Compositions and methods related to il27 receptor binding

Abstract

To provide IL27 receptor binding proteins.SOLUTION: The disclosure provides an IL27 receptor binding protein that specifically binds to IL27Rα subunit (IL27Rα) and glycoprotein 130 subunit (gp130), where the binding protein causes the multimerization of IL27Rα and gp130 when bound to IL27Rα and gp130 and comprises a single-domain antibody that specifically binds to IL27Rα and a single-domain antibody that specifically binds to gp130.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims priority to U.S. Provisional Patent Application No. 63 / 061,562, filed August 5, 2020; U.S. Provisional Patent Application No. 63 / 078,745, filed September 15, 2020; and U.S. Provisional Patent Application No. 63 / 135,884, filed January 11, 2021, the disclosures of which are hereby incorporated by reference in their entireties for all purposes.

Background Art

[0002] Background of the Disclosure Interleukin - 27 receptor (IL27R) is a type I cytokine receptor for interleukin - 27 (IL27). It is a heterodimer composed of the IL27Rα subunit and glycoprotein 130 (gp130). IL27 is expressed by antigen - presenting cells and induces the differentiation of diverse T - cell populations in the immune system. When IL27 binds to IL27R, signaling pathways such as the JAK - STAT pathway and the p38 MAPK pathway are turned on, inducing pro - inflammatory or anti - inflammatory responses involving different types of cells such as macrophages, dendritic cells, T cells, and B cells. The activated response may depend on the external environment of IL27.

[0003] IL - 27 is a heterodimeric cytokine consisting of two non - covalently linked subunits, p28 and EBI3. The p28 subunit belongs to the 4 - helix bundle cytokine family, while EBI3 is the shortest possible form of a soluble cytokine receptor having two typical cytokine - binding domains (Pflanz S, et al., Immunity. 2002 Jun;16(6):779 - 90 (Non - Patent Document 1)).

[0004] The main binding receptor for IL-27 is IL-27R1 (also known as TCCR- or WSX-1 receptor). IL-27 and IL-27R1 form a complex with a significant affinity (nM). Gp130 is a second receptor that binds to the IL-27 / IL-27R complex to form an active signaling complex. The binding of Gp130 to the IL-27 / IL-27R1 complex is much weaker than the interaction between IL-27 and IL-27R1 (Pflanz S, et al., J Immunol. 2004 Feb 15;172(4):2225-31 (Non-Patent Document 2)).

[0005] The IL-27 extracellular domain has five domains. The first two domains form the IL-27 binding domain. Typically, the loop between D1 and D2 provides most of the binding energy.

[0006] The other three domains are called fibronectin type III domains (Fn3). The sequences of each Fn3 domain are diverse.

[0007] The gp130 receptor has six domains. The top domain D1 of gp130 binds to p28 of IL-27. D2 and D3 contribute little to the binding of IL-27. The three domains near the membrane are Fn3 domains. The sequences of each Fn3 domain are diverse.

[0008] Although the structure of IL-27R is unknown, its domain structure is known. The structure of gp130 is known to be complexed with IL-6. Based on its structure, it is clear that the Fn3 domain does not contribute energetically to IL-27R complex formation. Rather, the structure of gp130 is such that domains 4 and 5 form a "C" at an 80% angle to each other. Certain residues in each of the "toll" receptors are conserved as in D4 and D5 in gp130. This indicates that all toll receptors of the gp130 family, including IL-27R, form this "C" structure (Yibin Xu, et al., J Biol Chem. 2010 Jul 9;285(28):21214-8 (Non-Patent Document 3)).

[0009] IL-27R has five extracellular domains. D1 and D2 are cytokine binding domains. D3, D4, and D5 are Fn3 domains. The D5 domain of IL-27R and the D6 domain of gp130 approach each other at the membrane due to the "C" shape of each receptor. This is necessary so that the receptor complex can induce JAK binding at the positions of the intracellular domains of both receptors.

Prior Art Documents

Non-Patent Documents

[0010]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Summary of the Invention

[0011] Summary of the Disclosure The present disclosure provides compositions useful in cell receptor pairing to produce desirable effects useful in the treatment of diseases in mammalian subjects.

[0012] Several advantages arise from the binding molecules described herein. IL-27, the natural ligand of IL-27R, brings gp130 and IL-27Ra into proximity (i.e., by its co-binding of IL-27). However, when IL-27 is used as a therapeutic agent in mammalian, particularly human, subjects, it may induce several harmful and undesirable effects by various mechanisms including the presence of gp130 and IL-27Ra on other cell types, and binding to gp130 and IL-27Ra on other cell types may result in undesirable effects and / or unwanted signaling in cells expressing gp130 and IL-27Ra. The present disclosure is directed to methods and compositions that modulate the multiple effects of gp130 and IL-27Ra binding such that desired therapeutic signaling occurs, particularly in desired cell or tissue subtypes, while minimizing unwanted activity and / or intracellular signaling.

[0013] In some embodiments, the IL-27R binding molecules described herein are partial agonists of the IL-27 receptor. In some embodiments, the binding molecules described herein are designed such that the binding molecule is a full agonist. In some embodiments, the binding molecules described herein are designed such that the binding molecule is a superagonist.

[0014] In some embodiments, the binding molecule provides maximal desired IL-27 intracellular signaling from binding to gp130 and IL-27Ra on the desired cell type while providing significantly less IL-27 signaling on other unwanted cell types. This can be, for example, having a different affinity for gp130 and IL-27Ra compared to the affinity of IL-27 for gp130 and IL-27Ra, or having a different E for gp130 and IL-27Ra maxThis can be achieved by the selection of binding molecules that cause

[0015] This disclosure provides a bivalent binding molecule that is an agonist of the IL-27 receptor, · a first single-domain antibody (sdAb) that specifically binds to the extracellular domain of gp130 (“anti-gp130 sdAb”), and · a second single-domain antibody that specifically binds to the extracellular domain IL-27Ra (“anti-IL-27Ra sdAb”) comprising · the anti-gp130 sdAb and the anti-IL-27Ra sdAb are stably associated, and when cells that initially express gp130 and IL-27Ra are contacted with an effective amount of the bivalent binding molecule, dimerization of gp130 and IL-27Ra occurs, resulting in intracellular signaling characteristic of the IL-27 receptor when activated by its native cognate IL-27. The bivalent binding molecule is provided. In some embodiments, one or both of the sdAbs are scFvs. In some embodiments, one or both of the sdAbs are VHHs.

[0016] In some embodiments, one sdAb of the bivalent binding molecule is an scFv and the other sdAb is a VHH.

[0017] In some embodiments, the first and second sdAbs are covalently linked via a chemical bond.

[0018] In some embodiments, the first and second sdAbs are provided as a single continuous polypeptide.

[0019] In some embodiments, the first and second sdAbs are provided as a single continuous polypeptide optionally comprising a polypeptide linker intervening between the amino acid sequences of the first and second sdAbs.

[0020] In some embodiments, the bivalent binding molecule optionally comprising a linker may optionally be expressed as a fusion protein with an additional amino acid sequence. In some embodiments, the additional amino acid sequence is a purification handle such as a chelating peptide, or an additional protein such as a subunit of an Fc molecule.

[0021] In one aspect, the present disclosure provides an IL27 receptor (IL27R) binding protein that specifically binds to the IL27Rα subunit (IL27Rα) and the glycoprotein 130 subunit (gp130), wherein the binding protein causes multimerization of IL27Rα and gp130 upon binding to IL27Rα and gp130, and the multimerization results in activation of JAK kinases associated with the intracellular domains of IL27Rα and gp130 and intracellular signal transduction, and the binding protein comprises a single domain antibody (sdAb) that specifically binds to IL27Rα (anti-IL27Rα sdAb) and an sdAb that specifically binds to gp130 (anti-gp130 sdAb). In some embodiments, multimerization of IL27Rα and gp130 can cause downstream signal transduction.

[0022] In some embodiments, the anti-IL27Rα sdAb is a V H H antibody (anti-IL27Rα V H H antibody), and / or the anti-gp130 sdAb is a V H H antibody (anti-gp130 V Han H antibody). In some embodiments, the anti-IL27Rα sdAb and the anti-gp130 sdAb are joined directly or by a peptide linker. In some embodiments, the peptide linker comprises 1 to 50 amino acids. In certain embodiments, the peptide linker comprises the sequence of GGGS (SEQ ID NO:108).

[0023] In some embodiments, the IL27R-binding protein comprises: a CDR1 having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to the sequence of CDR1 from the row of Table 1A, or having 0, 1, 2 or 3 amino acid changes, optionally conservative amino acid changes; a CDR2 having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to the sequence of CDR2 from the same row of Table 1A, or having 0, 1, 2 or 3 amino acid changes, optionally conservative amino acid changes; and a CDR3 having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to the sequence of CDR3 from the same row of Table 1A, or having 0, 1, 2 or 3 amino acid changes, optionally conservative amino acid changes, comprising a first V H H antibody; and CDR1 having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to the CDR4 sequence from the same row of Table 1A, or having 0, 1, 2 or 3 amino acid changes, optionally conservative amino acid changes; CDR2 having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to the CDR5 sequence from the same row of Table 1A, or having 0, 1, 2 or 3 amino acid changes, optionally conservative amino acid changes; and CDR3 having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to the CDR6 sequence from the same row of Table 1A, or having 0, 1, 2 or 3 amino acid changes, optionally conservative amino acid changes, comprising a second V H H antibody.

[0024] In some embodiments, the IL27R binding protein comprises a sequence that is at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identical to any one of the dual VHH dimer sequences shown in Table 1A.

[0025] In certain embodiments, the anti-IL27Rα V H H antibody comprises CDR1 having 0, 1, 2 or 3 amino acid changes relative to any one of the sequences of SEQ ID NOs: 193 - 198; CDR2 having 0, 1, 2 or 3 amino acid changes relative to any one of the sequences of SEQ ID NOs: 199 - 204; and CDR3 having 0, 1, 2 or 3 amino acid changes relative to any one of the sequences of SEQ ID NOs: 205 - 210. In a further embodiment, the anti-gp130 V HThe H antibody comprises CDR1 having 0, 1, 2 or 3 amino acid changes relative to any one of the sequences of SEQ ID NOs: 211 to 217; CDR2 having 0, 1, 2 or 3 amino acid changes relative to any one of the sequences of SEQ ID NOs: 218 to 224; and CDR3 having 0, 1, 2 or 3 amino acid changes relative to any one of the sequences of SEQ ID NOs: 225 to 231.

[0026] In certain embodiments, the IL27R binding protein is anti-IL27Rα V as set forth in the rows of Table 1 H CDR1, CDR2 and CDR3 in the H antibody and anti-gp130 V H comprising CDR1, CDR2 and CDR3 in the H antibody.

[0027] In some embodiments, the binding protein comprises anti-gp130 V linked to the N-terminus of a linker H and an H antibody, and anti-IL27Rα V linked to the C-terminus of the linker H and an H antibody. In some embodiments, the anti-gp130 V H The H antibody comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to any one of the sequences of SEQ ID NOs: 232 to 237. In some embodiments, the anti-IL27Rα V H The H antibody comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to any one of the sequences of SEQ ID NOs: 238 to 244.

[0028] In certain embodiments, the anti-gp130 V H each of the H antibody and the anti-IL27Rα V H The H antibody comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identity to the sequences set forth in the rows of Table 2A.

[0029] In certain embodiments, the binding protein comprises a sequence that is substantially identical to any one of the sequences of SEQ ID NOs: 1-42. In certain embodiments, the binding protein comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identity to any one of the sequences of SEQ ID NOs: 1-42.

[0030] In some embodiments, the binding protein comprises an anti-IL27Rα VH antibody linked to the N-terminus of a linker and an anti-gp130 VH antibody linked to the C-terminus of the linker. H In some embodiments, the binding protein comprises an anti-IL27Rα VH antibody linked to the N-terminus of a linker and an anti-gp130 VH antibody linked to the C-terminus of the linker. H In some embodiments, the anti-IL27Rα VH antibody comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to any one of the sequences of SEQ ID NOs: 245-251. In certain embodiments, the anti-gp130 VH antibody comprises a sequence having at least 90% sequence identity to any one of the sequences of SEQ ID NOs: 252-257. H In some embodiments, the anti-IL27Rα VH antibody comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to any one of the sequences of SEQ ID NOs: 245-251. In certain embodiments, the anti-gp130 VH antibody comprises a sequence having at least 90% sequence identity to any one of the sequences of SEQ ID NOs: 252-257. H In some embodiments, the anti-IL27Rα VH antibody comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to any one of the sequences of SEQ ID NOs: 245-251. In certain embodiments, the anti-gp130 VH antibody comprises a sequence having at least 90% sequence identity to any one of the sequences of SEQ ID NOs: 252-257.

[0031] In certain embodiments, each of the anti-IL27Rα VH antibody and the anti-gp130 VH antibody comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identity to the sequences set forth in the rows of Table 3A. H In certain embodiments, each of the anti-IL27Rα VH antibody and the anti-gp130 VH antibody comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identity to the sequences set forth in the rows of Table 3A. H In certain embodiments, each of the anti-IL27Rα VH antibody and the anti-gp130 VH antibody comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identity to the sequences set forth in the rows of Table 3A.

[0032] In certain embodiments, the binding protein comprises a sequence that is substantially identical to any one of the sequences of SEQ ID NOs: 43-84. In certain embodiments, the binding protein comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identity to any one of the sequences of SEQ ID NOs: 43-84.

[0033] In another aspect, the present disclosure provides an isolated nucleic acid encoding an IL27R binding protein described herein. In certain embodiments, the isolated nucleic acid comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to any one of the sequences of SEQ ID NO: 109 - 192 or a sequence from Table 1B. The present disclosure also provides an expression vector comprising the nucleic acid. The present disclosure also provides an isolated host cell comprising the expression vector.

[0034] In another aspect, the present disclosure provides a pharmaceutical composition comprising an IL27R binding protein described herein and a pharmaceutically acceptable carrier.

[0035] In another aspect, the present disclosure provides a method of treating an autoimmune or inflammatory disease, disorder or condition, a neoplastic disease, or a viral infection in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an IL27R binding protein described herein or a pharmaceutical composition described herein.

[0036] In some embodiments, the method further comprises administering one or more adjuvants selected from the group consisting of corticosteroids, Janus kinase inhibitors, calcineurin inhibitors, mTor inhibitors, IMDH inhibitors, biologics, vaccines and therapeutic antibodies. In certain embodiments, the therapeutic antibody is an antibody that binds to a protein selected from the group consisting of BLyS, CD11a, CD20, CD25, CD3, CD52, IgE, IL12 / IL23, IL17a, IL1β, IL4Rα, IL5, IL6R, integrin-α4β7, RANKL, TNFα, VEGF-A and VLA-4.

[0037] In certain embodiments, the disease, disorder or condition is selected from viral infection, Helicobacter pylori infection, HTLV, organ rejection, graft-versus-host disease, autoimmune thyroid disease, multiple sclerosis, allergy, asthma, neurodegenerative diseases including Alzheimer's disease, systemic lupus erythematosus (SLE), autoinflammatory diseases, inflammatory bowel disease (IBD), Crohn's disease, diabetes, cartilage inflammation, arthritis, rheumatoid arthritis, juvenile arthritis, juvenile rheumatoid arthritis, juvenile rheumatoid arthritis, polyarticular juvenile rheumatoid arthritis, systemic juvenile rheumatoid arthritis, juvenile ankylosing spondylitis, juvenile enteropathic arthritis, juvenile reactive arthritis, juvenile Reiter's syndrome, SEA syndrome, juvenile dermatomyositis, juvenile psoriatic arthritis, juvenile scleroderma, juvenile systemic lupus erythematosus, juvenile vasculitis, oligoarticular rheumatoid arthritis, polyarticular rheumatoid arthritis, systemic rheumatoid arthritis, ankylosing spondylitis, enteropathic arthritis, reactive arthritis, Reiter's syndrome, SEA syndrome, psoriasis, psoriatic arthritis, dermatitis (eczema), exfoliative dermatitis or atopic dermatitis, pityriasis rubra pilaris, pityriasis rosea, parapsoriasis, pityriasis lichenoides, lichen planus, lichen nitidus, ichthyosiform dermatitis, keratosis, skin diseases, alopecia areata, pyoderma gangrenosum, vitiligo, pemphigoid, urticaria, porokeratosis, rheumatoid arthritis, seborrheic dermatitis, solar dermatitis, seborrheic keratosis, actinic keratosis, photoinduced keratosis, follicular keratosis, acne vulgaris, keloid, nevus, mole, condyloma or warts including genital condyloma, and human papillomavirus (HPV) infection.

[0038] The IL27R binding protein described herein is useful in the treatment of neoplastic diseases, such as cancer (e.g., solid tumor cancer; e.g., non-small cell lung cancer (NSCLC), renal cell carcinoma (RCC) or melanoma) and / or infectious diseases (e.g., bacterial infections and viral infections (e.g., viral infections caused by hepatitis C virus (HCV), human papillomavirus (HPV) or human immunodeficiency virus (HIV))) in a subject in need thereof. The IL27R binding protein is a CD8 + T cell, CD4 +Binds to T cells and / or regulatory T (Treg) cells and activates them. The IL27R binding protein can induce different levels of downstream signaling in different cell types. For example, by varying the length of the linker between the anti-IL27Rα V H H antibody and the anti-gp130 V H H antibody, the IL27R binding protein can cause a higher level of downstream signaling in the desired cell type compared to the undesired cell type. In some embodiments, by varying the length of the linker, the IL27R binding protein can cause a higher level of downstream signaling in T cells (e.g., CD8 + T cells) compared to the level of downstream signaling in other cells. In other embodiments, different anti-IL27Rα V H H antibodies having different binding affinities and different anti-gp130 V H H antibodies having different binding affinities can be combined to create different IL27R binding proteins. Further, by varying the orientation of the two antibodies in the binding protein, different binding proteins (i.e., anti-IL27Rα V H H antibody-linker-anti-gp130 V H H antibody, or anti-gp130 V H H antibody-linker-anti-IL27Rα V H H antibody) can be created. Different IL27R binding proteins can be screened to find the ideal binding protein that causes a higher level of downstream signaling in the desired cell type compared to the undesired cell type. In some embodiments, the level of downstream signaling in T cells (e.g., CD8 + T cells) is at least 1.1, 1.5, 2, 3, 5, or 10 times the level of downstream signaling in other cells.

[0039] In particular, the IL27R binding protein binds to CD8 + T cells and activates CD8 + T cells. In some embodiments, the IL27R binding protein is CXCR5+ CD8 + binds to T cells and CXCR5 + CD8 + T cells are activated. IL27 can promote and maintain the rapid division of memory-like CXCR5 + CD8 + T cells during, for example, viral infection. It is known that CXCR5 + CD8 + T cells can maintain the T cell response during persistent infection or cancer and drive the proliferative burst of CD8 + T cells after anti-PD1 treatment. Therefore, the IL27R-binding proteins described herein are useful for maintaining and enhancing self-renewing T cells in neoplastic diseases such as chronic infections and cancer. [Invention 1001] An IL27 receptor (IL27R)-binding protein that specifically binds to the L27Rα subunit (IL27Rα) and the glycoprotein 130 subunit (gp130), which causes multimerization of IL27Rα and gp130 upon binding to IL27Rα and gp130, and comprising a single-domain antibody (sdAb) that specifically binds to IL27Rα (anti-IL27Rα sdAb) and an sdAb that specifically binds to gp130 (anti-gp130 sdAb), the binding protein. [Invention 1002] The IL27R-binding protein of Invention 1001, wherein the anti-IL27Rα sdAb is a V H H antibody (anti-IL27Rα V H H antibody) and / or the anti-gp130 sdAb is a V H H antibody (anti-gp130 V H H antibody). [Invention 1003] The IL27R-binding protein according to any one of Inventions 1001 to 1002, wherein the anti-IL27Rα sdAb and the anti-gp130 sdAb are linked by a peptide linker. [Invention 1004] The IL27R-binding protein of the present invention 1003, wherein the peptide linker contains 1 to 50 amino acids. [The present invention 1005] The IL27R-binding protein of the present invention 1004, wherein the peptide linker contains the sequence of GGGS (SEQ ID NO:108). [The present invention 1006] Anti-IL27Rα V H The IL27R-binding protein according to any one of the present inventions 1002 to 1005, wherein the anti-IL27Rα VH antibody has CDR1 having 0, 1, 2 or 3 amino acid changes relative to any one of the sequences of SEQ ID NOs: 193 to 198; CDR2 having 0, 1, 2 or 3 amino acid changes relative to any one of the sequences of SEQ ID NOs: 199 to 204; and CDR3 having 0, 1, 2 or 3 amino acid changes relative to any one of the sequences of SEQ ID NOs: 205 to 210. [The present invention 1007] Anti-gp130 V H The IL27R-binding protein according to any one of the present inventions 1002 to 1006, wherein the anti-gp130 VH antibody has CDR1 having 0, 1, 2 or 3 amino acid changes relative to any one of the sequences of SEQ ID NOs: 211 to 217; CDR2 having 0, 1, 2 or 3 amino acid changes relative to any one of the sequences of SEQ ID NOs: 218 to 224; and CDR3 having 0, 1, 2 or 3 amino acid changes relative to any one of the sequences of SEQ ID NOs: 225 to 231. [The present invention 1008] The IL27R-binding protein according to any one of the present inventions 1002 to 1005, comprising the following: A first V H H antibody that has at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to the CDR1 sequence from the horizontal row of Table 1A, or has 0, 1, 2 or 3 amino acid changes, optionally conservative amino acid changes; a CDR2 that has at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to the CDR2 sequence from the same horizontal row of Table 1A, or has 0, 1, 2 or 3 amino acid changes, optionally conservative amino acid changes; and a CDR3 that has at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to the CDR3 sequence from the same horizontal row of Table 1A, or has 0, 1, 2 or 3 amino acid changes, optionally conservative amino acid changes; and a second V H H antibody that has at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to the CDR1 sequence from the horizontal row of Table 1A, or has 0, 1, 2 or 3 amino acid changes, optionally conservative amino acid changes; a CDR2 that has at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to the CDR2 sequence from the same horizontal row of Table 1A, or has 0, 1, 2 or 3 amino acid changes, optionally conservative amino acid changes; and a CDR3 that has at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to the CDR3 sequence from the same horizontal row of Table 1A, or has 0, 1, 2 or 3 amino acid changes, optionally conservative amino acid changes. [Invention 1009] An IL27R-binding protein of the present invention 1008, comprising a sequence that is at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identical to any one of the dual VHH dimer sequences shown in Table 1A. [The present invention 1010] CDR1, CDR2 and CDR3 in the anti-IL27Rα V H H antibody as described in the horizontal rows of Table 1, and CDR1, CDR2 and CDR3 in the anti-gp130 V H An IL27R-binding protein of any one of the present inventions 1002 to 1009, comprising H antibody. [The present invention 1011] An anti-gp130 V H H antibody linked to the N-terminus of the linker, and an anti-IL27Rα V H An IL27R-binding protein of any one of the present inventions 1001 to 1014, comprising H antibody linked to the C-terminus of the linker. [The present invention 1012] An anti-gp130 V H An IL27R-binding protein of the present invention 1011, wherein the H antibody comprises a sequence having at least 90% sequence identity with any one of the sequences of SEQ ID NOs: 232 to 237. [The present invention 1013] An anti-IL27Rα V H An IL27R-binding protein of the present invention 1011, wherein the H antibody comprises a sequence having at least 90% sequence identity with any one of the sequences of SEQ ID NOs: 238 to 244. [The present invention 1014] An anti-gp130 V H Each of the H antibody and the anti-IL27Rα V H An IL27R-binding protein of the present invention 1011, wherein each H antibody comprises a sequence having at least 90% identity with the sequences described in the horizontal rows of Table 2A. [The present invention 1015] An IL27R-binding protein of the present invention 1011, comprising a sequence having at least 90% identity with any one of the sequences of SEQ ID NOs: 1 to 42. [The present invention 1016] The IL27R-binding protein of the present invention 1001, comprising an anti-IL27Rα VHH antibody linked to the N-terminus of a linker and an anti-gp130 VHH antibody linked to the C-terminus of the linker. [The present invention 1017] The anti-IL27Rα V H The IL27R-binding protein of the present invention 1016, wherein the H antibody comprises a sequence having at least 90% sequence identity with any one of the sequences of SEQ ID NOs: 245 to 251. [The present invention 1018] The anti-gp130 V H The IL27R-binding protein of the present invention 1016, wherein the H antibody comprises a sequence having at least 90% sequence identity with any one of the sequences of SEQ ID NOs: 252 to 257. [The present invention 1019] The anti-IL27Rα V H The H antibody and the anti-gp130 V H The IL27R-binding protein of the present invention 1016, wherein each of the H antibodies comprises a sequence having at least 90% identity with the sequences listed in the rows of Table 3A. [The present invention 1020] The IL27R-binding protein of the present invention 1016, comprising a sequence having at least 90% identity with any one of the sequences of SEQ ID NOs: 43 to 84. [The present invention 1021] An isolated nucleic acid encoding the IL27R-binding protein of any one of the present inventions 1001 to 1020. [The present invention 1022] The isolated nucleic acid of the present invention 1021, comprising a sequence having at least 90% sequence identity with any one of the sequences of SEQ ID NOs: 109 to 192 or the sequences in Table 1B. [The present invention 1023] An expression vector comprising the nucleic acid of the present invention 1021. [The present invention 1024] An isolated host cell comprising the vector of the present invention 1023. [The present invention 1025] A pharmaceutical composition comprising any one of the IL27R-binding proteins 1001 to 1020 of the present invention and a pharmaceutically acceptable carrier. [The present invention 1026] A method of treating an autoimmune or inflammatory disease, disorder or condition, neoplastic disease, or viral infection in a subject in need thereof, comprising administering a therapeutically effective amount of any one of the IL27R-binding proteins 1001 to 1020 of the present invention or the pharmaceutical composition of the present invention 1025 to the subject. [The present invention 1027] The method of the present invention 1026, further comprising administering one or more adjuvants selected from the group consisting of corticosteroids, Janus kinase inhibitors, calcineurin inhibitors, mTor inhibitors, IMDH inhibitors, biologics, vaccines and therapeutic antibodies. [The present invention 1028] The method of the present invention 1027, wherein the therapeutic antibody is an antibody that binds to a protein selected from the group consisting of BLyS, CD11a, CD20, CD25, CD3, CD52, IgEIL12 / IL23, IL17a, IL1β, IL4Rα, IL5, IL6R, integrin-α4β7, RANKL, TNFα, VEGF-A and VLA-4. [The present invention 1029] The disease, disorder or condition is selected from viral infection, Helicobacter pylori infection, HTLV, organ rejection, graft-versus-host disease, autoimmune thyroid disease, multiple sclerosis, allergy, asthma, neurodegenerative diseases including Alzheimer's disease, systemic lupus erythematosus (SLE), autoinflammatory diseases, inflammatory bowel disease (IBD), Crohn's disease, diabetes, cartilage inflammation, arthritis, rheumatoid arthritis, juvenile arthritis, juvenile rheumatoid arthritis, juvenile rheumatoid arthritis, polyarticular juvenile rheumatoid arthritis, systemic juvenile rheumatoid arthritis, juvenile ankylosing spondylitis, juvenile enteropathic arthritis, juvenile reactive arthritis, juvenile Reiter's syndrome, SEA syndrome, juvenile dermatomyositis, juvenile psoriatic arthritis, juvenile scleroderma, juvenile systemic lupus erythematosus, juvenile vasculitis, oligoarticular rheumatoid arthritis, polyarticular rheumatoid arthritis, systemic rheumatoid arthritis, ankylosing spondylitis, enteropathic arthritis, reactive arthritis, Reiter's syndrome, SEA syndrome, psoriasis, psoriatic arthritis, dermatitis (eczema), exfoliative dermatitis or atopic dermatitis, pityriasis rubra pilaris, pityriasis rosea, pityriasis lichenoides, lichen planus, lichen nitidus, ichthyosiform dermatitis, keratosis, skin diseases, alopecia areata, pyoderma gangrenosum, vitiligo, pemphigoid, urticaria, porokeratosis, rheumatoid arthritis, seborrheic dermatitis, solar dermatitis, seborrheic keratosis, actinic keratosis, photoinduced keratosis, follicular keratosis, acne vulgaris, keloid, nevus, mole, condyloma or warts including condyloma acuminata, and human papillomavirus (HPV) infection, and any of the methods of the present invention from 1026 to 1028.

