Compositions and methods related to IL27 receptor binding

Abstract

This provides an IL27 receptor-binding protein. [Solution] An IL27 receptor-binding protein that specifically binds to the IL27Rα subunit (IL27Rα) and the glycoprotein 130 subunit (gp130), comprising a single-domain antibody that causes polymerization of IL27Rα and gp130 upon binding to IL27Rα and gp130, and a single-domain antibody that specifically binds to IL27Rα and gp130.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority to U.S. Provisional Patent Application No. 63 / 061,562 filed on 5 August 2020, U.S. Provisional Patent Application No. 63 / 078,745 filed on 15 September 2020, and U.S. Provisional Patent Application No. 63 / 135,884 filed on 11 January 2021, the disclosures thereof incorporated herein by reference in their entirety for all purposes. [Background technology]

[0002] Background of Disclosure The 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 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 activated, inducing pro-inflammatory or anti-inflammatory responses involving different cell types, including macrophages, dendritic cells, T cells, and B cells. The activated response may be dependent on the external environment of IL27.

[0003] IL-27 is a heterodimeric cytokine consisting of two non-covalent 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 with two typical cytokine-binding domains (Pflanz S, et al., Immunity. 2002 Jun;16(6):779-90 (Non-Patent Literature 1)).

[0004] The primary receptor for IL-27 is IL-27R1 (also known as the TCCR- or WSX-1 receptor). IL-27 and IL-27R1 form a complex with considerable 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 Literature 2)).

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

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

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

[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 this structure, it is clear that the Fn3 domain does not energetically contribute to IL-27R complex formation. Rather, the structure of gp130 shows that domains 4 and 5 form a "C" at an 80% angle to each other. Certain residues in each "Tall" receptor are conserved, as are D4 and D5 in gp130. This indicates that all Toll receptors in 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 Literature 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 are located close together at the membrane site due to the "C" shape of each receptor. This is necessary for the receptor complex to induce JAK binding at the site of the intracellular domains of both receptors. [Prior art documents] [Non-patent literature]

[0010] [Non-Patent Document 1] Pflanz S, et al., Immunity. 2002 Jun;16(6):779-90 [Non-Patent Document 2] Pflanz S, et al., J Immunol. 2004 Feb 15;172(4):2225-31 [Non-Patent Document 3] Yibin Xu, et al., J Biol Chem. 2010 Jul 9;285(28):21214-8 [Overview of the Initiative]

[0011] Summary of Disclosure This disclosure provides compositions useful in cell receptor pair formation to produce desirable effects useful in the treatment of diseases in mammals.

[0012] Several advantages arise from the binding molecules described herein. IL-27, the natural ligand for IL-27R, brings gp130 and IL-27Ra into close proximity (i.e., by its co-binding). However, when IL-27 is used as a therapeutic agent in mammals, particularly in human subjects, it may induce several adverse and undesirable effects through 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 undesirable signaling in cells expressing gp130 and IL-27Ra. This disclosure relates to methods and compositions for modulating multiple effects of gp130 and IL-27Ra binding so that desired therapeutic signaling occurs, particularly in desired cell or tissue subtypes, while minimizing undesirable 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 to be complete agonists. In some embodiments, the binding molecules described herein are designed to be superagonists.

[0014] In some embodiments, the binding molecule provides maximum desired IL-27 intracellular signaling from binding to gp130 and IL-27Ra on desired cell types, while providing significantly less IL-27 signaling on other undesirable cell types. This is because, for example, the binding molecule has a different affinity for gp130 and IL-27Ra compared to the affinity of IL-27 on gp130 and IL-27Ra, or a different affinity for gp130 and IL-27Ra. maxThis can be achieved by selecting the binding molecule that triggers the desired activity. Since different cell types respond to ligand binding to their homologous receptors with different sensitivities, regulating the affinity of the dimeric ligand (or its individual binding sites) to the IL-27 receptor compared to wild-type IL-27 binding can promote the stimulation of desired activity while reducing unwanted activity on non-target cells.

[0015] This disclosure relates to a divalent ligation 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 ("anti-IL-27Ra sdAb") that specifically binds to the extracellular domain IL-27Ra. Includes, The anti-gp130 sdAb and anti-IL-27Ra sdAb are stably associated, and when cells initially expressing gp130 and IL-27Ra are first brought into contact with an effective amount of the divalent binding molecule, dimerization of gp130 and IL-27Ra occurs, leading to intracellular signaling specific to the IL-27 receptor when activated by its native congener IL-27. The present invention provides a divalent bond molecule. In some embodiments, one or both of the sdAb atoms are scFv. In some embodiments, one or both of the sdAb atoms are VHH.

[0016] In some embodiments, one sdAb of the divalent bond molecule is scFv, and the other sdAb is VHH.

[0017] In some embodiments, the first and second sdAb are covalently bonded via chemical bonds.

[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 that may optionally contain polypeptide linkers interposed between the amino acid sequences of the first and second sdAbs.

[0020] In some embodiments, the divalent bond molecule, which may optionally contain a linker, may optionally be expressed as a fusion protein with a further amino acid sequence. In some embodiments, the further amino acid sequence is a purified handle such as a chelated peptide, or a further 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 induces the multimerization of IL27Rα and gp130 upon binding, and this multimerization leads to the activation of JAK kinases associated with the intracellular domains of IL27Rα and gp130, as well as intracellular signaling, wherein 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, the multimerization of IL27Rα and gp130 can induce downstream signaling.

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

[0023] In some embodiments, the IL27R-binding protein includes: CDR1 having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity with respect to the CDR1 sequence from the row in 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 with respect to the CDR2 sequence from the same row in Table 1A, or having 0, 1, 2, or 3 amino acid changes, optionally conservative amino acid changes; and CDR3 having at least 90% The first V contains a CDR3 having sequence identity of (for example, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) or having 0, 1, 2, or 3 amino acid changes, optionally conservative amino acid changes. 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, including 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 includes CDR1 having 0, 1, 2, or 3 amino acid changes for any one sequence from SEQ ID NO: 211 to 217; CDR2 having 0, 1, 2, or 3 amino acid changes for any one sequence from SEQ ID NO: 218 to 224; and CDR3 having 0, 1, 2, or 3 amino acid changes for any one sequence from SEQ ID NO: 225 to 231.

[0026] In a particular embodiment, the IL27R-binding protein is the anti-IL27Rα V protein listed in the row of Table 1. H CDR1, CDR2, and CDR3 in H antibodies, as well as anti-gp130 V H This includes CDR1, CDR2, and CDR3 in the H antibody.

[0027] In some embodiments, the binding protein is linked to the N-terminus of the linker with anti-gp130 V H H antibody and anti-IL27Rα V linked to the C-terminus of the linker H It contains H antibody. In some embodiments, anti-gp130 V H The H antibody contains 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 SEQ ID NO:232-237. In some embodiments, it is an anti-IL27Rα V antibody. H The H antibody contains a sequence that has at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity with one of the sequences with SEQ ID NO: 238-244.

[0028] In certain embodiments, anti-GP130 V H H antibody and anti-IL27Rα V H Each H antibody contains a sequence that is at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the sequences listed in the rows of Table 2A.

[0029] In a particular embodiment, the binding protein includes a sequence that is substantially identical to any one of the sequences with SEQ ID NO: 1 to 42. In a particular embodiment, the binding protein includes a sequence that has at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity with any one of the sequences with SEQ ID NO: 1 to 42.

[0030] In some embodiments, the binding protein is linked to the N-terminus of the linker with anti-IL27Rα V H H antibody and anti-gp130 V linked to the C-terminus of the linker H It contains H antibody. In some embodiments, it contains anti-IL27Rα V H The H antibody contains 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 with SEQ ID NO: 245-251. In a particular embodiment, anti-gp130 V H The H antibody contains a sequence that has at least 90% sequence identity with one of the sequences with SEQ ID NO: 252-257.

