with an anti-human IL-4Rα antibody. The present disclosure provides a method for producing a conjugate, the method comprising conjugating a compound of formula IVb with an anti-human IL-4Rα antibody. The present disclosure provides a method for producing a conjugate, the method comprising conjugating a compound of formula IVc with an anti-human IL-4Rα antibody. The present disclosure provides a method for producing a conjugate, the method comprising conjugating a compound of formula IVd with an anti-human IL-4Rα antibody.

[0171] In some embodiments, the conjugate produced is a conjugate of Formula I.

[0172] The present disclosure provides a method for producing a conjugate, the method comprising the steps of:

[0173] (a) reducing an anti-human IL-4Rα antibody with a reducing agent to produce a reduced anti-human IL-4R antibody, wherein the anti-human IL-4Rα antibody comprises one or more engineered cysteine ​​residues;

[0174] (b) oxidizing the reduced anti-human IL-4Rα antibody with an oxidizing agent to generate an oxidized anti-human IL-4R antibody; and

[0175] (c) contacting the oxidized anti-human IL-4Rα antibody with a compound of the present disclosure to produce a conjugate.

[0176] The present disclosure provides a method for producing a conjugate, the method comprising the steps of:

[0177] (a) reducing an anti-human IL-4Rα antibody with a reducing agent, wherein the anti-human IL-4Rα antibody comprises one or more engineered cysteine ​​residues;

[0178] (b) oxidizing the anti-human IL-4Rα antibody with an oxidizing agent to produce an oxidized anti-human IL-4R antibody;

[0179] as well as

[0180] (c) contacting the oxidized anti-human IL-4Rα antibody with a compound of the following formula

[0181]

[0182] to produce a conjugate.

[0183] In some embodiments, the reducing agent is dithiothreitol. In some embodiments, the oxidizing agent is dehydroascorbic acid. In some embodiments, the reducing agent is dithiothreitol and the oxidizing agent is dehydroascorbic acid.

[0184] The present disclosure also provides antibodies or antigen-binding fragments thereof produced by any of the methods described herein.

[0185] In an embodiment, the present disclosure also provides a pharmaceutical composition comprising a conjugate of Formula I described herein or a pharmaceutically acceptable salt thereof or an antibody, a nucleic acid or a vector and one or more pharmaceutically acceptable carriers, diluents or excipients. In an embodiment, the present disclosure also provides a pharmaceutical composition comprising a conjugate of Formula I or a pharmaceutically acceptable salt thereof and one or more pharmaceutically acceptable carriers, diluents or excipients. In an embodiment, the present disclosure also provides a pharmaceutical composition comprising a conjugate of Formula I and one or more pharmaceutically acceptable carriers, diluents or excipients. In an embodiment, the present disclosure also provides a method for preparing a pharmaceutical composition, the method comprising mixing a conjugate of Formula I or a pharmaceutically acceptable salt thereof with one or more pharmaceutically acceptable carriers, diluents or excipients. In an embodiment, the present disclosure also includes novel intermediates and methods for synthesizing conjugates of Formula I. DETAILED DESCRIPTION

[0186] Unless otherwise indicated, the term "IL-4Rα" as used herein refers to any natural mature IL-4Rα produced by processing of the IL-4Rα precursor protein in a cell. Unless otherwise indicated, the term includes IL-4Rα from any vertebrate source, including mammals, such as primates (e.g., humans and cynomolgus monkeys or rhesus monkeys). The term also includes naturally occurring IL-4Rα variants, such as splice variants or allelic variants. Exemplary amino acid sequences of human IL-4Rα are known in the art, such as the UniProt reference sequence P24394 (SEQ ID NO: 39). Exemplary amino acid sequences of cynomolgus monkey IL-4Rα are also known in the art, such as the NCBI reference sequence XP_005591572.2 (SEQ ID NO: 40). The term "human IL-4Rα" is used herein to collectively refer to all known human IL-4Rα isoforms and polymorphic forms. The sequence numbering used herein is based on mature proteins without signal peptides.

[0187] Unless otherwise indicated, the term "IL-4R" as used herein refers to the complex of the IL-4Rα subunit and the common γ chain (type I receptor) and / or the complex of the IL-4Rα subunit and IL-13Rα1 (type II receptor).

[0188] Unless otherwise indicated, the term "IL-4" as used herein refers to any natural mature IL-4 produced by the processing of IL-4 precursor protein in cells. Unless otherwise indicated, the term includes IL-4 from any vertebrate source, including mammals, such as primates (e.g., humans and cynomolgus monkeys or rhesus monkeys). The term also includes naturally occurring IL-4 variants, such as splice variants or allelic variants. The exemplary amino acid sequence of human IL-4 is known in the art, such as UniProt reference sequence P05112 (SEQ ID NO: 17). The term "human IL-4" is used herein to collectively refer to all known human IL-4 isoforms and polymorphic forms.

[0189] Unless otherwise indicated, the term "IL-13" as used herein refers to any natural mature IL-13 produced by the processing of IL-13 precursor protein in cells. Unless otherwise indicated, the term includes IL-13 from any vertebrate source, including mammals, such as primates (e.g., humans and cynomolgus monkeys or rhesus monkeys). The term also includes naturally occurring IL-13 variants, such as splice variants or allelic variants. The exemplary amino acid sequence of human IL-13 is known in the art, such as UniProt reference sequence P35225 (SEQ ID NO: 18). The term "human IL-13" is used herein to collectively refer to all known human IL-13 isoforms and polymorphic forms.

[0190] Unless otherwise indicated, the term "CD23" as used herein refers to any natural mature CD23 produced by the processing of CD23 precursor protein in cells. Unless otherwise indicated, the term includes CD23 from any vertebrate source, including mammals, such as primates (e.g., humans and cynomolgus monkeys or rhesus monkeys). The term also includes naturally occurring CD23 variants, such as splice variants or allelic variants. The exemplary amino acid sequence of human CD23 is known in the art, such as the UniProt reference sequence P06734 (SEQ ID NO: 41). The term "human CD23" is used herein to collectively refer to all known human CD23 isoforms and polymorphic forms.

[0191] As used herein, the term "IL-4R-associated disorder" refers to a disorder associated with IL-4R-mediated signaling, such as a disorder associated with IL-4R type 1 and IL-4R type II signaling. Such IL-4R-associated disorders may, for example, include immunoinflammatory disorders. Such immunoinflammatory disorders may include type 2 inflammatory disorders as disclosed herein. IL-4R-associated disorders may also include cancer.

[0192] As used herein, the terms "modulate" or "modulate" and the like refer to changing or altering a measurable value, and include changing or altering such a measurable value upward (i.e., upmodulate or upmodulating) or downward (i.e., downmodulate / downmodulating or downmodulating).

[0193] As used herein, the term "antibody" refers to an immunoglobulin molecule that binds an antigen. Embodiments of antibodies include monoclonal antibodies, polyclonal antibodies, human antibodies, humanized antibodies, chimeric antibodies, bispecific or multispecific antibodies, or conjugated antibodies. These antibodies may belong to any class (e.g., IgG, IgE, IgM, IgD, IgA) and any subclass (e.g., IgG1, IgG2, IgG3, IgG4). Embodiments of the present disclosure also include antibody fragments or antigen binding fragments, and the term "antibody fragment or antigen binding fragment" includes at least a portion of an antibody that retains the ability to interact with an antigen, such as, for example, Fab, Fab', F(ab')2, Fv fragments, scFv, scFab, disulfide-linked Fv (sdFv), Fd fragments, or linear antibodies, which may, for example, be fused to Fc regions or IgG heavy chain constant regions.

[0194] An exemplary antibody is an immunoglobulin G (IgG) type antibody, which is composed of four polypeptide chains: two heavy chains (HC) and two light chains (LC) cross-linked by interchain disulfide bonds. The amino terminal portion of each of the four polypeptide chains contains a variable region of about 100 to 125 or more amino acids that is primarily responsible for antigen recognition. The carboxyl terminal portion of each of the four polypeptide chains contains a constant region that is primarily responsible for effector function. Each heavy chain is composed of a heavy chain variable region (VH) and a heavy chain constant region. The heavy chain constant region refers to the region of the antibody that contains the Fc region and CH1 domain of the antibody heavy chain. Each light chain is composed of a light chain variable region (VL) and a light chain constant region. IgG isotypes can be further divided into subclasses (e.g., IgG1, IgG2, IgG3, and IgG4). The numbering of amino acid residues in the constant region is based on the EU index as in Kabat. Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., Bethesda, MD: USDept. of Health and Human Services, Public Health Service, National Institutes of Health (1991). The terms EU index number or EU number are used interchangeably herein.

[0195] VH and VL regions can be further subdivided into hypervariable regions known as complementary determining regions (CDRs), which are interspersed with more conservative regions known as framework regions (FRs). CDRs are exposed to the surface of the protein and are important regions of the antigen-binding specificity of antibodies. Each VH and VL is composed of three CDRs and four FRs, which are arranged in the following order from amino-terminal to carboxyl-terminal: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In this article, the three CDRs of the heavy chain are referred to as "HCDR1, HCDR2 and HCDR3", and the three CDRs of the light chain are referred to as "LCDR1, LCDR2 and LCDR3". These CDRs contain most of the residues that form specific interactions with the antigen. The assignment of amino acid residues to CDRs can be performed according to well-known schemes, including those described in Kabat (Kabat et al., "Sequences of Proteins of Immunological Interest," National Institutes of Health, Bethesda, Md. (1991)), Chothia (Chothia et al., "Canonical structures for the hypervariable regions of immunoglobulins," Journal of Molecular Biology, 196, 901-917 (1987); Al-Lazikani et al., "Standard conformations for the canonical structures of immunoglobulins," Journal of Molecular Biology, 273, 927-948 (1997)), North (North et al., "A New Clustering of Antibody CDR Loop Conformations," Journal of Molecular Biology, 274, 937-951 (1998)), and the like. Biology, 406, 228-256 (2011)) or IMGT (International Immunogenetics Database available at www.imgt.org; see Lefranc et al., Nucleic Acids Res. 1999; 27:209-212). The combination of IMGT and North CDR definitions is used for the exemplified anti-human IL-4Rα antibodies as described herein.

[0196] As used herein, the term "Fc region" refers to the region of an antibody that includes the CH2 and CH3 domains of an antibody heavy chain. Optionally, the Fc region may include a portion or the entire hinge region of an antibody heavy chain. Biological activities such as effector functions may be attributed to the Fc region, which vary with the antibody isotype. Examples of antibody effector functions include Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP), C1q binding, complement-dependent cytotoxicity (CDC), phagocytosis, downregulation of cell surface receptors (e.g., B cell receptors), and B cell activation.

[0197] As used herein, the term "epitope" refers to the amino acid residues in an antigen that are bound by an antibody. An epitope can be a linear epitope, a conformational epitope, or a hybrid epitope. The term "epitope" can be used to refer to a structural epitope. According to some embodiments, a structural epitope can be used to describe an antigen region covered by an antibody (e.g., the footprint of an antibody when it binds to an antigen). In some embodiments, a structural epitope can be described as an amino acid residue of an antigen within a specified proximity of an amino acid residue of an antibody (e.g., within a specified number of angstroms). The term "epitope" can also be used to refer to a functional epitope. According to some embodiments, a functional epitope can be used to describe an amino acid residue of an antigen that interacts with an amino acid residue of an antibody in a manner that contributes to the binding energy between an antigen and an antibody. An epitope can be determined according to different experimental techniques (also referred to as "epitope mapping techniques"). It should be understood that the determination of an epitope can vary based on the different epitope mapping techniques used, and can also vary with the different experimental conditions used, such as due to conformational changes or cleavage of an antigen induced by specific experimental conditions. Epitope mapping techniques are known in the art (e.g., Rockberg and Nilvebrant, Epitope Mapping Protocols: Methods in Molecular Biology, Humana Press, 3rd ed. 2018; Holst et al., Molecular Pharmacology 1998, 53(1): 166-175), and include, but are not limited to, X-ray crystallography, nuclear magnetic resonance (NMR) spectroscopy, site-directed mutagenesis, species exchange mutagenesis, alanine scanning mutagenesis, steric hindrance mutagenesis, hydrogen-deuterium exchange (HDX), and cross-blocking assays.

[0198] As used herein, unless otherwise indicated, the term "binding" refers to the ability of a protein or molecule to form a chemical bond or attractive interaction with another protein or molecule that brings the two proteins or molecules into proximity as determined by common methods known in the art.

[0199] As used herein, the term "nucleic acid" refers to a polymer of nucleotides, including molecules containing single-stranded and / or double-stranded nucleotides, such as DNA, cDNA and RNA molecules, incorporating natural, modified and / or analog nucleotides. The polynucleotides of the present disclosure may also include substrates incorporated therein, for example, by DNA or RNA polymerase or synthesis reactions.

[0200] Embodiments of the present disclosure include conjugates in which a polypeptide (e.g., an anti-human interleukin-4 receptor alpha antibody) is conjugated to one or more drug moieties, such as 2 drug moieties, 3 drug moieties, 4 drug moieties, 5 drug moieties, or more drug moieties. As described herein, the drug moiety can be conjugated to the polypeptide at one or more sites in the polypeptide. In certain embodiments, the conjugate has an average drug to antibody ratio (DAR) (molar ratio) in the range of 2 to 5, or 3 to 5, or 3 to 4. In certain embodiments, the conjugate has an average DAR of 3 to 4. In certain embodiments, the conjugate has an average DAR of about 3. In certain embodiments, the conjugate has an average DAR of about 4.

[0201] As used herein, it is understood that the conjugates of Formula I encompass conjugates of Formulas Ia, Ib, Ic, Id, Ie and If, and all references herein to the conjugates of Formula I are understood to include conjugates of Formulas Ia, Ib, Ic, Id, Ie and If. It will also be understood by those skilled in the art that the conjugates of Formula I including the conjugates of Formulas Ia, Ib, Ic, Id, Ie and If may also be referred to as anti-human IL-4Rα antibody glucocorticoid conjugates ("anti-human IL-4RαAb GC conjugates").