Brief Description of the Drawings

[0040]

Figure 1

Figure 2

Figure 3

Figure 4

DETAILED DESCRIPTION OF THE INVENTION

[0041] DETAILED DESCRIPTION OF THE DISCLOSURE I. INTRODUCTION The present disclosure provides compositions useful in the dimerization of cell receptors to produce desirable effects useful in the treatment of diseases. Generally, provided is a binding protein comprising a first domain that binds to IL27Rα and a second domain that binds to gp130 such that upon contact with cells expressing IL27Rα and gp130, the binding protein causes a functional association of IL27Rα and gp130, thereby resulting in functional dimerization of the receptor and downstream signaling.

[0042] Several advantages result from the binding proteins described herein. IL27, the natural ligand of IL27R, brings IL27Rα and gp130 into proximity (i.e., by its simultaneous binding of IL27). However, when IL27 is used as a therapeutic agent in mammalian, particularly human, subjects, it may also induce some harmful and undesirable effects by various mechanisms including the presence of IL27Rα and gp130 on other cell types, and binding to IL27Rα and gp130 on other cell types may result in undesirable effects and / or unwanted signaling in cells expressing IL27Rα and gp130. The present disclosure is directed to methods and compositions that modulate the multiple effects of IL27Rα and gp130 binding such that desired therapeutic signaling occurs, particularly in desired cell or tissue subtypes, while minimizing unwanted activity and / or intracellular signaling.

[0043] In some embodiments, the binding proteins described herein are designed such that the binding protein provides maximal desired IL27 intracellular signaling from binding to IL27Rα and gp130 on the desired cell type while providing significantly less IL27 signaling on other unwanted cell types. This can be achieved, for example, by having a different affinity for IL27Rα and gp130 compared to the affinity of IL27 for IL27Rα and gp130, or by having a different E for IL27Rα and gp130 maxThis can be achieved by selecting the binding protein that causes it. Since different cell types respond with different sensitivities to the binding of a ligand to its cognate receptor, by modulating the affinity of the dimeric ligand (or its individual binding moieties) for the IL27 receptor as compared to wild-type IL27 binding, stimulation of the desired activity is promoted while reducing unwanted activity on non-target cells. Several methods are available for measuring downstream signaling activity. For example, in some embodiments, JAK / STAT signaling can be measured by the presence of phosphorylated receptor and / or phosphorylated STAT. In other embodiments, the expression of one or more downstream genes whose expression levels can be affected by the level of downstream signaling caused by the binding protein can also be measured.

[0044] Interleukin 27 (IL27) Structure IL27 is a member of the IL-12 cytokine family. IL27 is a heterodimeric cytokine composed of two subunits: IL27A (also called IL-27p28) and IL27B (also called Epstein-Barr virus-induced gene 3 or "EBI3"). Human p28 (hIL27A) is expressed as a 243-amino acid preprotein containing a 28-amino acid signal sequence, which is removed after translation to yield a 215-amino acid mature protein. UniProtKB - Q8NEV9 (IL27A_HUMAN). The mature form of p28 (without the signal peptide) has the following amino acid sequence: TIFF2025108521000002.tif22128

[0045] Human IL27B (hIL27B) is expressed as a 229-amino acid preprotein containing a 20-amino acid signal sequence, which is removed after translation to yield a 209-amino acid mature protein. UniProtKB - Q14213 (IL27B_HUMAN). The mature form of hIL27B (without the signal peptide) has the following amino acid sequence: TIFF2025108521000003.tif22128

[0046] Interleukin 27 (IL27) Receptor IL27 elicits intracellular signaling through its interaction with a heterodimeric receptor consisting of IL-27Rα (or IL27RA) and gp130. Binding of IL27 to its receptor activates signaling pathways including the JAK / STAT pathway and the p38 MAPK pathway. IL27 stimulates both pro-inflammatory and anti-inflammatory responses in different cell types such as macrophages, dendritic cells, T cells, and B cells. The type of response is environment-dependent.

[0047] Human IL27 receptor subunit α (hIL27RA) is expressed as a 636-amino acid preprotein containing a 32-amino acid signal sequence, which is removed after translation to yield a 604-amino acid mature protein. UniProtKB - Q6UWB1 (I27RA_HUMAN)

[0048] The mature form of hIL27RA (without the signal peptide) has the following amino acid sequence: TIFF2025108521000004.tif59128

[0049] The extracellular domain of hIL27RA (IL27RA-ECD) is a 484-amino acid polypeptide corresponding to amino acids 33 - 516 of the hIL27RA preprotein and has the following amino acid sequence: TIFF2025108521000005.tif50128

[0050] The human gp130 receptor subunit (hGP130), also known as the IL6 receptor β subunit. UniProtKB - P40189 (IL6RB_HUMAN). hGP130 is expressed as a 918-amino acid preprotein containing a 22-amino acid signal sequence, which is removed after translation to yield an 896-amino acid mature protein. The mature form of hGP130 has the following amino acid sequence: TIFF2025108521000006.tif87128

[0051] The extracellular domain of hGP130 (hGP130-ECD) is a 597-amino acid polypeptide corresponding to amino acids 23 to 619 of the hGP130 preprotein and has the following amino acid sequence: TIFF2025108521000007.tif59128

[0052] IL27 Activity IL27 is expressed by antigen-presenting cells. hIL27 induces the differentiation of diverse populations of T cells of the immune system and also upregulates IL10. hIL27 is pro-inflammatory and anti-inflammatory, capable of regulating T helper cell development, suppressing T cell proliferation, stimulating cytotoxic T cell activity, inducing B cell isotype switching, and having diverse effects on innate immune cells. Among its target cells are CD4 T helper cells that can differentiate into type 1 effector cells (TH1), type 2 effector cells (TH2), and IL17-producing helper T cells (TH17).

[0053] T Cell Differentiation IL27 plays an important role in differentiation through the induction or suppression of T cell subtypes including Th1, Th2, Th17, Tr1, and Treg cells. IL-27 is highly involved in differentiation through the induction or suppression of each T cell subset. Th1 cells expressing interferon gamma (IFNg) are created by STAT1 signaling via the expression of T-bet and signature Th1 genes in response to IL27. Th2 cells expressing IL4 are inhibited by IL27 through the transcription factor GATA-3. Th17 cells expressing IL17, IL22, and GM-CSF are inhibited by IL27 through the expression of STAT1 and the transcription factor RORγt. Treg cells are inhibited by IL27 through STAT1 and STAT3.

[0054] IL27 drives the rapid clonal expansion of naive CD4 T cells rather than memory CD4 T cells. IL27 also potently synergizes with IL-12 to induce interferon-γ / IFN-γ production in naive CD4 T cells and binds to the cytokine receptor WSX-1 / TCCR. Another important role of IL-27 is its anti-tumor activity and anti-angiogenic activity that activates the production of anti-angiogenic chemokines.

[0055] Induction of IL10 Tr1 cells that express IL-10 are induced by IL-27 through the transcription factor c-Maf, resulting in an anti-inflammatory response. The main activity of IL-10 is the suppression of the inflammatory response. The transcription factors STAT1 and STAT3 that specifically bind to IL-27α are also involved. Activation of STAT3 by IL-27 leads to an increase in IL-10 secretion from Treg cells.

[0056] II. Definitions To facilitate the understanding of the present disclosure, certain terms and phrases are defined not only below but also throughout this specification. The definitions provided herein are non-limiting and should be read in consideration of the knowledge that would be known to those skilled in the art.

[0057] Before describing the methods and compositions, it should be understood that the present invention is not limited to the specific methods or compositions described and can, of course, vary. It should also be understood that the technical terms used herein are for the purpose of describing aspects only and are not intended to be limiting.

[0058] When a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit, between the upper and lower limits of the range is specifically disclosed, unless the context clearly dictates otherwise. Smaller ranges between any of the disclosed values or intervening values within a disclosed range are each included in the present invention. The upper and lower limits of these smaller ranges may independently be included in or excluded from the smaller range, and any limits specifically excluded in the disclosed range, any range that includes either or both of these limits or neither of these limits is also included in the present invention. When the disclosed range includes either or both of these limits, ranges excluding either or both of the included limits are also included in the present invention.

[0059] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, several potential and preferred methods and materials will now be described. All publications mentioned herein are incorporated herein by reference for the purpose of disclosing and describing the methods and / or materials in connection with which the publications are cited.

[0060] It should be noted that, as used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a cell" includes a plurality of such cells, and reference to "a peptide" includes reference to one or more peptides and their equivalents, e.g., polypeptides known to those skilled in the art.

[0061] Publications discussed herein are provided only for their disclosure prior to the filing date of the present application. No admission is made that the present invention is not entitled to antedate such publications by virtue of prior invention. Further, the dates of the publications presented may be different from the actual publication dates which may need to be independently confirmed.

[0062] Unless otherwise indicated, parts are by weight, molecular weights are weight average molecular weights, temperatures are in degrees Celsius (°C), and pressures are at or near atmospheric pressure. Standard abbreviations are used, including: bp = base pair; kb = kilobase; pl = picoliter; s or sec = second; min = minute; h or hr = hour; AA or aa = amino acid; kb = kilobase; nt = nucleotide; pg = picogram; ng = nanogram; μg = microgram; mg = milligram; g = gram; kg = kilogram; dl or dL = deciliter; μl or μL = microliter; ml or mL = milliliter; l or L = liter; μM = micromolar; mM = millimolar; M = molar; kDa = kilodalton; i.m. = intramuscularly; i.p. = intraperitoneally; SC or SQ = subcutaneously; QD = once daily; BID = twice daily; QW = once weekly; QM = once monthly; HPLC = high performance liquid chromatography; BW = body weight; U = unit; ns = not statistically significant; PBS = phosphate buffered saline; PCR = polymerase chain reaction; HSA = human serum albumin; MSA = mouse serum albumin; DMEM = Dulbecco's modified Eagle's medium; EDTA = ethylenediaminetetraacetic acid.

[0063] Throughout the present disclosure, it will be recognized that amino acids are referred to according to either the one-letter code or the three-letter code. For the convenience of the reader, the one-letter amino acid code and the three-letter amino acid code are provided in the table below.

[0064] (Table) Amino Acid Abbreviations TIFF2025108521000008.tif97128

[0065] Standard methods in molecular biology are described in the scientific literature (see, e.g., Sambrook and Russell (2001) Molecular Cloning, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; and Ausubel, et al. (2001) Current Protocols in Molecular Biology, Vols. 1-4, John Wiley and Sons, Inc. New York, N.Y., which describes cloning and DNA mutagenesis in bacterial cells (Vol. 1), cloning in mammalian cells and yeast (Vol. 2), expression of complex carbohydrates and proteins (Vol. 3), and bioinformatics (Vol. 4)). This scientific literature describes not only methods for protein purification, including immunoprecipitation, chromatography, electrophoresis, centrifugation, and crystallization, but also chemical analysis, chemical modification, post-translational modification, production of fusion proteins, and protein glycosylation (see, e.g., Coligan, et al. (2000) Current Protocols in Protein Science, Vols. 1-2, John Wiley and Sons, Inc., NY).

[0066] Activate : As used herein, the term "activate" is used with respect to a receptor or receptor complex and reflects a biological effect resulting from the binding of an agonist ligand to the receptor in response to ligand binding, either directly and / or through involvement in a multi-component signaling cascade.

[0067] ActivityAs used herein, the term "activity" is used to describe, with respect to a molecule, the properties of that molecule with respect to a test system (e.g., an assay) or its biological or chemical properties (e.g., the degree of binding of that molecule to another molecule) or the physical properties of a substance or cell (e.g., modification of cell membrane potential). Examples of such biological functions include, but are not limited to, the catalytic activity of a biological agent, the ability to stimulate intracellular signaling, gene expression, cell proliferation, and the ability to modulate immune activities such as the inflammatory response. "Activity" is typically expressed as the level of biological activity per unit of the agent being tested, such as [catalytic activity] / [mg protein], [immune activity] / [mg protein], international units of activity (IU), [STAT5 phosphorylation] / [mg protein], [T cell proliferation] / [mg protein], plaque forming units (pfu), etc. As used herein, the term "proliferative activity" refers to the activity that promotes cell growth and replication.

[0068] Administer / Administration The terms "administer" and "administering" are used interchangeably herein to refer to the act of contacting a subject, including in vitro, in vivo, or ex vivo, with an agent (e.g., an ortholog, an IL2 ortholog, an engineered cell expressing an ortholog receptor, an engineered cell expressing an ortholog IL2 receptor, a CAR-T cell expressing an ortholog IL2 receptor, a chemotherapeutic agent, an antibody, or a pharmaceutical formulation comprising one or more of the foregoing) to a target cell, tissue, organ, or biological fluid. Administration of the agent can be accomplished by any of a variety of methods approved in the art, including, but not limited to, topical administration, intravascular injection (including intravenous or intraarterial infusion), intradermal injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, inhalation, etc. The term "administer" includes not only contact of the agent with a cell, tissue, or organ, but also contact of the agent with a liquid in contact with the cell, tissue, or organ.

[0069] Affinity: As used herein, the term "affinity" refers to the degree of specific binding of a first molecule (e.g., ligand) to a second molecule (e.g., receptor), and is measured by the equilibrium dissociation constant (KD), which is the ratio of the dissociation rate constant (Koff) between the molecule and its target to the association rate constant (Kon) between the molecule and its target.

[0070] Agonist : As used herein, the term "agonist" refers to a first agent that specifically binds to a second agent (the "target") and interacts with the target to cause or promote an enhancement of the activation of the target. In some cases, the agonist regulates cell activation, enhances activation, sensitizes cells to activation by a second agent, or upregulates the expression of one or more genes, proteins, ligands, receptors, biological pathways that can result in cell proliferation, or pathways that result in cell death such as cell cycle arrest or apoptosis. In some embodiments, the agonist is an activator of a receptor protein that binds to the receptor, modifies the state of the receptor, and results in a biological response. This response mimics the effect of the receptor's endogenous activator. The term "agonist" includes partial agonists, full agonists, and superagonists. An agonist may be described as a "full agonist" when such agonist produces a substantially complete biological response (i.e., a response associated with the natural ligand / receptor binding interaction) induced by the receptor under study, or as a partial agonist. In contrast to an agonist, an antagonist can specifically bind to a receptor but typically does not result in a signal cascade initiated by the receptor and can modify the action of an agonist at that receptor. An inverse agonist is an agent that produces a pharmacological response opposite to that of an agonist. A "superagonist" is a type of agonist that is capable of producing a greater maximal response than the endogenous agonist for a target receptor, and thus has an activity greater than 100% of the natural ligand. A superagonist is typically a synthetic molecule that exhibits a response greater than 110%, alternatively greater than 120%, alternatively greater than 130%, alternatively greater than 140%, alternatively greater than 150%, alternatively greater than 160%, or alternatively greater than 170% of the evaluable quantitative or qualitative parameter of the molecule in its natural form when evaluated at a similar concentration in an equivalent assay.

[0071] Antagonist As used herein, the terms "antagonist" or "inhibitor" refer to a molecule that counteracts the action of an agonist. An antagonist prevents, reduces, inhibits, or neutralizes the activity of an agonist, and an antagonist can also prevent, inhibit, or reduce the constitutive activity of a target, such as a target receptor, even in the absence of a specified agonist. An inhibitor is, for example, a molecule that decreases, blocks, prevents, delays activation, inactivates, desensitizes, or down-regulates a gene, protein, ligand, receptor, biological pathway, or cell.

[0072] Antibody As used herein, the term "antibody" collectively refers to: (a) glycosylated and non-glycosylated immunoglobulins that specifically bind to a target molecule (including, but not limited to, mammalian immunoglobulin classes IgG1, IgG2, IgG3, and IgG4), and (b) IgG(1-4) delta C that competes with the immunoglobulin from which it is derived for binding to the target molecule H 2, F(ab')2, Fab, ScFv, V H 、V LAn immunoglobulin derivative including, but not limited to, tetrabody, tribody, diabody, dsFv, F(ab')3, scFv-Fc and (scFv)2. The term "antibody" is not limited to immunoglobulins derived from any particular mammalian species, and includes antibodies from mice, humans, horses and camels (e.g., human antibodies). The term "antibody" includes not only antibodies isolatable from natural origin or from animals after immunization with an antigen, but also monoclonal antibodies, bispecific antibodies, trispecific antibodies, chimeric antibodies, humanized antibodies, human antibodies, CDR grafted, veneered, or deimmunized (e.g., to remove T cell epitopes) antibodies, i.e., engineered antibodies. The term "human antibody" includes not only antibodies obtained from humans, but also antibodies obtained from transgenic mammals containing human immunoglobulin genes such that when stimulated with an antigen, the transgenic animals produce antibodies including the amino acid sequence characteristics of antibodies produced by humans. The term "antibody" should not be construed as limited to any particular synthetic means, and includes not only natural antibodies isolatable from natural origin, but also antibodies isolated from transgenic animals that are transgenic with respect to human immunoglobulin genes or hybridomas prepared therefrom, antibodies isolated from host cells transformed with a nucleic acid construct that results in expression of the antibody, antibodies isolated from combinatorial antibody libraries including phage display libraries, i.e., engineered antibody molecules prepared by "recombinant" means.

[0073] Binding molecule: As used herein, the term "binding molecule" refers to a bivalent molecule capable of binding to the extracellular domains of two cell surface receptors. In some embodiments, the binding molecule specifically binds to two different receptors (or domains or subunits thereof), such that these receptors (or domains or subunits) are maintained in close proximity to each other such that these receptors (or domains or subunits) including their domains (e.g., intracellular domains) interact with each other to result in downstream signal transduction.

[0074] CDRAs used herein, the terms "CDR" or "complementary determining region" are intended to mean the discontinuous antigen-binding combining sites found within the variable regions of both heavy chain immunoglobulin polypeptides and light chain immunoglobulin polypeptides. CDRs are described by Kabat et al., J. Biol. Chem. 252:6609-6616 (1977); Kabat, et al., U.S. Dept. of Health and Human Services publication entitled "Sequences of proteins of immunological interest" (1991) (also referred to herein as "Kabat 1991" or "Kabat"); Chothia, et al. (1987) J. Mol. Biol. 196:901-917 (also referred to herein as "Chothia"); and MacCallum, et al. (1996) J. Mol. Biol. 262:732-745, where these definitions include amino acid residue overlaps or subsets when compared to each other. Nevertheless, the application of any of the definitions for referring to the CDRs of an antibody or chimeric antibody or variants thereof is intended to be within the scope of this term as defined and used herein. The term "Chothia numbering" as used herein is recognized in the art and refers to a system of numbering amino acid residues based on the positions of structural loop regions (Chothia et al. 1986, Science 233:755-758; Chothia & Lesk 1987, JMB 196:901-917; Chothia et al. 1992, JMB 227:799-817). For the purposes of the present disclosure, unless otherwise specified, the positioning of CDR2 and CDR3 in the variable region of an antibody follows the Kabat numbering or simply "Kabat". The positioning of CDR1 in the variable region of an antibody follows a hybrid of the Kabat and Chothia numbering schemes.

[0075] Clonal trait: A clonal trait is defined as an aggregate of binding molecules derived from the same B cell progenitor cell. As used herein, the term "clonal trait" refers to an aggregate of antigen-binding molecules that belong to the same germline lineage, have the same CDR3 length, and have 70% or more homology in the CDR3 sequence.

[0076] Equivalent As used herein, the term "equivalent" is used to describe the degree of difference between two measurements of an evaluable quantitative or qualitative parameter. For example, if the difference between a first measurement of an evaluable quantitative parameter and a second measurement of an evaluable parameter does not exceed the range within which one of ordinary skill in the art would recognize that there is no actual statistical significant difference between the two results in that situation, these two measurements would be considered "equivalent". In some cases, if the difference of one measurement from another measurement is less than 30%, alternatively less than 25%, alternatively less than 20%, alternatively less than 15%, alternatively less than 10%, alternatively less than 7%, alternatively less than 5%, alternatively less than 4%, alternatively less than 3%, alternatively less than 2%, or less than 1%, these measurements may be considered "equivalent". In certain embodiments, if the difference of a measurement from a reference standard is less than 15%, alternatively less than 10%, or alternatively less than 5%, this measurement is equivalent to the reference standard.

[0077] As used herein, the term "downstream signaling" refers to an intracellular signaling process caused by the interaction of two or more cell surface receptors that are in proximity to each other.

[0078] Effective Concentration (EC)As used herein, the term "effective concentration" or its abbreviation "EC" is used interchangeably to refer to the concentration of an agent (e.g., hIL2 mutein) in an amount sufficient to effect a change in a given parameter in a test system. The abbreviation "E" refers to the magnitude of a given biological effect observed in the test system when the test system is exposed to the test substance. When the magnitude of the response is expressed as a function of the concentration of the test substance ("C"), the abbreviation "EC" is used. In the context of a biological system, the term Emax refers to the maximum magnitude of a given biological effect observed in response to a saturating concentration of an activating test substance. When a subscript is attached to the abbreviation EC (e.g., EC 40 , EC 50 , etc.), the subscript refers to the percent of Emax of the biological effect observed at that concentration. For example, the concentration of such a test substance sufficient to result in the induction of such a measurable biological parameter in a test system that is 30% of the maximum level of the measurable biological parameter in response to the test substance is referred to as the "EC 30 " of the test substance with respect to such a biological parameter. Similarly, the term "EC 100 " is used to mean the effective concentration of such an agent that results in a maximum (100%) response of a measurable parameter in response to the agent. Similarly, the term EC 50 (commonly used in the field of pharmacodynamics) refers to the concentration of an agent sufficient to result in a maximum 50% change in a measurable parameter. The term "saturating concentration" refers to the maximum possible amount of a test substance that can be dissolved in a standard volume of a particular solvent (e.g., water) under standard conditions of temperature and pressure. In pharmacodynamics, the saturating concentration of a drug is typically used to mean the drug concentration sufficient for all available receptors to be occupied by the drug, and EC 50 is the drug concentration to give a maximum half effect. The EC of a particular effective concentration of a test substance may be abbreviated with respect to a particular parameter and test system.