[0031] In certain embodiments, anti-IL27Rα V H H antibody and anti-gp130 V H Each H antibody contains a sequence that has at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity with the sequences listed in the rows of Table 3A.

[0032] In a particular embodiment, the binding protein includes a sequence that is substantially identical to any one of the sequences with SEQ ID NO: 43-84. In a particular embodiment, the binding protein includes a sequence that has at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity with any one of the sequences with SEQ ID NO: 43-84.

[0033] In another aspect, the Disclosure provides isolated nucleic acids encoding the IL27R-binding protein described herein. In certain embodiments, the isolated nucleic acids include 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 from SEQ ID NO: 109–192 or a sequence from Table 1B. The Disclosure similarly provides an expression vector containing said nucleic acid. The Disclosure similarly provides isolated host cells containing said expression vector.

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

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

[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, IgEIL12 / IL23, IL17a, IL1β, IL4Rα, IL5, IL6R, integrin-α4β7, RANKL, TNFα, VEGF-A, and VLA-4.

[0037] In certain aspects, a disease, disorder, or condition is a viral infection, such as Helicobacter pylori. 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, chondritis, arthritis, rheumatoid arthritis, juvenile arthritis, juvenile rheumatoid arthritis, juvenile rheumatoid arthritis, polyarticular juvenile rheumatoid arthritis, systemic juvenile juvenile rheumatoid arthritis, juvenile ankylosing spondylitis, juvenile enteroarthritis, juvenile reactive arthritis, juvenile Reiter's syndrome, SEA syndrome, juvenile dermatomyositis, juvenile psoriatic arthritis, juvenile scleroderma, juvenile systemic lupus erythematosus, juvenile vasculitis, small-joint rheumatoid arthritis, polyarticular rheumatoid arthritis, polyarticular rheumatoid arthritis Rheumatoid arthritis, systemic rheumatoid arthritis, ankylosing spondylitis, enteritis-induced 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 lichenoidis, lichen planus, lichen patella, ichthyosis-like dermatitis, keratosis, skin diseases, alopecia areata The following are selected from pyoderma gangrenosum, vitiligo, bullous pemphigoid, urticaria, porokeratosis, rheumatoid arthritis, seborrheic dermatitis, photodermatitis, seborrheic keratosis, senile keratosis, actinic keratosis, actin-induced keratosis, follicular keratosis, acne vulgaris, keloids, nevi, warts, verrucae, including condyloma or genital warts, and human papillomavirus (HPV) infection.

[0038] The IL27R-binding proteins described herein are useful in the treatment of neoplasms, such as cancer (e.g., solid tumors; e.g., non-small cell lung cancer (NSCLC), renal cell carcinoma (RCC), or melanoma) and / or infectious diseases (e.g., bacterial and viral infections (e.g., viral infections caused by hepatitis C virus (HCV), human papillomavirus (HPV), or human immunodeficiency virus (HIV))) in subjects where such treatment is needed. IL27R-binding proteins are CD8 + T cells, CD4 +It binds to and activates T cells and / or T regulatory (Treg) cells. IL27R-binding proteins can induce different levels of downstream signaling in different cell types. For example, anti-IL27Rα V in IL27R-binding proteins. H H antibody and anti-gp130 V H By altering the linker length between the IL27R-binding protein and the H antibody, the IL27R-binding protein can induce higher levels of downstream signaling in desired cell types compared to undesired cell types. In some embodiments, by altering the linker length, the IL27R-binding protein can induce higher levels of downstream signaling in T cells (e.g., CD8) compared to other cells. + In another embodiment, different anti-IL27Rα V cells with different binding affinities can be used to induce higher levels of downstream signaling in T cells. H H antibodies and different anti-gp130 V antibodies with different binding affinities H By combining H antibodies, different IL27R-binding proteins can be created. Furthermore, by changing the orientation of the two antibodies in the binding protein, different binding proteins (i.e., anti-IL27Rα V) can be created. H H antibody-linker-anti-gp130 V H H antibody, or anti-gp130 V H H antibody-linker-anti-IL27Rα V H It is also possible to produce H antibodies. By screening different IL27R-binding proteins, it is possible to find the ideal binding protein that elicits higher levels of downstream signaling in the desired cell type compared to the undesired cell type. In some embodiments, T cells (e.g., CD8) + The level of downstream signaling in T cells is at least 1.1, 1.5, 2, 3, 5, or 10 times higher than the level of downstream signaling in other cells.