[0202] The anti-human IL-4Rα antibody GC conjugates disclosed herein can be formulated as pharmaceutical compositions that are administered by any route that makes the conjugate bioavailable, including, for example, intravenous or subcutaneous administration. Such pharmaceutical compositions can be prepared using techniques and methods known in the art (see, for example, Remington: The Science and Practice of Pharmacy, edited by A. Adejare, 23rd edition, published in 2020, Elsevier Science).

[0203] As used herein, the terms "treating", "treatment" or "to treat" include inhibiting, slowing down, stopping, controlling, delaying or reversing the progression or severity of existing symptoms or disorders, or improving existing symptoms or disorders, but do not necessarily mean completely eliminating existing symptoms or disorders. Treatment includes administering proteins or nucleic acids or vectors or compositions to treat symptoms or disorders in patients (especially humans).

[0204] As used herein, the term "inhibit" or "inhibiting" refers to, for example, reducing, lowering, slowing, decreasing, stopping, destroying, eliminating, antagonizing or blocking a biological response or activity, but does not necessarily mean complete elimination of the biological response or activity.

[0205] As used herein, the term "subject" refers to mammals, including but not limited to humans, chimpanzees, apes, monkeys, cows, horses, sheep, goats, pigs, rabbits, dogs, cats, rats, mice, guinea pigs, etc. Preferably, the subject is a human.

[0206] As used herein, the term "effective amount" refers to the amount or dosage of the conjugate of the present invention or its pharmaceutically acceptable salt, which provides the desired effect in the subject receiving diagnosis or treatment after single or multiple administration to the subject. As used herein, the term "effective amount" further refers to the amount or dosage of the conjugate of the present invention or its pharmaceutically acceptable salt, which will induce the desired biological or medical response of the subject, for example, reducing or inhibiting protein activity, or improving symptoms, alleviating symptoms, slowing or delaying disease progression, or preventing diseases, etc. In a non-limiting embodiment, the term "effective amount" refers to the necessary amount (in terms of dosage and for time period and for administration mode) or dosage of the conjugate of the present invention or its pharmaceutically acceptable salt when administered to a subject, which can effectively at least partially alleviate, inhibit, prevent and / or improve the condition, disorder or disease to achieve the desired therapeutic result. The effective amount is also the amount in which any toxic or harmful effect or dosage of the conjugate of the present invention or its pharmaceutically acceptable salt of the present invention is exceeded by the beneficial effect.

[0207] The effective amount can be determined by one skilled in the art by using known techniques and by observing the results obtained under similar circumstances. In determining the effective amount for a patient, the attending diagnostician will consider many factors, including, but not limited to: the species of the patient; the size, age, and general health of the patient; the specific disease or disorder involved; the extent or severity of the disease or disorder; the response of the individual patient; the specific conjugate being administered; the mode of administration; the bioavailability characteristics of the administered preparation; the dosage regimen selected; the use of concomitant medications; and other relevant circumstances.

[0208] Included within the scope of the present invention are pharmaceutically acceptable salts of the conjugates of Formula I. Pharmaceutically acceptable salts of the conjugates of the present invention (such as the conjugates of Formula I) can be formed under standard conditions known in the art. See, for example, Berge, SM et al., "Pharmaceutical Salts," Journal of Pharmaceutical Sciences, 66: 1-19, (1977).

[0209] Table 1: Abbreviations and definitions

[0210] the term definition ACN Acetonitrile aq Aqueous solution C18 Octadecylsilane cP Centipoise DCM Dichloromethane DIPEA N,N-Diisopropylethylamine DMF N,N-Dimethylformamide DMSO Dimethyl sulfoxide dppf 1,1'-Bis(diphenylphosphino)ferrocene ES / MS Electrospray mass spectrometry EtOAc Ethyl acetate HATU Azabenzotriazole tetramethyl urea hexafluorophosphate HIC Hydrophobic Interaction Chromatography HPLC High performance liquid chromatography LDA Lithium diisopropylamide MeOH Methanol MS Mass spectrometry MTBE Methyl tert-butyl ether m / z Mass-to-charge ratio NMR Nuclear Magnetic Resonance Pet Ether Petroleum ether RP-HPLC Reversed Phase HPLC rt Room temperature satd saturation THF Tetrahydrofuran

[0211] The conjugates of the present invention or their salts can be easily prepared by a variety of procedures known to those of ordinary skill in the art, some of which are described in the preparation and in the following examples. It is recognized by those of ordinary skill in the art that the specific synthetic steps of each route can be combined in different ways, or combined with steps from different schemes, to prepare the conjugates of the present invention or their salts. The product of each step can be recovered by conventional methods well known in the art, including extraction, evaporation, precipitation, chromatography, filtration, trituration and crystallization. Unless otherwise stated, all substituents are as previously defined. Reagents and starting materials are readily available to those of ordinary skill in the art. The following preparations, examples and determinations further illustrate the present invention, but should not be construed as limiting the scope of the present invention in any way. BRIEF DESCRIPTION OF THE DRAWINGS

[0212] Figure 1 The X-ray crystal structure of the Fab portion of Ab9 shown bound to IL-4Rα ECD is superimposed with the crystal structure of the dupilumab Fab portion with Crystal Kappa design in complex with human IL-4Rα (pdb accession number 6WGL).

[0213] Figure 2 The positions of the functional epitope amino acid residues in the crystal structure of the Ab10 Fab portion with Crystal Kappa design in complex with human IL-4Rα ECD are shown (human IL-4Rα residues Asp66 and Asp125).

[0214] Figure 3 The X-ray crystal structure of the Fab portion of Ab1 shown bound to IL-4Rα ECD is superimposed with the crystal structure of the dupilumab Fab portion with Crystal Kappa design in complex with human IL-4Rα (pdb accession number 6WGL).

[0215] Figure 4 Shown are the human IL-4Rα amino acid residue positions Asp66, Asp67, and Asp125 (all identified in the structural epitope; additionally, Asp66 was identified in the functional epitope) in the crystal structure of the Ab1 Fab portion with Crystal Kappa design in complex with the human IL-4Rα ECD.

[0216] Figure 5 The Ab1 GC conjugate of Example 1b was shown to inhibit anti-CD40 induced B cell proliferation.

[0217] Figure 6A-6BThe inhibition of IL-4-induced CD23 expression (6A) and GC-induced CD163 expression (6B) in myeloid cells by the Ab1 GC conjugate of Example 1b is shown.

[0218] Figure 7A-7C It was shown that the Ab1 GC conjugate of Example 1b significantly inhibited the IL-4Rα-mediated MDC (7A), GM-CSF (7B) and glucocorticoid receptor-mediated IL-5 (7C) cytokine secretion.

[0219] Figure 8 It was shown that the Ab1 GC conjugate of Example 1b did not significantly induce ADCC activity.

[0220] Fig. 9 It was shown that the Ab1 GC conjugate of Example 1b did not induce CDC activity in Daudi cells.

[0221] Figure 10A-10C Differential Scanning Calorimetry (DSC) thermograms of the exemplified Abl GC conjugate of Example 1b in PBS pH 7.2 (10A), acetate pH 5 (10B), and histidine pH 6 (10C) are shown.

[0222] Preparation 1

[0223] 6-Bromo-2-fluoro-3-methoxybenzaldehyde

[0224]

[0225] Two reactions were carried out in parallel. At -78 ° C, in 30 minutes, LDA (2M, 730 mL) was slowly added to a solution of 4-bromo-2-fluoro-1-methoxybenzene (250 g, 1.2 mol) in THF (1500 mL). After another 30 minutes, at -78 ° C, in 30 minutes, DMF (140 mL, 1.8 mol) was slowly added. After 1 hour, the two reactions were combined, and the mixture was diluted with citric acid aqueous solution (2000 mL) and extracted with EtOAc (1500 mL × 2). The combined organic layer was washed with saturated NaCl aqueous solution (1000 mL) and concentrated under reduced pressure to obtain a residue. The residue was ground with petroleum ether (1000 mL) at room temperature within 12 hours to obtain the title compound (382 g, 67% yield). ES / MS m / z 233.9 (M+H).

[0226] Preparation 2

[0227] 2-Fluoro-3-methoxy-6-methylbenzaldehyde

[0228]

[0229] Three reactions were carried out in parallel. 6-bromo-2-fluoro-3-methoxybenzaldehyde (120g, 5.3mol), methylboric acid (47g, 7.9mol), Pd(dppf)Cl2 (12g, 0.02mol) and Cs2CO3 (340g, 1.1mol) were added to a mixture of 1,4-dioxane (600mL) and water (120mL). The mixture was stirred at 120°C. After 12 hours, the three reactions were combined, and the mixture was diluted with saturated NH4Cl aqueous solution (1000mL) and extracted with MTBE (1500mL×2). The combined organic layer was washed with saturated NaCl aqueous solution (1000mL) and concentrated under reduced pressure to obtain a residue. The residue was purified by silica gel chromatography, eluted with 40:1 petroleum ether:EtOAc to obtain the title compound (180g, 59%). ES / MS m / z 169.3 (M+H).

[0230] Preparation 3

[0231] 2-Fluoro-3-hydroxy-6-methylbenzaldehyde

[0232]

[0233] 2-Fluoro-3-methoxy-6-methylbenzaldehyde (175 g, 1.0 mol) was added to DCM (1050 mL). BBr3 (200 mL, 2.1 mol) was slowly added to the solution at 0 °C. The reaction was stirred at room temperature. After 1 hour, the mixture was diluted with saturated NaHCO3 aqueous solution (1000 mL) until pH = 7-8, and then extracted with MTBE (1500 mL × 2). The combined organic layers were washed with saturated NaCl aqueous solution (1000 mL) and concentrated under reduced pressure to obtain the title compound (110 g, 68%). ES / MS m / z 154.9 (M+H).

[0234] Preparation 4

[0235] tert-Butyl N-[3-[(2-fluoro-3-formyl-4-methyl-phenoxy)methyl]phenyl]carbamate

[0236]

[0237] 2-Fluoro-3-hydroxy-6-methylbenzaldehyde (130g, 0.84mol), tert-butyl (3-(bromomethyl)phenyl)carbamate (200g, 0.70mol) and potassium carbonate (350g, 2.5mol) were added to acetonitrile (780mL) at room temperature and then heated to 50°C. After 5 hours, the reaction was diluted with water (600mL) and extracted with EtOAc (800mL×2). The combined organic layer was washed with saturated aqueous NaCl solution (800mL) and concentrated under reduced pressure to obtain a residue. The residue was purified by silica gel chromatography, eluted with 50:1 petroleum ether:EtOAc to obtain a crude product. The crude product was ground with MTBE (500mL) at room temperature for 30 minutes to obtain the title compound (103g, 32%). ES / MS m / z382.1(M+Na).

[0238] Preparation 5

[0239] (6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(3-((3-aminobenzyl)oxy)-2-fluoro-6-methylphenyl)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-1,2,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-4H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-4-one

[0240]

[0241] Perchloric acid (70% aqueous solution, 4.8 mL) was added to a suspension of (8S,9S,10R,11S,13S,14S,16R,17S)-11,16,17-trihydroxy-17-(2-hydroxyacetyl)-10,13-dimethyl-7,8,9,11,12,14,15,16-octahydro-6H-cyclopenta[a]phenanthrene-3-one (4.4 g, 12 mmol, also known as "16α-hydroxyprednisolone") and tert-butyl N-[3-[(2-fluoro-3-formyl-4-methyl-phenoxy)methyl]phenyl]carbamate (4.0 g, 11 mmol, Preparation 4) in acetonitrile (110 mL) at -10°C and warmed to room temperature. After 1 hour, DMF (10 mL) was added to the suspension at room temperature. After 18 hours, the reaction was quenched with saturated NaHCO3 aqueous solution and extracted with 9:1 DCM:isopropanol. The organic layers were combined, dried over MgSO4, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by reverse phase chromatography, eluted with 1:1 NH4HCO3 aqueous solution (10mM+5% MeOH):ACN to give the title compound, peak 1 (1.72g, 25%). ES / MS m / z 618.6 (M+H). 1 H NMR(400.13MHz,d6-DMSO)δ0.93-0.87(m,6H),1.40(s,3H),1.71-1.60(m,1H),1.89-1.76(m,4H),2.1 8-2.12(m,2H),2.29(s,4H),4.23-4.17(m,1H),4.32-4.30(m,1H),4.50-4.43(m,1H),4.81(d,J=3.2Hz ,1H),4.98-4.95(m,3H),5.16-5.10(m,3H),5.61(s,1H),5.95(s,1H),6.18-6.15(m,1H),6.53-6.48(m ,2H),6.58(s,1H),6.90-6.86(m,1H),6.99(t,J=7.7Hz,1H),7.12(t,J=8.5Hz,1H),7.33-7.30(m,1H).

[0242] Preparation 6

[0243] (6aR,6bS,7S,8aS,8bS,10S,11aR,12aS,12bS)-10-(3-((3-aminobenzyl)oxy)-2-fluoro-6-

[0244] methylphenyl)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-1,2,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-4H-naphtho[2',1':4,5]indeno[1,2-d][1,3]

[0245] Dioxol-4-one (also referred to herein as GC1)

[0246]

[0247] From Preparation 5, the residue was purified by reverse phase chromatography eluting with 1:1 aqueous NH4HCO3 (10 mM + 5% MeOH):ACN to give the title compound, Peak 2 (1.24 g, 18%). ES / MS m / z 618.6 (M+H). 1 H NMR (400.13 MHz, d6-DMSO) 1 H NMR(400.13MHz, DMSO):0.88(s,3H),1.24-1.12(m,2H),1.40(s,3H),1.69-1.56(m,1H),1.91-1.76(m,4H),2.08-2.01(m ,2H),2.22(s,3H),2.39-2.29(m,1H),3.18(d,J=5.2Hz,1H),4.12-4.00(m,1H),4.37-4.30(m,2H),4.79(d,J=3.1Hz,1H) ,5.00-4.93(m,2H),5.10-5.06(m,3H),5.31(d,J=6.7Hz,1H),5.95(s,1H),6.18(dd,J=1.8,10.1Hz,1H),6.34(s,1H),6. 53-6.48(m,2H),6.58(s,1H),6.87(d,J=8.5Hz,1H),6.99(t,J=7.7Hz,1H),7.09(t,J=8.5Hz,1H),7.33(d,J=10.1Hz,1H).