[0079] Extracellular DomainAs used herein, the term "extracellular domain" or its abbreviation "ECD" refers to the portion of a cell surface protein (e.g., a cell surface receptor) that is outside the plasma membrane of a cell. The term "ECD" can include the extracellular portion of a transmembrane protein or the extracellular portion of a cell surface (or membrane-bound protein).

[0080] Identity: As used herein, the term "percent (%) sequence identity" or "substantially identical" when used in the context of a nucleic acid or polypeptide refers to a sequence having at least 50% sequence identity to a reference sequence. Alternatively, the percent sequence identity can be any integer from 50% to 100%. In some embodiments, the sequence has at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the reference sequence when determined by BLAST using standard parameters as follows. In the case of sequence comparison, usually one sequence serves as the reference sequence against which the test sequence is compared. When using a sequence comparison algorithm, the test sequence and the reference sequence are input into a computer, the coordinates of the subsequences are specified if necessary, and the program parameters of the sequence algorithm are specified. Default program parameters may be used, or other parameters may be specified. The sequence comparison algorithm then calculates the percent sequence identity of the test sequence to the reference sequence based on the program parameters. The comparison window includes a reference to any one segment of the number of consecutive positions, e.g., a segment of at least 10 residues. In some embodiments, the comparison window has 10 to 600 residues, e.g., about 10 to about 30 residues, about 10 to about 20 residues, about 50 to about 200 residues, or about 100 to about 150 residues, and after the two sequences are optimally aligned therein, the sequences can be compared to the reference sequence at the same number of consecutive positions. Algorithms suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms described in Altschul et al. (1990) J. Mol. Biol. 215: 403-410 and Altschul, et al. (1977) Nucleic Acids Res. 25: 3389-3402, respectively. Software for performing BLAST analysis is publicly available through the website of the National Center for Biotechnology Information (NCBI) of the United States.This algorithm involves first identifying high-scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence that match or satisfy a positive-valued threshold score T when aligned with words of the same length in the database array. T is referred to as the threshold for adjacent word scores (Altschul et al., supra). These initial hits for adjacent words serve as seeds to initiate a search for longer HSPs that contain them. Then, for each sequence, the word hits are extended in both directions as long as the sum of the alignment scores can be increased. For nucleotide sequences, the parameters M (reward score for pairs of matching residues; always >0) and N (penalty score for mismatching residues; always <0) are used to calculate the sum of the scores. For amino acid sequences, a scoring matrix is used to calculate the sum of the scores. The extension of word hits in each direction stops when the sum of the alignment scores drops by an amount X from its maximum achieved value; when the sum of the scores becomes zero or less due to the accumulation of alignments of one or more negative-scoring residues; or when the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) functions similarly but, by default, uses a word size (W) of 28, an expectation value (E) of 10, M = 1, N = -2, and comparison of both strands. For amino acid sequences, the BLASTP program, by default, uses a word size (W) of 3, an expectation value (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989)). The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin & Altschul, Proc. Nat'l. Acad. Sci. USA 90:5873-5787 (1993)).One measure of similarity provided by the BLAST algorithm is the minimum sum probability (P(N)), which provides an indication of the probability that a match between two nucleotide or amino acid sequences occurs by chance. For example, an amino acid sequence has a minimum sum probability of less than about 0.01, more preferably about 10, in a comparison of the test amino acid sequence with a reference amino acid sequence. -5 less, most preferably about 10 -20 If it is less, it is considered to be similar to the reference sequence.

[0081] As used herein, the terms "interleukin 27 receptor" or "IL27R" refer to a heterodimeric receptor formed by the subunits IL27Rα (IL27Rα) and glycoprotein 130 (gp130) and bound by the ligand IL27. The human sequence of IL27Rα is described as UniProt ID number Q6UWB1. The human sequence of gp130 is described as UniProt ID number Q13514.

[0082] Intracellular Signaling: As used herein, the terms "intracellular signaling" and "downstream signaling" are used interchangeably to refer to the process of cell signaling caused by the interaction of the intracellular domains (ICDs) of two or more cell surface receptors in close proximity to each other. In the receptor complex via the JAK / STAT pathway, the association of the ICDs of the receptor subunits brings the JAK domains of the ICDs into proximity, initiating a phosphorylation cascade in which STAT molecules are phosphorylated and translocate to the nucleus to associate with specific nucleic acid sequences, resulting in the activation and expression of specific genes within the cell. The binding molecules of the present disclosure, when activated by their natural cognate IL27, provide intracellular signaling characteristic of the IL-27 receptor. Several methods are available for measuring downstream signaling activity. For example, in some embodiments, JAK / STAT signaling can be measured by the presence of phosphorylated receptor and / or phosphorylated STAT. In other embodiments, the expression of one or more downstream genes whose expression levels can be affected by the level of downstream signaling induced by the binding molecule can also be measured.

[0083] Ligand As used herein, the term "ligand" refers to a molecule that exhibits specific binding to a receptor and causes a change in the biological activity of the receptor such that it results in a change in the activity of the receptor to which it binds. In one embodiment, the term "ligand" refers to a molecule or complex thereof that can act as an agonist or antagonist of a receptor. As used herein, the term "ligand" encompasses both natural and synthetic ligands. "Ligand" also includes small molecules, such as peptide mimics of cytokines and peptide mimics of antibodies. The complex of a ligand and a receptor is referred to as a "ligand-receptor complex".

[0084] As used herein, the term "linker" refers to a connection between two elements, such as between protein domains. A linker can be a covalent bond or a peptide linker. The term "bond" refers to a chemical bond, such as an amide bond or a disulfide bond, or any type of bond resulting from a chemical reaction, such as a chemical conjugation. The term "peptide linker" refers to an amino acid or polypeptide that can be utilized to connect two protein domains so as to provide space and / or mobility between the two protein domains.

[0085] Regulate As used herein, terms such as "modulate", "modulation", etc. refer to the ability of a test substance to have a positive or negative, or direct or indirect, effect on a response in a biological system or a system including a biochemical pathway.

[0086] Multimerization : As used herein in the context of the structure of a polypeptide, "N-terminus" (or "amino terminus") and "C-terminus" (or "carboxyl terminus") refer to the amino-terminal most and carboxyl-terminal most ends of the polypeptide, respectively, whereas "N-terminal side" and "C-terminal side" refer to the relative positions in the amino acid sequence of the polypeptide in the N-terminal and C-terminal directions, respectively, and can include the residues of the N-terminus and C-terminus. The terms "immediately N-terminal side" or "immediately C-terminal side" are used to refer to the position of a first amino acid residue relative to a second amino acid residue when the first and second amino acid residues are covalently bonded to provide a continuous amino acid sequence.

[0087] N-Terminal : As used herein, the term "multimerization" refers to the proximity of two or more cell surface receptors, or domains or subunits thereof, such that the receptors, or domains or subunits thereof, can act on each other to cause intracellular signal transduction.

[0088] Nucleic Acid : Terms such as "nucleic acid", "nucleic acid molecule", and "polynucleotide" are used interchangeably herein to refer to polymeric forms of nucleotides of any length that are deoxyribonucleotides or ribonucleotides or analogs thereof. Non-limiting examples of polynucleotides include linear or circular nucleic acids, messenger RNA (mRNA), complementary DNA (cDNA), recombinant polynucleotides, vectors, probes, primers, etc. (and the).

[0089] Functionally Linked : The term "functionally linked" is used herein to refer to the relationship between nucleic acid sequences encoding different functions that, when combined in a single nucleic acid sequence, can provide a nucleic acid that, when introduced into a cell, can result in transcription and / or translation of a specific nucleic acid sequence within the cell. For example, the DNA of a signal sequence is functionally linked to the DNA of a polypeptide when it is expressed as a preprotein involved in the secretion of the polypeptide; a promoter or enhancer is functionally linked to a coding sequence when it affects the transcription of the sequence; or a ribosome binding site is functionally linked to a coding sequence when it is arranged to promote translation. Generally, "functionally linked" means that the linked DNA sequences are contiguous, and in the case of a secretion leader, are contiguous and in the correct reading frame. However, certain genetic elements, such as enhancers, need not be contiguous with the sequences that produce their effect.

[0090] Partial Agonist: As used herein, the term "partial agonist" refers to a molecule that specifically binds to and activates a given receptor, but has only partial activation of the receptor compared to a full agonist. A partial agonist may exhibit both agonist and antagonist effects. For example, when both a full agonist and a partial agonist are present, the partial agonist acts as a competitive antagonist by competing with the full agonist for binding to the receptor, resulting in a net reduction in receptor activation compared to the contact of the receptor with the full agonist in the absence of the partial agonist. Clinically, partial agonists can be used to activate a receptor when there is an insufficient amount of endogenous ligand to give a response below the desired maximum, or to reduce overstimulation of the receptor when there is an excess amount of endogenous ligand. The maximum response (Emax) produced by a partial agonist is called its intrinsic activity and can be expressed on a percentage scale where a full agonist produces a 100% response. In some embodiments, the IL-27 binding molecule has a reduced E max compared to max . E max reflects the maximum response level in a cell type that can be obtained by a ligand (e.g., a binding molecule or a natural cytokine described herein such as IL-27). In some embodiments, the IL-27 binding molecule described herein has at least 1% (e.g., 1% - 100%, 10% - 100%, 20% - 100%, 30% - 100%, 40% - 100%, 50% - 100%, 60% - 100%, 70% - 100%, 80% - 100%, 90% - 100%, 1% - 90%, 1% - 80%, 1% - 70%, 1% - 60%, 1% - 50%, 1% - 40%, 1% - 30%, 1% - 20% or 1% - 10%) of the E max caused by IL-27. In other embodiments, the E max of the IL-27 binding molecule described herein is the E maxLarger than (e.g., at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% or 50% larger). In some embodiments, by varying the linker length of the IL-27 binding molecule, the E max of the IL-27 binding molecule can be varied. The IL-27 binding molecule results in E max in the most desired cell type and can result in reduced E max in other cell types.

[0091] Polypeptide : As used herein, the terms "polypeptide", "peptide" and "protein" are used interchangeably herein to refer to a polymeric form of amino acids of any length that can include genetically encoded and non-genetically encoded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having modified polypeptide backbones. These terms include, but are not limited to, fusion proteins having heterologous amino acid sequences; fusion proteins having heterologous and homologous leader sequences; fusion proteins having or not having an N-terminal methionine residue; fusion proteins with immunologically tagged proteins; fusion proteins of immunologically active proteins (e.g., antigenic diphtheria or tetanus toxin fragments), etc.

[0092] As used herein, terms such as "prevent," "preventing," "prevention" generally relate to a subject having a predisposition to a particular disease, disorder, or condition due to genetic, empirical, or environmental factors, and refer to an action initiated with respect to the subject prior to the occurrence of the disease, disorder, condition, or others (e.g., as determined by the absence of clinical symptoms) to temporarily or permanently prevent, suppress, inhibit, or reduce the risk of the subject developing the disease, disorder, condition, or others, or to delay their occurrence. In a particular example, the terms "prevent," "preventing," "prevention" are also used to refer to delaying the progression from the current state of a disease, disorder, or condition to a more detrimental state.

[0093] Proximity: As used herein, the term "proximity" refers to the spatial proximity or physical distance between two cell surface receptors, or domains or subunits thereof, after a binding molecule as described herein binds to the two cell surface receptors, or domains or subunits thereof. In some embodiments, after the binding molecule binds to a cell surface receptor, or a domain or subunit thereof, the spatial proximity between the cell surface receptors, or domains or subunits thereof can be, for example, less than about 500 angstroms, such as a distance of about 5 angstroms to about 500 angstroms. In some embodiments, the spatial proximity is less than about 5 angstroms, less than about 20 angstroms, less than about 50 angstroms, less than about 75 angstroms, less than about 100 angstroms, less than about 150 angstroms, less than about 250 angstroms, less than about 300 angstroms, less than about 350 angstroms, less than about 400 angstroms, less than about 450 angstroms, or less than about 500 angstroms. In some embodiments, the spatial proximity is less than about 100 angstroms. In some embodiments, the spatial proximity is less than about 50 angstroms. In some embodiments, the spatial proximity is less than about 20 angstroms. In some embodiments, the spatial proximity is less than about 10 angstroms. In some embodiments, the spatial proximity ranges from about 10 to 100 angstroms, about 50 to 150 angstroms, about 100 to 200 angstroms, about 150 to 250 angstroms, about 200 to 300 angstroms, about 250 to 350 angstroms, about 300 to 400 angstroms, about 350 to 450 angstroms, or about 400 to 500 angstroms. In some embodiments, the spatial proximity is less than about 250 angstroms, alternatively less than about 200 angstroms, alternatively less than about 150 angstroms, alternatively less than about 120 angstroms, alternatively less than about 100 angstroms, alternatively less than about 80 angstroms, alternatively less than about 70 angstroms, or alternatively less than about 50 angstroms.

[0094] Receptor : As used herein, the term "receptor" refers to a polypeptide having a domain that specifically binds a ligand, where binding of the ligand results in a change in at least one biological property of the polypeptide. In some embodiments, the receptor is a "soluble" receptor not associated with the cell surface. In some embodiments, the receptor is a cell surface receptor that includes an extracellular domain (ECD) and a membrane-associated domain that serves to anchor the ECD to the cell surface. In some embodiments of the cell surface receptor, the receptor is a transmembrane polypeptide typically comprising an intracellular domain (ICD) and an extracellular domain (ECD) linked by a transmembrane domain, often referred to as the transmembrane domain (TM). Binding of the ligand to the receptor results in a conformational change in the receptor and a measurable biological effect. In some cases where the receptor is a transmembrane polypeptide comprising an ECD, TM, and ICD, binding of the ligand to the ECD results in a measurable intracellular biological effect mediated by one or more domains of the ICD in response to binding of the ligand to the ECD. In some embodiments, the receptor is a component of a multi-component complex for promoting intracellular signal transduction. For example, a ligand can bind to a cell surface molecule not associated with any intracellular signal transduction alone, but upon binding of the ligand, promotes the formation of a multimeric complex that results in intracellular signal transduction.

[0095] Recombinant: As used herein, the term "recombinant" is used as an adjective to describe a method by which a polypeptide, nucleic acid, or cell has been modified using recombinant DNA technology. A recombinant protein is a protein produced using recombinant DNA technology and may be so designated (e.g., rhIL2) using the abbreviation of the lower case "r" to indicate the method by which the protein was produced. Similarly, a cell is said to be "recombinant" if it has been modified by the incorporation (e.g., transfection, transduction, infection) of exogenous nucleic acid (e.g., ssDNA, dsDNA, ssRNA, dsRNA, mRNA, viral or non-viral vector, plasmid, cosmid, etc.) using recombinant DNA technology. Techniques and protocols for recombinant DNA technology, such as those found in Sambrook, et al. (1989) Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Plainview, N.Y.) and other standard molecular biology laboratory manuals, are well known in the art.

[0096] Response : For example, the term "response" of a cell, tissue, organ, or organism encompasses measurable biochemical or physiological parameters (e.g., concentration, density, adhesion, proliferation, activation, phosphorylation, migration, enzyme activity, gene expression level, gene expression rate, energy consumption rate, level of differentiation or qualitative or quantitative changes in state, where the changes are correlated with activation, stimulation or treatment, or internal mechanisms such as genetic programming). In certain contexts, terms such as "activation", "stimulation", etc. refer to cell activation regulated not only by external or environmental factors but also by internal mechanisms. In contrast, terms such as "inhibition", "down-regulation", etc. refer to the opposite effect.

[0097] Single Domain Antibody (sdAb): The term "single domain antibody" or "sdAb" refers to an antibody having a single (only one) monomeric variable antibody domain. An sdAb can selectively bind to a specific antigen. The V, further defined below H H antibody is an example of an sdAb.

[0098] Specifically Bind : As used herein, the term "specifically binds" refers to the degree of selectivity or affinity by which one molecule binds to another molecule. With respect to a binding pair (e.g., a binding molecule / receptor, ligand / receptor, antibody / antigen, antibody / ligand, antibody / receptor binding pair described herein), the first molecule of the binding pair is said to specifically bind to the second molecule of the binding pair if the first molecule of the binding pair does not bind in significant amounts to other components present in the sample. The affinity of the first molecule for the second molecule is at least 2-fold greater, alternatively at least 5-fold greater, alternatively at least 10-fold greater, alternatively at least 20-fold greater, or alternatively at least 100-fold greater than the affinity of the first molecule for other components present in the sample, the first molecule of the binding pair is said to specifically bind to the second molecule.

[0099] Stably Associate: As used herein, the terms "stably associate" or "in stable association" are used to refer to the various means by which one molecule (e.g., a polypeptide) can associate with another molecule over a long period of time. Stable association of one molecule to another can be effected by a variety of means including covalent bonds and non-covalent interactions. In some embodiments, stable association of two molecules can be effected by covalent bonds such as peptide bonds. In other embodiments, stable association of two molecules can be effected by non-covalent interactions. Examples of non-covalent interactions that can provide stable association between two molecules include electrostatic interactions (e.g., hydrogen bonds, ionic bonds, halogen bonds, dipole-dipole interactions, van der Waals forces and π-effects including cation-π, anion-π and π-π interactions) and hydrophobic / hydrophilic interactions. In some embodiments, stable association of the sdAbs of the bivalent binding molecules of the present disclosure can be effected by non-covalent interactions. In one embodiment, non-covalent stable association of the sdAbs of the bivalent binding molecule can be achieved by conjugation of the sdAb to a "knob-into-hole" modified Fc monomer. The Fc "knob" monomer stably associates non-covalently with the Fc "hole" monomer. Conjugation of a "Fc knob" monomer of a first sdAb that specifically binds to the extracellular domain of the first subunit of a heterodimeric receptor and conjugation of a "Fc hole" monomer of a second sdAb that specifically binds to the extracellular domain of the second subunit of the heterodimeric receptor results in stable association of the first and second sdAbs. The knob-into-hole modification is more fully described in Ridgway, et al. (1996) Protein Engineering 9(7):617-621, as well as U.S. Patent No. 5,731,168, issued March 24, 1998, U.S. Patent No. 7,642,228, issued January 5, 2010, U.S. Patent No. 7,695,936, issued April 13, 2010 and U.S. Patent No. 8,216,805, issued July 10, 2012.The knob-into-hole modification refers to a modification at the interface between two immunoglobulin heavy chains in the CH3 domain, where: i) an amino acid residue in the CH3 domain of the first heavy chain is replaced by an amino acid residue having a larger side chain (e.g., tyrosine or tryptophan), creating a protrusion from the surface ("knob"), and ii) an amino acid residue in the CH3 domain of the second heavy chain is replaced by an amino acid residue having a smaller side chain (e.g., alanine or threonine), thereby creating a cavity ("hole") within the interface in the second CH3 domain, and the protruding side chain ("knob") of the first CH3 domain is accommodated by the cavity in the second CH3 domain. In one embodiment, the "knob-into-hole modification" includes the amino acid substitutions T366W and optionally S354C in one antibody heavy chain, and the amino acid substitutions T366S, L368A, Y407V and optionally Y349C in the other antibody heavy chain. Further, the Fc domain may be modified by the introduction of cysteine residues at position S354 of one chain and position Y349 of the other chain, which results in a stabilizing disulfide bridge between the two antibody heavy chains in the Fc region (Carter, et al. (2001) Immunol Methods 248, 7-15). The knob-into-hole format is used to promote the expression of a first polypeptide (e.g., an IL27Rα-binding sdAb) on a first Fc monomer having a "knob" modification and a second polypeptide on a second Fc monomer carrying a "hole" modification for the expression of a heterodimeric polypeptide conjugate.

[0100] Subject : The terms "recipient", "individual", "subject", and "patient" are used interchangeably herein and refer to any mammalian subject, particularly a human, for whom diagnosis, treatment, or therapy is desired. For purposes of treatment, "mammal" refers to any animal classified as a mammal, including humans, domestic and agricultural animals, and show animals, sport animals, or pets, such as dogs, horses, cats, cows, sheep, goats, pigs, etc. In some embodiments, the mammal is a human.

[0101] Substantially As used herein, the term "substantially" refers to a component, level, value, number, frequency, percentage, dimension, size, amount, weight, or length that is 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more of a referenced amount, level, value, number, frequency, percentage, dimension, size, amount, weight, or length. In one embodiment, "substantially the same" refers to a component, level, value, number, frequency, percentage, dimension, size, amount, weight, or length that produces an effect that is approximately the same as a referenced amount, level, value, number, frequency, percentage, dimension, size, amount, weight, or length, for example, a physiological effect.

[0102] Affected with : As used herein, the term "afflicted" refers to a determination made by a physician with respect to a subject, based on available information accepted in the art for the identification of a disease, disorder, or condition, including, but not limited to, X-rays, CT scans, conventional clinical diagnostic tests (such as blood cell counts), genomic data, protein expression data, immunohistochemistry, that the subject is in need of treatment or would benefit from treatment. The term "afflicted" is typically used with a specific medical condition, such as "afflicted with a neoplastic disease," and refers to a subject in whom a neoplasm has been diagnosed.

[0103] Therapeutically Effective Amount: As used herein, the term "therapeutically effective amount" refers to an amount of a single dose, as part of a series of doses, administered alone or as part of a pharmaceutical composition or treatment regimen, that can have any detectable positive effect on any symptom, aspect, or characteristic of a disease, disorder, or condition when administered to a subject. A therapeutically effective amount can be confirmed by measuring the relevant physiological effect, which can be adjusted with respect to the dosing regimen and in response to diagnostic analyses such as the subject's condition. Parameters for evaluation to determine a therapeutically effective amount of an agent include, but are not limited to, signs such as age, weight, sex, general health status, ECOG score, observable physiological parameters, blood levels, blood pressure, electrocardiogram, computed tomography, X-ray, and others, and are determined by a physician using approved diagnostic criteria in the art. Alternatively or additionally, other parameters commonly evaluated in a clinical setting, such as normalization of body temperature, heart rate, blood chemistry, blood pressure, cholesterol level, or any symptom, aspect, or characteristic of a disease, disorder, or condition, modification of biomarker levels, increase in survival period, extension of progression-free survival period, extension of time to progression, increase in time to treatment failure, extension of event-free survival period, extension of time to next treatment, improvement in response rate, improvement in response duration, etc., may be monitored to determine whether a therapeutically effective amount of an agent has been administered to a subject, and these parameters are relied upon by clinicians in the art to evaluate improvement in the condition of a subject in response to administration of an agent.

[0104] Treat: Terms such as "treating", "treatment", and "to treat" refer to actions (e.g., administering a binding molecule or a pharmaceutical composition comprising the same as described herein) initiated with respect to a subject after a disease, disorder, or condition, or a symptom thereof, has been diagnosed, observed, or otherwise detected in the subject, and which serve to transiently or permanently remove, reduce, suppress, alleviate, or reverse at least one of the underlying causes of such a disease, disorder, or condition that is afflicting the subject, or at least one of the symptoms associated with such a disease, disorder, or condition. Treatment includes actions taken with respect to a subject having a disease, which actions result in inhibition of the disease in the subject (e.g., halting the progression of a disease, disorder, or condition or reversing one or more symptoms associated therewith).

[0105] VHH : As used herein, the term "V H H" refers to a type of sdAb having a single monomeric heavy chain variable antibody domain. Such antibodies can be found in or produced from camelid mammals (e.g., camels, llamas) that originally lack light chains. V H H can be obtained from the immunization of camelids (including camels, llamas, and alpacas) (see, e.g., Hamers-Casterman, et al. (1993) Nature 363:446-448), or by screening a library constructed with a V H H framework (e.g., a phage library). Antibodies having a given specificity can be derived from non-mammalian sources such as V H H obtained from the immunization of cartilaginous fish including, but not limited to, sharks. In certain embodiments, the bispecific V H H 2 in the binding molecule has a V H H with an equilibrium dissociation constant between V H H and a receptor of, for example, about 10-6 Greater than M, alternatively about 10 -8 Greater than M, alternatively about 10 -10 Greater than M, alternatively about 10 -11 Greater than M, alternatively about 10 -10 Greater than M, about 10 -12 When greater than M, it binds to a receptor (e.g., the first or second receptor of a natural or non-natural receptor pair). Standardized protocols for the generation of single domain antibodies from camels are well known in the scientific literature. See, for example, Vincke, et al (2012) Chapter 8 in Methods in Molecular Biology , Walker, J. editor (Humana Press, Totowa NJ). Specific binding can be evaluated using techniques known in the art including, but not limited to, competitive ELISA, BIACORE® assays and / or KINEXA® assays. In some embodiments, the V H H can be humanized to include human framework regions. Examples of human germline cell lines that can be used to make humanized V H H include, but are not limited to, VH3-23 (e.g., UniProt ID: P01764), VH3-74 (e.g., UniProt ID: A0A0B4J1X5), VH3-66 (e.g., UniProt ID: A0A0C4DH42), VH3-30 (e.g., UniProt ID: P01768), VH3-11 (e.g., UniProt ID: P01762) and VH3-9 (e.g., UniProt ID: P01782).