[0039] In particular, IL27R-binding proteins are CD8 + Binds to T cells, CD8 + It activates T cells. In some embodiments, the IL27R-binding protein is CXCR5+ CD8 + Binds to T cells, CXCR5 + CD8 + It activates T cells. IL27, for example, during viral infection, memory-like CXCR5 + CD8 + It is known that CXCR5 can promote and maintain the rapid division of T cells. + CD8 + T cells maintain the T cell response during persistent infection or cancer, and CD8 after anti-PD1 treatment. + It can drive a proliferative explosion of T cells. Therefore, the IL27R-binding proteins described herein are useful for maintaining and enhancing self-regenerating T cells in chronic infections and neoplastic diseases such as cancer. [Invention 1001] An IL27 receptor (IL27R) binding protein that specifically binds to the L27Rα subunit (IL27Rα) and the glycoprotein 130 subunit (gp130), When binding to IL27Rα and gp130, it causes polymerization of IL27Rα and gp130, and This includes 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] Anti-IL27Rα sdAb is V H H antibody (anti-IL27Rα V H (H antibody) and / or anti-gp130 sdAb is V H H antibody (anti-gp130 V H The IL27R-binding protein of the present invention 1001 is an H antibody. [Invention 1003] An IL27R-binding protein according to any of invention 1001 to 1002, wherein an anti-IL27Rα sdAb and an anti-gp130 sdAb are linked together 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. [Invention 1005] The IL27R-binding protein of the present invention 1004, wherein the peptide linker contains the sequence GGGS (SEQ ID NO:108). [Invention 1006] Anti-IL27Rα V H An IL27R-binding protein according to any of invention 1002 to 1005, wherein the H antibody comprises CDR1 having 0, 1, 2, or 3 amino acid changes to any one sequence of SEQ ID NO: 193 to 198; CDR2 having 0, 1, 2, or 3 amino acid changes to any one sequence of SEQ ID NO: 199 to 204; and CDR3 having 0, 1, 2, or 3 amino acid changes to any one sequence of SEQ ID NO: 205 to 210. [Invention 1007] anti-gp130v H An IL27R-binding protein according to any of Invention 1002 to 1006, wherein the H antibody comprises CDR1 having 0, 1, 2, or 3 amino acid changes to any one sequence of SEQ ID NO: 211 to 217; CDR2 having 0, 1, 2, or 3 amino acid changes to any one sequence of SEQ ID NO: 218 to 224; and CDR3 having 0, 1, 2, or 3 amino acid changes to any one sequence of SEQ ID NO: 225 to 231. [Invention 1008] IL27R-binding proteins of any of invention 1002 to 1005, including the following: CDR1 having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity with respect to the CDR1 sequence from the row in 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 with respect to the CDR2 sequence from the same row in Table 1A, or having 0, 1, 2, or 3 amino acid changes, optionally conservative amino acid changes; and CDR3 having at least 90% The first V contains a CDR3 having sequence identity of (for example, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) or having 0, 1, 2, or 3 amino acid changes, optionally conservative amino acid changes. H H antibody; and CDR1 has at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity with respect to the sequence of CDR4 from the same row in Table 1A, or has 0, 1, 2, or 3 amino acid changes, optionally conservative amino acid changes; CDR2 has at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity with respect to the sequence of CDR5 from the same row in Table 1A, or has 0, 1, 2, or 3 amino acid changes, optionally conservative amino acid changes; and CDR6 has at least 90% A second V containing a CDR3 having sequence identity of (for example, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) or having 0, 1, 2, or 3 amino acid changes, optionally conservative amino acid changes. H H antibody. [Invention 1009] The 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 double VHH dimer sequences shown in Table 1A. [Invention 1010] The row in Table 1 lists the anti-IL27Rα V H CDR1, CDR2, and CDR3 in H antibodies, as well as anti-gp130 V H An IL27R-binding protein according to any of the inventions 1002 to 1009, comprising CDR1, CDR2, and CDR3 in the H antibody. [Invention 1011] Anti-GP130 V bonded to the N-terminus of the linker H H antibody and anti-IL27Rα V linked to the C-terminus of the linker H An IL27R-binding protein according to any of the present invention 1001 to 1014, comprising an H antibody. [Invention 1012] anti-gp130v H The IL27R-binding protein of the present invention 1011, wherein the H antibody contains a sequence having at least 90% sequence identity with one of the sequences SEQ ID NO:232~237. [Invention 1013] Anti-IL27Rα V H The IL27R-binding protein of the present invention 1011, wherein the H antibody contains a sequence having at least 90% sequence identity with one of the sequences SEQ ID NO:238~244. [Invention 1014] anti-gp130v H H antibody and anti-IL27Rα V H The IL27R-binding protein of the present invention 1011, wherein each H antibody contains a sequence having at least 90% identity with the sequences listed in the horizontal rows of Table 2A. [Invention 1015] The IL27R-binding protein of the present invention 1011, comprising a sequence having at least 90% identity with one of the sequences with SEQ ID NO:1 to 42. [Invention 1016] The IL27R-binding protein of the present invention 1001, 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. [Invention 1017] Anti-IL27Rα V H The IL27R-binding protein of the present invention 1016, wherein the H antibody contains a sequence having at least 90% sequence identity with one of the sequences SEQ ID NO:245~251. [Invention 1018] anti-gp130v H The IL27R-binding protein of the present invention 1016, wherein the H antibody contains a sequence having at least 90% sequence identity with one of the sequences SEQ ID NO:252~257. [Invention 1019] Anti-IL27Rα V H H antibody and anti-gp130 V H The IL27R-binding protein of the present invention 1016, wherein each H antibody contains a sequence having at least 90% identity with the sequences listed in the horizontal rows of Table 3A. [Invention 1020] The IL27R-binding protein of the present invention 1016, comprising a sequence having at least 90% identity with one of the sequences with SEQ ID NO:43-84. [Invention 1021] An isolated nucleic acid encoding any of the IL27R-binding proteins of invention 1001 to 1020. [Invention 1022] An isolated nucleic acid of the present invention 1021, comprising one of the sequences from SEQ ID NO: 109 to 192 or a sequence having at least 90% sequence identity with a sequence in Table 1B. [Invention 1023] An expression vector comprising the nucleic acid of the present invention 1021. [Invention 1024] Isolated host cells containing the vector of Invention 1023. [Invention 1025] A pharmaceutical composition comprising an IL27R-binding protein according to any of invention 1001 to 1020 and a pharmaceutically acceptable carrier. [Invention 1026] A method for treating an autoimmune or inflammatory disease, disorder or condition, neoplasm or viral infection in a subject requiring treatment, comprising the step of administering a therapeutically effective amount of any IL27R-binding protein of Invention 1001 to 1020 or a pharmaceutical composition of Invention 1025 to a subject. [Invention 1027] The method of the present invention 1026, further comprising the step of 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. [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. [Invention 1029] The aforementioned disease, disorder, or condition is caused by a viral infection, such as Helicobacter pylori. 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, chondritis, arthritis, rheumatoid arthritis, juvenile arthritis, juvenile rheumatoid arthritis, juvenile rheumatoid arthritis, polyarticular juvenile rheumatoid arthritis, systemic juvenile juvenile rheumatoid arthritis, juvenile ankylosing spondylitis, juvenile enteroarthritis, juvenile reactive arthritis, juvenile Reiter's syndrome, SEA syndrome, juvenile dermatomyositis, juvenile psoriatic arthritis, juvenile scleroderma, juvenile systemic lupus erythematosus, juvenile vasculitis, small-joint rheumatoid arthritis, polyarticular rheumatoid arthritis, all Rheumatoid arthritis, ankylosing spondylitis, enteritis 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 lichenoid, lichen planus, lichen patella, ichthyosis-like dermatitis, keratoderma, skin diseases, alopecia areata, pyoderma gangrenosum, vitiligo, bullous pemphigoid A method of any of items 1026 to 1028 of the present invention, selected from urticaria, porokeratosis, rheumatoid arthritis, seborrheic dermatitis, photodermatitis, seborrheic keratosis, senile keratosis, actinic keratosis, photoinduced keratosis, follicular keratosis, acne vulgaris, keloids, nevi, warts, verrucae, including condyloma or genital warts, and human papillomavirus (HPV) infection. [Brief explanation of the drawing]

[0040] [Figure 1]Figure 1 of the attached drawings provides a schematic diagram of one embodiment of a divalent binding molecule of the present disclosure, which is depicted as interacting with a cell membrane (10)-bound heterodimer receptor comprising a first single-domain antibody (1) and a second single-domain antibody (3) and a linker (2), with respect to a first receptor subunit comprising an extracellular domain (4), a transmembrane domain (5), and an intracellular domain (6), and a second first receptor subunit comprising an extracellular domain (7), a transmembrane domain (8), and an intracellular domain (9), wherein the intracellular domain (6) of the first receptor and the intracellular domain (9) of the second receptor on the divalent binding molecule are within a proximal distance (11). [Figure 2] Figure 2 of the attached drawings provides schematic diagrams of two exemplary configurations of the divalent bonded molecule of the present disclosure. Panel A provides a schematic diagram of an exemplary single polypeptide chain divalent bonded molecule comprising a first single-domain antibody (1) and a second single-domain antibody (3) and a linker (2) in the amino-to-carboxyl direction. Panel B provides a schematic diagram of a divalent bonded molecule comprising a first single-domain antibody (1) and a second single-domain antibody (3) and a linker (2), as well as a knob-into-hole Fc domain, the Fc domain comprising a first subunit (13) which is an Fc knob and a second subunit (14) which is an Fc hole, and in the figure, the divalent bonded molecule is covalently bonded to the Fc domain subunit via an IgG hinge sequence (12). [Figure 3]Figure 3 of the attached drawings provides schematic diagrams of two exemplary configurations of the divalent bond molecule of the present disclosure. Panel A provides a schematic diagram of an exemplary divalent bond molecule construct comprising two divalent bond molecules, each attached to a subunit of a knob-into-hole Fc domain, wherein the construct comprises two polypeptide chains, the first polypeptide chain comprising a first single-domain antibody (1), a linker (2), and a second single-domain antibody (3), an IgG hinge sequence (12), and an Fc knob subunit (13) in the amino-to-carboxyl direction, and the second polypeptide chain comprising a first single-domain antibody (1), a linker (2), and a second single-domain antibody (3), an IgG hinge sequence (12), and an Fc hole subunit (14) in the amino-to-carboxyl direction, wherein the first and second polypeptides are in a stable associated state via the interaction of the knob-into-hole Fc domain. Panel B provides a schematic diagram of an alternative configuration of a bivalent bonded molecular construct comprising two polypeptides, wherein the first polypeptide chain comprises a first single-domain antibody (1), a linker (2), and a second single-domain antibody (3), an IgG hinge sequence (12), and an Fc knob subunit (13), arranged in the amino-to-carboxyl direction, and the second polypeptide chain comprises a first second single-domain antibody (3), a linker (2), and a first single-domain antibody (1), an IgG hinge sequence (12), and an Fc hole subunit (14), arranged in the amino-to-carboxyl direction, and the first and second polypeptides are in a stable associated state via knob-into-hole Fc domain interactions. [Figure 4]Panel A of Figure 4 provides an alternative schematic diagram of the configuration of the divalent binding molecule of this disclosure, comprising two polypeptides, each containing one single-domain antibody attached to each subunit of the knob-into-hole Fc domain, wherein the first polypeptide comprises a first single-domain antibody (1), an IgG hinge sequence (12), and an Fc knob subunit (13) in the amino-to-carboxyl direction, and the second polypeptide comprises a second single-domain antibody (3), an IgG hinge sequence (12), and an Fc hole subunit (13) in the amino-to-carboxyl direction, and the first and second single-domain antibodies are in a stable associated state via the interaction of the knob-into-hole Fc domain. Panel B of Figure 4 provides a schematic diagram of the binding molecule, wherein the binding domain is a single-domain antibody linked via a transition metal coordination covalent complex. As illustrated, the binding molecule comprises two polypeptide subunits: a first subunit containing a first single-domain antibody (1) is attached to a first chelated peptide (17) by a first linker (15); and a second subunit containing a second single-domain antibody (3) is attached to a second chelated peptide (18) by a second linker (16). The first chelated peptide (17) and the second chelated peptide (18) form a coordination-covalent complex with a single transition metal ion ("M"). The transition metal ion can be in a kinetically unstable or kinetically inert oxidation state. [Modes for carrying out the invention]