[0248] Preparation 7

[0249] (3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanoyl)-L-alanyl-L-alanine

[0250]

[0251] To a solution of N-succinimidyl 3-maleimidopropionate (5.0 g, 19 mmol) and L-alanyl-L-alanine (3.4 g, 21 mmol) in DMF (25 mL) was added DIPEA (3.1 mL, 18 mmol), and the mixture was stirred at room temperature overnight. The reaction mixture was concentrated under reduced pressure to give a residue, which was purified by silica gel chromatography, eluting with 2% acetic acid in EtOAc to give the title compound (4.0 g, 69%). ES / MS m / z 312.3 (M+H).

[0252] Preparation 8

[0253] 3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N-((S)-1-(((S)-1-((3-((2-fluoro-3-((6aR,6bS,7S,8aS,8bS,10S,11aR,12aS,12bS)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4, 6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)-4-methylphenoxy)methyl)phenyl)amino)-1-oxopropan-2-yl)amino)-1-oxopropan-2-yl)propanamide (also referred to herein as "GC-L")

[0254]

[0255] To (6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(3-((3-aminobenzyl)oxy)-2-fluoro-6-methylphenyl)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-1,2,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-4H-naphtho[2',1':4,5]indeno[1 ,2-d][1,3]dioxol-4-one (GC1, 24g, 39mmol, see Preparation 6) and 3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propionyl)-L-alanyl-L-alanine (15g, 47mmol, see Preparation 7) in DMF (250mL) solution was added 2,6-lutidine (11mL, 97mmol), followed by HATU (17g, 43mmol). The mixture was stirred at 0-5°C for 5 minutes, then the cooling bath was removed and the mixture was stirred for 2 hours. The mixture was diluted with EtOAc. The organic solution was washed with three parts of water, one part of saturated NaCl aqueous solution, dried over Na2SO4 (MeOH was added to help dissolve), filtered and evaporated to give the crude product. The crude product was purified by silica gel chromatography using a gradient of 1% to 10% MeOH in DCM to give the title compound (24g, 68%). ES / MS m / z 911.4 (M+H). 1 H NMR(400.13MHz,DMSO):d 9.88(s,1H),8.20(d,J=7.1Hz,1H),8.11(d,J=7.2Hz,1H),7.68(s,1H),7.60-7.58(m,1H),7.34-7.29(m,2H),7.14-7.09(m, 2H),7.00(s,2H),6.89(d,J=8.4Hz,1H),6.34(s,1H),6.18(dd,J=1.8,10.0Hz,1H),5.95(s,1H),5.76(s,1H),5.31(d,J=6.8 Hz,1H),5.13-5.04(m,3H),4.78(d,J=3.1Hz,1H),4.41-4.30(m,4H),4.10-4.00(m,1H),3.61(t,J=7.3Hz,2H),2.42-2.31(m ,3H),2.22(s,3H),2.11-2.01(m,2H),1.91-1.78(m,5H),1.40(s,3H),1.31(d,J=7.2Hz,3H),1.19-1.11(m,5H),0.88(s,3H).

[0256] Preparation 9

[0257] 3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N-((S)-1-(((S)-1-((3-((2-fluoro-3-((6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4- Oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)-4-methylphenoxy)methyl)phenyl)amino)-1-oxopropan-2-yl)amino)-1-oxopropan-2-yl)propanamide

[0258]

[0259] In a manner analogous to the procedure described in Preparation 8, the compound of Preparation 9 was prepared from (6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(3-((3-aminobenzyl)oxy)-2-fluoro-6-methylphenyl)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-1,2,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-4H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-4-one (see Preparation 5) and 3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanoyl)-L-alanyl-L-alanine (see Preparation 7). ES / MS m / z 911.4(M+H). 1HNMR(500.11MHz,DMSO):d 9.88(s,1H),8.23-8.20(m,1H),8.11(d,J=7.2Hz,1H),7.69(s,1H),7.59(d,J=8.0Hz,1H),7.33-7.28(m,2H),7.15-7.08(m, 2H),7.00(s,2H),6.91-6.89(m,1H),6.17(dd,J=1.7,10.1Hz,1H),5.94(s,1H),5.61(s,1H),5.16-5.12(m,3H),4.98-4.96( m,1H),4.81(d,J=3.1Hz,1H),4.49-4.36(m,6H),3.61(t,J=7.3Hz,2H),2.41(t,J=7.3Hz,2H),2.30-2.29(m,4H),2.17-2.15 (m,2H),1.88-1.77(m,4H),1.69-1.61(m,1H),1.40(s,3H),1.31(d,J=7.2Hz,3H),1.18(d,J=7.2Hz,3H),0.93-0.87(m,6H).

[0260] Example

[0261] Example 1. Production of anti-human IL-4Rα antibody GC conjugate

[0262] Example 1a. Generation and Engineering of Anti-Human IL-4Rα Antibodies

[0263] Antibody production: To develop antibodies specific for human IL-4Rα, transgenic mice with human immunoglobulin variable regions were immunized with the Fc-tagged extracellular domain (ECD) of human IL-4Rα and boosted alternately with human and cynomolgus Fc-tagged IL-4RαECD proteins. Screening was performed with histidine-tagged human and cynomolgus IL-4RαECD to identify cross-reactivity, and IL-4 blocking antibodies were identified in the absence or presence of excess soluble IL-4. Cross-reactive antibodies were cloned as Fabs, expressed and purified by standard procedures, and tested for blocking activity against IL-4 and IL-13 in the reporter cell line human embryonic kidney (HEK)-blue IL-4 / IL-13 (InvivoGen). Antibodies were selected and engineered in their CDRs, variable domain framework regions, and IgG isotypes to improve characteristics such as affinity, stability, solubility, viscosity, hydrophobicity, and reduced aggregation.

[0264] The amino acid sequence of human IL-4RαECD is provided by SEQ ID NO:15, the amino acid sequence of cynomolgus monkey IL-4RαECD is provided by SEQ ID NO:16, the amino acid sequence of human IL-4 is provided by SEQ ID NO:17, and the amino acid sequence of human IL-13 is provided by SEQ ID NO:18.

[0265] The antibodies of the present invention can be synthesized and purified by well-known methods. If two vectors are used, or a single vector system encoding heavy and light chains, a predetermined HC:LC vector ratio can be used to transiently or stably transfect suitable host cells (such as Chinese hamster ovary cells (CHO)) with an expression system for secreting antibodies. Commonly used techniques can be used to purify clarified culture media that have secreted antibodies.

[0266] Antibody engineering for affinity and biophysical properties: The IL-4Rα antibody Ab10 was engineered as a Fab in a mammalian cell expression vector using a high-throughput, site-specific, saturation mutagenesis protocol to discover mutations that improve affinity and / or biophysical properties (such as stability, solubility, viscosity, hydrophobicity, aggregation, serum protein binding, thermal stability, or chemical stability).

[0267] Briefly, CDR mutagenesis and atypical germline residues in the framework regions of Ab10 were evaluated. CDR analysis identified key CDR substitutions: LCDR3 H91W, N92S, which significantly improved the affinity of the resulting antibody from 10 -9 M range to 10 -11M range. Further analysis and experiments identified the following key residue modifications: A23V, N92S, I31H in VH; G28D in VL, which improve thermal stability in heat challenge ELISA while maintaining affinity. In addition, it was found that amino acid residue substitutions A23V, I58V in VH and G28D in VL reduced self-association and hydrophobicity while maintaining affinity. It was found that amino acid residue substitutions I31H in VH reduced serum protein binding. Certain antibodies were further engineered to eliminate deamidation by replacing asparagine in HC framework 3 (N72) with aspartic acid (N72D). In summary, 7 key amino acid residues were identified and engineered into Ab1, Ab2, Ab3, Ab4, Ab5, Ab6, Ab7 and Ab8 as follows: A23V, I31H, I58V, N72D in VH and G28D, H91W, N92S in VL, which significantly improved the affinity and biophysical properties of anti-human IL-4Rα antibodies (as shown below), such as thermal stability, reduced self-association, hydrophobicity and / or serum protein binding, without negatively affecting other functions or biophysical properties of the antibody. Ab9 was engineered as follows by amino acid residue substitution: I31H, I58V, N72D in VH and H91W, N92S in VL. Table 2 shows the CDR amino acid sequences of the exemplified antibodies. The exemplified antibodies were generated with different IgG main chains (including those provided in Table 3).

[0268] Antibody hinge and Fc backbone selection: Anti-human IL-4Rα antibodies Ab1, Ab2, Ab3, Ab4, Ab5, Ab9 and Ab10 were engineered to include an S228P mutation that stabilizes the hinge and prevents arm exchange. Wild-type IgG4 domains were used together with human kappa constant domains to complete the constructs. Antibodies were synthesized, expressed and purified essentially as described above.

[0269] Human IgG1A and / or human IgG4P backbones were selected for the exemplified antibodies because they provide unexpected advantages in binding to B cells and myeloid cells. As shown below, the exemplified antibodies were found to have greater binding potency to B cells when compared to the effector null antibody, thus indicating that the Fc portion of the exemplified antibodies that were not engineered as effector null antibodies positively affects B cell binding of the anti-IL-4Rα antibody.

[0270] Antibody constant region engineering for improved viscosity: The heavy chain constant region of the anti-human IL-4Rα antibody was further engineered by charge balance to improve viscosity and alleviate potential electrostatic interactions between the Fab and constant regions of the antibody. Five key amino acid residues in the CH1, CH2, and CH3 domains of IgG4 were identified as affecting the viscosity of anti-human IL-4Rα antibodies: 1) E137 (CH1 domain), 2) D203 (CH1 domain), 3) Q274 (CH2 domain), 4) Q355 (CH3 domain), and 5) E419 (CH3 domain). Antibodies with various combinations of these amino acid substitutions were generated to significantly improve the viscosity of the antibodies, including the combination for Ab1: Q274K (CH2 domain), Q355R (CH3 domain), and E419Q (CH3 domain). Similar positions of the five amino acids in the hIgG1 constant region are different and were found to affect the overall pI of each domain.

[0271] Table 2: CDR amino acid sequences of exemplary anti-human IL-4Rα antibodies

[0272]

[0273]

[0274] Table 3: Amino acid sequences of exemplary anti-human IL-4Rα antibodies

[0275] IL-4Rα Antibody HC LC VH V L Ab1 SEQ ID NO:9 SEQ ID NO:10 SEQ ID NO:7 SEQ ID NO:8 Ab2 SEQ ID NO:50 SEQ ID NO:10 SEQ ID NO:7 SEQ ID NO:8 Ab3 SEQ ID NO:37 SEQ ID NO:10 SEQ ID NO:7 SEQ ID NO:8 Ab4 SEQ ID NO:31 SEQ ID NO:10 SEQ ID NO:7 SEQ ID NO:8 Ab5 SEQ ID NO:35 SEQ ID NO:10 SEQ ID NO:7 SEQ ID NO:8 Ab6 SEQ ID NO:33 SEQ ID NO:10 SEQ ID NO:7 SEQ ID NO:8 Ab7 SEQ ID NO:13 SEQ ID NO:10 SEQ ID NO:7 SEQ ID NO:8 Ab8 SEQ ID NO:52 SEQ ID NO:10 SEQ ID NO:7 SEQ ID NO:8 Ab9 SEQ ID NO:46 SEQ ID NO:47 SEQ ID NO:44 SEQ ID NO:45 Ab10 SEQ ID NO:27 SEQ ID NO:28 SEQ ID NO:25 SEQ ID NO:26

[0276] Example 1b. Generation of anti-human IL-4Rα Ab1 GC conjugate (n=4)

[0277]

[0278] wherein n is 4; and

[0279] Ab is Ab1.

[0280] The exemplified anti-human IL-4RαAb1 (see Tables 2 and 3) was first reduced in the presence of a 40-fold molar excess of dithiothreitol (DTT) at 37°C for 2 hours or at 21°C for >16 hours. This initial reduction step is used to remove various capping groups, including cysteine ​​and glutathione, which are bound to the engineered cysteines at positions 124 and 378 of the heavy chain during expression. After the reduction step, the sample was purified by a desalting resin to remove the cysteine ​​cap as well as the reducing agent. A subsequent 2-hour oxidation step was performed at room temperature (approximately 21°C) in the presence of a 10-fold molar excess of dehydroascorbic acid (DHAA) to reform the native interchain disulfide and hinge disulfide pairs between the light and heavy chains. After the 2 h oxidation step, 4-8 molar equivalents of the glucocorticoid receptor agonist payload-linker ("GC-L"), 3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N-((S)-1-(((S)-1-((3-((2-fluoro-3-((6aR,6bS,7S,8aS,8bS,10S,11aR,12aS prepared in Preparation 8, were added using a 10 mM stock solution in DMSO. ,12bS)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)-4-methylphenoxy)methyl)phenyl)amino)-1-oxopropan-2-yl)amino)-1-oxopropan-2-yl)propanamide. The sample is then incubated at room temperature for 30-60 minutes to allow efficient conjugation of LP with engineered cysteine. Subsequent polishing steps such as size exclusion chromatography (SEC) or tangential flow filtration (TFF) are then used to buffer exchange the sample into a suitable formulation buffer and remove DMSO and any excess linker-payload.

[0281] Drug to Antibody Ratio (DAR) Assessment: To evaluate the average number of linkers-payloads present on the final conjugates, two analytical methods were used, including: 1) reverse phase (RP) HPLC and 2) time of flight (TOF) mass spectrometry. Both methods require an initial sample reduction step, which includes the addition of dithiothreitol (DTT) to a final concentration of approximately 10 mM, followed by incubation at 42°C for 5 minutes.