[0106] V H H 2 : As used herein, "V H H 2 " and "bispecific V H H 2The terms "and "VHH dimer" are used interchangeably to refer to a subtype of binding molecule of the present disclosure in which both the first and second sdAbs are VHHs and the first V H H binds to the first receptor, or a domain or subunit thereof, and the second V H H binds to the second receptor, or a domain or subunit thereof.

[0107] Wild-Type As used herein, the terms "wild-type" or "WT" or "native" are used to refer to an amino acid or nucleotide sequence that is found in nature and has not been modified by human hand.

[0108] III. IL27 Receptor-Binding Protein The IL27 receptor (IL27R) comprises an IL27Rα subunit (IL27Rα) and a glycoprotein 130 subunit (gp130). Provided herein are IL27R-binding proteins that specifically bind to IL27Rα and gp130. In some embodiments, the IL27R-binding protein binds to mammalian cells that express both IL27Rα and gp130. In some embodiments, the IL27R-binding protein can be a bispecific V H H 2 as described below.

[0109] The IL27R-binding protein has a first V H H (anti-IL27Rα V H H antibody) that binds to IL27Rα and a second V H H (anti-gp130 V H H antibody) that binds to gp130, and causes dimerization and downstream signaling of the two receptor subunits when binding to cells expressing IL27Rα and gp130, such as CD8 + T cells, CD4 + T cells, and / or regulatory T (Treg) cells, a bispecific V H H 2It can be.

[0110] V H H is a type of single domain antibody (sdAb) that contains a single monomeric variable antibody domain. Like a full-length antibody, it can selectively bind to a specific antigen. V H The complementarity determining regions (CDRs) of V H H are within the polypeptide of a single domain. V

[0111] Exemplary V H H has a molecular weight of approximately 12 - 15 kDa, which is much smaller than that of a conventional mammalian antibody (150 - 160 kDa) composed of two heavy chains and two light chains. V H H is originally found in or can be produced from mammals of the family Camelidae (e.g., camels, llamas, dromedaries, alpacas, and guanacos) that originally lack light chains. Descriptions of sdAb and V H H can be found, for example, in De Greve et al., Curr Opin Biotechnol. 61:96 - 101, 2019; Ciccarese, et al., Front Genet. 10:997, 2019; Chanier and Chames, Antibodies (Basel) 8(1), 2019; and De Vlieger et al., Antibodies (Basel) 8(1), 2018.

[0112] Bispecific V H H 2 In some embodiments, to prepare a binding protein that is, two V H H can be synthesized separately and then linked together by a linker. Alternatively, bispecific V H H 2 can be synthesized as a fusion protein. V H Hs with different binding activities and receptor targets are paired to form bispecific V H H 2can be made. This fusion protein can be screened for signal transduction in cells that possess one or both of the relevant receptors.

[0113] In some embodiments, the bispecific V H H 2 comprises: a CDR1 having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to the sequence of CDR1 from the row of Table 1A, or having 0, 1, 2 or 3 amino acid changes, optionally conservative amino acid changes; a CDR2 having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to the sequence of CDR2 from the same row of Table 1A, or having 0, 1, 2 or 3 amino acid changes, optionally conservative amino acid changes; and a CDR3 having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to the sequence of CDR3 from the same row of Table 1A, or having 0, 1, 2 or 3 amino acid changes, optionally conservative amino acid changes, comprising a first V H H antibody; and CDR1 having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to the CDR4 sequence from the same row of Table 1A, or having 0, 1, 2 or 3 amino acid changes, optionally conservative amino acid changes; CDR2 having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to the CDR5 sequence from the same row of Table 1A, or having 0, 1, 2 or 3 amino acid changes, optionally conservative amino acid changes; and CDR3 having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to the CDR6 sequence from the same row of Table 1A, or having 0, 1, 2 or 3 amino acid changes, optionally conservative amino acid changes, comprising a second V H H antibody. In some embodiments, the bispecific V H H 2 comprises a sequence that is at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identical to any one of the bispecific VHH dimer sequences shown in Table 1A.

[0114] In some embodiments, the V H H described herein can be humanized to include a human framework region. Examples of human germline cell lines that can be used to make humanized V H H include, but are not limited to, VH3-23 (e.g., UniProt ID: P01764), VH3-74 (e.g., UniProt ID: A0A0B4J1X5), VH3-66 (e.g., UniProt ID: A0A0C4DH42), VH3-30 (e.g., UniProt ID: P01768), VH3-11 (e.g., UniProt ID: P01762) and VH3-9 (e.g., UniProt ID: P01782).

[0115] (Table 1A) TIFF2025108521000009.tif240167TIFF2025108521000010.tif241167TIFF2025108521000011.tif241167TIFF2025108521000012.tif240167TIFF2025108521000013.tif241167TIFF2025108521000014.tif236167TIFF2025108521000015.tif241167TIFF2025108521000016.tif236167TIFF2025108521000017.tif240167TIFF2025108521000018.tif241167TIFF2025108521000019.tif240167TIFF2025108521000020.tif240167TIFF2025108521000021.tif241167TIFF2025108521000022.tif236167TIFF2025108521000023.tif241167TIFF2025108521000024.tif236167TIFF2025108521000025.tif240167TIFF2025108521000026.tif241167TIFF2025108521000027.tif240167TIFF2025108521000028.tif241167TIFF2025108521000029.tif241167TIFF2025108521000030.tif236167TIFF2025108521000031.tif241167TIFF2025108521000032.tif236167TIFF2025108521000033.tif240167TIFF2025108521000034.tif241167TIFF2025108521000035.tif240167TIFF2025108521000036.tif241167TIFF2025108521000037.tif232167TIFF2025108521000038.tif240167TIFF2025108521000039.tif241167TIFF2025108521000040.tif240167TIFF2025108521000041.tif240167TIFF2025108521000042.tif241167TIFF2025108521000043.tif240167TIFF2025108521000044.tif241167TIFF2025108521000045.tif236167TIFF2025108521000046.tif241167TIFF2025108521000047.tif241167TIFF2025108521000048.tif236167TIFF2025108521000049.tif240167TIFF2025108521000050.tif241167TIFF2025108521000051.tif240167TIFF2025108521000052.tif241167TIFF2025108521000053.tif240167TIFF2025108521000054.tif241167TIFF2025108521000055.tif241167TIFF2025108521000056.tif236167TIFF2025108521000057.tif241167TIFF2025108521000058.tif241167TIFF2025108521000059.tif240167TIFF2025108521000060.tif241167TIFF2025108521000061.tif236167TIFF2025108521000062.tif241167TIFF2025108521000063.tif236167TIFF2025108521000064.tif240167TIFF2025108521000065.tif241167TIFF2025108521000066.tif241167TIFF2025108521000067.tif240167TIFF2025108521000068.tif241167TIFF2025108521000069.tif236167TIFF2025108521000070.tif241167TIFF2025108521000071.tif236167TIFF2025108521000072.tif240167TIFF2025108521000073.tif241167TIFF2025108521000074.tif240167TIFF2025108521000075.tif240167TIFF2025108521000076.tif241167TIFF2025108521000077.tif236167TIFF2025108521000078.tif241167TIFF2025108521000079.tif236167TIFF2025108521000080.tif240167TIFF2025108521000081.tif241167TIFF2025108521000082.tif240167TIFF2025108521000083.tif241167TIFF2025108521000084.tif241167TIFF2025108521000085.tif236167TIFF2025108521000086.tif241167TIFF2025108521000087.tif236167TIFF2025108521000088.tif240167TIFF2025108521000089.tif241167TIFF2025108521000090.tif240167TIFF2025108521000091.tif241167TIFF2025108521000092.tif232167TIFF2025108521000093.tif240167TIFF2025108521000094.tif241167TIFF2025108521000095.tif240167TIFF2025108521000096.tif240167TIFF2025108521000097.tif241167TIFF2025108521000098.tif240167TIFF2025108521000099.tif241167TIFF2025108521000100.tif236167TIFF2025108521000101.tif241167TIFF2025108521000102.tif241167TIFF2025108521000103.tif236167TIFF2025108521000104.tif240167TIFF2025108521000105.tif241167TIFF2025108521000106.tif240167TIFF2025108521000107.tif241167TIFF2025108521000108.tif240167TIFF2025108521000109.tif241167TIFF2025108521000110.tif241167TIFF2025108521000111.tif236167TIFF2025108521000112.tif241167TIFF2025108521000113.tif241167TIFF2025108521000114.tif240167TIFF2025108521000115.tif241167TIFF2025108521000116.tif236167TIFF2025108521000117.tif241167TIFF2025108521000118.tif236167TIFF2025108521000119.tif240167TIFF2025108521000120.tif241167TIFF2025108521000121.tif241167TIFF2025108521000122.tif240167TIFF2025108521000123.tif241167TIFF2025108521000124.tif236167TIFF2025108521000125.tif241167TIFF2025108521000126.tif236167TIFF2025108521000127.tif240167TIFF2025108521000128.tif241167TIFF2025108521000129.tif240167TIFF2025108521000130.tif240167TIFF2025108521000131.tif241167TIFF2025108521000132.tif236167TIFF2025108521000133.tif241167TIFF2025108521000134.tif236167TIFF2025108521000135.tif240167TIFF2025108521000136.tif241167TIFF2025108521000137.tif240167TIFF2025108521000138.tif241167TIFF2025108521000139.tif241167TIFF2025108521000140.tif236167TIFF2025108521000141.tif241167TIFF2025108521000142.tif236167TIFF2025108521000143.tif240167TIFF2025108521000144.tif241167TIFF2025108521000145.tif240167TIFF2025108521000146.tif241167TIFF2025108521000147.tif232167TIFF2025108521000148.tif240167TIFF2025108521000149.tif241167TIFF2025108521000150.tif240167TIFF2025108521000151.tif240167TIFF2025108521000152.tif241167TIFF2025108521000153.tif240167TIFF2025108521000154.tif241167TIFF2025108521000155.tif236167TIFF2025108521000156.tif241167TIFF2025108521000157.tif241167TIFF2025108521000158.tif236167TIFF2025108521000159.tif240167TIFF2025108521000160.tif241167TIFF2025108521000161.tif240167TIFF2025108521000162.tif241167TIFF2025108521000163.tif240167TIFF2025108521000164.tif241167TIFF2025108521000165.tif241167TIFF2025108521000166.tif236167TIFF2025108521000167.tif241167TIFF2025108521000168.tif241167TIFF2025108521000169.tif240167TIFF2025108521000170.tif241167TIFF2025108521000171.tif236167TIFF2025108521000172.tif241167TIFF2025108521000173.tif236167TIFF2025108521000174.tif240167TIFF2025108521000175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[0116] In some embodiments, the IL27R binding proteins described herein (e.g., in Table 1A) are encoded by an isolated nucleic acid that is substantially identical to any one of the sequences in Table 1B below. In some embodiments, the IL27R binding proteins described herein (e.g., an IL27R binding protein comprising the sequence of Table 1A) are encoded by an isolated nucleic acid comprising a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to the sequences in Table 1B below.

[0117] (Table 1B) TIFF2025108521000229.tif186158TIFF2025108521000230.tif237158TIFF2025108521000231.tif236158TIFF2025108521000232.tif236158TIFF2025108521000233.tif237158TIFF2025108521000234.tif236158TIFF2025108521000235.tif237158TIFF2025108521000236.tif232158TIFF2025108521000237.tif236158TIFF2025108521000238.tif237158TIFF2025108521000239.tif236158TIFF2025108521000240.tif237158TIFF2025108521000241.tif236158TIFF2025108521000242.tif236158TIFF2025108521000243.tif237158TIFF2025108521000244.tif237158TIFF2025108521000245.tif232158TIFF2025108521000246.tif237158TIFF2025108521000247.tif237158TIFF2025108521000248.tif236158TIFF2025108521000249.tif237158TIFF2025108521000250.tif237158TIFF2025108521000251.tif236158TIFF2025108521000252.tif237158TIFF2025108521000253.tif237158TIFF2025108521000254.tif236158TIFF2025108521000255.tif237158TIFF2025108521000256.tif237158TIFF2025108521000257.tif236158TIFF2025108521000258.tif237158TIFF2025108521000259.tif232158TIFF2025108521000260.tif236158TIFF2025108521000261.tif237158TIFF2025108521000262.tif232158TIFF2025108521000263.tif237158TIFF2025108521000264.tif237158TIFF2025108521000265.tif236158TIFF2025108521000266.tif237158TIFF2025108521000267.tif236158TIFF2025108521000268.tif236158TIFF2025108521000269.tif237158TIFF2025108521000270.tif237158TIFF2025108521000271.tif237158TIFF2025108521000272.tif236158TIFF2025108521000273.tif236158TIFF2025108521000274.tif237158TIFF2025108521000275.tif237158TIFF2025108521000276.tif237158TIFF2025108521000277.tif232158TIFF2025108521000278.tif237158TIFF2025108521000279.tif237158TIFF2025108521000280.tif237158TIFF2025108521000281.tif232158TIFF2025108521000282.tif236158TIFF2025108521000283.tif237158TIFF2025108521000284.tif237158TIFF2025108521000285.tif237158TIFF2025108521000286.tif237158TIFF2025108521000287.tif236158TIFF2025108521000288.tif237158TIFF2025108521000289.tif237158TIFF2025108521000290.tif237158TIFF2025108521000291.tif237158TIFF2025108521000292.tif232158TIFF2025108521000293.tif237158TIFF2025108521000294.tif237158TIFF2025108521000295.tif237158TIFF2025108521000296.tif236158TIFF2025108521000297.tif237158TIFF2025108521000298.tif237158TIFF2025108521000299.tif236158TIFF2025108521000300.tif237158TIFF2025108521000301.tif237158TIFF2025108521000302.tif236158TIFF2025108521000303.tif237158TIFF2025108521000304.tif237158TIFF2025108521000305.tif236158TIFF2025108521000306.tif237158TIFF2025108521000307.tif237158TIFF2025108521000308.tif236158TIFF2025108521000309.tif237158TIFF2025108521000310.tif237158TIFF2025108521000311.tif236158TIFF2025108521000312.tif236158TIFF2025108521000313.tif237158TIFF2025108521000314.tif237158TIFF2025108521000315.tif232158TIFF2025108521000316.tif236158TIFF2025108521000317.tif237158TIFF2025108521000318.tif232158TIFF2025108521000319.tif236158TIFF2025108521000320.tif237158TIFF2025108521000321.tif237158TIFF2025108521000322.tif232158TIFF2025108521000323.tif236158TIFF2025108521000324.tif237158TIFF2025108521000325.tif237158TIFF2025108521000326.tif236158TIFF2025108521000327.tif236158TIFF2025108521000328.tif237158TIFF2025108521000329.tif237158TIFF2025108521000330.tif236158TIFF2025108521000331.tif237158TIFF2025108521000332.tif237158TIFF2025108521000333.tif236158TIFF2025108521000334.tif236158TIFF2025108521000335.tif237158TIFF2025108521000336.tif232158TIFF2025108521000337.tif237158TIFF2025108521000338.tif237158TIFF2025108521000339.tif236158TIFF2025108521000340.tif237158TIFF2025108521000341.tif237158TIFF2025108521000342.tif236158TIFF2025108521000343.tif237158TIFF2025108521000344.tif236158TIFF2025108521000345.tif236158TIFF2025108521000346.tif237158TIFF2025108521000347.tif232158TIFF2025108521000348.tif236158TIFF2025108521000349.tif237158TIFF2025108521000350.tif237158TIFF2025108521000351.tif232158TIFF2025108521000352.tif236158TIFF2025108521000353.tif237158TIFF2025108521000354.tif237158TIFF2025108521000355.tif232158TIFF2025108521000356.tif237158TIFF2025108521000357.tif237158TIFF2025108521000358.tif236158TIFF2025108521000359.tif237158TIFF2025108521000360.tif237158TIFF2025108521000361.tif236158TIFF2025108521000362.tif236158TIFF2025108521000363.tif237158TIFF2025108521000364.tif237158TIFF2025108521000365.tif236158TIFF2025108521000366.tif237158TIFF2025108521000367.tif237158TIFF2025108521000368.tif236158TIFF2025108521000369.tif237158TIFF2025108521000370.tif237158TIFF2025108521000371.tif236158TIFF2025108521000372.tif236158TIFF2025108521000373.tif237158TIFF2025108521000374.tif237158TIFF2025108521000375.tif236158TIFF2025108521000376.tif237158TIFF2025108521000377.tif237158TIFF2025108521000378.tif236158TIFF2025108521000379.tif237158TIFF2025108521000380.tif232158TIFF2025108521000381.tif236158TIFF2025108521000382.tif237158TIFF2025108521000383.tif236158TIFF2025108521000384.tif236158TIFF2025108521000385.tif22158.

[0118] In some embodiments, the bispecific V H H 2 comprises the following: An anti-gp130 VH antibody comprising a CDR1 having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to any one of the sequences of SEQ ID NOs: 193 - 198, or having 0, 1, 2 or 3 amino acid changes, optionally conservative amino acid changes; a CDR2 having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to any one of the sequences of SEQ ID NOs: 199 - 204, or having 0, 1, 2 or 3 amino acid changes, optionally conservative amino acid changes; and a CDR3 having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to any one of the sequences of SEQ ID NOs: 205 - 210, or having 0, 1, 2 or 3 amino acid changes, optionally conservative amino acid changes; H and an anti-IL27Rα VH antibody comprising a CDR1 having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to any one of the sequences of SEQ ID NOs: 211 - 217, or having 0, 1, 2 or 3 amino acid changes, optionally conservative amino acid changes; a CDR2 having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to any one of the sequences of SEQ ID NOs: 218 - 224, or having 0, 1, 2 or 3 amino acid changes, optionally conservative amino acid changes; and a CDR3 having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to any one of the sequences of SEQ ID NOs: 225 - 231, or having 0, 1, 2 or 3 amino acid changes, optionally conservative amino acid changes. H H antibody.

[0119] In certain embodiments, the bispecific V H H 2 is an anti-gp130 V antibody comprising CDR1, CDR2 and CDR3 as described in each row of Table 1 below. H H antibody and anti-IL27Rα V antibody containing CDR1, CDR2 and CDR3 H In some embodiments, the anti-gp130 V H antibody H CDR1, CDR2 and CDR3 in H antibody and anti-IL27Rα V H Each of the CDR1, CDR2 and CDR3 in the H antibody independently comprises at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to the sequence set forth in each row of Table 1, or may have 0, 1, 2 or 3 amino acid changes, optionally conservative amino acid changes.

[0120] In some embodiments, the bispecific V H H 2 , anti-gp130 V H H antibody and C-terminal anti-IL27Rα V H In some embodiments, the bispecific VH antibody is H H 2 IL27Rα V H H antibody and C-terminal anti-gp130 V H Contains H antibodies.

[0121] (Table 1) TIFF2025108521000386.tif218163TIFF2025108521000387.tif218163TIFF2025108521000388.tif78163

[0122] Anti-GP130-Linker-Anti-Il27Rα V H H Bispecific V H H 2 From the N-terminus to the C-terminus, the first V H H (anti-gp130 VH an H antibody), a linker, and a second VH that binds to IL27Rα (an anti-IL27Rα VH H antibody). In other words, the linker connects the C-terminus of the anti-gp130 VH in the binding protein to the N-terminus of the anti-IL27Rα VH in the binding protein. In some embodiments, a purified peptide, such as a 6-histidine peptide ((His)6 (SEQ ID NO: 1531) or His-tag), may or may not be included in the bispecific VH H In certain embodiments, the bispecific VH H described herein comprises an anti-gp130 VH antibody having a sequence with at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to any one of the sequences of SEQ ID NOs: 232 - 237; and an anti-IL27Rα VH antibody having a sequence with at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to any one of the sequences of SEQ ID NOs: 238 - 244 H In certain embodiments, the bispecific VH H described herein comprises an anti-gp130 VH antibody and an anti-IL27Rα VH antibody described in each row of Table 2A below or Table 1A above. In some embodiments, in each row of Table 2A, the anti-gp130 VH antibody and the anti-IL27Rα VH antibody 2 can be included or not included.

[0123] In certain embodiments, the bispecific VH H described herein 2 comprises an anti-gp130 VH antibody having a sequence with at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to any one of the sequences of SEQ ID NOs: 232 - 237; and an anti-IL27Rα VH antibody having a sequence with at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to any one of the sequences of SEQ ID NOs: 238 - 244 H In certain embodiments, the bispecific VH H described herein comprises an anti-gp130 VH antibody and an anti-IL27Rα VH antibody described in each row of Table 2A below or Table 1A above. In some embodiments, in each row of Table 2A, the anti-gp130 VH antibody and the anti-IL27Rα VH antibody

[0124] In certain embodiments, the bispecific VH H described herein 2 comprises an anti-gp130 VH antibody and an anti-IL27Rα VH antibody described in each row of Table 2A below or Table 1A above. In some embodiments, in each row of Table 2A, the anti-gp130 VH antibody and the anti-IL27Rα VH antibody H In certain embodiments, the bispecific VH H described herein H comprises an anti-gp130 VH antibody and an anti-IL27Rα VH antibody described in each row of Table 2A below or Table 1A above. In some embodiments, in each row of Table 2A, the anti-gp130 VH antibody and the anti-IL27Rα VH antibody HEach H antibody can independently include at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity with the sequences described in each row of Table 2A. In some embodiments, bispecific V H H 2 can include a linker (e.g., the linker described in Section IV) between the anti-gp130 V H H antibody and the anti-IL27Rα V H H antibody described in each row of Table 2A below. In certain embodiments, the linker is GGGS (SEQ ID NO:108) or (GGGS)n (SEQ ID NO:1532), (GGS)nG (SEQ ID NO:1533), (GGGGS)n (SEQ ID NO:1534) as described elsewhere herein. The sequence of the anti-gp130 V H H is N-terminal to the linker, and the sequence of the anti-IL27Rα V H H is C-terminal to the linker. Examples of linkers are further described in Section IV below. The CDR sequences of each V H H are underlined.

[0125] (Table 2A) TIFF2025108521000389.tif180150TIFF2025108521000390.tif225150TIFF2025108521000391.tif220150TIFF2025108521000392.tif249150

[0126] In certain embodiments, bispecific V H H 2 includes a sequence that is substantially identical to any one of the sequences of SEQ ID NOs: 1-42. Such bispecific V H H 2can have a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity with any one of the sequences of SEQ ID NOs: 1-42, as shown in Table 2B below. In each of the sequences of SEQ ID NOs: 1-42, the linker GGGS (SEQ ID NO: 108) is in bold. The sequence of anti-gp130 V H H is N-terminal to the linker, and the sequence of anti-IL27Rα V H H is C-terminal to the linker. Each V H H's CDR sequences are underlined.

[0127] (Table 2B) TIFF2025108521000393.tif107150TIFF2025108521000394.tif224150TIFF2025108521000395.tif224150TIFF2025108521000396.tif224150TIFF2025108521000397.tif135150

[0128] In some embodiments, the IL27R-binding proteins described herein (e.g., IL27R-binding proteins comprising any one of the sequences of SEQ ID NOs: 1-42) are encoded by an isolated nucleic acid that is substantially identical to any one of the sequences of SEQ ID NOs: 109-150, as described in Table 2C below. In some embodiments, the IL27R-binding proteins described herein (e.g., IL27R-binding proteins comprising any one of the sequences of SEQ ID NOs: 1-42) are encoded by an isolated nucleic acid comprising a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity with any one of the sequences of SEQ ID NOs: 109-150, as described in Table 2C below.

[0129] (Table 2C) TIFF2025108521000398.tif244150TIFF2025108521000399.tif232150TIFF2025108521000400.tif232150TIFF2025108521000401.tif232150TIFF2025108521000402.tif228150TIFF2025108521000403.tif224150TIFF2025108521000404.tif232150TIFF2025108521000405.tif232150TIFF2025108521000406.tif232150TIFF2025108521000407.tif232150

[0130] Anti-IL27Rα V H H-Linker-Anti-GP130 V H H Bispecific V H H 2 In the direction from the N-terminus to the C-terminus, a first V that binds to IL27Rα H H (Anti-IL27Rα V H H antibody), a linker, and a second V that binds to gp130 H H (Anti-gp130 V H H antibody) can be included. In other words, the linker connects the C-terminus of the Anti-IL27Rα V H H in the binding protein to the N-terminus of the Anti-gp130 V H H in the binding protein. In some embodiments, a purified peptide, such as a 6-histidine peptide ((His)6 (SEQ ID NO:1531) or His-tag) or an Fc tag can be included in the bispecific V H H 2 In certain embodiments, the bispecific V described herein

[0131] In certain embodiments, the bispecific V described herein H H 2comprises an anti-IL27Rα V H H antibody having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity with any one of the sequences of SEQ ID NOs: 245 - 251; and an anti-gp130 V H H antibody having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity with any one of the sequences of SEQ ID NOs: 252 - 257.