[0041] Detailed explanation of disclosure I. Introduction This disclosure provides compositions useful in cell receptor pairing to produce desirable effects useful in the treatment of diseases. Generally, binding proteins are provided, comprising a first domain that binds to IL27Rα and a second domain that binds to gp130, such that when the binding protein comes into contact with cells expressing IL27Rα and gp130, the binding protein induces the functional association of IL27Rα and gp130, thereby resulting in functional dimerization of the receptors and downstream signaling.

[0042] Several advantages arise from the binding proteins described herein. IL27, the natural ligand for IL27R, brings IL27Rα and gp130 into close proximity (i.e., by its co-binding). However, when IL27 is used as a therapeutic agent in mammals, particularly in human subjects, it may induce several adverse and undesirable effects through 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 undesirable signaling in cells expressing IL27Rα and gp130. This disclosure aims to provide methods and compositions for modulating the multiple effects of IL27Rα and gp130 binding so that desired therapeutic signaling occurs, particularly in desired cell or tissue subtypes, while minimizing undesirable activity and / or intracellular signaling.

[0043] In some embodiments, the binding proteins described herein are designed to provide maximum desired intracellular IL27 signaling from binding to IL27Rα and gp130 on desired cell types, while providing significantly less IL27 signaling on other undesired cell types. This is because, for example, the binding proteins have different affinities to IL27Rα and gp130 compared to the affinity of IL27 to IL27Rα and gp130, or different affinities to IL27Rα and gp130. maxThis can be achieved by selecting the binding protein that triggers the desired activity. Since different cell types respond to ligand binding to their homologous receptors with different sensitivities, regulating the affinity of the dimeric ligand (or its individual binding sites) to the IL27 receptor compared to wild-type IL27 binding promotes the stimulation of desired activity while reducing unwanted activity on non-target cells. Several methods are available to measure downstream signaling activity. For example, in some embodiments, JAK / STAT signaling can be measured by the presence of phosphorylated receptors and / or phosphorylated STAT. In other embodiments, the expression of one or more downstream genes whose expression levels may be influenced by the level of downstream signaling triggered 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 known as IL-27p28) and IL27B (also known as 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 post-translation to become 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: TIFF2026090564000002.tif22128

[0045] Human IL27B (hIL27B) is expressed as a 229-amino acid preprotein containing a 20-amino acid signal sequence, which is then removed post-translation to become 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: TIFF2026090564000003.tif22128

[0046] Interleukin-27 (IL27) receptor: IL27 induces intracellular signaling through its interaction with a heterodimeric receptor consisting of IL-27Rα (or IL27RA) and gp130. IL27 binding to the IL27 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, including macrophages, dendritic cells, T cells, and B cells. The type of response is environment-dependent.

[0047] The human IL27 receptor subunit α (hIL27RA) is expressed as a 636-amino acid preprotein containing a 32-amino acid signal sequence, and is post-translationally removed to become 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: TIFF2026090564000004.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: TIFF2026090564000005.tif50128

[0050] The human gp130 receptor subunit (hGP130) is 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, and is removed post-translation to become an 896-amino acid mature protein. The mature form of hGP130 has the following amino acid sequence: TIFF2026090564000006.tif87128

[0051] The extracellular domain of hGP130 (hGP130-ECD) is a 597-amino acid polypeptide corresponding to amino acids 23-619 of the hGP130 preprotein, and has the following amino acid sequence: TIFF2026090564000007.tif59128

[0052] IL27 activity IL27 is expressed by antigen-presenting cells. hIL27 induces differentiation of diverse populations of T cells in the immune system and also upregulates IL10. hIL27 has pro-inflammatory and anti-inflammatory properties, regulating T helper cell development, suppressing T cell proliferation, stimulating cytotoxic T cell activity, inducing B cell isotype switching, and exerting 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 IL-27 plays a crucial role in differentiation through the induction or suppression of T cell subtypes, including Th1, Th2, Th17, Tr1, and Treg cells. IL-27 is significantly involved in differentiation through the induction or suppression of each T cell subset. Th1 cells expressing interferon-gamma (IFNg) are produced in response to IL-27 via STAT1 signaling through the expression of T-bet and signature Th1 genes. IL-4 expressing Th2 cells are inhibited by IL-27 via the transcription factor GATA-3. Th17 cells expressing IL-17, IL-22, and GM-CSF are inhibited by IL-27 through the expression of STAT1 and the transcription factor RORγt. Treg cells are inhibited by IL-27 via STAT1 and STAT3.

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

[0055] IL10 induction IL-10-expressing Tr1 cells are induced by IL-27 via the transcription factor c-Maf, leading to an anti-inflammatory response. The primary activity of IL-10 is the suppression of the inflammatory response. The transcription factors STAT1 and STAT3, which specifically bind to IL-27α, are also involved. Activation of STAT3 by IL-27 leads to increased IL-10 secretion from Treg cells.

[0056] II. Definition To facilitate understanding of this disclosure, certain terms and phrases are defined not only below but throughout this specification. The definitions provided herein are not limiting and should be read with consideration to the knowledge that a person skilled in the art would possess.

[0057] Before describing the methods and compositions described herein, it should be understood that the present invention is not limited to the specific methods or compositions described and is, of course, subject to change. It should also be understood that the technical terms used herein are intended to describe embodiments only and are not intended to limit them.

[0058] Where a range of values ​​is provided, unless the context explicitly indicates otherwise, it is understood that each intermediate value between the upper and lower limits of this range, up to one-tenth of the lower limit, is also specifically disclosed. Any smaller range between any indicated value or intermediate value within the indicated range and any other indicated value or intermediate value within the indicated range is 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 if there are any limits that are specifically excluded in the indicated range, each range in which one or both of these limits are included in the smaller range, or in which neither is included, is also included in the present invention. If the indicated range includes one or both of these limits, the range that excludes one or both of the limits that they include is also included in the present invention.

[0059] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art to which the present invention pertains. Any methods and substances similar to or equivalent to those described herein may be used in the practice or testing of the present invention, but several potential and preferred methods and substances are described herein. All publications referenced herein are incorporated herein by reference to disclose and describe the methods and / or substances in which the publications are cited in connection therewith.

[0060] It should be noted that, as used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural references unless the context otherwise explicitly indicates otherwise. For example, a reference to “cell” includes multiple such cells, and a reference to “peptide” includes one or more peptides and their equivalents, such as polypeptides known to those skilled in the art.

[0061] The publications discussed herein are provided only if their disclosures precede the filing date of this application. Nothing contained herein should be construed as acknowledging that the present invention is not granted prior rights to such publications by prior art. Furthermore, the publication dates presented may differ from the actual publication dates, which may need to be verified separately.