[0282] Reverse phase HPLC method: 1 mg / mL of anti-human IL-4Rα antibody GC conjugate sample was reduced by incubating the sample at 42°C for 5 minutes in the presence of 10 mM DTT. Ten to thirty micrograms of reduced anti-human IL-4Rα antibody GC conjugate sample were injected onto a Phenyl 5PW, 4.6 mm × 7.5 cm, 10 μM column (Tosh part number 0008043). Buffer A consisted of an aqueous solution of 0.1% trifluoroacetic acid (TFA), while buffer B consisted of an acetonitrile (ACN) solution of 0.1% trifluoroacetic acid (TFA). Prior to sample injection, the column was equilibrated in 20% B buffer, followed by a gradient of 28% B to 40% B in approximately 8.5 column volumes. The average DAR was determined by calculating the contribution of each individual DAR species by multiplying the fractional percentage by the DAR number of each contributing species. Since this value is based on the reduced sample and represents only half of the molecule, this number is multiplied by 2 to account for the complete antibody GC conjugate. The DAR calculations for the anti-human IL-4Rα Ab1 GC conjugate of Example 1b are provided in Table 4.

[0283] Table 4. Comparison of eCys conjugation of Example 1b using the percentage of each DAR species from the reduced samples Quantification of the average DAR of the species.

[0284]

[0285]

[0286] Time-of-flight mass spectrometry method: 8 μg of reduced sample was injected onto a Poroshell 300sb-C3 2.1×2.5 mm, 5 μM column (Agilent part number 821075-924). Buffer A consisted of 0.1% trifluoroacetic acid (TFA) in water, while buffer B consisted of 0.1% trifluoroacetic acid (TFA) in acetonitrile (ACN). Prior to sample injection, the column was equilibrated in 0% B buffer, followed by a gradient of 10% B to 80% B in approximately 28 column volumes. The average DAR was determined by calculating the contribution of each individual DAR species by multiplying the fractional percentage by the DAR number of each contributing species. Since this value is based on a reduced sample and represents only half of a molecule, this number is multiplied by 2 to account for the intact antibody.

[0287] The DAR calculations for the anti-human IL-4Rα Ab1 GC conjugate of Example 1b are provided in Table 5.

[0288] Table 5: Analysis of Example 1b using fractional percentages based on total ion counts from time-of-flight mass spectrometry Quantification of the average DAR of the eCys conjugates.

[0289]

[0290]

[0291] Example 1c. Generation of anti-human IL-4Rα Ab1 GC conjugate (n=3)

[0292]

[0293] wherein n is 3; and

[0294] Ab is Ab1.

[0295] The conjugate of Example 1c was prepared in a manner similar to the procedure described in Example 1b, using a lower molar ratio of GC-L, 3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N-((S)-1-(((S)-1-((3-((2-fluoro-3-((6aR,6bS,7S,8aS,8bS,10S,11aR,12aS,12bS)-7-hydroxy-8b- (2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)-4-methylphenoxy)methyl)phenyl)amino)-1-oxopropan-2-yl)amino)-1-oxopropan-2-yl)propanamide with Ab1. For example, using a corresponding GC-L:Ab1 molar ratio of 3.2:1 would yield a final DAR of about 3.

[0296] Example 1d. Sulfosuccinimide hydrolysis: The sulfosuccinimide ring of compound Formula Ie can be hydrolyzed under conditions well known in the art, as shown in Scheme 1 below (see, for example, WO 2017 / 210471, paragraph 001226) to provide a ring-opened product of Formula If.

[0297] Solution 1

[0298]

[0299] Furthermore, the above-mentioned thiosuccinimide ring of the compound of Formula Ie can undergo at least partial hydrolysis in vivo and under standard or well-known formulation conditions to provide the ring-opened product of Formula If.

[0300] Example 2. Determination of the structure and functional epitope of anti-human IL-4Rα antibody

[0301] Example 2a. Determination of the structural epitope of Ab9 Fab by X-ray crystallography. The physical epitope of the Fab of anti-IL-4RαAb9 on human IL-4Rα was determined by identifying the interaction interface between human IL-4Rα and the exemplified antibody. In brief, in order to determine the structural epitope, human IL-4RαECD was co-crystallized with the Fab portion of Ab9. The structure of Ab9 Fab in complex with IL-4Rα was determined by generating a hexa-histidine-tagged IgG1 variant of the heavy chain truncated after the CH1 domain and a "Crystal Kappa" form of the light chain of Ab9 Fab (see, Lieu et al., "Rapid and Robust Antibody FabFragment Crystallization Utilizing Edge-to-edge Beta-sheet Packing," PLoS One, 15 (9) (2020), which is incorporated herein by reference in its entirety). The Ab9 variants were co-expressed with a hexa-histidine-tagged version of the human IL-4Rα ECD containing the C182L mutation, and the complex was then purified by immobilized metal affinity chromatography and screened using a standard commercially available crystallization screen. Crystals were obtained and x-ray diffraction data were collected at the Advanced Photon Source. The diffraction data were reduced and solved by molecular replacement and refined to produce the exemplified 2.8 angstrom structure of the Ab9 Fab and IL-4Rα ECD complex. From the resulting crystal structure, any IL-4Rα amino acid residue within 4.5 angstroms of an atom of the co-crystallized Ab9 Fab was counted as part of the epitope (using PyMOL visualization software ).

[0302] PyMOL analysis demonstrated that the IL-4Rα amino acid residues within 4.5 angstroms of Ab9 Fab in the crystal structure complex (with respect to SEQ ID NO: 15) comprise the structural epitope of the exemplified antibody. Specifically, the analysis determined that the structural epitope comprises the following amino acid residues: Asp at position 12, Met at position 14, Ser at position 15, Ile at position 16, Tyr at position 37, Leu at position 39, Phe at position 41, Leu at position 43, Glu at position 45, His at position 47, Thr at position 48, Cys at position 49, Ile at position 50, His at position 62, Leu at position 64, Met at position 65, Asp at position 66, Asp at position 67, Val at position 69, Asp at position 72, Arg at position 99, Pro at position 121, Pro at position 123, Pro at position 124, Asp at position 125. This analysis further determined that the structural epitope spans domains 1 and 2 of the N-terminal fibronectin type III domain of IL-4Rα. In addition, this analysis determined that the following amino acid residues of the structural epitope are located in domain 2 of the N-terminal fibronectin type III domain of IL-4Rα: R99, P121, P123, P124, D125.

[0303] Furthermore, superposition of the crystal structures of the exemplified Ab9 Fab and the Crystal Kappa designed Dupilumab Fab in complex with human IL-4Rα (pdb accession number 6WGL) showed that Ab9 Fab binds to a novel epitope on IL-4Rα when compared to Dupilumab ( Figure 1 ).

[0304] Furthermore, alignment of the exemplified IL-4RαAb9 Fab:IL-4Rα complex crystal structure with published complexes of IL-4 and IL-13 and their corresponding receptors (pdb accession numbers 3BPN and 3BPO) on the IL-4Rα component in each structure (using PyMOL visualization software) showed that the exemplified Ab9 Fab antibody epitope overlaps with the IL-4 and IL-13 binding sites of IL-4Rα, thus indicating that when the Fab variant portion of the exemplified antibody binds to IL-4Rα, the binding of the exemplified antibody to IL-4Rα will physically block the binding of IL-4 and IL-13 cytokines to IL-4Rα.

[0305] Example 2b. Functional epitopes of Ab10. The functional epitopes of the exemplified anti-human IL-4Rα Ab10 were determined by ELISA. Briefly, thirty surface amino acid residue substitutions were introduced into the hexa-histidine-tagged human IL-4Rα extracellular domain (ECD) as follows: K2D, E6R, K22D, P26R, T31R, F41A, L42G, L43G, E45R, G56R, D66R, A71R, Q82G, K87D, E94R, H107A, D108R, P124R, D125R, D143R, R148D, L155R, R160D, S164R, S168R, Q181R, P192R, K195D or H197G. Each mutant protein with a single amino acid residue substitution as described above was transiently expressed in CHO cells and purified using standard fixed metal affinity chromatography techniques. The ELISA plate was coated with 1 μg / mL goat anti-human κ antibody (SouthernBiotech, catalog number 2060-01) in PBS at 4°C overnight, then washed 3 times in PBST and blocked with PBS casein at room temperature for 30 minutes. The plate was then washed 3 times with PBST, and the exemplified anti-human IL-4Rα antibody Ab6 was added to the wells at a final concentration of 1 μg / mL in PBS-casein and incubated for 1 hour. The plate was washed 3 times with PBST, the IL-4Rα mutant protein was serially diluted 3 times from 1 μg / mL in PBS-casein, and added to the plate at 50 μL / well, and incubated at room temperature for 1 hour. The plate was washed 3 times with PBST, and a 5000-fold dilution of an anti-histidine tag antibody HRP conjugate (R&D Systems, catalog number MAB050H) in PBS-casein was added and incubated at room temperature for 1 hour. The plate was washed 3 times, TMB substrate (Pierce, catalog number 34021) was added according to the manufacturer's instructions, the reaction was quenched with H2SO4, and the absorbance at 450nm was read on an ELISA plate reader. When compared to a control antibody, the functional epitope of the antibody was determined to correspond to the mutant amino acid residues of the wells that did not show a binding signal or showed a significantly reduced binding signal.

[0306] The results in Table 6A show that the functional epitope of the exemplified anti-human IL-4Rα Ab10 contains amino acid residues D66 and D125. Among the amino acid residues identified in the structural epitope, the substitution of amino acid residues D66R and D125R on IL-4Rα showed a significant negative effect on the binding of Ab10 to the mutant IL-4Rα, respectively. Specifically, the substitution of amino acid residue D66 of IL-4Rα to arginine reduced the binding of Ab10 to the mutant IL-4Rα to less than that of the control (0.04 OD 450 and 0.14OD 450In addition, amino acid residue D125 was substituted with arginine, which is located near amino acid residue D66 on the crystal structure of IL-4Rα (see Figure 2 ), also shows 0.59OD 450 The remaining amino acid substitutions were either within the range of positive binding or outside the defined structural epitopes.

[0307] Table 6A: Functional epitope determination of exemplary anti-human IL-4Rα antibody Ab10

[0308]

[0309]

[0310] Example 2c. Determination of the structural epitope of Ab1 Fab by X-ray crystallography. The physical epitope of the Fab of Ab1 anti-human IL-4Rα antibody on human IL-4Rα was determined essentially as described above. Crystals were obtained and x-ray diffraction data were collected at the Advanced Photon Source. The diffraction data was reduced and solved by molecular replacement and refined to produce a 2.49 angstrom structure of the exemplified Fab and IL-4Rα ECD complex. From the resulting crystal structure, any IL-4Rα amino acid residue within 4.5 angstroms of an atom of the co-crystallized Fab was counted as part of the epitope (using Molecular Operating Environment (MOE) visualization, modeling, and simulation software [Chemical Computing Group]; Coot (General Public License); and PyMOL visualization software ).

[0311] MOE, Coot and PyMOL analysis demonstrated that the IL-4Rα amino acid residues within 4.5 angstroms of the exemplified Fab in the crystal structure complex (with respect to SEQ ID NO: 15) comprise a structural epitope. Specifically, the analysis determined that the structural epitope comprises the following amino acid residues: Asp at position 12, Met at position 14, Ser at position 15, Ile at position 16, Leu at position 39, Phe at position 41, Leu at position 42, Thr at position 48, Cys at position 49, Ile at position 50, Glu at position 52, His at position 62, Leu at position 64, Met at position 65, Asp at position 66, Asp at position 67, Val at position 68, Val at position 69, Asp at position 72, Arg at position 99, Pro at position 121, Pro at position 123, Pro at position 124, Asp at position 125, Pro at position 192. Asp at position 66 coordinates well with the heavy chain of Ab1 Fab, with The Asp at position 67 has The analysis determined that the structural epitope spans domains 1 and 2 of the N-terminal fibronectin type III domain of IL-4Rα. In addition, the analysis determined that the following amino acid residues of the structural epitope are located in domain 2 of the N-terminal fibronectin type III domain of IL-4Rα: R99, P121, P123, P124, D125, P192.

[0312] Superposition of the crystal structures of the exemplified anti-human IL-4Rα Ab1 Fab and the CrystalKappa designed Dupilumab Fab in complex with human IL-4Rα (pdb accession number 6WGL) showed that the anti-human IL-4Rα Ab1 binds to a novel epitope on IL-4Rα when compared to Dupilumab ( Figure 3 ).

[0313] Alignment of the exemplified anti-human IL-4RαAb1 Fab:IL-4Rα complex crystal structure with the published complexes of IL-4 and IL-13 and their corresponding receptors (pdb accession numbers 3BPN and 3BPO) on IL-4Rα in each structure (using PyMOL visualization software) shows that the exemplified anti-human IL-4RαAb Fab antibody epitope overlaps with the IL-4 and IL-13 binding sites of IL-4Rα. This suggests that binding of the exemplified Ab1 will physically block the binding of IL-4 and IL-13 cytokines to IL-4Rα.

[0314] Example 2d. Functional epitopes of Ab1. The functional epitopes of the exemplified human IL-4Rα antibody Ab1 were determined by ELISA. Briefly, thirty surface amino acid residue substitutions were introduced into the hexa-histidine-tagged human IL-4Rα extracellular domain (ECD) as follows: K2D, E6R, K22D, P26R, T31R, F41A, L42G, L43G, E45R, E52R, G56R, D66R, A71R, Q82G, K87D, E94R, H107A, D108R, P124R, D125R, D143R, R148D, L155R, R160D, S164R, S168R, Q181R, P192R, K195D or H197G. Each mutant protein with a single amino acid residue substitution as described above was transiently expressed in CHO cells and purified using standard fixed metal affinity chromatography techniques. The ELISA plate was coated with 1 μg / mL goat anti-human IgG Fc antibody (Jackson ImmunoResearch Laboratories, catalog number 109-005-098) in PBS at 4°C overnight, then washed 3 times in PBST and blocked with PBS casein for 1 hour at room temperature. The plate was then washed 3 times with PBST, and the exemplified human IL-4Rα antibody was added to the wells at a final concentration of 1 μg / mL in PBS-casein and incubated for 1 hour at room temperature. The plate was washed 3 times with PBST, and the IL-4Rα mutant protein was serially diluted 5-fold in PBS-casein from 1 μg / mL for 3 points and added to the plate at 50 μL / well and incubated for 1 hour at room temperature. The plate was washed 3 times with PBST, and a 1000-fold dilution of an anti-histidine tag antibody HRP conjugate (R&D Systems, catalog number MAB050H) in PBS-casein was added and incubated at room temperature for 45 minutes. The plate was washed 3 times, TMB substrate (Pierce, catalog number 34028) was added according to the manufacturer's instructions, the reaction was quenched with H2SO4, and the absorbance at 450nm was read on an ELISA plate reader. When compared to the wild-type control, the functional epitope of the antibody was determined to correspond to the mutant amino acid residues of the wells that did not show a binding signal or showed a significantly reduced binding signal.