[0132] In certain embodiments, the bispecific V H H 2 comprises the anti-IL27Rα V H H antibody and the anti-gp130 V H H antibody described in each row of Table 3A below or Table A above. In some embodiments, in each row of Table 3A, the anti-IL27Rα V H H antibody and the anti-gp130 V H H antibody can each independently comprise at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity with the sequences described in each row of Table 3A. In some embodiments, the bispecific V H H 2 can comprise a linker (e.g., the linker described in Section IV) between the anti-IL27Rα V H H antibody and the anti-gp130 V H H antibody. In certain embodiments, the linker is GGGS (SEQ ID NO: 108). The sequence of the anti-IL27Rα V H H is N-terminal to the linker and the sequence of the anti-gp130 V H H is C-terminal to the linker. Examples of linkers are further described in Section IV below. The CDR sequences of each V H H are underlined.

[0133] (Table 3A) TIFF2025108521000408.tif26150TIFF2025108521000409.tif220150TIFF2025108521000410.tif225150TIFF2025108521000411.tif220150TIFF2025108521000412.tif188150

[0134] In certain embodiments, the bispecific V H H 2 comprises a sequence that is substantially identical to any one of the sequences of SEQ ID NOs: 43 - 84. Such bispecific V H H 2 can have a sequence identity of at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) with any one of the sequences of SEQ ID NOs: 43 - 84, as shown in Table 3B below. In each of the sequences of SEQ ID NOs: 43 - 84, the linker GGGS (SEQ ID NO: 108) is in bold. The sequence of the anti - IL27Rα V H H is N - terminal to the linker, and the sequence of the anti - gp130 V H H is C - terminal to the linker. The CDR sequences of each V H H are underlined.

[0135] (Table 3B) TIFF2025108521000413.tif188150TIFF2025108521000414.tif228150TIFF2025108521000415.tif232150TIFF2025108521000416.tif212150

[0136] In some embodiments, the IL27R binding proteins described herein (e.g., an IL27R binding protein comprising any one of the sequences of SEQ ID NO: 43-84) are encoded by an isolated nucleic acid that is substantially identical to any one of the sequences of SEQ ID NO: 151-192, as set forth in Table 3C below. In some embodiments, the IL27R binding proteins described herein (e.g., an IL27R binding protein comprising any one of the sequences of SEQ ID NO: 43-84) are encoded by an isolated nucleic acid comprising a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to any one of the sequences of SEQ ID NO: 151-192, as set forth in Table 3C below.

[0137] (Table 3C) TIFF2025108521000417.tif177150TIFF2025108521000418.tif232150TIFF2025108521000419.tif232150TIFF2025108521000420.tif232150TIFF2025108521000421.tif224150TIFF2025108521000422.tif232150TIFF2025108521000423.tif232150TIFF2025108521000424.tif232150TIFF2025108521000425.tif232150TIFF2025108521000426.tif232150TIFF2025108521000427.tif177150

[0138] Additional IL27R binding proteins Furthermore, additional IL27R binding proteins are the three CDR sequences underlined in the anti-IL27Rα V H H antibody described in Table 4 below, and any one of the anti-gp130 V of SEQ ID NO: 232-237 HThe H antibody can contain three CDR sequences underlined. Further IL27R-binding proteins can be anti-IL27Rα V H H antibodies (e.g., any one of the sequences described in Table 4 below) and anti-gp130 V H H antibodies (e.g., any one of SEQ ID NOs: 232 - 237). In some embodiments, the binding protein can be an anti-IL27Rα V H H antibody at the N-terminus and an anti-gp130 V H H antibody at the C-terminus. In some embodiments, the binding protein can be an anti-gp130 V H H antibody at the N-terminus and an anti-IL27Rα V H H antibody at the C-terminus. In some embodiments, the binding protein can be an anti-IL27Rα V H H antibody and an anti-gp130 V H H antibody, and can contain a linker (e.g., any one of SEQ ID NOs: 85 - 108 (e.g., SEQ ID NO: 108)) between them. In certain embodiments, the binding protein contains a purification tag, such as a 6-histidine peptide (His)6 (SEQ ID NO: 1531) (His-tag).

[0139] (Table 4) TIFF2025108521000428.tif158150TIFF2025108521000429.tif64150

[0140] The IL27R-binding protein can contain three CDR sequences underlined in the anti-IL27Rα V H H antibody and three CDR sequences underlined in the anti-gp130 V H H antibody. In some embodiments, the IL27R-binding protein can be an anti-IL27Rα V H H antibody and an anti-gp130 V H H antibody as described in each row of Table 5 below. In some embodiments, the binding protein can be an anti-IL27Rα V H H antibody at the N-terminus and an anti-gp130 VH It contains an anti-H antibody. In some embodiments, the binding protein has an anti-gp130 V H anti-H antibody at the N-terminus and an anti-IL27Rα V H anti-H antibody at the C-terminus. In some embodiments, the binding protein has an anti-IL27Rα V H anti-H antibody and an anti-gp130 V H anti-H antibody, and contains a linker (e.g., any one of SEQ ID NOs: 85 to 108 (e.g., SEQ ID NO: 108)) between the anti-IL27Rα V

[0141] (Table 5) TIFF2025108521000430.tif60150TIFF2025108521000431.tif223150TIFF2025108521000432.tif223150TIFF2025108521000433.tif21150

[0142] Furthermore, the IL27R binding protein can contain a mouse anti-IL27Rα V H anti-H antibody and a mouse anti-gp130 V H anti-H antibody. In some instances, due to sequence or structural similarity between extracellular domains of receptors from various mammalian species, immunization with an antigen derived from the IL27Rα or gp130 of a first mammalian species can provide antibodies that specifically bind to receptors of one or more additional mammalian species. Such antibodies are termed "cross-reactive". For example, immunizing camels with a human-derived antigen (e.g., hIL27Rα-ECD) can generate antibodies that cross-react with mouse and human receptors. Assessment of cross-reactivity of antibodies against receptors from other mammalian species can be readily determined by one of ordinary skill in the art using methods for assessment of binding affinity and / or specific binding described elsewhere herein, such as flow cytometry or SPR. As a result, the use of the terms "human IL27Rα VHH" or "hIL27Rα VHH" is understood to simply indicate that the species of the IL27Rα antigen used for immunization of the camel from which the VHH is derived is human IL27Rα, and is not to be construed as limiting with respect to the specific binding affinity of the VHH for IL27Rα molecules of other mammalian species. Similarly, the use of the terms "mouse IL27Rα VHH" or "mIL27Rα VHH" is understood to simply indicate that the species of the IL27Rα antigen used for immunization of the camel from which the VHH is derived is mouse IL27Rα, and is not to be construed as limiting with respect to the specific binding affinity of the VHH for IL27Rα molecules of other mammalian species. In some embodiments, the IL27R binding protein has the mouse anti-IL27Rα V H anti-H antibody described below, the three CDR sequences underlined in the anti-H antibody sequence, and the mouse anti-gp130 V H anti-H antibody described below, the three CDR sequences underlined in the anti-H antibody sequence. In some embodiments, the IL27R binding protein has the mouse anti-IL27Rα V H anti-H antibody described below and the mouse anti-gp130 V H anti-H antibody described below. In some embodiments, the binding protein has a mouse anti-IL27Rα V H anti-H antibody at the N-terminus and a mouse anti-gp130 V HIt contains an H antibody. In some embodiments, the binding protein has a murine anti-gp130 V at the N-terminus H H antibody and a murine anti-IL27Rα V at the C-terminus H H antibody. In some embodiments, the binding protein is a murine anti-IL27Rα V H H antibody and a murine anti-gp130 V H H antibody and contains a linker (e.g., any one of SEQ ID NOs: 85 to 108 (e.g., SEQ ID NO: 108)) therebetween. In certain embodiments, the binding protein contains a purification tag, such as a 6-histidine peptide (His)6 (SEQ ID NO: 1531) (His-tag).

[0143] Murine anti-IL27Rα V H Examples of H antibody sequences: TIFF2025108521000434.tif188150TIFF2025108521000435.tif68150

[0144] Murine anti-gp130 V H Examples of H antibody sequences: TIFF2025108521000436.tif146150TIFF2025108521000437.tif224150TIFF2025108521000438.tif228150TIFF2025108521000439.tif220150TIFF2025108521000440.tif68150

[0145] In some embodiments, the V H H described herein can be humanized to contain a human framework region. Humanized V HExamples of human germ cell lines that can be used to generate H include, but are not limited to, VH3-23 (e.g., UniProt ID: P01764), VH3-74 (e.g., UniProt ID: A0A0B4J1X5), VH3-66 (e.g., UniProt ID: A0A0C4DH42), VH3-30 (e.g., UniProt ID: P01768), VH3-11 (e.g., UniProt ID: P01762), and VH3-9 (e.g., UniProt ID: P01782).

[0146] In some embodiments, the IL27R binding protein has an E max that is reduced compared to max E max reflects the maximum response level in the cell type that can be obtained by a ligand (e.g., a binding molecule or a natural cytokine (e.g., IL27) described herein). In some embodiments, the IL27R binding protein described herein has an E max of at least 1% (e.g., 1% - 100%, 10% - 100%, 20% - 100%, 30% - 100%, 40% - 100%, 50% - 100%, 60% - 100%, 70% - 100%, 80% - 100%, 90% - 100%, 1% - 90%, 1% - 80%, 1% - 70%, 1% - 60%, 1% - 50%, 1% - 40%, 1% - 30%, 1% - 20%, or 1% - 10%) of the E max induced by IL27. In other embodiments, the E max of the IL27R binding protein described herein is greater than (e.g., at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% greater than) the E max of the native ligand IL27. In some embodiments, by varying the linker length of the IL27R binding protein, the E max of the IL27R binding protein can be varied. The IL27R binding protein provides an Emax This can result in...

[0147] IV. Linkers As described above, the binding domains of the binding proteins of the present disclosure may be contiguously linked (e.g., the first V H The C-terminal amino acid of H binds the second V in the protein. H The binding domains of the binding protein may be optionally joined by a linker (eg, to the N-terminal amino acid of H). A linker is a link between two elements, e.g., protein domains. The bispecific V H H 2 In the binding protein, the linker is a linker between two V H H. The linker can be a covalent bond or a peptide linker. In some embodiments, the linker between two V H H is directly (i.e., via a covalent bond) linked to two V in the binding protein. H The length of the linker between the H and the two V of the binding protein H The linker can be used to adjust the proximity of H. By varying the length of the linker, the overall size and length of the binding protein can be tailored to bind to a specific cellular receptor or its domain or subunit. For example, if the binding protein is designed to bind to two receptors or their domains or subunits located near each other on the same cell, a short linker can be used. In another example, if the binding protein is designed to bind to two receptors or their domains or subunits located on two different cells, a long linker can be used.

[0148] In some embodiments, the linker is a peptide linker. The peptide linker can comprise from 1 to 50 amino acids (e.g., from 2 to 50, from 5 to 50, from 10 to 50, from 15 to 50, from 20 to 50, from 25 to 50, from 30 to 50, from 35 to 50, from 40 to 50, from 45 to 50, from 2 to 45, from 2 to 40, from 2 to 35, from 2 to 30, from 2 to 25, from 2 to 20, from 2 to 15, from 2 to 10, from 2 to 5 amino acids). The linker can be a synthetic polymer, such as a chemical linker like a polyethylene glycol (PEG) polymer.

[0149] In some embodiments, the linker connects the C-terminus of the first VH in the binding protein to the N-terminus of the second VH in the binding protein. In other embodiments, the linker connects the C-terminus of the second VH in the binding protein to the N-terminus of the first VH in the binding protein. H H in the binding protein to the N-terminus of the second VH H H in the binding protein. In other embodiments, the linker H H in the binding protein to the N-terminus of the first VH H H in the binding protein.

[0150] Suitable peptide linkers are known in the art and include, for example, peptide linkers containing mobile amino acid residues such as glycine and serine. In certain embodiments, the peptide linker can comprise a motif, such as a multiple or repeating motif, of GS, GGS, GGGGS (SEQ ID NO:85), GGGGGG (SEQ ID NO:86), GGSG (SEQ ID NO:87) or SGGG (SEQ ID NO:88). In certain embodiments, the peptide linker comprises from 2 to 12 amino acids with a GS motif, such as It can include TIFF2025108521000441.tif11155. In certain other embodiments, the peptide linker can include 3 to 12 amino acids containing the GGS motif, such as GGS, GGSGGS (SEQ ID NO:94), GGSGGSGGS (SEQ ID NO:95), and GGSGGSGGSGGS (SEQ ID NO:96). In yet other embodiments, the peptide linker can include 4 to 20 amino acids containing the GGSG (SEQ ID NO:87) motif, such as It can include TIFF2025108521000442.tif18150. In other embodiments, the peptide linker can include the GGGGS (SEQ ID NO:85) motif, such as GGGGSGGGGS (SEQ ID NO:101) or GGGGSGGGGSGGGGS (SEQ ID NO:102).

[0151] Examples of flexible linkers include glycine polymers (G)n, glycine-alanine polymers, alanine-serine polymers, glycine-serine polymers (e.g., (GmSo)n (SEQ ID NO:1535), (GSGGS)n (SEQ ID NO:1536), (GmSoGm)n (SEQ ID NO:1537), (GmSoGmSoGm)n (SEQ ID NO:1538), (GSGGSm)n (SEQ ID NO:1539), (GSGSmG)n (SEQ ID NO:1540), (GGS)nG (SEQ ID NO:1541) and (GGGSm)n (SEQ ID NO:1542), and combinations thereof, where m, n and o are each independently selected from integers of at least 1 to 20, such as 1 to 18, 216, 3 to 14, 4 to 12, 5 to 10, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10), and other flexible linkers. Glycine and glycine-serine polymers are relatively unstructured and can function as neutral tethers between components. Examples of flexible linkers include, but are not limited to, GGSG (SEQ ID NO:87), GGSGG (SEQ ID NO:103), GSGSG (SEQ ID NO:104), GSGGG (SEQ ID NO:105), GGGSG (SEQ ID NO:106), and GSSSG (SEQ ID NO:107).

[0152] As further examples of flexible linkers, glycine polymers (G)n or glycine-serine polymers (e.g., (GS)n (SEQ ID NO:1543), (GSGGS)n (SEQ ID NO:1544), (GGGS)n (SEQ ID NO:1545) and (GGGGS)n (SEQ ID NO:1546), where n = 1 to 50, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 10 to 20, 20 to 30, 30 to 50) can be mentioned. Exemplary flexible linkers include, but are not limited to, GGGS (SEQ ID NO:108), GGGGS (SEQ ID NO:85), GGSG (SEQ ID NO:87), GGSGG (SEQ ID NO:103), GSGSG (SEQ ID NO:104), GSGGG (SEQ ID NO:105), GGGSG (SEQ ID NO:106) and GSSSG (SEQ ID NO:107).

[0153] V. Modifications for Extending the Duration of Action In Vivo The binding proteins described herein can be modified to provide for an extended lifespan in vivo and / or an extended duration of action in a subject. In some embodiments, the binding protein can be conjugated to a carrier molecule to provide desired pharmacological properties such as an extended half-life. In some embodiments, the binding protein can be covalently attached to the Fc domain of IgG, albumin, or other molecules to extend the half-life, for example, by pegylation, glycosylation, etc., as known in the art.

[0154] In some embodiments, the binding protein is conjugated to a functional domain of an Fc-fusion chimeric polypeptide molecule. Fc fusion conjugates have been shown to increase the systemic half-life of biologics, and thus biologic products can require less frequent dosing. Fc binds to the neonatal Fc receptor (FcRn) in endothelial cells lining blood vessels, and upon binding, the Fc fusion molecule is protected from degradation and re-released into circulation, keeping the molecule in circulation longer. This Fc binding is thought to be the mechanism by which endogenous IgG retains its long plasma half-life. Recent Fc-fusion technologies link a single copy of a biologic to the Fc region of an antibody to optimize the pharmacokinetic and pharmacodynamic properties of the biologic compared to conventional Fc-fusion conjugates. The “Fc region” useful for preparation of Fc fusions can be a native or synthetic polypeptide homologous to the IgG C-terminal domain produced by digestion of IgG with papain. The IgG Fc has a molecular weight of approximately 50 kDa. The binding proteins described herein can be conjugated to the entire Fc region or to a smaller portion that retains the ability to extend the circulating half-life of the chimeric polypeptide of which it is a part. In addition, the full-length or fragmented Fc region can be a variant of the wild-type molecule. In a typical presentation, each monomer of the dimeric Fc can carry a heterologous polypeptide, and the heterologous polypeptides can be the same or different.

[0155] In some embodiments, when the binding proteins described herein are to be administered in the form of an Fc fusion, particularly where the polypeptide chains conjugated to each subunit of the Fc dimer are different, the Fc fusion can be engineered to have a "knob-into-hole modification." The knob-into-hole modification is fully described by Ridgway, et al. (1996) Protein Engineering 9(7):617-621 and U.S. Patent No. 5,731,168, issued March 24, 1998. The knob-into-hole modification refers to a modification at the interface between two immunoglobulin heavy chains in the CH3 domain, where: i) an amino acid residue in the CH3 domain of the first heavy chain is replaced by an amino acid residue with a larger side chain (e.g., tyrosine or tryptophan), creating a protrusion from the surface ("knob"), and ii) an amino acid residue in the CH3 domain of the second heavy chain is replaced by an amino acid residue with a smaller side chain (e.g., alanine or threonine), thereby creating a cavity ("hole") at the interface in the second CH3 domain, and the protruding side chain ("knob") of the first CH3 domain is accommodated by the cavity in the second CH3 domain. In one embodiment, the "knob-into-hole modification" includes the amino acid substitutions T366W and optionally S354C on one of the antibody heavy chains, and the amino acid substitutions T366S, L368A, Y407V and optionally Y349C on the other antibody heavy chain. Further, the Fc domain may be modified by the introduction of cysteine residues at positions S354 and Y349, which results in a stabilizing disulfide bridge between the two antibody heavy chains in the Fc region (Carter, et al. (2001) Immunol Methods 248, 7-15). The knob-into-hole format is used to promote the expression of a first polypeptide on a first Fc monomer having a "knob" modification and a second polypeptide on a second Fc monomer carrying a "hole" modification for the expression of a heterodimeric polypeptide conjugate.

[0156] In some embodiments, the binding protein can be conjugated to one or more water-soluble polymers. Examples of water-soluble polymers useful in the practice of the present disclosure include polyethylene glycol (PEG), polypropylene glycol (PPG), polysaccharides (polyvinylpyrrolidone, copolymers of ethylene glycol and propylene glycol, poly(oxyethylated polyol), polyolefin alcohol), polysaccharides), poly-alpha-hydroxy acids), polyvinyl alcohol (PVA), polyphosphazene, polyoxazoline (POZ), poly(N-acryloylmorpholine), or combinations thereof.

[0157] In some embodiments, the binding protein can be conjugated to, or "PEGylated" with, one or more polyethylene glycol molecules. Although the methods or sites for attaching PEG to the binding protein can vary, in certain embodiments, PEGylation does not change, or minimally changes, the activity of the binding protein.

[0158] In some examples, when utilizing the VHH sequences described herein in the preparation of the IL27 binding molecules of the present disclosure, the VHHs carry an N-terminal glutamine ("1Q") residue. It has been observed that the N-terminal glutamine residue cyclizes spontaneously to form pyroglutamic acid (pE) under or near physiological conditions. (See, e.g., Liu, et al (2011) J. Biol. Chem. 286(13): 11211-11217). In some embodiments, the formation of pyroglutamic acid complicates N-terminal PEG conjugation, particularly when aldehyde chemistry is used for N-terminal PEGylation. As a result, when PEGylating the IL27R binding molecules of the present disclosure, particularly when aldehyde chemistry is utilized, the IL27Rα binding molecule carrying an amino acid (e.g., 1Q) at position 1 is either substituted with another amino acid at position 1 or deleted at position 1 (e.g., des-1Q). In some embodiments, the IL27R binding molecules of the present disclosure include an amino acid substitution selected from the group Q1E and Q1D.

[0159] In some embodiments, for example, selective PEGylation of binding proteins can be utilized by incorporation of unnatural amino acids having side chains that facilitate selective PEG conjugation. The specific PEGylation site can be selected such that PEGylation of the binding protein does not affect its binding to the target receptor.

[0160] In certain embodiments, the increase in half-life is greater than any decrease in biological activity. PEGs suitable for conjugation to polypeptide sequences are generally water-soluble at room temperature and have the general formula R(O-CH2-CH2)nO-R where R is hydrogen or a protecting group such as an alkyl or alkanol group and n is an integer from 1 to 1000. n When R is a protecting group, it generally has from 1 to 8 carbons. The PEG conjugated to the polypeptide sequence can be linear or branched. Branched PEG derivatives, "star PEGs" and multi-arm PEGs are contemplated by the present disclosure.

[0161] The molecular weight of PEG used in the present disclosure is not limited to any particular range. The PEG component of the conjugate protein can have a molecular mass greater than about 5 kDa, greater than about 10 kDa, greater than about 15 kDa, greater than about 20 kDa, greater than about 30 kDa, greater than about 40 kDa, or greater than about 50 kDa. In some embodiments, the molecular mass is from about 5 kDa to about 10 kDa, from about 5 kDa to about 15 kDa, from about 5 kDa to about 20 kDa, from about 10 kDa to about 15 kDa, from about 10 kDa to about 20 kDa, from about 10 kDa to about 25 kDa, or from about 10 kDa to about 30 kDa. The linear or branched PEG molecule has a molecular weight of from about 2,000 to about 80,000 daltons, alternatively from about 2,000 to about 70,000 daltons, alternatively from about 5,000 to about 50,000 daltons, alternatively from about 10,000 to about 50,000 daltons, alternatively from about 20,000 to about 50,000 daltons, alternatively from about 30,000 to about 50,000 daltons, alternatively from about 20,000 to about 40,000 daltons, or alternatively from about 30,000 to about 40,000 daltons. In one embodiment of the present disclosure, the PEG is a 40 kD branched PEG containing two 20 kD arms.

[0162] The present disclosure also contemplates conjugate compositions in which the PEGs have different n values and thus different PEGs are present in certain ratios. For example, some compositions include mixtures of conjugates with n = 1, 2, 3, and 4. In some compositions, the rate of the conjugate with n = 1 is 18 - 25%, the rate of the conjugate with n = 2 is 50 - 66%, the rate of the conjugate with n = 3 is 12 - 16%, and the rate of the conjugate with n = 4 is up to 5%. Such compositions can be produced by reaction conditions and purification methods known in the art. Chromatography may be used to separate conjugate fractions, and then, for example, the fraction containing the conjugate with the desired number of PEGs attached is identified and purified to be free of unmodified protein sequences and conjugates with other numbers of PEGs attached.

[0163] PEGs suitable for conjugation to polypeptide sequences are generally water-soluble at room temperature and have the general formula R(O-CH2-CH2) n O-R, where R is hydrogen or a protecting group such as an alkyl or alkanol group, and n is an integer from 1 to 1000. When R is a protecting group, it generally has 1 to 8 carbons.

[0164] Two widely used first-generation activated monomethoxy PEGs (mPEGs) are succinimidyl carbonate PEG (SC-PEG; see, e.g., Zalipsky, et al. (1992) Biotehnol. Appl. Biochem 15:100-114) and benzotriazole carbonate PEG (BTC-PEG; see, e.g., Dolence et al., U.S. Patent No. 5,650,234), which preferentially react with lysine residues to form carbamate bonds but are also known to react with histidine and tyrosine residues. The use of PEG-aldehyde linkers targets a single site at the N-terminus of the polypeptide through reductive amination.

[0165] PEGylation most often occurs at the α-amino group at the N-terminus of the polypeptide, the ε-amino group on the side chain of lysine residues, and the imidazole group on the side chain of histidine residues. Since most recombinant polypeptides have a single α-amino group as well as several ε-amino groups and imidazole groups, depending on the chemical nature of the linker, a number of positional isomers can be generated. General PEGylation strategies known in the art can be applied herein.

[0166] PEG can be attached to the binding proteins of the present disclosure by a terminal reactive group (“spacer”) that mediates the bond between the free amino or carboxyl group of one or more polypeptide sequences and polyethylene glycol. PEG having a spacer that can be attached to a free amino group includes N-hydroxysuccinimide polyethylene glycol, which can be prepared by activating the succinic acid ester of polyethylene glycol with N-hydroxysuccinimide.

[0167] In some embodiments, PEGylation of the binding protein is facilitated by incorporating non-natural amino acids bearing unique side chains to promote site-specific PEGylation. Incorporating non-natural amino acids into a polypeptide to provide a functional moiety for achieving site-specific PEGylation of such polypeptides is known in the art. See, for example, Ptacin et al., PCT International Application No. PCT / US2018 / 045257, filed August 3, 2018, and published as International Publication No. WO 2019 / 028419A1 on February 7, 2019.