[0062] Unless otherwise specified, parts are by weight, molecular weight is weight-average molecular weight, temperature is degrees Celsius (°C), and pressure is atmospheric pressure or near atmospheric pressure. Standard abbreviations are used, including the following: 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 = micromoles; mM = millimoles; M = mole; kDa = kilodalton; im = intramuscular; ip = intraperitoneal; SC or SQ = subcutaneous; 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 medium; EDTA = ethylenediaminetetraacetic acid.

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

[0064] (Table) Amino acid abbreviations TIFF2026090564000008.tif97128

[0065] Standard methods in molecular biology are described in scientific literature (see, for example, Sambrook and Russell (2001) Molecular Cloning, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; and Ausubel, et al. (2001) Current Protocols in Molecular Biology, Vols. 1-4, John Wiley and Sons, Inc. New York, NY, 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, fusion protein production, and protein glycosylation (see, for example, 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 “activates” is used in reference to a receptor or receptor complex and reflects the biological effects resulting from the binding of an agonist ligand to a receptor in response to ligand binding, both directly and / or through involvement in a multi-component signaling cascade.

[0067] Activity:As used herein, the term “activity” is used to describe, with respect to a molecule, its properties in relation to a test system (e.g., an assay), or its biological or chemical properties (e.g., the degree of binding of the 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, its ability to stimulate intracellular signaling, gene expression, cell proliferation, and its ability to modulate immune activity such as inflammatory responses. “Activity” is typically expressed as the level of bioactivity per unit of the agent being tested, such as [catalytic activity] / [mg protein], [immune activity] / [mg protein], international units (IU) of activity, [STAT5 phosphorylation] / [mg protein], [T cell proliferation] / [mg protein], or plaque-forming units (pfu). As used herein, the term “proliferative activity” means the activity that promotes cell proliferation and replication.

[0068] Administer / Administer: The terms “administer” and “administer” are used interchangeably herein to mean the act of contacting a subject, including, in vitro, in vivo, or ex vivo, with the active substance (e.g., orthologs, IL2 orthologs, engineered cells expressing ortholog receptors, engineered cells expressing ortholog IL2 receptors, CAR-T cells expressing ortholog IL2 receptors, chemotherapeutic agents, antibodies, or pharmaceutical formulations comprising one or more of the above). Administration of the active substance may be achieved by any of the various methods approved in the Art, including, but not limited to, topical administration, intravascular injection (including intravenous or intra-arterial injection), intradermal injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, and inhalation. The term “administer” includes contact between the active substance and the cells, tissues, or organs, as well as contact between the active substance and the fluid in contact with the cells, tissues, or organs.

[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” means a first active substance that specifically binds to a second active substance (“target”) and interacts with the target to cause or promote the enhancement of the target’s activation. In some cases, an agonist is an activator of a receptor protein that modulates, enhances, sensitizes a cell to activation by the second active substance, or upregulates one or more genes, proteins, ligands, receptors, biological pathways that may result in the expression of a gene, protein, ligand, receptor, cell proliferation, or cell death, such as cell cycle arrest or apoptosis. In some embodiments, an agonist is an active substance that binds to a receptor, modifies the state of the receptor, and results in a biological response that mimics the effect of the receptor’s endogenous activator. The term “agonist” includes partial agonists, full agonists, and superagonists. Agonists may be described as "full agonists" or "partial agonists" when such agonists produce a substantially complete biological response induced by the receptor under study (i.e., a response associated with the innate ligand / receptor binding interaction). In contrast to agonists, antagonists can bind specifically to a receptor but typically do not result in a signaling cascade initiated by the receptor, and can modify the action of the agonist at that receptor. Reverse agonists are agents that produce a pharmacological response opposite to that of the agonist. "Superagonists" are a type of agonist capable of producing a greater maximal response to a target receptor than the endogenous agonist, and therefore have greater activity than 100% of the innate ligand. A superagonist is typically a synthetic molecule that, when evaluated at similar concentrations in an equivalent assay, 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% in an evaluable quantitative or qualitative parameter of the molecule in its native form.

[0071] Antagonist: As used herein, the terms “antagonist” or “inhibitor” refer to molecules that counteract the action of an agonist. Antagonists prevent, reduce, inhibit, or neutralize the activity of an agonist, and antagonists can also prevent, inhibit, or reduce the constitutive activity of a target, such as a target receptor, even in the absence of a specific agonist. Inhibitors are molecules that reduce, block, prevent, delay, inactivate, desensitize, or downregulate genes, proteins, ligands, receptors, biological pathways, or cells.

[0072] antibody: As used herein, the term “antibody” collectively means: (a) glycosylated and nonglycosylated immunoglobulins (including, but not limited to, mammalian immunoglobulin classes IgG1, IgG2, IgG3, and IgG4) that specifically bind to a target molecule; and (b) IgG(1-4) delta C2 that competes for binding to the target molecule with the immunoglobulin from which it originates. H 2, F(ab')2, Fab, ScFv, V H , V LThe term "antibody" refers to immunoglobulin derivatives, including but not limited to tetrabodies, triabodies, diabodies, dsFv, F(ab')3, scFv-Fc, and (scFv)2. The term "antibody" is not limited to immunoglobulins derived from any specific mammalian species, but includes antibodies from mice, humans, horses, and camels (e.g., human antibodies). The term "antibody" includes not only antibodies that can be isolated from naturally occurring sources or from animals after immunization with antigens, but also engineered antibodies, including monoclonal antibodies, bispecific antibodies, trispecific antibodies, chimeric antibodies, humanized antibodies, human antibodies, CDR-transplanted, veneered, or deimmunized antibodies (e.g., to remove T cell epitopes). 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 animal produces antibodies that have the amino acid sequence characteristics of antibodies produced by humans. The term “antibody” should not be interpreted as being limited to any specific synthetic means, and includes not only naturally occurring antibodies that can be isolated from natural sources, but also manipulated antibody molecules prepared by “recombination” means, including 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 nucleic acid constructs that result in antibody expression, and antibodies isolated from combinatorial antibody libraries, including phage display libraries.

[0073] Binding molecule: As used herein, the term “binding molecule” means 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 their domains or subunits) as a result, these receptors (or domains or subunits) are maintained at close proximity to each other, including their domains (e.g., intracellular domains), to enable them to interact with each other and facilitate downstream signaling.

[0074] CDR:As used herein, the terms “CDR” or “complementarity-determining region” are intended to mean a discontinuous antigen-binding site found within the variable region of both heavy-chain immunoglobulin polypeptides and light-chain immunoglobulin polypeptides. CDR is described by Kabat et al., J. Biol. Chem. 252:6609-6616 (1977); Kabat, et al., US Dept. of Health and Human Services publication entitled "Sequences of proteins of immunological interest" (1991) (hereinafter also referred to as "Kabat 1991" or "Kabat"); Chothia, et al. (1987) J. Mol. Biol. 196:901-917 (hereinafter also referred to as "Chothia"); and MacCallum, et al. (1996) J. Mol. Biol. 262:732-745, where these definitions include overlaps or subsets of amino acid residues when compared to one another. Nevertheless, the application of any definition to mean the CDR of an antibody or transplanted antibody or its variants is intended to be within the scope of this term as defined and used herein. As used herein, the term “Chothia numbering” refers to a system recognized in the art that numbers amino acid residues based on their position in 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 this disclosure, unless otherwise specified, the positioning of CDR2 and CDR3 in the variable region of an antibody follows 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: Clonal trait is defined as a collection of binding molecules derived from the same B cell progenitor cell. The term “clonal trait” is used herein to mean a collection of antigen-binding molecules belonging to the same germline, having the same CDR3 length, and exhibiting 70% or more homology in their CDR3 sequences.