[0315] The results in Table 6B show that the functional epitope of the exemplary anti-human IL-4Rα antibody Ab1 contains amino acid residue D66. Substitution of the amino acid residue D66R on IL-4Rα reduced the binding of Ab1 to D66R IL-4Rα to below that of the negative control (0.047 OD 450 and 0.063OD 450The amino acid residue D66 is located near the structural epitope residues D67 and D125 in the IL-4Rα crystal structure ( Figure 4 The remaining amino acid substitutions were within the range of positive binding or outside the defined structural epitopes.

[0316] Table 6B: Functional epitope determination of exemplary anti-human IL-4Rα antibody Ab1

[0317]

[0318] Example 3. Binding efficacy of the exemplary anti-human IL-4Rα antibody and IL-4RαAb1 GC conjugate of Example 1b

[0319] Example 3a. ELISA binding: The binding potency of the exemplified anti-human IL-4Rα Ab1 and IL-4Rα Ab1 GC conjugates to human and cynomolgus monkey IL-4Rα was measured using a competitive Meso Scale Discovery (MSD) ELISA binding assay. A constant final concentration of Ab1 or conjugate (10 pM) was mixed with a 3-fold dilution series of human or cynomolgus monkey IL-4Rα to obtain a final starting concentration of 10 nM, and the mixture was incubated at 37°C for 4 days. A 96-well multi-array plate (Meso Scale Diagnostics, catalog number L15XA-3) was coated overnight at 4°C with 0.5 μg / mL hexa-histidine-tagged human or cynomolgus monkey IL-4Rα ECD in phosphate-buffered saline (PBS). After coating, 200 μL PBST (with 0.05% The plate was washed 10 times with PBST (20% PBS) and blocked with 300 μL / well PBS casein blocking buffer (Pierce, catalog number 37528) at 37°C for 1 hour. The plate was then washed 10 times as above, and 50 μL of the pre-incubated antibody: IL-4Rα dilution series was transferred to the wells and incubated at 37°C for 150 seconds with shaking at 300 rpm. The plate was washed 10 times with PBST, 50 μL of 1 μg / mL anti-human antibody sulfo-tag 20 (Meso Scale Diagnostics, catalog number R32AJ-1) was added to the plate for determination of Table 7a, and anti-human NHPκ light chain sulfo-tag 20 (Meso Scale Diagnostics, catalog number D20TF-6) was added to the plate for determination of Table 7b, and the plate was incubated at 37°C for 30 minutes with shaking at 300 rpm. The plate was washed 10 times with PBST, 150 μL / well of 1X Read Buffer T was added to the wells, and the ... minutes after the addition of the buffer. The apparent K was determined by fitting a sigmoidal curve to the electrochemiluminescence (ECL) response relative to log (soluble IL-4Rα concentration) using GraphPad Prism 9. D The data were plotted using normalized ECL values. The results in Table 7a are representative data from a single experiment, and the results in Table 7b are representative data from the average of 3 independent experiments performed in duplicate.

[0320] The exemplary results in Tables 7a and 7b show that the exemplary anti-IL-4Rα antibody and Ab1 GC conjugates have comparable K D Binds to human and cynomolgus monkey IL-4Rα.

[0321] Table 7a. Exemplary binding affinities of exemplary anti-human IL-4Rα antibodies for human and cynomolgus monkey IL-4Rα

[0322]

[0323]

[0324] Table 7b: Exemplary binding of the exemplary anti-human IL-4Rα conjugates of Example 1b to human and cynomolgus IL-4Rα Affinity

[0325]

[0326] Example 3b. Binding to B cells and T cells: The exemplified anti-human IL-4Rα antibodies were tested for binding to B cells and T cells in a fluorescence activated cell sorting (FACS) assay. TM Human PBMCs were isolated from human blood samples by density gradient centrifugation using GE Healthcare plus. Freshly isolated PBMCs were centrifuged at 2 × 10 6 The cells were resuspended at 100 μL / well and allowed to stand at room temperature for 15 minutes before being plated into a round-bottom 96-well plate. The cells were neutralized and FACS buffer (PBS containing 2% fetal bovine serum, from ) and washed. The Alexa Fluor 500 was conjugated to Alexa Fluor 500 according to the manufacturer's protocol (Thermo Fisher Scientific). The exemplary anti-human IL-4Rα antibody of 647 and the corresponding control IgG antibody were added to the wells at 66.67 nM and diluted 4-fold in duplicate. Then an equal volume of 2X antibody mixture was added to the wells, which mixture contained: human TruStain FcX TM 、FITC anti-human CD3 antibody、Alexa 700 anti-human CD4 antibodies (all from ) and CD20 monoclonal antibody (2H7), PerCP-Cyanine5.5 (ThermoFisher Scientific). The cells were incubated at 4°C for 30 minutes, washed twice with FACS buffer, and resuspended in a final volume of 100 μL of FACS buffer. The viability dye Sytox TM blue (Thermo Fisher Scientific) and analyzed by flow cytometry (LSRFortessa TM X-20; BD BIOSCIENCES) analyzed samples. Data were analyzed using FlowJo software and statistical analysis was performed using GraphPad Prism 9. Data represent the mean ± SEM of the percentage of IL-4Rα expressing cells from CD20 B cells and CD4 positive T cell populations from six donors. Curves were generated by fitting a sigmoidal curve of log(Ab concentration) relative to the percentage of positive IL-4Rα expressing cells from a single cell population.

[0327] The results in Table 8A from a representative experiment show that the exemplified anti-human IL-4Rα Ab1 and Ab7 bind to human PBMC-isolated B cells with comparable affinity (EC values ​​of 0.15 nM and 0.14 nM, respectively). 50 ) and CD4 + T cells (EC 26.3 nM and 28.7 nM, respectively) 50 ) on IL-4Rα.

[0328] In addition, the results in Table 8B from a representative experiment comparing the exemplified effector empty anti-human IL-4Rα Ab8 with Ab7 showed that when compared with Ab7 (EC 50 ) compared to the effector-empty Ab8, which had an unexpectedly reduced affinity for B cells (EC of 1.07 nM). 50 ), indicating that the Fc portion of the antibody can affect the binding of the exemplified IL-4Rα antibody to B cells. Ab7 and Ab8 share the same CDR amino acid sequence.

[0329] Table 8A: Binding of exemplary anti-human IL-4Rα antibodies to B cells and T cells

[0330]

[0331] Table 8B: Binding of exemplary human IL-4Rα antibodies to B cells

[0332]

[0333] Example 4. In vitro functional characterization of anti-human IL-4Rα Ab GC conjugate

[0334] Example 4a. Blocking activity of anti-human IL-4Rα Ab1 on cell-based IL-4 and IL-13 cytokines: The antagonistic activity of the exemplified anti-human IL-4Rα antibody on IL-4 and IL-13 was measured using HEK-Blue IL-4R and IL-13R expressing cell lines (InvivoGen). HEK-Blue cells were cultured at 5×10 4 Cells / well were plated in 50 μL of growth medium in poly-lysine coated plates overnight. Anti-human IL-4Rα antibodies were prepared in 4-fold dilutions in growth medium in Greiner 96-well low protein binding plates starting at 20 μg / mL. The dilution series was mixed with an equal volume of recombinant human IL-4 or IL-13 (Eli Lilly) in growth medium. 50 μL of the mixture was then added to the plate with HEK-Blue cells to a final concentration of 100 pg / mL human IL-4 or 10 ng / mL human IL-13, and the plates were then incubated overnight at 37°C in a tissue culture incubator. 20 μL of supernatant from the overnight incubated plate was transferred to a 96-well tissue culture treated plate, and 180 μL of QUANTI-Blue was added to each well. TM (InvivoGen), and the mixture was incubated at 37°C for 45 minutes. Secreted embryonic alkaline phosphatase (SEAP) activity was measured at 650 nm on a SpectraMax microplate reader (Molecular Devices). Results are reported as optical density (OD) at 650 nm, and statistical analysis was performed using GraphPad Prism 9. IC was generated by fitting a sigmoidal curve of log(Ab concentration) relative to OD at 650 nm for each exemplified antibody. 50 and curves.

[0335] The results showed that the exemplified anti-human IL-4Rα antibodies Ab1, Ab7, and Ab9 inhibited IL-4 and IL-13-induced SEAP activity in a dose-dependent manner. For IL-4 inhibition, IC 50 0.08nM, 0.07nM and 0.03nM respectively; for IL-13 inhibition, IC 50 They were 0.67 nM, 0.51 nM and 0.24 nM respectively (Table 9).

[0336] Table 9. Inhibition of cell-based IL-4 and IL-13 by exemplary anti-human IL-4Rα antibodies

[0337]

[0338] Example 4b. Internalization of anti-human IL-4Rα Ab1 GC conjugate: The ability of the exemplified anti-human IL-4Rα Ab1 GC conjugate of Example 1b to bind to IL-4Rα and internalize into Daudi cells was evaluated. The pH sensitive marker pHrodo was used according to the manufacturer's protocol. TM iFL microlabeling kit (Invitrogen # P36014) was used to label 100 μg of F(ab')2 goat anti-human IgG Fcγ (Jackson Immuno Research Labs # 109-006-098). Daudi cells (ATCC # CCL-213) were cultured at 1×10 6 Cells / mL were resuspended in culture medium and 100 μL were plated in a 96-well plate. The exemplified anti-human IL-4RαAb1 GC conjugate and anti-human IL-4RαAb were incubated with an equal concentration of pHrodo-labeled F(ab')2 goat anti-human IgG at 4°C for 30 minutes to form a complex, then serially diluted 10-fold for a 3-point curve and added to Daudi cells and incubated at 37°C in a CO2 incubator for 25 hours. After incubation, the plate was centrifuged at 400×g for 3 minutes, and the supernatant was aspirated. The antibody mixture containing CD20 monoclonal antibody (2H7), PerCP-Cyanine5.5 (eBioscience TM ) and TruStain FcX TM (BioLegend) in d-PBS with 2% fetal bovine serum. The stained cells were then incubated at 4°C for 30 minutes. The cells were then washed 2-3 times with 100 μL-150 μL of d-PBS with 2% FBS. The cells were resuspended in 100 μL of d-PBS with 2% FBS and 5 μL of SYTOX TM Blue dead cell stain (Invitrogen) was added to appropriate wells and mixed by pipetting. TM Plates were analyzed on an X-20 cell analyzer. Analysis was performed using FlowJo software. Curves were generated by plotting concentration relative to pHrodo geometric mean fluorescence intensity (gMFI) using GraphPad Prism 9.

[0339] The results in Table 10 show that the anti-human IL-4RαAb1 GC conjugate of Example 1b binds and internalizes into Daudi cells in a dose-dependent manner, with MFIs of 859, 509, and 397 at 10 μg / mL, 1 μg / mL, and 0.1 μg / mL, respectively. The internalization of the anti-human IL-4RαAb1 GC conjugate was comparable to that of the unconjugated Ab1. This indicates that the binding and internalization functions of the anti-human IL-4RαAb1 GC conjugate are not affected by the conjugation of anti-human IL-4RαAb1 to glucocorticoid.

[0340] Table 10. Internalization of the exemplary anti-human IL-4Rα Ab1 GC conjugate of Example 1b into Daudi cells

[0341]

[0342] Example 4c. Inhibition of IL-4 and IL-13-induced pSTAT6 phosphorylation in human PBMCs: The exemplified anti-human IL-4RαAb1 GC conjugate and anti-human IL-4RαAb1 of Example 1b were evaluated for inhibition of IL-4 and IL-13-mediated IL-4R pSTAT6 phosphorylation in primary B cells and / or T cells. TM Human PBMCs were isolated from human blood samples by density gradient centrifugation using GE Healthcare. The isolated cells were resuspended at 100-300 million cells in 100 mL of complete medium (containing 10% FBS, 1% penicillin-streptomycin solution (from ) and 1% GlutaMAX TM and 0.1% β-mercaptoethanol (from Gibco TM ) in RPMI-1640) and stimulated overnight with 2 μg / mL PHA (SIGMA), 0.5 μg / mL LPS (SIGMA) and 100 ng / mL recombinant human IL-6. The cells were washed with fresh medium and cultured at 5×10 4 to 2×10 5 Cells / well were plated in 96-well round-bottom plates 100 μL of complete medium containing the exemplified antibodies diluted at 10 μg / mL in 4-fold dilutions and 11-point titration. The cells were incubated with the samples at room temperature for 30 minutes and then stimulated with human recombinant IL-4 (20 ng / mL final concentration) or human recombinant IL-13 (100 ng / mL final concentration, R&D SYSTEMS) for 12 minutes at room temperature. The stimulation was stopped by adding 120 μL of 1X Lyse / Fix buffer (BD BIOSCIENCES) for 5 minutes, then the plate was centrifuged at 2000 rpm for 2 minutes, and the supernatant was aspirated. The cell pellet was resuspended in 100 μL of ice-cold methanol (SIGMA) and placed on ice for 20 minutes and washed with DPBS containing 2% FBS. Wash. Resuspend cells in 50 μL of a cocktail of antibodies against the following proteins: CD4, CD33, CD8, and CD3 (Thermo Fisher Scientific), phosphorylated STAT6 The cells were incubated with 1% STAT6 and CD20 (BDBIOSCIENCES) and incubated at room temperature for 30 minutes, followed by washing with DPBS containing 2% FBS. Cell samples were analyzed using a flow cytometer. FlowJo software was used for analysis, and GraphPad Prism 9 was used for statistical analysis. The curves were generated by fitting a sigmoidal curve of the log (Ab concentration) of the percentage of inhibition of phosphorylated STAT6 relative to each individual donor cell population (n=2).