[0168] The PEG conjugated to the polypeptide sequence can be linear or branched. Branched PEG derivatives, "star PEGs" and multi-arm type PEGs are contemplated by the present disclosure. In certain embodiments, the PEG useful in the practice of the present disclosure is 10 kDa linear PEG-aldehyde (e.g., Sunbright® ME-100AL, NOF America Corporation, One North Broadway, White Plains,USA, NY 10601), 10 kDa linear PEG-NHS ester (e.g., Sunbright® ME-100CS, Sunbright® ME-100AS, Sunbright® ME-100GS, Sunbright® ME-100HS, NOF), 20 kDa linear PEG-aldehyde (e.g., Sunbright® ME-200AL, NOF), 20 kDa linear PEG-NHS ester (e.g., Sunbright® ME-200CS, Sunbright® ME-200AS, Sunbright® ME-200GS, Sunbright® ME-200HS, NOF), 20 kDa 2-arm branched PEG-aldehyde containing two 10 kDa linear PEG molecules, 20 kDA PEG-aldehyde (e.g., Sunbright® GL2-200AL3, NOF), 20 kDa 2-arm branched PEG-NHS ester containing two 10 kDA linear PEG molecules, 20 kDA PEG-NHS ester (e.g., Sunbright® GL2-200TS, Sunbright® GL200GS2, NOF), 40 kDa 2-arm branched PEG-aldehyde containing two 20 kDA linear PEG molecules, 40 kDA PEG-aldehyde (e.g., Sunbright® GL2-400AL3), 40 kDa 2-arm branched PEG-NHS ester containing two 20 kDA linear PEG molecules, 40 kDA PEG-NHS ester (e.g., Sunbright® GL2-400AL3, Sunbright® GL2-400GS2, NOF), linear 30 kDa PEG-aldehyde (e.g., Sunbright® ME-300AL) and linear 30 kDa PEG-NHS ester.,

[0169] In some embodiments, the linker can be used to join the binding protein and the PEG molecule. Suitable linkers generally include a modified polypeptide sequence and a "flexible linker" of sufficient length to allow some movement between the linked components and molecules. The linker molecule is generally about 6 to 50 atoms in length. The linker molecule can be, for example, arylacetylene, an ethylene glycol oligomer containing 2 to 10 monomer units, diamine, diacid, amino acid, or a combination thereof. Suitable linkers can be readily selected and can be of any suitable length, such as 1 amino acid in length (e.g., Gly), 2, 3, 4, 5, 6, 7, 8, 9, 10, 10 - 20, 20 - 30, 30 - 50 or more than 50 amino acids in length. Examples of flexible linkers are described in Section IV. Further, multimers of these linker sequences (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 10 - 20, 20 - 30 or 30 - 50) can be linked together to provide a flexible linker that can be used to conjugate two molecules. Instead of a polypeptide linker, the linker can be a chemical linker, such as a PEG-aldehyde linker. In some embodiments, the binding protein is acetylated at the N-terminus by an enzymatic reaction with N-terminal acetyltransferase and, for example, acetyl CoA. Alternatively, or in addition to N-terminal acetylation, the binding protein can be acetylated at one or more lysine residues by an enzymatic reaction with, for example, lysine acetyltransferase. See, for example, Choudhary et al. (2009) Science 325 (5942):834 - 840.

[0170] In other embodiments, the binding protein can be modified to include an additional polypeptide sequence that functions as an antigenic tag, such as a FLAG sequence. The FLAG sequence is recognized by biotinylated anti-FLAG antibodies as described herein (see, for example, Blanar et al. (1992) Science 256:1014 and LeClair, et al. (1992) PNAS-USA 89:8145). In some embodiments, the binding protein further includes a C-terminal c-myc epitope tag.

[0171] In some embodiments, the binding protein is expressed as a fusion protein with an albumin molecule (e.g., human serum albumin) known in the art to facilitate long-term exposure in vivo.

[0172] In some embodiments, the binding proteins of the disclosure (including fusion proteins of the binding proteins) are expressed as fusion proteins having one or more transition metal chelating polypeptide sequences. Incorporation of such transition metal chelating domains facilitates purification by immobilized metal affinity chromatography (IMAC) as described in Smith et al., U.S. Patent No. 4,569,794, issued February 11, 1986. Examples of transition metal chelating polypeptides useful in the practice of the disclosure are described in Smith et al., supra and Dobeli et al., U.S. Patent No. 5,320,663, issued May 10, 1995, the entire teachings of which are incorporated herein by reference. A particular transition metal chelating polypeptide useful in the practice of the disclosure is a peptide comprising 3 to 6 contiguous histidine residues (SEQ ID NO:1547), such as the 6-histidine peptide (His)6 (SEQ ID NO:1531), which is often referred to in the art as a "His tag".

[0173] The aforementioned fusion protein can be easily produced by recombinant DNA methods known in the art by constructing a recombinant vector that contains, in-frame, a nucleic acid sequence encoding a binding protein, optionally further containing a nucleic acid sequence encoding a linker or spacer polypeptide in-frame with a nucleic acid sequence encoding a fusion partner at either the N-terminal or C-terminal position of the binding protein.

[0174] VI. Pharmaceutical Compositions The binding proteins of the present disclosure can be administered to a subject in a pharmaceutically acceptable dosage form. Preferred formulations depend on the intended method of administration and therapeutic use. The pharmaceutical dosage forms of the binding proteins described herein include physiologically acceptable carriers that are essentially non-toxic and non-therapeutic. Examples of such carriers include ion exchangers, alumina, aluminum stearate, lecithin, serum proteins such as human serum albumin, buffers such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulose-based substances, and PEG. Carriers for topical or gel-based forms of the polypeptide include polysaccharides such as sodium carboxymethylcellulose or methylcellulose, polyvinylpyrrolidone, polyacrylates, polyoxyethylene-polyoxypropylene block polymers, PEG, polymeric amino acids, amino acid copolymers, and lipid aggregates (such as oil droplets or liposomes).

[0175] The pharmaceutical composition may contain a pharmaceutically acceptable, non-toxic carrier, excipient, stabilizer or diluent, which are defined as vehicles commonly used for formulating pharmaceutical compositions for administration to animals or humans. The diluent is selected so as not to affect the biological activity of the combination. Acceptable carriers, excipients or stabilizers are non-toxic to the recipient at the dosages and concentrations employed, and include buffering agents such as phosphates, citrates and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (e.g., octadecyidimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzetonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides, and other saccharides including glucose, mannose or dextrin; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or nonionic surfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG).

[0176] Formulations for in vivo administration are typically sterile. Sterilization of the compositions of the present disclosure can be readily achieved by filtration through sterile filtration membranes.

[0177] Typically, the composition is prepared as an injectable, either as a liquid solution or suspension; solid forms suitable for solution or suspension in a liquid vehicle can also be prepared prior to injection. The preparation can also be emulsified or encapsulated in microparticles such as liposomes or polylactide, polyglycolide or copolymers for enhanced adjuvant effect, as discussed above (Langer, Science 249: 1527, 1990 and Hanes, Advanced Drug Delivery Reviews 28: 97-119, 1997). The agents of the present disclosure can be administered in the form of depot injections or implant preparations formulated in such a way as to allow for sustained or pulsed release of the active ingredient. The pharmaceutical composition is generally sterile and substantially isotonic and is formulated in full compliance with all proper manufacturing standards (GMP) of the US Food and Drug Administration.

[0178] Administration of the binding proteins described herein can be accomplished through any of a variety of approved methods in the art, including, but not limited to, topical, intravascular injection (including intravenous or intra-arterial infusion), intradermal injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, intracranial injection, intratumoral injection, intranodular injection, transdermal, transmucosal, iontophoretic delivery, intralymphatic injection (Senti and Kundig (2009) Current Opinions in Allergy and Clinical Immunology 9(6):537-543), intragastric infusion, intraprostatic injection, intracapsular infusion (e.g., bladder), respiratory inhalers including nebulizers, intraocular injection, intraperitoneal injection, intralesional injection, intraovarian injection, intracerebral infusion or injection, intracerebroventricular injection (ICVI), etc. In some embodiments, administration includes administration of the binding protein itself (e.g., parenterally), as well as administration of a recombinant vector (e.g., viral or non-viral vector) to cause in situ expression of the binding protein in a subject. Alternatively, cells such as cells isolated from a subject can be recombinantly modified to express the binding proteins of the present disclosure.

[0179] The dosage of the pharmaceutical composition depends on factors including the route of administration, the disease being treated, and the physical characteristics of the subject, such as age, weight, and general health. Typically, the amount of the binding protein included within a single dose can be an amount that effectively prevents, delays, or treats the disease without inducing significant toxicity. The pharmaceutical compositions of the present disclosure can include dosages of the binding protein described herein in the range of 0.01 to 500 mg / kg (e.g., 0.01 to 450 mg, 0.01 to 400 mg, 0.01 to 350 mg, 0.01 to 300 mg, 0.01 to 250 mg, 0.01 to 200 mg, 0.01 to 150 mg, 0.01 to 100 mg, 0.01 to 50 mg, 0.01 to 10 mg, 0.01 to 1 mg, 0.1 to 500 mg / kg, 1 to 500 mg / kg, 5 to 500 mg / kg, 10 to 500 mg / kg, 50 to 500 mg / kg, 100 to 500 mg / kg, 150 to 500 mg / kg, 200 to 500 mg / kg, 250 to 500 mg / kg, 300 to 500 mg / kg, 350 to 500 mg / kg, 400 to 500 mg / kg, or 450 to 500 mg / kg) and, in more specific embodiments, from about 1 to about 100 mg / kg (e.g., from about 1 to about 90 mg / kg, from about 1 to about 80 mg / kg, from about 1 to about 70 mg / kg, from about 1 to about 60 mg / kg, from about 1 to about 50 mg / kg, from about 1 to about 40 mg / kg, from about 1 to about 30 mg / kg, from about 1 to about 20 mg / kg, from about 1 to about 10 mg / kg, from about 10 to about 100 mg / kg, from about 20 to about 100 mg / kg, from about 30 to about 100 mg / kg, from about 40 to about 100 mg / kg, from about 50 to about 100 mg / kg, from about 60 to about 100 mg / kg, from about 70 to about 100 mg / kg, from about 80 to about 100 mg / kg, or from about 90 to about 100 mg / kg).In some embodiments, the pharmaceutical composition of the present disclosure may comprise an amount of the binding protein described herein in the range of 0.01 - 20 mg / kg (e.g., 0.01 - 15 mg / kg, 0.01 - 10 mg / kg, 0.01 - 8 mg / kg, 0.01 - 6 mg / kg, 0.01 - 4 mg / kg, 0.01 - 2 mg / kg, 0.01 - 1 mg / kg, 0.01 - 0.1 mg / kg, 0.01 - 0.05 mg / kg, 0.05 - 20 mg / kg, 0.1 - 20 mg / kg, 1 - 20 mg / kg, 2 - 20 mg / kg, 4 - 20 mg / kg, 6 - 20 mg / kg, 8 - 20 mg / kg, 10 - 20 mg / kg, 15 - 20 mg / kg). The dosage can be adjusted by a physician according to conventional factors such as the degree of the disease and different parameters of the subject.

[0180] The pharmaceutical composition containing the binding protein described herein can be administered to a subject in need thereof, for example, once or multiple times (e.g., 1 - 10 times or more) daily, weekly, monthly, twice a year, annually, or as medically necessary. The dosage can be provided in either a single - dose or a multiple - dose regimen. The timing between administrations may decrease as the medical condition improves or increase as the patient's health status deteriorates. The treatment course may be a single - dose administration or a multiple - dose administration over a certain period. In some embodiments, a single - dose is used. In some embodiments, two or more divided doses are administered over a period of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 21, 28, 30, 60, 90, 120, or 180 days. Each dose administered in such a divided - dosing protocol may be the same or different for each administration. A multiple - day dosing protocol over a certain period can be provided by one of ordinary skill in the art (e.g., a physician) by monitoring the administration, taking into account the subject's response to the treatment, including side effects of the treatment and its modulation, as discussed above.

[0181] VII. Indications Immune diseases The present disclosure further provides a method of treating a subject suffering from a disease, disorder or condition by administering a therapeutically effective amount of an IL27R binding protein of the present disclosure (or a nucleic acid encoding an IL27R binding protein, including a recombinant virus encoding the IL27R binding protein). Disorders suitable for treatment with the IL27R binding proteins of the present disclosure (including pharmaceutically acceptable formulations comprising IL27R binding proteins and / or nucleic acid molecules encoding them, including recombinant viruses encoding such IL27R binding proteins) include viral infections (e.g., AIDS, influenza, chronic HCV, chronic hepatitis B, C or D viral hepatitis), Helicobacter pylori infection, HTLV, organ rejection, graft-versus-host disease, autoimmune thyroid disease, multiple sclerosis, allergies, asthma, neurodegenerative diseases including Alzheimer's disease, systemic lupus erythematosus (SLE), autoinflammatory diseases, inflammatory bowel disease (IBD), Crohn's disease, diabetes including type 1 or type 2 diabetes, inflammation, autoimmune diseases, atopic diseases, tumor-associated autoimmune diseases, cartilage inflammation, arthritis, rheumatoid arthritis, juvenile arthritis, juvenile rheumatoid arthritis, polyarticular juvenile rheumatoid arthritis, systemic juvenile rheumatoid arthritis, juvenile ankylosing spondylitis, juvenile enteropathic arthritis, juvenile reactive arthritis, juvenile Reiter's syndrome, SEA syndrome (Seronegativity Enthesopathy Arthropathy Syndrome), juvenile dermatomyositis, juvenile psoriatic arthritis, juvenile scleroderma, juvenile systemic lupus erythematosus, juvenile vasculitis, oligoarticular rheumatoid arthritis, polyarticular rheumatoid arthritis, systemic rheumatoid arthritis, ankylosing spondylitis, enteropathic arthritis, reactive arthritis, Reiter's syndrome, SEA syndrome (Seronegativity, Enthesopathy, Arthropathy Syndrome), including, but not limited to, inflammatory diseases or autoimmune diseases.

[0182] Other examples of proliferative and / or differentiative disorders suitable for treatment with the IL27R binding proteins of the present disclosure (including pharmaceutically acceptable formulations comprising IL27R binding proteins and / or nucleic acid molecules encoding such IL27R binding proteins, including recombinant viruses encoding such IL27R binding proteins) include, but are not limited to, skin disorders. Skin disorders can be associated with abnormal activity of cells or cell populations in the dermis, epidermis or subcutaneous layer, or abnormalities at the dermo-epidermal junction. For example, skin disorders can be associated with abnormal activity of keratinocytes (e.g., hyperproliferative basal keratinocytes and immediately suprabasal keratinocytes), melanocytes, Langerhans cells, Merkel cells, immune cells, and other cells found in one or more of the epidermal layers, such as the basal layer (stratum germinativum), spinous layer, granular layer, lucid layer or stratum corneum. In other embodiments, the disorder can be associated with abnormal activity of dermal cells found in the dermis, such as the papillary layer or reticular layer, such as dermal endothelium, fibroblasts, immune cells (e.g., mast cells or macrophages).

[0183] Examples of skin disorders include psoriasis, psoriatic arthritis, dermatitis (eczema), such as exfoliative dermatitis or atopic dermatitis, pityriasis rubra pilaris, pityriasis rosea, parapsoriasis, pityriasis lichenoides, lichen planus, lichen nitidus, ichthyosiform dermatitis, keratoderma, skin diseases, alopecia areata, pyoderma gangrenosum, vitiligo, pemphigoid (e.g., ocular cicatricial pemphigoid or bullous pemphigoid), urticaria, porokeratosis, rheumatoid arthritis with overproliferation and inflammation of epithelial-related cells lining the joint capsule, dermatitis such as seborrheic dermatitis and solar dermatitis, seborrheic keratosis, senile keratosis, actinic keratosis, photoinduced keratosis and follicular keratosis, acne vulgaris, prevention of keloid and keloid formation, nevus, mole, warts including condyloma or verruca acuminata, and human papillomavirus (HPV) infections such as genital warts, leukoderma, lichen planus, and keratitis. The skin disorder can be dermatitis, such as atopic dermatitis or allergic dermatitis, or psoriasis.

[0184] The compositions of the present disclosure (including pharmaceutical formulations comprising an IL27R-binding protein and / or nucleic acid molecules encoding them, including recombinant viruses encoding such IL27R-binding proteins) can also be administered to patients suffering from (or susceptible to) psoriasis or psoriatic disorders. The term "psoriasis" is intended to have its medical meaning, i.e., a disease that mainly affects the skin, causing raised, thickened, scaly, non-wounded lesions. These lesions are usually well-defined erythematous papules covered with overlapping shiny scales. The scales are usually silver or slightly opaque. Nail involvement occurs frequently, resulting in pitting, nail detachment, thickening, and discoloration. Psoriasis may be associated with arthritis and can be disabling. Hyperproliferation of keratinocytes, along with epidermal inflammation and reduced keratinocyte differentiation, is a major feature of psoriatic epidermal hyperplasia. Multiple mechanisms have been proposed to explain the keratinocyte hyperproliferation that characterizes psoriasis. Abnormal cellular immunity is also thought to be involved in the pathogenesis of psoriasis. Examples of psoriatic disorders include chronic plaque psoriasis, vulgaris psoriasis, moderate to severe vulgaris psoriasis, guttate psoriasis, eruptive psoriasis, erythrodermic psoriasis, generalized pustular psoriasis, annular pustular psoriasis, or localized pustular psoriasis.

[0185] Combination of an IL27R-binding protein with additional therapeutic agents for autoimmune diseases: The present disclosure provides for the use of the IL27R-binding proteins of the present disclosure in combination with one or more additional active agents ("adjuvant agents") in the treatment of autoimmune diseases. As used herein, the term "adjuvant agent" includes agents that can be administered or introduced separately, e.g., agents that are separately formulated for a separate administration that can be administered or introduced in combination with an IL27R-binding protein (e.g., can be provided in a kit) and / or therapies.

[0186] As used herein, the term "in combination with" when used in connection with the administration of multiple agents to a subject refers to the administration of a first agent and at least one additional (i.e., second, third, fourth, fifth, etc.) agent to the subject. For the purposes of the present invention, if the biological effect resulting from the administration of the first agent persists in the subject at the time of administration of the second agent, such that the therapeutic effect of the first agent and the therapeutic effect of the second agent overlap, then one agent (e.g., an IL27R binding protein) is considered to be administered in combination with a second agent (e.g., a therapeutic autoimmune antibody such as Humira®). For example, a therapeutic antibody may be administered every two weeks by IV infusion (e.g., adalimumab in the treatment of Crohn's disease), whereas the IL27R binding proteins of the present disclosure can be administered more frequently, e.g., daily, BID, or weekly. However, if the administration of the first agent (e.g., entanercept) provides a therapeutic effect over a long period of time and the administration of the second agent (e.g., an IL27R binding protein) provides its therapeutic effect, and the therapeutic effect of the first agent persists, such that the first agent may have been administered at a time significantly removed (e.g., days or weeks) from the time of administration of the second agent, the second agent is still considered to be administered in combination with the first agent. In one aspect, one agent is considered to be administered in combination with a second agent if the first agent and the second agent are administered simultaneously (within 30 minutes of each other), concurrently, or sequentially. In some aspects, the first agent is considered to be administered "concurrently" with the second agent if the first agent and the second agent are administered within about 24 hours of each other, preferably within about 12 hours of each other, preferably within about 6 hours of each other, preferably within about 2 hours of each other, or preferably within about 30 minutes of each other. It should also be understood that the term "in combination with" applies to situations where the first agent and the second agent are co-formulated as a single pharmaceutically acceptable formulation and the co-formulation is administered to the subject.In certain embodiments, for example, if one agent is administered before one or more other agents, the IL27R binding protein and co-agent are administered or applied sequentially. In other embodiments, for example, when two or more agents are administered simultaneously or approximately simultaneously, the IL27R binding protein and co-agent are administered together; the two or more agents may be present in two or more separate formulations or combined as a single formulation (i.e., a co-formulation). Regardless of whether the agents are administered sequentially or simultaneously, they are considered to be administered in combination for the purposes of this disclosure.

[0187] In some embodiments, the adjuvant agent is one or more agents selected from the group consisting of corticosteroids (including but not limited to prednisone, budesonide, prednisolone), Janus kinase inhibitors (including but not limited to tofacitinib (Xeljanz®)), calcineurin inhibitors (including but not limited to cyclosporine and tacrolimus), mTor inhibitors (including but not limited to sirolimus and everolimus), IMDH inhibitors (including but not limited to azathioprine, leflunomide and mycophenolate), biological agents such as abatacept (Orencia®) or etanercept (Enbrel®), and therapeutic antibodies. Examples of therapeutic antibodies that can be administered as adjuvant agents in combination with the IL27R binding protein of the present disclosure in the treatment of autoimmune diseases include anti-CD25 antibodies (such as daclizumab and basiliximab), anti-VLA-4 antibodies (such as natalizumab), anti-CD52 antibodies (such as alemtuzumab), anti-CD20 antibodies (such as rituximab, ocrelizumab), anti-TNF antibodies (such as infliximab and adalimumab), anti-IL6R antibodies (such as tocilizumab), anti-TNFα antibodies (such as adalimumab (Humira®), golimumab and infliximab), anti-integrin-α4β7 antibodies (such as vedolizumab), anti-IL17a antibodies (such as brodalumab or secukinumab), anti-IL4Rα antibodies (such as dupilumab), anti-RANKL antibodies, IL6R antibodies, anti-IL1β antibodies (such as canakinumab), anti-CD11a antibodies (such as efalizumab), anti-CD3 antibodies (such as muramonab), anti-IL5 antibodies (such as mepolizumab, reslizumab), anti-BLyS antibodies (such as belimumab); and anti-IL12 / IL23 antibodies (such as ustekinumab), but are not limited thereto.

[0188] Many therapeutic antibodies have been approved for clinical use against autoimmune diseases. Examples of antibodies approved by the US Food and Drug Administration (FDA) for use in the treatment of autoimmune diseases in subjects suffering from an autoimmune disease, which can be administered as adjuvants in combination with the IL27R binding proteins (and optionally additional adjuvants) of the present disclosure for the treatment of the indicated autoimmune diseases, are provided in Table 4.

[0189] (Table 4) TIFF2025108521000443.tif207159

[0190] Treatment of neoplastic diseases The present disclosure provides a method of using an IL-27R binding molecule (or a nucleic acid encoding an IL-27R binding molecule, including a recombinant vector encoding an IL-27R binding molecule, and eukaryotic and prokaryotic cells modified to express the IL-27R binding molecule) in the treatment of a subject suffering from a neoplastic disease disorder or condition by administration of a therapeutically effective amount of the IL-27R binding molecule.

[0191] Neoplasms suitable for treatment: The compositions and methods of the present disclosure are useful in the treatment of subjects suffering from neoplastic diseases characterized by the presence of a neoplasm, including benign and malignant neoplasms and neoplastic diseases.

[0192] Examples of benign neoplasms suitable for treatment using the compositions and methods of the present disclosure include, but are not limited to, adenomas, fibromas, hemangiomas, and lipomas. Examples of premalignant neoplasms suitable for treatment using the compositions and methods of the present disclosure include, but are not limited to, hyperplasia, dysplasia, metaplasia, and atypia. Examples of malignant neoplasms suitable for treatment using the compositions and methods of the present disclosure include, but are not limited to, cancers (cancers arising from epithelial tissues such as the skin or tissues lining the internal organs), leukemias, lymphomas, and sarcomas typically derived from bone marrow, muscle, blood vessels, or connective tissue. The term neoplasm also includes virus-induced neoplasms such as warts and EBV-induced diseases (i.e., infectious mononucleosis), as well as hyperproliferative vascular diseases including scar formation, intimal smooth muscle cell hyperplasia, restenosis, and vascular occlusion.

[0193] The term "neoplastic disease" includes breast cancer, sarcomas (including, but not limited to, osteosarcoma, angiosarcoma, and fibrosarcoma), leukemias, lymphomas, genitourinary cancers (including, but not limited to, ovarian, urethral, bladder, and prostate cancers), gastrointestinal cancers (including, but not limited to, colon, esophageal, and stomach cancers), lung cancer, myeloma, pancreatic cancer, liver cancer, kidney cancer, endocrine cancers, skin cancers, as well as brain or central nervous system (CNS) and peripheral nervous system tumors including gliomas and neuroblastomas, astrocytomas, myelodysplastic syndromes, malignant or benign, cervical intraepithelial neoplasia, intestinal polyposis, oral leukoplakia, histiocytosis, hypertrophic scars including keloid scars, hemangiomas, hyperplastic arteriolar stenosis, psoriasis, inflammatory arthritis, hyperkeratosis including arthritis, and papulosquamous eruptions including cancers characterized by solid and non-solid tumors including, but not limited to.

[0194] The term neoplastic disease includes cancer. The term "cancer" refers to malignant diseases of epithelial or endocrine tissues, including respiratory cancers, digestive system cancers, genitourinary system cancers, testicular cancer, breast cancer, prostate cancer, endocrine system cancers, and melanoma. The term neoplastic disease includes adenocarcinoma. "Adenocarcinoma" refers to a cancer that originates from glandular tissue or in which the tumor cells form recognizable glandular structures.

[0195] As used herein, the term "hematopoietic neoplastic disorder" refers to a neoplastic disease arising from hematopoietic origin, such as from the bone marrow, lymphoid or erythroid lineages, or their progenitor cells, and involving hyperplastic / neoplastic cells.