[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, two measurements would be considered “equivalent” if the difference between a first measurement of an evaluable quantitative parameter and a second measurement of the same parameter does not exceed a range that a person skilled in the art would recognize as not actually producing a statistically significant difference between the two results in the given context. In some cases, measurements may be considered “equivalent” if the difference between one measurement and another 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%. In certain embodiments, a measurement is equivalent to a reference standard if the difference between it and a reference standard is less than 15%, alternatively less than 10%, or alternatively less than 5%.

[0077] As used herein, the term “downstream signaling” refers to an intracellular signaling process triggered by the interaction of two or more cell surface receptors located in close proximity to each other.

[0078] Effective concentration (EC):Where used herein, the term “effective concentration” or its abbreviation “EC” is used interchangeably to refer to the concentration of an active substance (e.g., hIL2 mutein) sufficient to produce a change in a given parameter in the 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. The abbreviation “EC” is used when the magnitude of the response is expressed as a factor of the concentration of the test substance ("C"). In the context of biological systems, the term Emax refers to the maximum magnitude of a given biological effect observed in response to the saturation concentration of the activated test substance. When the abbreviation EC is followed by a subscript (e.g., EC 40 , EC 50 (e.g., the subscript indicates the percentage of the biological effect observed at that concentration relative to Emax. For example, a concentration of such a test substance sufficient to result in the induction of such a measurable biological parameter in a test system is 30% of the maximum level of the measurable biological parameter in response to the test substance. 30 It is said that "EC 100 The term "effective concentration" is used to mean the effective concentration of such an active substance that results in the maximum (100%) response of a measurable parameter in response to the active substance. Similarly, EC 50 The term "saturation concentration" (commonly used in the field of pharmacodynamics) refers to the concentration of an active substance sufficient to result in a maximum half-volume (50%) change in a measurable parameter. The term "saturation 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 temperature and pressure conditions. In pharmacodynamics, the saturation concentration of a drug is typically used to mean a drug concentration sufficient to occupy all available receptors, and EC 50 This is the drug concentration required to produce the maximum half-dose effect. The EC of a specific effective concentration of a test substance may be omitted for certain parameters and test systems.

[0079] Extracellular domain:As 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) located outside the plasma membrane of a cell. The term “ECD” may include the extracellular portion of a transmembrane protein or the extracellular portion of a cell surface (or membrane-bound protein).

[0080] identityWhere used herein, the terms “percent (%) sequence identity” or “substantially identical” in the context of nucleic acids or polypeptides mean a sequence that has at least 50% sequence identity with a reference sequence. Alternatively, percent sequence identity can be any integer from 50% to 100%. In some embodiments, a 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 with a reference sequence when determined by BLAST using standard parameters, as described below. In sequence comparison, typically one sequence acts as the reference sequence, and the test sequence is compared against it. When using a sequence comparison algorithm, the test sequence and reference sequence are entered into a computer, the coordinates of subsequences are specified as needed, and the program parameters for the sequence algorithm are specified. Default program parameters may be used, or different parameters may be specified. The sequence comparison algorithm then calculates the percentage sequence identity of the test sequence to the reference sequence based on program parameters. The comparison window includes a reference to any one segment of any 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, within which the two sequences can be optimally aligned before being compared to the reference sequence at the same number of consecutive positions. Suitable algorithms for determining the percentage of 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).This algorithm first identifies high-scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence that match when aligned with words of the same length in the database sequence or satisfy a certain positive threshold score T. T is called the adjacent word score threshold (Altschul et al., see above). These initial adjacent word hits act as seeds to initiate a search for longer HSPs that contain them. Then, word hits are extended in both directions for each sequence as long as the sum of the alignment scores can be increased. For nucleotide sequences, the sum of scores is calculated using parameters M (reward score for matching residue pairs; always > 0) and N (penalty score for mismatched residues; always < 0). For amino acid sequences, a scoring matrix is ​​used to calculate the sum of scores. The extension of word hits in each direction is stopped when the sum of the alignment scores decreases by 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 alignment. The BLASTN program (for nucleotide sequences) functions similarly, but uses a word size (W) of 28, an expected value (E) of 10, M=1, N=-2, and comparison of both strands as defaults. For amino acid sequences, the BLASTP program uses a word size (W) of 3, an expected value (E) of 10, and the BLOSUM62 scoring matrix as defaults (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989)). The BLAST algorithm also performs statistical analysis of the similarity between two sequences (see, for example, 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 indicator of the probability that a match between two nucleotide or amino acid sequences occurs by chance. For example, for amino acid sequences, the minimum sum probability in the comparison between a test amino acid sequence and a reference amino acid sequence is less than about 0.01, more preferably about 10. -5 Less than, most preferably about 10 -20 If it is less than [a certain value], it is considered similar to the reference array.

[0081] As used herein, the terms “interleukin-27 receptor” or “IL27R” refer to a heterodimeric receptor formed by the subunit IL27Rα (IL27Rα) and glycoprotein 130 (gp130), which is 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 signal transductionWhere used herein, the terms “intracellular signaling” and “downstream signaling” are used interchangeably to refer to cellular signaling processes triggered by the interaction of the intracellular domains (ICDs) of two or more cell surface receptors in close proximity to each other. In receptor complexes via the JAK / STAT pathway, the association of ICDS of receptor subunits brings the JAK domains of the ICDs into close proximity, causing the STAT molecule to be phosphorylated and translocate to the nucleus, where it associates with a specific nucleic acid sequence, initiating a phosphorylation cascade that results in the activation and expression of a specific gene within the cell. The binding molecule of this disclosure, when activated by its native congener IL-27, provides intracellular signaling characteristic of the IL-27 receptor. Several methods are available to measure downstream signaling activity. For example, in some embodiments, JAK / STAT signaling can be measured by the presence of a phosphorylated receptor and / or phosphorylated STAT. In other embodiments, the expression of one or more downstream genes, whose expression levels may be influenced by the level of downstream signaling triggered 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 that receptor, resulting 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” includes both natural and synthetic ligands. “Ligands” also include small molecules, such as peptide mimetic molecules of cytokines and peptide mimetic molecules of antibodies. A ligand-receptor complex is referred to as a “ligand-receptor complex.”

[0084] As used herein, the term “linker” means a link between two elements, for example, between protein domains. A linker may be a covalent bond or a peptide linker. The term “bond” means a chemical bond, for example, an amide bond or a disulfide bond, or any type of bond resulting from a chemical reaction, for example, a chemical conjugation. The term “peptide linker” means an amino acid or polypeptide that can be used to link two protein domains to provide space and / or mobility between them.

[0085] Adjust: As used herein, terms such as “regulate” and “modulate” refer to the ability of a test substance to positively or negatively, directly or indirectly, influence a response in a system, including a biological system or a biochemical pathway.

[0086] Multimerization As used herein, the term "multimerization" means that two or more cell surface receptors, or their domains or subunits, are brought into close proximity to each other so that they can interact with each other to induce intracellular signaling.

[0087] N-terminus As used herein in the context of polypeptide structure, “N-terminus” (or “amino-terminus”) and “C-terminus” (or “carboxyl-terminus”) refer to the amino-terminus and carboxyl-terminus of the polypeptide, respectively, while “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 may include the N-terminal and C-terminal residues, respectively. The terms “directly N-terminal side” or “directly C-terminal side” are used to refer to the position of the first amino acid residue relative to the second amino acid residue when the first and second amino acid residues are covalently bonded to provide a continuous amino acid sequence.

[0088] nucleic acid The terms "nucleic acid," "nucleic acid molecule," and "polynucleotide" are used interchangeably herein to refer to polymeric forms of nucleotides of any length, such as deoxyribonucleotides, ribonucleotides, or their analogues. Non-limiting examples of polynucleotides include linear or cyclic nucleic acids, messenger RNA (mRNA), complementary DNA (cDNA), recombinant polynucleotides, vectors, probes, primers, etc.