[0343] The results in Table 11A show that the exemplified anti-human IL-4RαAb1 GC conjugate of Example 1b inhibited CD4+ T cells in a dose-dependent manner (IC 50 ) and B cells (IC of 0.041 μg / mL 50 ) IL-4-induced STAT6 phosphorylation.

[0344] The results in Table 11B show that the exemplified anti-human IL-4RαAb1 GC conjugate of Example 1b inhibited IL-13-induced STAT6 phosphorylation in B cells in a dose-dependent manner (IC of 0.064 μg / mL). 50 ). This demonstrates the ability of human IL-4RαAb1 GC conjugate to block IL-4 and IL-13 signaling through IL-4R.

[0345] Table 11A. Effects of IL-4 induction in human T cells and B cells by exemplified anti-human IL-4RαAb1GC conjugates of Example 1b Inhibition of STAT6 phosphorylation

[0346]

[0347] Table 11B. Effect of the exemplified anti-human IL-4RαAb1 GC conjugate of Example 1b on IL-13-induced proliferation in human B cells Inhibition of STAT6 phosphorylation

[0348]

[0349] Example 4d. Inhibition of IL-4-induced B cell proliferation: The exemplified anti-human IL-4RαAb1 GC conjugate of Example 1b and anti-human IL-4RαAb1 and Ab7 were evaluated for inhibition of B cell proliferation in primary B cells isolated from human PBMCs. TM Human PBMCs were isolated from human blood samples by STEMCELL density gradient centrifugation according to the manufacturer's protocol ( TM Technologies) by using EasySep TM The negative selection of the Human Naive B Cell Enrichment Kit was used to isolate primary B cells from PBMC suspension. The isolated human primary B cells were cultured at 1×10 6 Cells / mL were resuspended and plated in complete medium (containing 10% fetal bovine serum, 1XMEM-non-essential amino acids, 1 mM sodium pyruvate, 1X penicillin-streptomycin solution (all from ) and 1XGlutaMAX TM (Gibco TM ), 0.1% β-mercaptoethanol (LIFE TECHNOLOGIES) in RPMI-1640. Anti-human IL-4RαAb1 GC conjugate, anti-human IL-4RαAb1 and Ab7,

[0350] (6aR,6bS,7S,8aS,8bS,10S,11aR,12aS,12bS)-10-(3-((3-aminobenzyl)oxy)-2-fluoro-6-methylphenyl)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-1,2,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-4H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-4-one (compound of Preparation 6, also referred to herein as "GC1") or isotype control were pretreated with cells for 0.5 to 1 hour at 66.67 nM diluted 4-fold and 10-point titration. The cells were stimulated with human CD40 / TNFRSF5 antibody (200 ng / mL; R&D SYSTEMS) and IL-4 recombinant human protein (5 ng / mL; R&D SYSTEMS) at 37°C and 5% CO2 for 2 days. 3 H]-thymidine (1 mCi thymidine / well; ) were pulsed at 37°C for 18 h and counted by microplate counter (MicroBeta2; )Measurement[ 3 H]-thymidine incorporation levels were measured and expressed as cells counts per minute (CCPM). Statistical analysis was performed using GraphPad Prism 9 and curves were generated by fitting a sigmoidal curve of log(Ab concentration) relative to the mean percent inhibition of CCPM compared to CD40 stimulation alone from both donors.

[0351] The results in Table 12A show that the exemplified anti-human IL-4RαAb1 GC conjugate of Example 1b inhibited IL-4-induced B cell proliferation in a dose-dependent manner (IC of 1.41 nM). 50 ), compared with unconjugated anti-human IL-4RαAb1 (IC 50 GC1 alone was comparable to the isotype control, i.e. no significant inhibition was observed.

[0352] Table 12B shows representative results of the inhibition of IL-4-induced B cell proliferation by anti-human IL-4Rα Ab1 and Ab7.

[0353] Table 12A. Effect of the exemplary anti-human IL-4Rα Ab1 GC conjugate of Example 1b on IL-4-induced B cell proliferation inhibition

[0354]

[0355] Table 12B. Inhibition of IL-4-induced B cell proliferation by exemplary anti-human IL-4Rα antibodies

[0356]

[0357] Example 4e. Glucocorticoid receptor-mediated inhibition of anti-CD40-induced B cell proliferation: The inhibition of B cell proliferation by the exemplified anti-human IL-4RαAb1GC conjugate and anti-human IL-4RαAb1 of Example 1b was evaluated in primary B cells isolated from human PBMCs. TM Human PBMCs were isolated from human blood samples by STEMCELL density gradient centrifugation according to the manufacturer's protocol ( TM Technologies) by using EasySep TM The negative selection of the Human Naive B Cell Enrichment Kit was used to isolate primary B cells from PBMC suspension. The isolated human primary B cells were cultured at 1×10 6The cells were resuspended at 100 cells / mL and plated in complete medium (containing 10% fetal bovine serum, 1X MEM-non-essential amino acids, 1 mM sodium pyruvate, 1X penicillin-streptomycin solution (all from ) and 1X GlutaMAX TM (Gibco TM ), 0.1% β-mercaptoethanol (RPMI-1640 from LIFE TECHNOLOGIES). Cells were pretreated for 0.5 h to 1 h with anti-human IL-4RαAb1, anti-human IL-4RαAb1 GC conjugate, compound of Preparation 6 (GC1), or isotype control at 100 nM dilution 4-fold and 9-point titration. Cells were stimulated with human CD40 / TNFRSF5 antibody (200 ng / mL; R&D SYSTEMS) at 37°C and 5% CO2 for 2 days. Cells were then treated with [ 3 H]-thymidine (1 mCi thymidine / well; ) were pulsed at 37°C for 18 h and counted by microplate counter (MicroBeta2; )Measurement[ 3 H]-thymidine incorporation levels were measured and expressed as cells counts per minute (CCPM). Statistical analysis was performed using GraphPad Prism 9 and curves were generated by fitting a sigmoidal curve to the log(Ab concentration) relative to the mean percent inhibition of CCPM compared to no stimulation from 2 donors.

[0358] Table 13 and Figure 5 The results in Example 1b showed that the exemplified anti-human IL-4RαAb1 GC conjugate and GC1 alone inhibited anti-CD40-induced B cell proliferation in a dose-dependent manner (IC values ​​of 5.51 nM and 3.73 nM, respectively). 50 ). Minimal inhibition was observed with anti-human IL-4RαAb1, which was comparable to the individual isotypes. This shows that the GC in the anti-human IL-4RαAb1GC conjugate is effectively delivered into B cells and effectively modulates GC receptor agonists in B cells to inhibit B cell proliferation, independent of IL-4R-mediated inhibition of B cell proliferation.

[0359] Table 13. Glucocorticoid agonist receptor-mediated effects of the anti-human IL-4Rα Ab1 GC conjugate of Example 1b Inhibition of CD40-induced B cell proliferation

[0360]

[0361] Example 4f. Inhibition of IL-4-induced CD23 expression and CD163 expression on myeloid cells: The exemplified anti-human IL-4RαAb1 GC conjugate of Example 1b was evaluated for its induction of IL-4-induced CD23 expression and glucocorticoid-induced CD163 expression in myeloid cells. CD163 is expressed by monocytes and is associated with autoimmune disorders. Glucocorticoids have been shown to induce CD163 expression, which is thought to contribute to the anti-inflammatory effects of glucocorticoids. To determine the role of glucocorticoids in anti-human IL-4RαAb1 GC conjugates, the level of CD163 expression on the surface of monocytes in response to treatment with anti-human IL-4RαAb1 GC conjugates was evaluated.

[0362] Briefly, fresh LRS-WBC donors were obtained from the San Diego Blood Bank and used for standard Ficoll-Paque TM PBMCs were isolated by density gradient centrifugation using GE Healthcare plus. 5 Cells / well were seeded in a 96-well flat bottom plate. 50 μL of 3X serially diluted antibodies were added to the wells and incubated for 30 minutes at 37°C with 5% CO2. Then 50 μL of 3X stimulation of recombinant human IL-4 (R&D SYSTEMS) in complete medium was added to the wells to a final concentration of 10 ng / mL. The plates were incubated at 37°C with 5% CO2 for 48 hours, the cells were washed and resuspended in FACS buffer containing human TruStain FcX TM , Bright Purple 605 TM Anti-human CD163 antibody, Brilliant Violet 785 TM Anti-human CD33 antibody, FITC anti-human CD3 antibody (from ), CD20 monoclonal antibody (2H7) PerCP-Cyanine5.5 and CD23 monoclonal antibody (EBVCS2), APC (from THERMO FISHER SCIENTIFIC). The cells were incubated at 4°C for 30 minutes, washed twice with FACS buffer, and resuspended in a final volume of 100 μL of FACS buffer. The viability dye Sytox TM blue (Thermo Fisher Scientific) was added to the wells and analyzed by flow cytometry (LSRFortessa TM X-20; BD BIOSCIENCES) to analyze the samples. FlowJo software was used for data analysis. Myeloid cells were identified as Sytox TMData are presented as sigmoidal curve fits of the geometric mean fluorescence intensity (gMFI) of myeloid cells relative to log(Ab concentration) of 3 or 4 donors (mean ± SD) and statistical analysis was performed using GraphPad Prism 9.

[0363] Table 14 and Fig. 6A and Figure 6B The results in Table 1 show that the exemplified anti-human IL-4RαAb1 GC conjugate and anti-human IL-4RαAb1 of Example 1b have IC values ​​of 10.75 nM and 8.27 nM, respectively. 50 Inhibits IL-4-induced CD23 expression on myeloid cells ( Fig. 6A In addition, the results showed that the anti-human IL-4RαAb1 GC conjugate significantly increased CD163 expression at 1000nM, 250nM and 63nM (gMFI were 8462, 5984 and 2317, respectively) compared to the unconjugated Ab1 at 1000nM (gMFI was 941) ( Figure 6B ). This shows that the exemplified anti-human IL-4RαAb1 GC conjugate can both inhibit the IL4R-mediated response mediated by Ab1 and induce the GC receptor-mediated response in the same cells, demonstrating the dual functionality of the anti-human IL-4RαAb1 GC conjugate from the antibody and glucocorticoid, as well as the effective delivery of GC into cells by the antibody.

[0364] Table 14. Effects of the anti-human IL-4Rα Ab1GC conjugate of Example 1b on IL-4-induced CD23 expression in myeloid cells and Inhibition of GC-induced CD163 expression

[0365]

[0366] Example 4g. Induction of glucocorticoid-induced gene expression in Th2 differentiated T cells: The induction and expression of three glucocorticoid receptor-mediated genes (Tsc22d3, Fkbp5 and Zbtb16) and one cytokine were measured in primary human T cells differentiated ex vivo into a Th2 phenotype representing cells involved in type 2 inflammation and disease.

[0367] Human Th2 cells were differentiated in vitro by culturing purified naive CD4 T cells with anti-human CD3 (BioXCell #BE0001-2), anti-human CD28 (BioLegend #302934), anti-human IFNγ (R&D Systems #MAB285-500), recombinant human IL-2 (R&D Systems #202-IL-050 / CF), and recombinant human IL-4 (R&D Systems #6507-IL-100 / CF) for 14 days. Terminally differentiated Th2 cells were then rested in the absence of IL-4 for 12 hours prior to assay to restore IL-4R surface expression. Flow cytometric staining was used to assess cell purity on a BD LSRFortessa cell analyzer. Th2 cells were identified as CD4+ (anti-human CD4-eFluor-450, Fisher Scientific #48-0047-42), GATA3+ (anti-human GATA3-PerCP / Cyanine5.5, BioLegend #653812), and IL4R+ (anti-IL-4Rα antibody-Alexa Fluor-647, Lilly). 6 Th2 cells / well were treated with 100 nM IL4R-GC Ab1 and stimulated with human T-activator CD3 / CD28 Dynabeads (Fisher Scientific #11132D) at 37°C for 24 hours. Cells from each assay condition were lysed in RLT buffer (Qiagen #79216) and frozen at -80°C. RNA was then isolated using the RNeasy 96 kit (Qiagen #74181) according to the manufacturer's protocol. Data represent four technical replicates (n=4) for each assay condition.

[0368] Gene expression levels were determined using the NanoString platform and a custom genomic panel (Table 15) following the manufacturer's recommended protocol. 260The RNA isolated from the in vitro culture was quantified. RNA was diluted to 20 ng / μl in RNase-free water, and a total of 100 ng (in 5 μl) was used to prepare the NanoString column. According to the data processing method recommended by the manufacturer, the original mRNA counts were standardized using nSolver advanced analysis software (version 2.0.115). First, all mRNA counts were scaled to the sample binding density control, and then the experimental genes were further standardized to the housekeeping gene index according to the dynamic housekeeping gene selection of the entire data set, thereby minimizing the housekeeping gene difference. The standardized gene counts were converted to a Log2 scale, and the relative expression of the experimental treatment group was determined by subtracting the average Log2 expression of the "untreated" group. Statistical analysis was performed using GraphPad Prism 9. The results are reported as average log2 expression, which is standardized relative to the mean + SD (for all groups n=4) of the replicates of the untreated group for each replicate (for each gene in the treatment group). Differences were assessed using two-way analysis of variance (ANOVA) on normalized values ​​and compared to the untreated group using the Bonferroni correction for multiple comparisons, with a significance level of p < 0.001.