[0196] Myeloid neoplasms include, but are not limited to, myeloproliferative neoplasms, myeloid and lymphoid disorders with eosinophilia, myeloproliferative / myelodysplastic neoplasms, myelodysplastic syndromes, acute myeloid leukemia and related progenitor cell neoplasms, and acute leukemias of ambiguous lineage. Exemplary myeloid disorders suitable for treatment according to the present disclosure include, but are not limited to, acute promyelocytic leukemia (APML), acute myeloid leukemia (AML), and chronic myeloid leukemia (CML).

[0197] Lymphoid neoplasms include, but are not limited to, precursor lymphoid neoplasms, mature B cell neoplasms, mature T cell neoplasms, Hodgkin lymphoma, and immunodeficiency-related lymphoproliferative disorders. Exemplary lymphoid disorders suitable for treatment according to the present disclosure include acute lymphoblastic leukemia (ALL), including B cell acute lymphoblastic leukemia (ALL) and T cell ALL, chronic lymphocytic leukemia (CLL), prolymphocytic leukemia (PLL), hairy cell leukemia (HLL), and Waldenström macroglobulinemia (WM).

[0198] In some cases, hematopoietic neoplastic disorders are caused by acute leukemias of low differentiation (e.g., erythroleukemia and acute megakaryoblastic leukemia). As used herein, the term "hematopoietic neoplastic disorder" refers to malignant lymphomas including, but not limited to, non-Hodgkin lymphoma and its variants, peripheral T cell lymphoma, adult T cell leukemia / lymphoma (ATL), cutaneous T cell lymphoma (CTCL), large granular lymphocytic leukemia (LGL), Hodgkin disease, and Reed-Sternberg disease.

[0199] The determination of whether a subject "suffers from a neoplastic disease" is based on available information accepted in the art for identifying diseases, disorders, or conditions, including but not limited to X-rays, CT scans, conventional clinical diagnostic tests (such as blood cell counts, etc.), genomic data, protein expression data, immunohistochemistry, etc., and is made by a physician with respect to the subject, a determination that the subject will require treatment or will benefit from treatment.

[0200] Evaluation of anti-neoplastic efficacy: The determination of the effectiveness of the methods of the present disclosure in the treatment of cancer is generally related to the achievement of one or more parameters approved in the art, such as reduction of lesions, particularly metastatic lesions, reduction of metastases, reduction of tumor volume, improvement of the ECOG score, and others. The determination of the response to treatment can be evaluated through the measurement of biomarkers that can provide reproducible information useful in any aspect of IL-27R binding molecule therapy, including the presence and extent of the subject's response to such a treatment method and the presence and extent of adverse effects caused by such a treatment method. By way of example and not limitation, biomarkers include an increase in IFNγ, as well as upregulation of granzyme A, granzyme B, and perforin; an increase in the number and enhanced function of CD8+ T cells; an increase in IFNγ, an increase in ICOS expression on CD8+ T cells, and an increase in IL-10-expressing T Reg cells. Treatment response may be characterized by improvement in conventional measures of clinical efficacy, which may include complete response (CR), partial response (PR), stable disease (SD), etc. with respect to target lesions, as defined by RECIST, and not only complete response (CR), incomplete response / stable disease (SD), but also immune-related complete response (irCR), immune-related partial response (irPR), and immune-related stable disease (irSD) as defined by immune-related response criteria (irRC), which are considered by those skilled in the art to evidence effectiveness in the treatment of neoplastic diseases in mammalian (e.g., human) subjects.

[0201] Maintenance of serum concentration: In some aspects of the invention, the disclosure provides methods and compositions for treating and / or preventing a neoplastic disease, disorder or condition by administration of a therapeutically effective amount of an IL-27R binding molecule, wherein the serum concentration of the IL-27R binding molecule is maintained at a therapeutically effective concentration or above a therapeutically effective concentration for a majority of the period (i.e., longer than about 50% of the period, alternatively longer than about 60% of the period, alternatively longer than about 70% of the period, alternatively longer than about 80% of the period, alternatively longer than about 90% of the period) (e.g., at least 24 hours, alternatively at least 48 hours, alternatively at least 72 hours, alternatively at least 96 hours, alternatively at least 120 hours, alternatively at least 144 hours, alternatively at least 7 days, alternatively at least 10 days, alternatively at least 12 days, alternatively at least 14 days, alternatively at least 28 days, alternatively at least 45 days, alternatively at least 60 days, or longer).

[0202] Combination of an IL-27R binding molecule and a therapeutic adjuvant: The disclosure provides methods for using the IL-27R binding molecules of the disclosure in combination with one or more additional active agents (“adjuvant agents”). Such additional combinations are interchangeably referred to as “adjuvant combinations” or “adjuvant combination therapies,” and the therapeutic agents used in combination with the IL-27R binding molecules of the disclosure are referred to as “adjuvant agents.” As used herein, the term “adjuvant agent” includes agents that can be administered or introduced separately, e.g., agents that are separately formulated for a separate administration that can be administered or introduced in combination with the IL-27R binding molecule (e.g., provided in a kit) and / or therapies.

[0203] Chemotherapeutic agents: In some embodiments, the adjuvant agent is a chemotherapeutic agent. In some embodiments, the adjuvant agent is a “cocktail” of multiple chemotherapeutic agents. In some embodiments, the chemotherapeutic agent or cocktail is administered in combination with one or more physical methods (e.g., radiation therapy). The term “chemotherapeutic agent” includes alkylating agents such as thiotepa and cyclophosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodepa, carbocon, meturedepa and uredepa; ethyleneimines and methylamelamines including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide and trimethylolmelamine; nitrogen mustards such as chlorambucil, chloronaphazine, chlorophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, noburemabicin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosoureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine; antibiotics such as aclacinomycin, actinomycin, anthramycin, azaserine, bleomycin such as bleomycin A2, calicheamicin, carabicin, caminomycin, cardinophilin, chromomycin, dactinomycin, daunorubicin and demethoxy-daunomycin, 11-deoxydaunorubicin, 13-deoxydaunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, esorubicin, idarubicin and other derivatives, marcellomycin, mitomycin C, mitomycin such as N-methylmitomycin C; mycophenolic acid, nogalamycin, olivomycin, peplomycin, potfiromycin, puromycin, quelamycin, rhodomycin, streptozocin, streptonigrin, tubercidin, ubenimex, dinostatin, zorubicin; antimetabolites such as methotrexate and 5-fluorouracil (5-FU);Folic acid analogs such as denopterin, methotrexate, pteropterin, trimetrexate, dideazatetrahydrofolic acid, and folinic acid; purine analogs such as fludarabine, 6-mercaptopurine, thiampurine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, didoxyridine, doxifluridine, enocitabine, floxuridine, 5-FU; androgens such as calusterone, drostanolone propionate, epithiostanol, mepitiostane, testolactone; anti-adrenal agents such as aminoglutethimide, mitotane, trilostane; folic acid supplements such as folinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; amsacrine; bestrabucil; bisantrene; edatrexate; defofamine; dexamethasone; diaziquone; eflornithine; elliptinium acetate; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidamine; mitoguazone; mitoxantrone; mopidamol; nitracrine; pentostatin; phenamet; pirarubicin; podophyllinic acid; 2-ethylhydrazide; procarbazine; razoxane; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2"-trichloroethylamine; urethane; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside (Ara-C); cyclophosphamide; thiotepa; taxoids such as paclitaxel, nab-paclitaxel, and docetaxel; chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum and platinum coordination compounds such as cisplatin, oxaplatin, and carboplatin; vinblastine; etoposide (VP-16); ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; navelbine; novantrone; teniposide; daunomycin; aminopterin; zeloda; ibandronate;CPT11; topoisomerase inhibitor; difluoromethylornithine (DMFO); retinoic acid; esperamicin; capecitabine; taxanes such as paclitaxel, docetaxel, cabazitaxel; calminomycin, adriamycin such as 4'-epiadriamycin, 4-adriamycin-14-benzoate, adriamycin-14-octanoate, adriamycin-14-naphthaleneacetate; colchicine and pharmaceutically acceptable salts, acids, or derivatives of any of the foregoing, but not limited thereto.;

[0204] The term "chemotherapeutic agent" also includes, for example, antiestrogenic drugs such as tamoxifen, raloxifene, aromatase inhibitory 4(5)-imidazole, 4-hydroxytamoxifen, trioxifene, keoxifene, onapristone and toremifene; and antiandrogenic drugs such as flutamide, nilutamide, bicalutamide, leuprolide and goserelin; and antihormonal agents that act to modulate or inhibit the action of hormones on tumors, such as pharmaceutically acceptable salts, acids or derivatives of any of the foregoing.;

[0205] In some embodiments, the adjuvant agent is a cytokine or cytokine antagonist, such as IL-12, INFα, or anti-epidermal growth factor receptor, irinotecan; a tetrahydrofolate antagonist, such as pemetrexed; an antibody against a tumor antigen, a complex of a monoclonal antibody and a toxin, a T cell adjuvant, a bone marrow graft, or an antigen presenting cell (e.g., dendritic cell therapy), an anti-tumor vaccine, a replicable virus, a signal transduction inhibitor (e.g., Gleevec® or Herceptin®) or an immunomodulatory factor, a non-steroidal anti-inflammatory drug (NSAID), a cyclooxygenase-2 (COX-2) inhibitor, a steroid, a TNF antagonist (e.g., Remicade® and Enbrel®), interferon-β1a (Avonex®), and interferon-β1b (Betaseron®), as practiced in known chemotherapy treatment regimens including, but not limited to, TAC, FOLFOX, TPC, FEC, ADE, FOLFOX-6, EPOCH, CHOP, CMF, CVP, BEP, OFF, FLOX, CVD, TC, FOLFIRI, PCV, FOLFOXIRI, ICE-V, XELOX, and others readily recognized by those of skill in the art, and including, but not limited to, one or more combinations thereof, one or more chemical or biological agents identified in the art as being useful in the treatment of neoplastic diseases.

[0206] In some embodiments, the IL-27R binding molecule is administered in combination with a BRAF / MEK inhibitor, a kinase inhibitor, such as sunitinib, a PARP inhibitor, such as olaparib, an EGFR inhibitor, such as osimertinib (Ahn, et al. (2016) J Thorac Oncol 11:S115), an IDO inhibitor, such as epacadostat, and a oncolytic virus, such as talimogene laherparepvec (T-VEC).

[0207] Substance for use as an adjuvant in anti-tumor antigen antibody therapy In some embodiments, the "adjuvant substance" is a therapeutic antibody (including, but not limited to, bispecific T cell engager antibodies (BITE), bispecific affinity retargeting (DART) constructs, and trispecific killer engager (TriKE) constructs, including bispecific and trispecific antibodies that bind to one or more tumor-associated antigens).

[0208] In some embodiments, the therapeutic antibody is HER2 (e.g., trastuzumab, pertuzumab, ado-trastuzumab emtansine), nectin-4 (e.g., enfortumab), CD79 (e.g., polatuzumab vedotin), CTLA4 (e.g., ipilimumab), CD22 (e.g., moxetumomab pasudotox), CCR4 (e.g., mogamulizumab), IL23p19 (e.g., tildrakizumab), PDL1 (e.g., durvalumab, avelumab, atezolizumab), IL17a (e.g., ixekizumab), CD38 (e.g., daratumumab), SLAMF7 (e.g., elotuzumab), CD20 (e.g., rituximab, tositumomab, ibritumomab, and ofatumumab), CD30 (e.g., brentuximab vedotin), CD33 (e.g., gemtuzumab ozogamicin), CD52 (e.g., alemtuzumab), EpCam, CEA, fpA33, TAG-72, CAIX, PSMA, PSA, folate-binding protein, GD2 (e.g., dinutuximab), GD3, IL6 (e.g., siltuximab), GM2, Le y , VEGF (e.g., bevacizumab), VEGFR, VEGFR2 (e.g., ramucirumab), PDGFRα (e.g., olartumumab), EGFR (e.g., cetuximab, panitumumab, and necitumumab), ERBB2 (e.g., trastuzumab), ERBB3, MET, IGF1R, EPHA3, TRAIL R1, TRAIL R2, RANKL RAP, tenascin, integrin αVβ3, and integrin α4β1, and is an antibody that binds to at least one tumor antigen selected from the group consisting of

[0209] Examples of antibody therapeutic substances that are FDA-approved and can be used as adjuvants for the treatment of neoplastic diseases are provided in the following table.

[0210] (Table) Approved Antineoplastic Disease Antibodies and Indications TIFF2025108521000444.tif85150TIFF2025108521000445.tif171150

[0211] Physical methods In some embodiments, the adjuvant is one or more non-pharmacological modalities (e.g., local or systemic radiation therapy or surgery). By way of example, the present disclosure contemplates treatment regimens in which treatment with a treatment regimen comprising an IL-27R binding molecule and one or more adjuvants precedes or follows a radiation exposure phase. In some embodiments, the present disclosure further contemplates the use of an IL-27R binding molecule in combination with surgery (e.g., tumor resection). In some embodiments, the present disclosure further contemplates the use of an IL-27R binding molecule in combination with bone marrow transplantation, peripheral blood stem cell transplantation or other types of transplantation therapies.

[0212] Combination with immune checkpoint regulators: In some embodiments, the "adjuvant agent" is an immune checkpoint regulator for treating and / or preventing not only neoplastic diseases in a subject, but also diseases, disorders or conditions associated with neoplastic diseases. The term "immune checkpoint pathway" refers to the biological response induced by the binding of a first molecule (e.g., a protein such as PD1) expressed on an antigen-presenting cell (APC) to a second molecule (e.g., a protein such as PDL1) expressed on an immune cell (e.g., a T cell), which regulates the immune response via either stimulation of the immune response (e.g., upregulation of T cell activity) or inhibition (e.g., downregulation of T cell activity). Molecules involved in the formation of binding pairs that regulate the immune response are generally referred to as "immune checkpoints". The biological responses regulated by such immune checkpoint pathways are mediated by intracellular signaling pathways leading to downstream immune effector pathways such as cell activation, cytokine production, cell migration, secretion of cytotoxic factors, and antibody production. The immune checkpoint pathway is generally induced by the binding of a first cell surface-expressed molecule to a second cell surface molecule associated with the immune checkpoint pathway (e.g., the binding of PD1 to PDL1, the binding of CTLA4 to CD28, etc.). Activation of the immune checkpoint pathway can result in either stimulation or inhibition of the immune response.

[0213] As used herein, the term "immune checkpoint pathway regulator" refers to a molecule that inhibits or stimulates the activity of an immune checkpoint pathway in a biological system, including an immunocompetent mammal. An immune checkpoint pathway regulator may exert its effect by binding to an immune checkpoint protein (such as an immune checkpoint protein expressed on the surface of antigen-presenting cells (APCs) such as cancer cells and / or immune T effector cells), or may exert its effect on upstream and / or downstream reactions in the immune checkpoint pathway. For example, an immune checkpoint pathway regulator may regulate the activity of SHP2, a tyrosine phosphatase involved in PD-1 and CTLA-4 signaling. The term "immune checkpoint pathway regulator" includes both immune checkpoint pathway regulators (referred to herein as "immune checkpoint pathway inhibitors" or "immune checkpoint pathway antagonists") that can at least partially downregulate the function of inhibitory immune checkpoints, and immune checkpoint pathway regulators (referred to herein as "immune checkpoint pathway effectors" or "immune checkpoint pathway agonists") that can at least partially upregulate the function of stimulatory immune checkpoints.

[0214] Immune checkpoint regulators include, but are not limited to, immune checkpoint antagonists (e.g., antagonist antibodies) that bind to T cell inhibitory receptors including PD1 (also known as CD279), TIM3 (T cell membrane protein 3; also known as HAVcr2), BTLA (B and T lymphocyte attenuator; also known as CD272), VISTA (B7-H5) receptor, LAG3 (lymphocyte activation gene 3; also known as CD233), and CTLA4 (cytotoxic T lymphocyte-associated antigen 4; also known as CD152). In some embodiments, the immune checkpoint regulator is an agonist that induces a checkpoint pathway that results in stimulation of an immune response. Examples of such agonist immune checkpoint regulators include, but are not limited to, agonists that modulate the binding of ICOS-L to ICOS (CD278), B7-H6 to NKp30, CD155 to CD96, OX40L to OX40, CD70 to CD27, CD40 to CD40L, and GITRL to GITR. Examples of such positive immune checkpoint agonists include agonist antibodies that bind to T cell activation receptors such as ICOS (e.g., JTX-2011, Jounce Therapeutics), OX40 (e.g., MEDI6383, Medimmune), CD27 (e.g., balstilimab, Celldex Therapeutics), CD40 (e.g., dacetuzmumab CP-870,893, Roche, Chi Lob 7 / 4), HVEM, CD28, CD137, 4-1BB, CD226, and GITR (e.g., MEDI1873, Medimmune; INCAGN1876, Agenus), but are not limited thereto.

[0215] Exemplary negative immune checkpoint pathway inhibitors include, but are not limited to, programmed death-1 (PD1) pathway inhibitors, programmed death ligand-1 (PDL1) pathway inhibitors, TIM3 pathway inhibitors, and anti-cytotoxic T lymphocyte antigen 4 (CTLA4) pathway inhibitors.

[0216] In one aspect, the immune checkpoint pathway regulator is an antagonist of the negative immune checkpoint pathway that inhibits the binding of PD1 to PDL1 and / or PDL2 (a "PD1 pathway inhibitor"). The term PD1 pathway inhibitor includes monoclonal antibodies that interfere with the binding of PD1 to PDL1 and / or PDL2. Examples of commercially available PD1 pathway inhibitors useful as adjuvant agents in the treatment of neoplastic diseases include nivolumab (Opdivo® (registered trademark), BMS-936558, MDX1106, commercially available from Bristol Myers Squibb, Princeton NJ), pembrolizumab (Keytruda® (registered trademark) MK-3475, lambrolizumab, commercially available from Merck and Company, Kenilworth NJ), and atezolizumab (Tecentriq® (registered trademark), Genentech / Roche, South San Francisco CA), but are not limited to, antibodies that interfere with the binding of PD1 to PDL1 and / or PDL2. Additional PD1 pathway inhibitory antibodies include, but are not limited to, durvalumab (MEDI4736, Medimmune / AstraZeneca), pidilizumab (CT-011, CureTech), PDR001 (Novartis), BMS-936559 (MDX1105, Bristol Myers Squibb), and avelumab (MSB0010718C, Merck Serono / Pfizer); and SHR-1210 (Incyte) are in clinical development. Further antibody PD1 pathway inhibitors are described in U.S. Patent No. 8,217,149 (Genentech, Inc.) issued on July 10, 2012; U.S. Patent No. 8,168,757 (Merck Sharp and Dohme Corp.) issued on May 1, 2012, U.S. Patent No. 8,008,449 (Medarex) issued on August 30, 2011, and U.S. Patent No. 7,943,743 (Medarex, Inc.) issued on May 17, 2011.

[0217] The term PD1 pathway inhibitor is not limited to antagonist antibodies. Non-antibody biological PD1 pathway inhibitors are also in clinical development, including the PD-L2 IgG2a fusion protein AMP-224, the PDL2 fusion protein AMP-514 (Amplimmune and Glaxo SmithKline), aptamers (Wang, et al. (2018) 145:125-130), peptide PD1 pathway inhibitors (U.S. Patent No. 9,422,339 issued on August 23, 2016 and U.S. Patent No. 8,907,053 issued on December 9, 2014 to Sasikumar, et al.), small molecules (CA-170, AUPM-170, Aurigene / Curis; Sasikumar, et al., 1,2,4-oxadiazole and thiadiazole compounds as immunomodulators (PCT / IB2016 / 051266 filed on March 7, 2016 and published as WO2016142833A1 on September 15, 2016) and Sasikumar, et al. PCT / IB2016 / 051343 filed on March 9, 2016 and published as WO2016142886A2), BMS-1166 and Chupak LS and Zheng X. (2015) WO 2015 / 034820 A1, EP3041822 B1 granted on August 9, 2017; WO2015034820 A1; and Chupak, et al. 2015) WO 2015 / 160641 A2. WO 2015 / 160641 A2, Chupak, et al. Sharpe, et al. WO 2011082400 A2 published on July 7, 2011; are in clinical development according to U.S. Patent No. 7,488,802 issued on February 10, 2009.

[0218] In some embodiments, the IL-27R binding molecule is administered in combination with an antagonist of a negative immune checkpoint pathway that inhibits the binding of CTLA4 to CD28 (a "CTLA4 pathway inhibitor"). Examples of CTLA4 pathway inhibitors are well known in the art (see, e.g., U.S. Patent No. 6,682,736 (Abgenix), issued January 27, 2004; U.S. Patent No. 6,984,720 (Medarex, Inc.), issued May 29, 2007; U.S. Patent No. 7,605,238 (Medarex, Inc.), issued October 20, 2009).

[0219] In some embodiments, the IL-27R binding molecule is administered in combination with an antagonist of a negative immune checkpoint pathway that inhibits the ability of TIM3 to bind to a TIM3 activating ligand (a "TIM3 pathway inhibitor"). Examples of TIM3 pathway inhibitors are known in the art, and representative non-limiting examples are described in PCT International Patent Publication No. WO2016 / 144803, published September 15, 2016; Lifke et al., U.S. Patent Application Publication No. 20160257749 A1 (F. Hoffman-LaRoche), published September 8, 2016; Karunsky, U.S. Patent No. 9,631,026, issued April 27, 2017; Karunsky, Sabatos-Peyton et al., U.S. Patent No. 8,841,418, issued September 23, 2014; U.S. Patent No. 9,605,070; Takayanagi et al., U.S. Patent No. 8,552,156, issued October 8, 2013.

[0220] In some embodiments, the IL-27R binding molecule is administered in combination with an inhibitor of both LAG3 and PD1, as blockade of LAG3 and PD1 has been shown to synergistically reverse anergy between tumor-specific CD8+ T cells and virus-specific CD8+ T cells in the context of chronic infection. IMP321 (ImmuFact) has been evaluated in melanoma, breast cancer, and renal cell carcinoma. See generally, Woo et al., (2012) Cancer Res 72:917-27; Goldberg et al., (2011) Curr. Top. Microbiol. Immunol. 344:269-78; Pardoll (2012) Nature Rev. Cancer 12:252-64; Grosso et al., (2007) J. Clin. Invest. 117:3383-392.

[0221] In some embodiments, the IL-27R binding molecule is administered in combination with an A2aR inhibitor. A2aR inhibits T cell responses by stimulating CD4+ T cells to develop in the direction of T Reg cells. A2aR is particularly important in tumor immunity because the rate of cell death in tumors due to cell turnover is high, and dying cells release adenosine, a ligand for A2aR. In addition, deletion of A2aR has been associated with an enhanced, sometimes pathological, inflammatory response to infection. Inhibition of A2aR can be caused by administration of a molecule such as an antibody that blocks adenosine binding, or by an adenosine analog. Such agents can be used in combination with an IL-27R binding molecule for use in treatment disorders such as cancer and Parkinson's disease.

[0222] In some embodiments, the IL-27R binding molecule is administered in combination with an inhibitor of IDO (indoleamine 2,3-dioxygenase). IDO downregulates immune responses mediated via the oxidation of tryptophan, resulting in the inhibition of T cell activation and the induction of T cell apoptosis, creating an environment in which tumor-specific cytotoxic T lymphocytes are functionally inactivated or can no longer attack the subject's cancer cells. Indoximod (NewLink Genetics) is an IDO inhibitor that has been evaluated in metastatic breast cancer.

[0223] As described above, the present invention provides a method for treating a neoplastic disease (e.g., cancer) in a mammalian subject by administering an IL-27R binding molecule in combination with an agent that modulates at least one immune checkpoint pathway, the agent comprising an immune checkpoint pathway modulator that modulates two, three, or more immune checkpoint pathways.

[0224] In some embodiments, the IL-27R binding molecule is administered in combination with an immune checkpoint regulator that modulates multiple immune checkpoint pathways. The multiple immune checkpoint pathways can be modulated by administration of a multifunctional molecule that acts as a regulator of the multiple immune checkpoint pathways. Examples of such multiple immune checkpoint pathway regulators include, but are not limited to, bispecific or multispecific antibodies. Examples of multispecific antibodies capable of acting as regulators of multiple immune checkpoint pathways are known in the art. For example, U.S. Patent Application Publication No. 2013 / 0156774 describes bispecific and multispecific agents (e.g., antibodies) that target cells co-expressing PD1 and TIM3, and methods of using them. Moreover, dual blockade of BTLA and PD1 has been shown to enhance antitumor immunity (Pardoll, (April 2012) Nature Rev. Cancer 12:252-64). The present disclosure contemplates the use of an IL-27R binding molecule in combination with an immune checkpoint pathway regulator that targets multiple immune checkpoint pathways, including but not limited to, bispecific antibodies that bind both PD1 and LAG3. Thus, antitumor immunity can be enhanced at multiple levels, and combination strategies can be generated taking into account various mechanistic considerations.