[0089] Functionally connected The term “functionally linked” is used herein to refer to the relationship between nucleic acid sequences encoding different functions that, when combined with a single nucleic acid sequence, provide a nucleic acid that, when introduced into a cell, can result in the transcription and / or translation of a particular nucleic acid sequence within the cell. For example, the DNA of a signal sequence is functionally linked with the DNA of a polypeptide if it is expressed as a preprotein involved in the secretion of the polypeptide; a promoter or enhancer is functionally linked with a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is functionally linked with a coding sequence if it is positioned to facilitate translation. Generally, “functionally linked” means that the linked DNA sequences are contiguous, or contiguous in the case of a secretion leader, and that the reading phases are aligned. However, certain genetic elements, such as enhancers, do not need to be contiguous with respect to the sequence that produces their effect.

[0090] Partial agonistAs used herein, the term “partial agonist” refers to a molecule that specifically binds to and activates a given receptor, but exhibits only partial activation of the receptor compared to a full agonist. Partial agonists may exhibit both agonist and antagonist effects. For example, in the presence of both a full agonist and a partial agonist, 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 contact between the receptor and the full agonist in the absence of the partial agonist. Clinically, partial agonists can be used to activate a receptor in the presence of an insufficient amount of endogenous ligand to produce a desired submaximal response, or they can reduce receptor overstimulation in the presence of an excess amount of endogenous ligand. The maximum response (Emax) produced by a partial agonist is called its endogenous activity and may be expressed on a percentage scale compared to the 100% response produced by a full agonist. In some embodiments, the IL-27 binding molecule is caused by IL-27. max E reduced compared to max It has. E max This reflects the maximum response level in a cell type that can be obtained by a ligand (e.g., a binding molecule described herein or a native cytokine (e.g., IL-27)). In some embodiments, the IL-27 binding molecule described herein is used to evoke E triggered by IL-27. max It has at least 1% (for example, 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%). In other embodiments, the E of the IL-27 binding molecule described herein. max E is the natural ligand IL-27 maxLarger than (for example, at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% larger). In some embodiments, the E of the IL-27 binding molecule can be changed by altering the linker length of the IL-27 binding molecule. max It can change the IL-27 binding molecule in the most desired cell type. max This results in reduced E in other cell types. max It could lead to this.

[0091] Polypeptide As used herein, the terms “polypeptide,” “peptide,” and “protein” are interchangeable to refer to polymeric forms of amino acids of any length, including genetically encoded and non-genetically encoded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having a modified polypeptide backbone. 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; and fusion proteins with immunologically active proteins (e.g., antigenic diphtheria or tetanus toxin fragments).

[0092] As used herein, terms such as “prevent,” “prevention,” and “prevention” generally refer to actions initiated with respect to an object predisposed to a particular disease, disorder, or condition due to genetic, empirical, or environmental factors, to temporarily or permanently prevent, suppress, inhibit, or reduce the risk of the object developing the disease, disorder, condition, or other (determined, for example, by the absence of clinical symptoms), or to delay their onset. In certain specific cases, the terms “prevent,” “prevention,” and “prevention” may also be used to mean delaying the progression of a disease, disorder, or condition from its current state to a more harmful state.

[0093] Close proximityAs used herein, the term “proximity” means the spatial proximity or physical distance between two cell surface receptors, or their domains or subunits, after the binding molecule described herein has bound to those two cell surface receptors, or their domains or subunits. In some embodiments, the spatial proximity between cell surface receptors, or their domains or subunits, after the binding molecule has bound to those cell surface receptors, or their domains or subunits, may be, for example, less than about 500 angstroms, for example, a distance of about 5 angstroms to about 500 angstroms. In some embodiments, spatial proximity is less than approximately 5 angstroms, less than approximately 20 angstroms, less than approximately 50 angstroms, less than approximately 75 angstroms, less than approximately 100 angstroms, less than approximately 150 angstroms, less than approximately 250 angstroms, less than approximately 300 angstroms, less than approximately 350 angstroms, less than approximately 400 angstroms, less than approximately 450 angstroms, or less than approximately 500 angstroms. In some embodiments, spatial proximity is less than approximately 100 angstroms. In some embodiments, spatial proximity is less than approximately 50 angstroms. In some embodiments, spatial proximity is less than approximately 20 angstroms. In some embodiments, spatial proximity is less than approximately 10 angstroms. In some embodiments, spatial proximity extends to approximately 10–100 angstroms, approximately 50–150 angstroms, approximately 100–200 angstroms, approximately 150–250 angstroms, approximately 200–300 angstroms, approximately 250–350 angstroms, approximately 300–400 angstroms, approximately 350–450 angstroms, or approximately 400–500 angstroms. In some embodiments, spatial proximity is less than approximately 250 angstroms, alternatively less than approximately 200 angstroms, alternatively less than approximately 150 angstroms, alternatively less than approximately 120 angstroms, alternatively less than approximately 100 angstroms, alternatively less than approximately 80 angstroms, alternatively less than approximately 70 angstroms, or alternatively less than approximately 50 angstroms.

[0094] receptor As used herein, the term “receptor” means a polypeptide having a ligand-specific binding domain such that ligand binding results in a change in at least one biological property of the polypeptide. In some embodiments, the receptor is a “soluble” receptor that is not associated with the cell surface. In some embodiments, the receptor is a cell surface receptor comprising an extracellular domain (ECD) and a membrane-associated domain that acts to fix the ECD to the cell surface. In some embodiments of cell surface receptors, the receptor is a transmembrane polypeptide comprising an intracellular domain (ICD) and an extracellular domain (ECD) linked by a transmembrane domain typically called a transmembrane domain (TM). Binding of a ligand to a 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 the ligand binding to the ECD. In some embodiments, the receptor is a component of a multicomponent complex for promoting intracellular signaling. For example, a ligand can bind to a cell surface molecule that is not associated with any intracellular signaling pathway in isolation, but once the ligand binds, it promotes the formation of a multimeric complex that leads to intracellular signaling.

[0095] RecombinationWhere used herein, the term “recombinant” is used as an adjective to describe a polypeptide, nucleic acid, or a cell modified using recombinant DNA technology. Recombinant proteins are proteins produced using recombinant DNA technology and may be denoted as such (e.g., rhIL2) using the lowercase letter “r” to indicate the method by which the protein was produced. Similarly, if cells are modified using recombinant DNA technology by the incorporation of exogenous nucleic acids (e.g., ssDNA, dsDNA, ssRNA, dsRNA, mRNA, viral or nonviral vectors, plasmids, cosmids, etc.) (e.g., transfection, transduction, infection), then the cells are called “recombinant cells.” Techniques and protocols for recombinant DNA technology are well known in the art, such as those found in Sambrook, et al. (1989) Molecular Cloning: A Laboratory Manual (2nd ed., Cold Spring Harbor Laboratory Press, Plainview, NY) and other standard molecular biology laboratory manuals.

[0096] response For example, the term “response” of a cell, tissue, organ, or organism encompasses quantitative or qualitative changes in evaluable biochemical or physiological parameters (e.g., concentration, density, adhesion, proliferation, activation, phosphorylation, migration, enzyme activity, gene expression level, gene expression rate, energy expenditure rate, level or state of differentiation), where the changes correlate with activation, stimulation, or treatment, or with internal mechanisms such as genetic programming. In certain contexts, terms such as “activation” and “stimulation” refer to cellular activation regulated by internal mechanisms as well as external or environmental factors. In contrast, terms such as “inhibition” and “downregulation” refer to the opposite effect.

[0097] Single-domain antibody (sdAb)The term "single-domain antibody" or "sdAb" refers to an antibody that has a single (only one) monomeric variable antibody domain. sdAbs can selectively bind to specific antigens. Further definitions are given below. H H antibody is an example of an sdAb.