[0369] The results in Table 16 show that the anti-human IL-4RαAb1 GC conjugate of Example 1b induced the mRNA expression of glucocorticoid receptor-mediated genes Tsc22d3 (increased to 2.96 times, p value <0.001), Fkbp5 (increased to 1.81 times, p value <0.001), and Zbtb16 (increased to 1.29 times, p value <0.003) in Th2 differentiated T cells, and reduced the mRNA expression of cytokine IL-5 to 1 / 2.29, p value <0.0001. This shows that the anti-human IL4RαAb1 GC conjugate can induce GC / GR-mediated gene regulation and reduce IL-5 expression in cells representing cells involved in type 2 inflammation and disease.

[0370] Table 15. Genomic set of GC / GR gene regulation by the anti-human IL4Rα Ab1 GC conjugate of test example 1b

[0371] Gene name Accession Number Specify Tsc22d3 Hs NM_004089.3 Endogenous FKB5 NM_001145775.1 Endogenous Zbtb16 NM_006006.6 Endogenous IL5 NM_000879.2 Endogenous R4H NM_000968.4 Housekeeping TBP NM_003194.4 Housekeeping UBB Hs NM_001281718.1 Housekeeping Gapdh H NM_001256799.1 Housekeeping Tubb4a Hs NM_006087.2 Housekeeping G6PD NM_001042351.2 Housekeeping Gusb Hs NM_000181.3 Housekeeping ABCF1Hs NM_001025091.1 Housekeeping OAZ1 Hs NM_004152.2 Housekeeping POLR2A Hs NM_000937.4 Housekeeping BABAM1 Hs NM_014173.4 Housekeeping Hprt1 Hs NM_000194.2 Housekeeping

[0372] Table 16. The anti-human IL-4RαAb1GC conjugate of Example 1b regulates GC / GR gene expression in Th2 differentiated T cells Festival

[0373] Gene Mean difference (log2) 95% CI Adjusted P value Tsc22d3 2.959 2.226 to 3.692 <0.0001 FKB 1.807 1.074 to 2.541 <0.0001 Zbtb16 1.29 0.5571 to 2.023 0.0003 IL-5 -2.289 -3.023 to -1.556 <0.0001

[0374] Example 4h. Inhibition of cytokine secretion from human PBMCs: PBMCs were isolated from blood layered over Ficoll by centrifugation at 400×g for 30 minutes at room temperature. The PBMC layer was incubated with sterile RPMI-1640 medium ( Catalog number 10041CV) and centrifuged at 1500 rpm for 5 minutes at 4°C. Red blood cells were lysed by resuspending the cell pellet in 5 mL of ACK lysis buffer (GIBCO catalog number A1049201) and incubated at room temperature for 5 minutes. After RBC lysis, cells were washed and lysed at 1×10 6 Cells / mL were resuspended in warm complete medium (containing 10% FBS, 1% MEM non-essential amino acid solution, 1% penicillin-streptomycin solution (from ) and 1% GlutaMAX TM and 0.1% β-mercaptoethanol (from Gibco TM ) in RPMI-1640. The cells were cultured at 1×10 6 Cells / well were seeded in a 96-well flat-bottom plate. 50 μL of 4X control IgG, anti-human IL4RαAb1, anti-human IL4RαAb1 GC conjugate of Example 1b or GC1 alone (9-point curve, final concentration of 100 nM, with 3-fold serial dilution) was added to the well. Then, 50 μL of 4X stimulation mixture in complete medium was added to the well, the stimulation mixture consisting of 40 μg / mL PHA-M (phytohemagglutinin, M form; LIFE TECHNOLOGIES) and 400 ng / mL PMA (phorbol 12-myristate 13-acetate; TOCRIS). The plate was then incubated at 37°C with 5% CO2 for 48 hours. After the incubation period, the plate was centrifuged at 2000 rpm for 1 minute, and the top 100 μL supernatant was removed and stored at -80°C until ready for cytokine detection. Cytokine release was detected using the U-PLEX Custom Biomarker [Human] Multiplex Assay (MESO SCALE DISCOVERY) according to the manufacturer's protocol. Statistical analysis was performed using GraphPad Prism 9. Data are presented as sigmoidal curve fits of the mean of the percent inhibition of individual donors for each cytokine (n=2) relative to log(Ab concentration) and statistical analysis was performed using GraphPad Prism 9.

[0375] Table 17 and Figure 7A-7C The results in Example 1b show that the anti-human IL-4RαAb1 GC conjugate of Example 1b significantly inhibits IL-4R-mediated and glucocorticoid receptor-mediated cytokine secretion. Anti-human IL-4RαAb1 GC conjugate inhibits MDC secretion similarly to anti-human IL-4RαAb1 (79.7% and 74.7%, respectively, at 100 nM). Anti-human IL-4RαAb1 GC conjugate inhibits GM-CSF secretion more strongly than Ab1 alone (77.6% and 40.9%, respectively, at 100 nM, Figure 4B). In addition, anti-human IL-4RαAb1 GC conjugate and GC1 payload inhibited IL-5 secretion by 78.5% and 97.4%, respectively, at 100 nM ( Figure 4 C), while Ab1 had no effect (-13.3% at 100 nM). This data demonstrates the dual functionality of anti-human IL-4RαAb1 GC conjugate in inhibiting IL-4R-dependent and IL4R-independent cytokine secretion. This demonstrates the additional functionality and improved efficacy of anti-human IL-4RαAb1 GC conjugate in cytokine inhibition when compared to the antibody alone.

[0376] Table 17. Effects of the anti-human IL-4Rα Ab1 GC conjugate of Example 1b on IL-4R-mediated and GC-mediated cytokines Inhibition of secretion

[0377]

[0378]

[0379] Example 5. Effector function and Fcγ receptor of the exemplary anti-human IL-4Rα antibody GC conjugate of Example 1b Combination

[0380] Example 5a. Human Fcγ receptor binding. The binding affinity of the anti-IL-4Rα GC conjugate and anti-human IL-4Rα antibody exemplified in Example 1b to human Fcγ receptors was evaluated by surface plasmon resonance (SPR) analysis. Series S CM5 chips (Cytiva P / N BR100530) were prepared using the manufacturer's EDC / NHS amine coupling method (Cytiva P / N BR100050). Briefly, the surfaces of all four flow cells (FCs) were activated by injecting a 1:1 mixture of EDC / NHS at 10 μL / min for 7 minutes. Protein A (Calbiochem P / N 539202) was diluted to 100 μg / mL in 10 mM acetate, pH 4.5 buffer, and approximately 4000 RU was immobilized on all 4 FCs by injecting at a flow rate of 10 μL / min for 7 minutes. Unreacted sites were blocked by injecting ethanolamine at 10 μL / min for 7 minutes. 2×10 μL of glycine, pH 1.5, was injected to remove any non-covalently bound proteins. The running buffer was 1×HBS-EP+ (TEKNOVA, P / N H8022). FcγR extracellular domain (ECD)-FcγRI (CD64), FcγRIIA_131R and FcγRIIA_131H (CD32a), FcγRIIIA_158V, FcγRIIIA_158F (CD16a) and FcγRIIb (CD32b) were produced from stable CHO cells and purified using IgG Sepharose and size exclusion chromatography. For FcγRI binding, the antibody was diluted to 2.5 μg / mL in running buffer, and about 150 RU of each antibody (capture RU) was captured in FC 2 to 4. FC1 is a reference FC, so there is no capture antibody in FC1. FcγRI ECD was diluted to 200 nM in running buffer and then serially diluted two-fold to 0.78 nM in running buffer. Duplicate injections of each concentration were injected into all FCs at 40 μL / min for 120 seconds, followed by a dissociation period of 1200 seconds. Regeneration was performed by injecting 15 μL of 10 mM glycine, pH 1.5, at 30 μL / min on all FCs. Reference-subtracted data were collected as FC2 FC1, FC3-FC1, and FC4-FC1, and measurements were obtained at 25°C. Affinity (K) was calculated using the "1:1 (Langmuir) binding" model in the steady-state equilibrium analysis or BIA evaluation with Scrubber 2 Biacore evaluation software. D). For FcγRIIa, FcγRIIb and FcγRIIIa binding, the antibodies were diluted to 5 μg / mL in running buffer and approximately 500 RU of each antibody was captured in FC 2 to 4). FC1 is the reference FC. The Fcγ receptor ECD was diluted to 10 μM in running buffer and then serially diluted 2-fold to 39 nM in running buffer. Duplicate injections of each concentration were injected into all FCs at 40 μL / min for 60 seconds, followed by a 120 second dissociation phase. Regeneration was performed by injecting 15 μL of 10 mM glycine, pH 1.5, at 30 μL / min on all FCs. The reference-subtracted data was collected as FC2-FC1, FC3-FC1 and FC4-FC1, and measurements were obtained at 25°C. Affinity (K) was calculated using steady-state equilibrium analysis with Scrubber 2 Biacore evaluation software. D ). Each receptor was assayed at least twice.

[0381] The results in Table 18A show the binding affinity (K) of the exemplified anti-human IL-4Rα Ab1 GC conjugate of Example 1b to human FcγRI, FcγRIIa, FcγRIIb and FcγRIIIa receptor ECD. D ).

[0382] The results in Table 18B summarize the binding affinities (K) of the exemplary anti-human IL-4Rα antibodies Abl and Ab7 to human FcγRI, FcγRIIa, FcγRIIb, and FcγRIIIa receptor ECDs. D ).

[0383] Table 18A. Binding affinity of the exemplary anti-human IL-4Rα Ab1GC conjugate of Example 1b to human Fcγ receptors

[0384]

[0385] Table 18B. Binding affinity of exemplary anti-human IL-4Rα antibodies to human Fcγ receptors

[0386]

[0387] Example 5b. Antibody-dependent cellular cytotoxicity (ADCC): The in vitro ADCC assay of the exemplified anti-human IL-4RαAb1 GC conjugate of Example 1b was evaluated using a reporter gene-based ADCC assay. Briefly, Daudi cells (ATCC, #CCL-213) expressing human IL-4Rα and human CD20 were used as target cell lines and Jurkat cells expressing functional FcγRIIIa (V158)-NFAT-Luc (Eli Lilly and Company) were used as effector cell lines. All test molecules and cells were diluted in assay medium containing RPMI-1640 (without phenol red) with 0.1 mM non-essential amino acids (NEAA), 1 mM sodium pyruvate, 2 mM L-glutamine, 500 U / mL penicillin-streptomycin and 0.1% w / v BSA. The test antibody was first diluted to a 3X concentration of 30 nM and then serially diluted 7 times at a 1:4 ratio. 50 μL / well of each molecule was aliquoted in triplicate in a white opaque bottom 96-well plate (Costar, #3917). CD20 antibody was used as a positive control. Daudi target cells were then plated at 5×10 4 50 μL aliquots of 10 cells / well were added to the plate and incubated at 37°C for 1 hour. Next, Jurkat V158 cells were plated at 1.5 × 10 5 The cells / well were added to the wells with 50 μL aliquots, and incubated at 37°C for 4 hours, followed by the addition of 100 μL / well One-Glo luciferase substrate (Promega, #E8130). The contents of the plate were mixed at low speed using a plate oscillator, incubated at room temperature for 5 minutes, and the luminescent signal was read on a BioTek microplate reader (BioTek Instruments) using a 0.2cps integration. GraphPad Prism 9 was used to analyze the data, and the relative luminescent unit (RLU) of each antibody concentration was plotted in a scatter format of RLU with antibody concentration. The results represent two independent experiments.

[0388] Figure 8 The results in show that the exemplified anti-human IL-4RαAb1 GC conjugate of Example 1b lacks or has no ADCC activity when compared to the positive control. Similarly, representative results (not shown) of exemplified anti-human IL-4RαAb1, Ab4 and Ab7 show that the antibodies lack or have no significant ADCC activity when compared to the positive control.

[0389] Example 5c. Complement-dependent cytotoxicity (CDC): In vitro CDC assays of the exemplified antibodies were performed using Daudi cells (ATCC, #CCL-213). All test antibodies, complement, and cells were diluted in assay medium consisting of RPMI-1640 (without phenol red) with 0.1 mM non-essential amino acids (NEAA), 1 mM sodium pyruvate, 2 mM L-glutamine, 500 U / mL penicillin-streptomycin, and 0.1% w / v BSA. The test antibodies were first diluted to a 3X concentration of 600 nM and then serially diluted 7-fold at a 1:4 ratio. 50 μL / well of each antibody (including a CD20 positive control antibody) was aliquoted in triplicate in a white opaque bottom 96-well plate (Costar, #3917). 5×10 4 Daudi target cells were added to each well, 50 μL / well, and incubated for 1 hour at 37°C. Next, human serum complement (Quidel, #A113) thawed quickly in a 37°C water bath was diluted 1:6 in the assay medium and added to the assay plate with 50 μL / well. The plate was incubated for 2 hours at 37°C, and then 100 μL / well CellTiter Glo substrate (Promega, #G7571) was added. The contents of the plate were mixed at low speed using a plate oscillator, incubated at room temperature for 5 minutes, and the luminescent signal was read on a BioTek microplate reader (BioTek Instruments) using a 0.2cps integration. GraphPad Prism 9 was used to analyze the data, and the relative luminescent unit (RLU) of each antibody concentration was plotted in a scatter format of antibody concentration to RLU. The results represent two independent experiments.

[0390] Fig. 9 The results in show that the exemplified IL-4RαAb GC conjugate of Example 1b does not elicit a CDC response compared to the positive control. Similarly, representative results (not shown) for exemplified anti-human IL-4RαAb1, Ab4, and Ab7 show that the antibodies do not elicit a CDC response when compared to the positive control.

[0391] Example 6. Biophysical properties of the anti-human IL-4Rα Ab1GC conjugate of Example 1b

[0392] The biophysical properties of the exemplified anti-human IL-4Rα Ab1 GC conjugate of Example 1b were evaluated for developability.