[0225] In some embodiments, the IL-27R binding molecule can be administered in combination with two, three, four, or more checkpoint pathway regulators. Such combinations can be advantageous in that the immune checkpoint pathways can have distinct mechanisms of action, which provides an opportunity to attack the underlying disease, disorder, or condition from multiple distinct therapeutic angles.

[0226] It should be noted that the therapeutic response to immune checkpoint pathway inhibitors often appears much later than the response to traditional chemotherapy such as tyrosine kinase inhibitors. In some cases, it may take more than 6 months after the start of treatment with immune checkpoint pathway inhibitors before objective features of the therapeutic response are observed. Therefore, decisions regarding treatment with immune checkpoint pathway inhibitors in combination with the IL-27R binding molecules of the present disclosure must often be made over a longer progression-free period than traditional chemotherapy. The desired response can be any result that is considered favorable under this situation. In some embodiments, the desired response is prevention of the progression of the disease, disorder, or condition, whereas in other embodiments, the desired response is regression or stabilization of one or more features of the disease, disorder, or condition (e.g., reduction in tumor size). In still other embodiments, the desired response is reduction or elimination of one or more adverse effects associated with one or more of the agents of this combination.

[0227] Cell therapy agents and methods as adjuvants: In some embodiments, the methods of the present disclosure may include a combination of administration of an IL-27R binding molecule and an adjuvant agent in the form of a cell therapy for treating a neoplastic disease, an autoimmune disease, or an inflammatory disease. Examples of cell therapies that can be applied in combination with the methods of the present disclosure include, but are not limited to, one or more activated CAR-T cells, engineered TCR cells, tumor infiltrating lymphocytes (TILs), engineered T cell products including engineered Treg cells. Engineered T cell products are typically activated ex vivo prior to their administration to a subject and thus provide an upregulated level of CD25, and thus cell products comprising such activated engineered T cell types are applicable to further assist the administration of the CD25-biased IL-27R binding molecules described herein.

[0228] In some aspects of the methods of the present disclosure, the auxiliary agent is a "chimeric antigen receptor T cell" and a "CAR-T cell" that are used interchangeably to refer to T cells that have been recombinantly modified to express a chimeric antigen receptor. As used herein, the terms "chimeric antigen receptor" and "CAR" refer to a chimeric polypeptide comprising a plurality of functional domains arranged in the amino-terminal to carboxy-terminal direction in the sequence: (a) an antigen-binding domain (ABD), (b) a transmembrane domain (TD); and (c) one or more cytoplasmic signaling domains (CSD), where the aforementioned domains may optionally be linked by one or more spacer domains. A CAR may also further comprise a signal peptide sequence that is customarily removed during post-translational processing and presentation of the CAR on the cell surface of a cell transformed with an expression vector comprising a nucleic acid sequence encoding the CAR. CARs useful in the practice of the present invention are prepared according to principles well known in the art. See, for example, Eshhaar et al., U.S. Patent No. 7,741,465 B1, issued June 22, 2010; Sadelain, et al (2013) Cancer Discovery 3(4):388-398; Jensen and Riddell (2015) Current Opinions in Immunology 33:9-15; Gross, et al. (1989) PNAS(USA) 86(24):10024-10028; Curran, et al. (2012) J Gene Med 14(6):405-15. Examples of commercially available CAR-T cell products that can be modified to incorporate the orthogonal receptors of the present invention include axicabtagene ciloleucel (sold as Yescarta® by Gilead Pharmaceuticals) and tisagenlecleucel (sold as Kymriah® by Novartis).In some embodiments, the CAR-T carries a CAR that specifically binds to a cell surface molecule selected from the group consisting of GD2, BCMA, CD19, CD33, CD38, CD70, GD2, IL3Rα2, CD19, mesothelin, Her2, EpCam, Muc1, ROR1, CD133, CEA, EGRFRVIII, PSCA, GPC3, Pan-ErbB, and FAP, which is associated with tumor cells.

[0229] Physical methods : In some embodiments, the adjuvant is an antineoplastic physical method including, but not limited to, radiotherapy, cryotherapy, thermotherapy, surgery, laser ablation, and proton beam therapy.

[0230] Kits: The present disclosure also contemplates pharmaceutical compositions, IL-27R binding molecules, and kits containing such pharmaceutical compositions. Kits are generally in the form of a physical structure that houses various components as described below and can be utilized, for example, in the practice of the methods described above. The kit can include a pharmaceutical composition in a form ready for use and suitable for administration to a subject, or an IL-27R binding molecule in a form that requires preparation before administration, such as thawing, reconstitution, or dilution. If the IL-27R binding molecule is in a form that needs to be reconstituted by the user, the kit can also include a sterile container that provides a reconstitution medium containing a buffer, pharmaceutically acceptable excipients, and the like. The kits of the present disclosure can be designed for the conditions necessary to properly maintain the components contained therein (e.g., refrigeration or freezing). The kit may further contain a label or package insert that includes identification information about the components therein and instructions for their use. Each component of the kit can be housed within a separate container, and all of the various containers can be placed within a single package. The label or package insert can include manufacturer information such as lot numbers and expiration dates. The label or package insert can be incorporated, for example, into the physical structure housing the components, contained separately within the physical structure, or affixed to the components of the kit (e.g., ampoules, syringes, or vials). The label or package insert may be provided in a physical form or in a computer-readable medium. In some embodiments, the actual instructions are not present within the kit, and instead the kit provides means for obtaining instructions via an Internet site, including, for example, secure access by providing a password (or a scannable code such as a barcode or QR code on the container of the IL-27R binding molecule or the kit containing it) in accordance with administrative regulations (e.g., HIPAA) from a remote source.

[0231] Every maximum numerical limitation given throughout this specification is intended to include every lower numerical limitation as if such lower numerical limitations were expressly recited herein. Every minimum numerical limitation given throughout this specification is intended to include every higher numerical limitation as if such higher numerical limitations were expressly recited herein. Every numerical range given throughout this specification is intended to include every narrower numerical range that falls within such broader numerical range as if such narrower numerical ranges were all expressly recited herein.

[0232] None of the references cited herein are admitted to constitute prior art. The discussion of references presents what their authors assert, and the inventors reserve the right to challenge the accuracy and pertinence of the cited documents. It will be clearly understood that, although some sources of information, including articles from scientific journals, patent documents, and textbooks, are referred to herein, this reference does not admit that any of these documents form part of the common general knowledge in the art.

[0233] The aforementioned antibodies useful as adjuvants in the practice of the methods of the disclosure can be administered alone or in the form of any antibody-drug conjugate (ADC) comprising an antibody, a linker, and one or more drugs (e.g., 1, 2, 3, 4, 5, 6, 7, or 8 drugs) or in a modified (e.g., PEGylated) form.

[0234] In some embodiments, the adjuvant is a vaccine. The IL27R binding protein of the present invention can be administered to a subject in combination with a vaccine as an adjuvant to enhance the immune response to the vaccine according to the teachings of U.S. Patent No. 5,800,819, issued September 1, 1998 to Doyle et al. Examples of vaccines that can be combined with the IL27R binding protein of the present invention include HSV vaccine, Bordetella pertussis vaccine, Escherichia coli vaccine, pneumococcal vaccines including Prevnar® 13 and other polyvalent pneumococcal vaccines, diphtheria, tetanus and pertussis vaccines (such as Pediatrix®) and mixed vaccines including Pentacel®), varicella vaccine, Haemophilus influenzae type B vaccine, human papillomavirus vaccines such as Gardasil®), polio vaccine, leptospirosis vaccine, respiratory combination vaccines, Moraxella vaccines, live attenuated or inactivated virus vaccine products such as bovine respiratory disease vaccine (RSV), polyvalent human influenza vaccines such as Fluzone® and Quadravlent Fluzone®), feline leukemia vaccine, transmissible gastroenteritis vaccine, COVID-19 vaccine, and rabies vaccine.

[0235] For prophylactic use, the pharmaceutical composition or medicament is administered to a patient susceptible to or at risk of a disease in an amount sufficient to eliminate or reduce the risk of the disease, mitigate the severity thereof, or delay the onset of the disease, including the biochemical, histological and / or kinetic symptoms of the disease, its complications, and intermediate pathological phenotypes that appear during the development of the disease.

Example

[0236] Example 1 The camel was acclimatized in the research facility for at least 7 days before immunization. The antigen was diluted with 1×PBS (total antigen approximately 1 mg). The quality of the antigen was evaluated by SDS-PAGE to confirm the purity (e.g., over 80%). Initially, 10 mL of CFA (subsequently, IFA was used 6 times) was added to a mortar, and then 10 mL of the antigen in 1×PBS was slowly added to the mortar while grinding with a pestle. The antigen and CFA / IFA were ground until the components showed a milky white color and seemed difficult to disperse. The antigen emulsified in CFA was subcutaneously injected into at least 6 sites of the body of the camel, with approximately 2 mL injected into each site (a total of 10 mL per camel). By injecting into more sites and in larger amounts, a stronger immune response was generated. Immunization was performed 7 times every week (7 days). To avoid leakage of the emulsion, the injection needle was inserted into the subcutaneous cavity for 10 - 15 seconds each time of injection. Alternatively, leakage was also prevented by gently pulling the plunger of the syringe. Blood samples were collected 3 days after the 7th immunization.

[0237] 100 mL of blood was collected from the camel 3 days after the last injection of the immunization protocol. RNA was extracted from the blood and transcribed into cDNA. An approximately 900 bp reverse transcription sequence encoding the VH-CH1-hinge-CH2-CH3 construct was isolated from the desired fragment of approximately 700 bp encoding the VHH-hinge-CH2-CH3 species. The purified fragment of approximately 700 bp was amplified by nested PCR. The amplified sequence was digested with Pst1 and Not1. The approximately 400 bp PST1 / Not1 digested fragment was inserted into the Pst1 / Not1 digested pMECS phagemid vector so that the sequence encoding VHH was in-frame with the DNA sequence encoding the HA / His sequence. The sequence generated by PCR and the pMECS phagemid vector were digested with Pst I and Not I, and then ligated into pMECS / Nb recombinant. After ligation, the product was transformed into E. coli TG1 cells by electroporation. This transformant was enriched in the growth medium and then transferred to a 2YT + 2% glucose agar plate.

[0238] To identify VHHs that bind to IL27Rα, phage library biopanning was performed. IL27Rα was coated on 96-well plates, and the phage library was incubated in each well to allow phage-expressed IL27Rα-reactive VHHs to bind to IL27Rα on the plate. Non-specifically bound phages were washed away, and specifically bound phages were isolated. After selection, the enriched phage library expressing IL27Rα-reactive VHHs was amplified in TG1 cells. The above-described biopanning process was repeated for 2-3 rounds to enrich the library of VHHs selective for IL27Rα. Once biopanning was completed, three individual phage clones from the 96-well plates were isolated to perform periplasmic extract ELISA (PE-ELISA) on antigen-coated plates to identify positive VHH conjugates. Briefly, the antigen and PBS were coated on 96-well plates under the same conditions. Next, the wells were blocked at 37 °C for 1 hour. Then, 100 μl of the extracted antibody was added to each well and incubated for 1 hour. Thereafter, 100 μl of HRP-conjugated anti-tag polyclonal antibody was added to each well and incubated at 37 °C for 1 hour. The plates were developed with TMB substrate. The reaction was stopped by the addition of H2SO4. The absorbance at 450 nm was read using a microtiter plate reader, and antibodies with an absorbance in antigen-coated wells at least 3-fold higher than that of the PBS-coated control were specific binding molecules and were subjected to sequence analysis.

[0239] Example 2 - Recombinant production and purification The codon-optimized DNA insert was cloned into modified pcDNA3.4 (Genewiz) for small-scale expression in HEK293 cells in a 24-well plate. The binding protein was purified substantially according to the following procedure. Using a Hamilton Star automated system, 96 × 4 mL of the supernatant of a 4 × 24-well block was re-arrayed into a 4 × 96-well, 1 mL block. A PhyNexus micropipette tip (Biotage, San Jose CA) holding 80 μL of Ni-Excel IMAC resin (Cytiva) was equilibrated with wash buffer: PBS pH 7.4, 30 mM imidazole. The PhyNexus tip was immersed, and 14 cycles of 1 mL pipetting were repeated in all 4 × 96-well blocks. The PhyNexus tip was washed in a 2 × 1 mL block holding wash buffer. The PhyNexus tip was eluted in a 3 × 0.36 mL block holding elution buffer: PBS pH 7.4, 400 mM imidazole. The PhyNexus tip was regenerated in a 3 × 1 mL block of 0.5 M sodium hydroxide.

[0240] The purified protein eluate was quantified substantially using a Biacore® T200 according to the following procedure. 10 μL of the first 96 × 0.36 mL eluate was transferred to a Biacore® 96-well microplate and diluted to 60 μL in HBS-EP+ buffer (10 mM Hepes pH 7.4, 150 mM NaCl, 1 mM EDTA, 0.05% Tween 20). Each of the 96 samples was injected into a CM5 series S chip pre-functionalized with an anti-histidine capture antibody (Cytiva): injection was performed at 5 μL / min for 18 seconds. The capture level was recorded after 60 seconds of buffer wash. To eliminate surface variations between cells, a known V was used in each of the four Biacore chip flow cells HA standard curve of H concentration (270, 90, 30, 10, 3.3, 1.1 μg / mL) was obtained. Ninety-six captures were interpolated against the standard curve using a non-linear model that included specific and non-specific one-site binding. The concentration of the first elution block varied from 12 to 452 μg / mL corresponding to 4 to 149 μg. SDS-PAGE analysis of five randomly selected samples was performed to confirm that the molecular weight of the eluate corresponded to the expected value (approximately 30 kDa).

[0241] The protein concentration was normalized using a Hamilton Star automated system substantially according to the following procedure. The concentration values were imported into an Excel spreadsheet where the pipetting volume was calculated to perform a dilution to 50 μg / mL in 0.22 mL. The spreadsheet was imported into a dedicated Hamilton Star method that performed dilution pipetting using the first elution block and elution buffer as diluents. The finally normalized plate was sterile filtered using a 0.22 μm filter plate (Corning).

[0242] Example 3 All experiments were performed on a Biacore T200 instrument (Cytiva) equipped with a Protein A chip in 10 mM Hepes, 150 mM NaCl, 0.05% (v / v) polysorbate 20 (PS20) and 3 mM EDTA (HBS-EP+ buffer). The Mono-Fc VHH ligand was run at 5 μl / min for various times in the range of 18 to 300 seconds to reach the capture load described in the following table.

[0243] After ligand capture, a two-fold dilution series of the his-tagged cytokine receptor, containing at least five concentrations typically between 1 μM and 1 nM, was injected either in high performance mode or in single cycle kinetics mode. Surface regeneration was achieved by flowing 10 mM glycine-HCl, pH 1.5 (60 seconds, 50 μL / min). The buffer-subtracted sensogram was processed with Biacore T200 Evaluation Software and globally fitted to a 1:1 Langmuir binding model (setting bulk shift to zero) to extract the kinetics and affinity constants (ka, kd, KD). RMAX < 100 RU indicates a surface density suitable for kinetic analysis. The calculated value of Rmax was generated using the equation Rmax = load (RU) × valence of the ligand × (molecular weight of the analyte / molecular weight of the ligand). Surface activity was defined as the ratio of the experimental value of Rmax to the calculated value. See the table below for sample information and experimental results. Anti-hGP130 Mono-Fc VHH (ligand) that binds to hGP130-his (Sino Biological, catalog number 10974) TIFF2025108521000446.tif69151Anti-hIL27Ra Mono-Fc VHH (ligand) that binds to hIL27Ra-his (Origene, catalog number TP307012) TIFF2025108521000447.tif61151 * Both the association rate constant and the dissociation rate constant can be suppressed at Rmax > 100. If present, this effect is likely to be offset by the kinetic ratio, i.e., the affinity constant.

[0244] Example 4 Binding to all VHHs was confirmed by ELISA. One representative VHH from each clone trait was selected for further analysis by surface plasmon resonance using a Biacore T200. See below. TIFF2025108521000448.tif206150TIFF2025108521000449.tif154150

Claims

1. An IL27 receptor (IL27R) binding protein that specifically binds to the L27Rα subunit (IL27Rα) and the glycoprotein 130 subunit (gp130), which causes multimerization of IL27Rα and gp130 upon binding to IL27Rα and gp130, and comprises a single domain antibody (sdAb) that specifically binds to IL27Rα (anti-IL27Rα sdAb) and an sdAb that specifically binds to gp130 (anti-gp130 sdAb). Said binding protein.

2. The anti-IL27Rα sdAb is a V H H antibody (anti-IL27Rα V H H antibody), and / or the anti-gp130 sdAb is a V H H antibody (anti-gp130 V H H antibody), the IL27R-binding protein according to claim 1.

3. The IL27R binding protein according to any one of claims 1 to 2, wherein the anti-IL27Rα sdAb and the anti-gp130 sdAb are linked by a peptide linker.

4. The IL27R binding protein according to claim 3, wherein the peptide linker comprises 1 to 50 amino acids.

5. The IL27R binding protein according to claim 4, wherein the peptide linker comprises the sequence of GGGS (SEQ ID NO:108).

6. Anti-IL27Rα V H The IL27R-binding protein according to any one of claims 2 to 5, wherein the H antibody comprises CDR1 having 0, 1, 2 or 3 amino acid changes with respect to any one of the sequences of SEQ ID NOs: 193 to 198; CDR2 having 0, 1, 2 or 3 amino acid changes with respect to any one of the sequences of SEQ ID NOs: 199 to 204; and CDR3 having 0, 1, 2 or 3 amino acid changes with respect to any one of the sequences of SEQ ID NOs: 205 to 210.

7. Anti-gp130 V H The IL27R-binding protein according to any one of claims 2 to 6, wherein the H antibody comprises a CDR1 having 0, 1, 2 or 3 amino acid changes relative to any one of the sequences of SEQ ID NOs: 211 to 217; a CDR2 having 0, 1, 2 or 3 amino acid changes relative to any one of the sequences of SEQ ID NOs: 218 to 224; and a CDR3 having 0, 1, 2 or 3 amino acid changes relative to any one of the sequences of SEQ ID NOs: 225 to 231.

8. The IL27R binding protein according to any one of claims 2 to 5, comprising: A CDR1 having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to the CDR1 sequence from the row of Table 1A, or having 0, 1, 2 or 3 amino acid changes, optionally conservative amino acid changes; a CDR2 having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to the CDR2 sequence from the same row of Table 1A, or having 0, 1, 2 or 3 amino acid changes, optionally conservative amino acid changes; and a CDR3 having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to the CDR3 sequence from the same row of Table 1A, or having 0, 1, 2 or 3 amino acid changes, optionally conservative amino acid changes, comprising a first V H H antibody; and CDR1 having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to the CDR4 sequence from the same row of Table 1A, or having 0, 1, 2 or 3 amino acid changes, optionally conservative amino acid changes; CDR2 having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to the CDR5 sequence from the same row of Table 1A, or having 0, 1, 2 or 3 amino acid changes, optionally conservative amino acid changes; and CDR3 having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to the CDR6 sequence from the same row of Table 1A, or having 0, 1, 2 or 3 amino acid changes, optionally conservative amino acid changes, comprising a second V H H antibody.

9. The IL27R binding protein according to claim 8, comprising a sequence that is at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identical to any one of the dual VHH dimer sequences shown in Table 1A.

10. The CDR1, CDR2 and CDR3 in the anti-IL27Rα V H H antibody described in the horizontal row of Table 1 and the CDR1, CDR2 and CDR3 in the anti-gp130 V H The IL27R-binding protein according to any one of claims 2 to 9, comprising the CDR1, CDR2 and CDR3 in the H antibody.

11. An anti-gp130 VH antibody linked to the N-terminus of the linker and an anti-IL27Rα VH antibody linked to the C-terminus of the linker, the IL27R-binding protein according to any one of claims 1 to 14. H An anti-gp130 VH antibody linked to the N-terminus of the linker and an anti-IL27Rα VH antibody linked to the C-terminus of the linker, the IL27R-binding protein according to any one of claims 1 to 14. H An anti-gp130 VH antibody linked to the N-terminus of the linker and an anti-IL27Rα VH antibody linked to the C-terminus of the linker, the IL27R-binding protein according to any one of claims 1 to 14.

12. Anti-gp130 V H The IL27R binding protein according to claim 11, wherein the H antibody comprises a sequence having at least 90% sequence identity with any one of the sequences of SEQ ID NOs: 232 to 237.

13. Anti-IL27Rα V H The IL27R-binding protein according to claim 11, wherein the H antibody comprises a sequence having at least 90% sequence identity with any one of the sequences of SEQ ID NOs: 238 to 244.

14. Anti-gp130 V H H antibody and anti-IL27Rα V H The IL27R binding protein according to claim 11, wherein each of the H antibodies comprises a sequence having at least 90% identity with the sequences listed in the rows of Table 2A.

15. The IL27R binding protein according to claim 11, comprising a sequence having at least 90% identity to any one of the sequences of SEQ ID NOs: 1 to 42.

16. The IL27R binding protein according to claim 1, comprising an anti-IL27Rα VHH antibody linked to the N-terminus of the linker and an anti-gp130 VHH antibody linked to the C-terminus of the linker.

17. Anti-IL27Rα V H The IL27R-binding protein according to claim 16, wherein the H antibody comprises a sequence having at least 90% sequence identity with any one of the sequences of SEQ ID NOs: 245 to 251.

18. Anti-gp130 V H The IL27R binding protein according to claim 16, wherein the H antibody comprises a sequence having at least 90% sequence identity with any one of the sequences of SEQ ID NOs: 252 to 257.

19. Anti-IL27Rα V H H antibody and anti-gp130 V H The IL27R binding protein according to claim 16, wherein each of the H antibodies comprises a sequence having at least 90% identity with the sequences listed in the rows of Table 3A.

20. The IL27R binding protein according to claim 16, comprising a sequence having at least 90% identity to any one of the sequences of SEQ ID NOs: 43 to 84.

21. An isolated nucleic acid encoding the IL27R binding protein according to any one of claims 1 to 20.

22. The isolated nucleic acid according to claim 21, comprising an array of any one of SEQ ID NOs: 109 to 192 or an array having at least 90% sequence identity with the array in Table 1B.

23. An expression vector comprising the nucleic acid according to claim 21.

24. An isolated host cell comprising the vector according to claim 23.

25. A pharmaceutical composition comprising the IL27R binding protein according to any one of claims 1 to 20 and a pharmaceutically acceptable carrier.

26. A method for treating an autoimmune or inflammatory disease, disorder or condition, neoplastic disease, or viral infection in a subject in need thereof, comprising administering a therapeutically effective amount of the IL27R binding protein according to any one of claims 1 to 20 or the pharmaceutical composition according to claim 25 to the subject.

27. The method according to claim 26, further comprising administering one or more adjuvants selected from the group consisting of corticosteroids, Janus kinase inhibitors, calcineurin inhibitors, mTor inhibitors, IMDH inhibitors, biologics, vaccines and therapeutic antibodies.

28. The method according to claim 27, wherein the therapeutic antibody is an antibody that binds to a protein selected from the group consisting of BLyS, CD11a, CD20, CD25, CD3, CD52, IgEIL12 / IL23, IL17a, IL1β, IL4Rα, IL5, IL6R, integrin-α4β7, RANKL, TNFα, VEGF-A and VLA-4.

29. The method according to any one of claims 26 to 28, wherein the disease, disorder or condition is selected from viral infection, Helicobacter pylori infection, HTLV, organ rejection, graft-versus-host disease, autoimmune thyroid disease, multiple sclerosis, allergy, asthma, neurodegenerative diseases including Alzheimer's disease, systemic lupus erythematosus (SLE), autoinflammatory diseases, inflammatory bowel disease (IBD), Crohn's disease, diabetes, cartilage inflammation, arthritis, rheumatoid arthritis, juvenile arthritis, juvenile rheumatoid arthritis, juvenile rheumatoid arthritis, polyarticular juvenile rheumatoid arthritis, systemic juvenile rheumatoid arthritis, juvenile ankylosing spondylitis, juvenile enteropathic arthritis, juvenile reactive arthritis, juvenile Reiter's syndrome, SEA syndrome, juvenile dermatomyositis, juvenile psoriatic arthritis, juvenile scleroderma, juvenile systemic lupus erythematosus, juvenile vasculitis, oligoarticular rheumatoid arthritis, polyarticular rheumatoid arthritis, systemic rheumatoid arthritis, ankylosing spondylitis, enteropathic arthritis, reactive arthritis, Reiter's syndrome, SEA syndrome, psoriasis, psoriatic arthritis, dermatitis (eczema), exfoliative dermatitis or atopic dermatitis, pityriasis rubra pilaris, pityriasis rosea, pityriasis lichenoides, lichen planus, lichen nitidus, ichthyosiform dermatitis, keratosis, skin diseases, alopecia areata, pyoderma gangrenosum, vitiligo, pemphigoid, urticaria, porokeratosis, rheumatoid arthritis, seborrheic dermatitis, solar dermatitis, seborrheic keratosis, actinic keratosis, photoinduced keratosis, follicular keratosis, acne vulgaris, keloid, nevus, mole, condyloma or warts including verruca acuminata, and human papillomavirus (HPV) infection.

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