[0393] Example 6a. Thermal stability: Differential scanning calorimetry (DSC) was used to evaluate the stability of the exemplified antibodies against thermal denaturation. The melting onset temperature (Tonset) and thermal melting temperatures (TM1 and TM2) of the anti-human IL-4RαAb1 GC conjugate in PBS pH 7.2 buffer, acetate pH 5, and histidine pH 6 were obtained (Table 19). Although the thermal transition temperatures of each domain were not well resolved, Table 19 and Figure 10A-10C The data in show that conjugation of the exemplified linker-payload to the exemplified four engineered cysteines on yields acceptable thermal stability.

[0394] Example 6b. Aggregation under temperature stress: Solution stability of the exemplified anti-human IL-4RαAb1 GC conjugate over time was evaluated at approximately 100 mg / mL in a common 5 mM histidine pH 6.0 buffer with excipients. The concentrated samples were incubated at 5°C and 35°C for a period of 4 weeks, respectively. After incubation, the percentage of high molecular weight species (% HMW) in the samples was analyzed using size exclusion chromatography (SEC). The results in Table 20 show that conjugation of the exemplified linker-payload to the 4 engineered cysteines on Ab1 resulted in an acceptable level of aggregation of the anti-human IL-4RαAb1 GC conjugate over a period of 4 weeks at 35°C.

[0395] Table 19. Thermal stability of the exemplary anti-human IL-4RαAb1 GC conjugate of Example 1b (°C)

[0396]

[0397] Table 20. Biophysical properties of the exemplary anti-human IL-4Rα Ab1 GC conjugate of Example 1b

[0398]

[0399] Example 7. In vivo function of the anti-human IL-4RαAb1 GC conjugate of Example 1b

[0400] In vivo efficacy in a fully humanized mouse model of type IV hypersensitivity: A humanized mouse model of contact hypersensitivity was used to determine the in vivo activity of the anti-human IL-4RαAb1 GC conjugate anti-human IL-4RαAb1.

[0401] Immunodeficient NOG mice (huNOG-EXL, Taconic) expressing human GM-CSF and human IL-3 to support myeloid development were transplanted with human CD34+ hematopoietic stem cells isolated from human umbilical cord blood at 6 weeks of age. 20-24 weeks after the administration of stem cells, mice were evaluated for adequate human CD45 engraftment (>25% in the blood) and subjected to an oxazolone-induced contact hypersensitivity protocol. On day 0, mice grouped by weight were administered anti-human IL-4RαAb1 GC conjugate (n=8), anti-human IL-4RαAb1 (n=8) or control human IgG4P antibody (n=8) subcutaneously (SC) at 10 mg / kg. GC alone (n=6) was administered SC at 0.15 mg / kg 24 hours before sensitization and each challenge. On day 1, mice were anesthetized with 5% isoflurane, their abdomens were shaved, and 100 μL of 3% oxazolone in ethanol was applied to the shaved area. On the 6th day, mice were re-administered at the same dose, anesthetized, and then challenged both ears with 2% oxazolone in ethanol 24 hours after administration (10 μL / side / ear). The dose challenge paradigm was repeated weekly for a total of 3 attacks. The inflammatory response was determined by the difference in ear thickness before and 24 hours after each attack using a Miltenyi Biotec electronic caliper. P values ​​between groups were calculated by one-way ANOVA, followed by Tukey post hoc tests, and were considered significant if <0.05 (GraphPad Prism).

[0402] The results in Table 21 show that the anti-human IL-4RαAb1 GC conjugate Example 1b was able to significantly inhibit the in vivo inflammatory response to hapten-induced contact hypersensitivity compared to all other treatments after challenges 2 and 3. The IL-4RαAb1 GC conjugate reduced inflammation by 80% and 78% for challenges 2 and 3, respectively, compared to the isotype control. In addition, there was a trend for activity in the case of unconjugated Ab1, although Ab1 only significantly inhibited inflammation after challenge 2 (40% reduction) compared to the isotype. In addition, GC treatment using an equivalent dose and paradigm to the Ab1 GC conjugate was ineffective in the model, indicating that the in vivo activity is attributed to the Ab1 GC conjugate. These results indicate that the anti-human IL-4RαAb1 GC conjugate effectively delivers glucocorticoids to inflamed tissues and significantly abrogates the biological effects associated with type IV hypersensitivity reactions in the humanized mouse model, indicating that this anti-inflammatory response can be elicited in human subjects.

[0403] Table 21: Effect of the anti-human IL-4Rα Ab1 GC conjugate of Example 1b in the type IV hypersensitivity humanized mouse model In vivo effects

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[0405] *p < 0.05 vs isotype; ANOVA Tukey; ^p < 0.01 vs all; ANOVA Tukey

[0406] Sequence Listing

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Claims

1. A conjugate of the formula: Wherein Ab is an antibody that binds to human IL-4Rα, in for: And n is 1 to 5.

2. The conjugate of claim 1, wherein Ab comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises heavy chain complementarity determining regions HCDR1, HCDR2 and HCDR3, and the VL comprises light chain complementarity determining regions LCDR1, LCDR2 and LCDR3, wherein: The HCDR1 comprises SEQ ID NO: 1, 42 or 19; The HCDR2 comprises SEQ ID NO: 2 or 20; The HCDR3 comprises SEQ ID NO: 3; The LCDR1 comprises SEQ ID NO: 4 or 22; The LCDR2 comprises SEQ ID NO:5; and The LCDR3 comprises SEQ ID NO: 6 or 24; 3. The conjugate according to claim 1, wherein for:

4. The conjugate according to claim 1, wherein for:

5. The conjugate according to claim 1 or 3, wherein for:

6. The conjugate according to claim 1 or 4, wherein for:

7. The conjugate according to claim 1 or 3, wherein for:

8. The conjugate according to claim 1 or 3, wherein for:

9. The conjugate according to claim 1 or 4, wherein for:

10. The conjugate according to claim 1 or 4, wherein for:

11. The conjugate of any one of claims 1 to 10, wherein the Ab comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises heavy chain complementarity determining regions HCDR1, HCDR2 and HCDR3, and the VL comprises light chain complementarity determining regions LCDR1, LCDR2 and LCDR3, wherein: The HCDR1 comprises SEQ ID NO: 1, The HCDR2 comprises SEQ ID NO: 2, The HCDR3 comprises SEQ ID NO: 3, The LCDR1 comprises SEQ ID NO:4, The LCDR2 comprises SEQ ID NO: 5, and The LCDR3 comprises SEQ ID NO:

6.

12. The conjugate of claim 11, wherein the VH comprises SEQ ID NO: 7 and the VL comprises SEQ ID NO:

8.

13. The conjugate according to any one of claims 11 to 12, wherein the Ab comprises: i. A heavy chain (HC) comprising SEQ ID NO: 9 and a light chain (LC) comprising SEQ ID NO: 10; ii. A heavy chain (HC) comprising SEQ ID NO: 50 and a light chain (LC) comprising SEQ ID NO: 10; iii. A heavy chain (HC) comprising SEQ ID NO: 37 and a light chain (LC) comprising SEQ ID NO: 10; iv. a heavy chain (HC) comprising SEQ ID NO:31 and a light chain (LC) comprising SEQ ID NO:10; v. A heavy chain (HC) comprising SEQ ID NO: 35 and a light chain (LC) comprising SEQ ID NO: 10; vi. A heavy chain (HC) comprising SEQ ID NO: 33 and a light chain (LC) comprising SEQ ID NO: 10; vii. A heavy chain (HC) comprising SEQ ID NO: 13 and a light chain (LC) comprising SEQ ID NO: 10; or viii. A heavy chain (HC) comprising SEQ ID NO:52 and a light chain (LC) comprising SEQ ID NO:

10.

14. The conjugate of any one of claims 1 to 10, wherein the Ab comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises heavy chain complementarity determining regions HCDR1, HCDR2 and HCDR3, and the VL comprises light chain complementarity determining regions LCDR1, LCDR2 and LCDR3, wherein: The HCDR1 comprises SEQ ID NO:42, The HCDR2 comprises SEQ ID NO: 2, The HCDR3 comprises SEQ ID NO: 3, The LCDR1 comprises SEQ ID NO: 22, The LCDR2 comprises SEQ ID NO: 5, and The LCDR3 comprises SEQ ID NO:

6.

15. The conjugate of claim 14, wherein the VH comprises SEQ ID NO: 44 and the VL comprises SEQ ID NO:

45.

16. The conjugate of any one of claims 14 to 15, wherein the Ab comprises a heavy chain (HC) comprising SEQ ID NO: 46 and a light chain (LC) comprising SEQ ID NO:

47.

17. The conjugate of any one of claims 1 to 10, wherein the Ab comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises heavy chain complementarity determining regions HCDR1, HCDR2, and HCDR3, and the VL comprises light chain complementarity determining regions LCDR1, LCDR2, and LCDR3, wherein: The HCDR1 comprises SEQ ID NO: 19, The HCDR2 comprises SEQ ID NO: 20, The HCDR3 comprises SEQ ID NO: 3, The LCDR1 comprises SEQ ID NO: 22, The LCDR2 comprises SEQ ID NO: 5, and The LCDR3 comprises SEQ ID NO:

24.

18. The conjugate of claim 17, wherein the VH comprises SEQ ID NO: 25 and the VL comprises SEQ ID NO:

26.

19. The conjugate of any one of claims 17 to 18, wherein the Ab comprises a heavy chain (HC) comprising SEQ ID NO: 27 and a light chain (LC) comprising SEQ ID NO:

28.

20. The conjugate of any one of claims 1 to 12, 14 to 15, 17 to 18, wherein the Ab comprises a heavy chain and a light chain, wherein the heavy chain comprises: cysteine ​​at amino acid residue 124 (EU numbering); Cysteine ​​at amino acid residue 378 (EU numbering); or Cysteine ​​at amino acid residue 124 (EU numbering) and Cysteine ​​at amino acid residue 378 (EU numbering).

21. The conjugate of any one of claims 1 to 12, 14 to 15, 17 to 18, wherein the Ab comprises a heavy chain (HC) and a light chain (LC), wherein the HC is of human IgG4 isotype.

22. The conjugate of any one of claims 1 to 12, 14 to 15, 17 to 18, wherein the Ab comprises a heavy chain (HC) and a light chain (LC), wherein the HC is of human IgG1 isotype.

23. The conjugate according to any one of claims 1 to 22, wherein n is 2 to 5.

24. The conjugate according to any one of claims 1 to 22, wherein n is 3 to 5.

25. The conjugate according to any one of claims 1 to 22, wherein n is 3 to 4.

26. The conjugate of any one of claims 1 to 22, wherein n is about 4.

27. The conjugate of any one of claims 1 to 22, wherein n is about 3.

28. The conjugate of any one of claims 1 to 22, wherein n is about 2.

29. A pharmaceutical composition comprising the conjugate according to any one of claims 1 to 28 or a pharmaceutically acceptable salt thereof and one or more pharmaceutically acceptable carriers, diluents or excipients.

30. A method of treating an inflammatory disease in a subject in need thereof, the method comprising administering to the subject an effective amount of a conjugate according to claims 1 to 28 or a pharmaceutical composition according to claim 29.

31. The method of claim 30, wherein the inflammatory disease is a type 2 inflammatory disease.

32. The method of claim 31, wherein the type 2 inflammatory disease is selected from atopic dermatitis, eosinophilic esophagitis, nasal polyposis, asthma, chronic rhinosinusitis (CRS), allergic diseases, chronic obstructive pulmonary disease (COPD), or chronic spontaneous urticaria (CSU).

33. The method of claim 32, wherein the type 2 inflammatory disease is atopic dermatitis.

34. The conjugate of any one of claims 1 to 28 for use in therapy.

35. The conjugate according to any one of claims 1 to 28 or the pharmaceutical composition according to claim 29 for use in the treatment of an inflammatory disease.

36. The conjugate or pharmaceutical composition for use according to claim 35, wherein the inflammatory disease is a type 2 inflammatory disease.

37. The conjugate or pharmaceutical composition for use according to claim 36, wherein the type 2 inflammatory disease is selected from atopic dermatitis, eosinophilic esophagitis, nasal polyposis, asthma, chronic rhinosinusitis (CRS), allergic diseases, chronic obstructive pulmonary disease (COPD) or chronic spontaneous urticaria (CSU).

38. The conjugate or pharmaceutical composition for use according to claim 37, wherein the type 2 inflammatory disease is atopic dermatitis.

39. Use of the conjugate according to any one of claims 1 to 28 or the pharmaceutical composition according to claim 29 in the manufacture of a medicament for the treatment of an inflammatory disease.

40. The use according to claim 39, wherein the inflammatory disease is a type 2 inflammatory disease.

41. The use according to claim 40, wherein the type 2 inflammatory disease is selected from atopic dermatitis, eosinophilic esophagitis, nasal polyposis, asthma, chronic rhinosinusitis (CRS), allergic diseases, chronic obstructive pulmonary disease (COPD) or chronic spontaneous urticaria (CSU).

42. Use according to claim 41, wherein the type 2 inflammatory disease is atopic dermatitis.

43. The conjugate of any one of claims 1 to 28, wherein the Ab neutralizes human IL-4Rα.

44. The conjugate of any one of claims 1 to 28, wherein the Ab inhibits binding of human IL-4 to human IL-4Rα.

45. The conjugate of any one of claims 1 to 28, wherein the Ab inhibits binding of human IL-13 to human IL-4Rα.

46. ​​The conjugate of any one of claims 1 to 28, wherein the Ab is an internalizing antibody.

47. A method of producing a conjugate, wherein the conjugate comprises the conjugate of any one of claims 1 to 28.

48. The method according to claim 47, comprising the steps of: (a) reducing an anti-human IL-4Rα antibody with a reducing agent to produce a reduced anti-human IL-4R antibody, wherein the anti-human IL-4Rα antibody comprises one or more engineered cysteine ​​residues; (b) oxidizing the reduced anti-human IL-4Rα antibody with an oxidizing agent to produce an oxidized anti-human IL-4R antibody; and (c) contacting the oxidized anti-human IL-4R antibody with a compound of the formula to produce the conjugate.

49. The method of claim 48, wherein the reducing agent is dithiothreitol and the oxidizing agent is dehydroascorbic acid.

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