[0098] Specific binding As used herein, the term “specifically binds” refers to the degree of selectivity or affinity one molecule has for binding to another molecule. In relation to a binding pair (e.g., binding molecule / receptor, ligand / receptor, antibody / antigen, antibody / ligand, antibody / receptor binding pairs as described herein), if the first molecule of the binding pair does not bind in significant amounts to other components present in the sample, then the first molecule of the binding pair is said to bind specifically to the second molecule of the binding pair. The first molecule of the binding pair is said to bind specifically to the second molecule if its affinity for the second molecule is at least twice, alternatively at least five times, alternatively at least ten times, alternatively at least twenty times, or alternatively at least one hundred times greater than its affinity for other components present in the sample.

[0099] To meet regularlyAs used herein, the terms “stable association” or “in a stable association” are used to refer to 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 with another can be brought about by various means, including covalent and non-covalent interactions. In some embodiments, the stable association of two molecules can be brought about by covalent bonds, such as peptide bonds. In other embodiments, the stable association of two molecules can be brought about by non-covalent interactions. Examples of non-covalent interactions that can provide a 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 interactions, anion-π interactions, and π-π interactions) and hydrophobic / hydrophilic interactions. In some embodiments, the stable association of sdAb in the divalent bonded molecules of this disclosure can be brought about by non-covalent interactions. In one embodiment, the non-covalent stable association of a divalent sdAb 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 first sdAb that specifically binds to the extracellular domain of the first subunit of the heterodimer receptor to an "Fc knob" monomer, and conjugation of a second sdAb that specifically binds to the extracellular domain of the second subunit of the heterodimer receptor to an "Fc hole" monomer, results in the stable association of the first and second sdAbs. The knob-into-hole modification is further described in Ridgway, et al. (1996) Protein Engineering 9(7):617-621, as well as in U.S. Patent No. 5,731,168 issued on 24 March 1998, U.S. Patent No. 7,642,228 issued on 5 January 2010, U.S. Patent No. 7,695,936 issued on 13 April 2010, and U.S. Patent No. 8,216,805 issued on 10 July 2012.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 with an amino acid residue having a larger side chain (e.g., tyrosine or tryptophan), creating a protrusion ("knob") from the surface; and ii) an amino acid residue in the CH3 domain of the second heavy chain is replaced with 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, within which 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 amino acid substitution T366W and optionally amino acid substitution S354C in one antibody heavy chain, and amino acid substitution T366S, L368A, Y407V and optionally Y349C in the other antibody heavy chain. Furthermore, the Fc domain may be modified by introducing cysteine ​​residues at position S354 of one chain and position Y349 of the other chain, which results in a stabilizing disulfide crosslink between the two antibody heavy chains in the Fc region (Carter, et al. (2001) Immunol Methods 248, 7-15). The knob-into-hole configuration is used to promote the expression of a first polypeptide (e.g., IL27Rα-binding sdAb) on a first Fc monomer having a "knob" modification to promote the expression of a heterodimer polypeptide conjugate, and a second polypeptide on a second Fc monomer having a "hole" modification.

[0100] subject The terms “recipient,” “individual,” “subject,” and “patient” are used interchangeably herein and refer to any mammalian subject, in particular human, to whom diagnosis, treatment, or therapy is desired. For treatment purposes, “mammal” means any animal classified as a mammal, including humans, livestock and farm animals, and exhibition animals, competition animals, or companion animals, such as dogs, horses, cats, cattle, sheep, goats, and pigs. In some embodiments, mammal is human.

[0101] In effect: As used herein, the term “substantially” means a quantity, 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 the quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length of the reference. In one embodiment, “substantially the same” means an effect that is approximately the same as the quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length of the reference, for example, a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length that produces a physiological effect.

[0102] I am suffering As used herein, the term “suffering” means a determination made by a physician with respect to a subject, based on information accepted in the art, that the subject requires or would benefit from treatment, for the identification of a disease, disorder, or condition, including but not limited to X-ray, CT scan, conventional clinical diagnostic tests (e.g., blood count), genomic data, protein expression data, and immunohistochemistry. The term “suffering” is typically used with a specific medical condition, such as “suffering from a neoplasm,” to refer to a subject diagnosed with the presence of a neoplasm.

[0103] Therapeutic effective doseWhere used herein, the term “therapeutic dose” is used to refer to a single dose of an active substance administered to a subject that, when administered to the subject, can have any detectable positive effect on any symptom, aspect, or feature of a disease, disorder, or condition, either as part of a series of doses, alone, or as part of a pharmaceutical composition or therapeutic regimen. The therapeutic dose can be determined by measuring the relevant physiological effect, which may be adjusted in relation to the drug regimen and in accordance with diagnostic analysis such as the condition of the subject. Parameters for evaluation to determine the therapeutic dose of an active substance are determined by a physician using diagnostic criteria approved in the art, including but not limited to age, weight, sex, overall health status, ECOG score, observable physiological parameters, blood levels, blood pressure, electrocardiogram, computed tomography, radiography, and other indicators. Alternatively or additionally, other parameters typically evaluated in the clinical context, such as body temperature, heart rate, normalization of blood chemistry, normalization of blood pressure, normalization of cholesterol levels, or any symptoms, phases, or characteristics of the disease, disorder, or condition, modification of biomarker levels, increased survival, extended progression-free survival, extended time to progression, increased time to treatment failure, extended event-free survival, extended time to subsequent treatment, improved response rate, and improved duration of response, may be monitored to determine whether a therapeutically effective dose of the active ingredient was administered to the subject, and these parameters are relied upon by clinicians in the art to assess the improvement in the subject's condition in response to administration of the active ingredient.

[0104] Treat: Terms such as "treating," "treatment," and "treat" refer to an action (e.g., administering a binding molecule described herein, or a pharmaceutical composition comprising the same) that is initiated with respect to a subject after a disease, disorder, or condition, or symptoms thereof, have been diagnosed, observed, or otherwise detected in the subject, and that temporarily or permanently removes, reduces, suppresses, alleviates, or cures 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 suffering from a disease, which actions result in inhibition of the disease in the subject (e.g., halting the progression of the disease, disorder, or condition, or restoring one or more symptoms associated therewith).

[0105] VHH : As used herein, "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 a binding molecule has a V H H with an equilibrium dissociation constant between the 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 camelids are well known in the scientific literature. For example, see 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 described herein 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 "VHH dimer" and "VHH dimer" refer to a state where both the first and second sdAb are VHH, and the first VHH dimer binds to the first receptor, or its domain or subunit. H H, and a second V that binds to a second receptor, or its domain or subunit. H The term H is used interchangeably to refer to a subtype of the binding molecule of this disclosure.

[0107] Wild type As used herein, the terms “wild-type,” “WT,” or “natural” are used to mean an amino acid or nucleotide sequence that is found in nature and has not been modified by human intervention.

[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 expressing both IL27Rα and gp130. In some embodiments, the IL27R-binding protein has bispecificity as described below. H H 2 It can be.

[0109] The IL27R-binding protein is the first V that binds to IL27Rα. H H (anti-IL27Rα V H (H antibody) and the second V that binds to gp130 H H (anti-gp130 V H Cells that possess (H antibody) and express IL27Rα and gp130, for example, CD8 + T cells, CD4 + When bispecific V cells bind to T cells and / or T regulatory (Treg) cells, they trigger dimerization of two receptor subunits and downstream signaling. H H 2It can be.

[0110] V H H is a type of single-domain antibody (sdAb) containing a single monomeric variable antibody domain. Like full-length antibodies, it can selectively bind to specific antigens. V H The complementarity-determining region (CDR) of H is located within a single-domain polypeptide. H H can be manipulated from heavy chain antibodies found in camelid animals.

[0111] Example V H H has a molecular weight of approximately 12-15 kDa, which is much smaller than conventional mammalian antibodies (150-160 kDa) composed of two heavy chains and two light chains. H H is found in or can be produced from camelid mammals (e.g., camels, llamas, dromedaries, alpacas, and guanacos) that originally lack a light chain. sdAb and V H Descriptions of 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 order to prepare the binding protein, in some embodiments, two V H H can be synthesized separately and then joined together with a linker. Alternatively, bispecificity V H H 2 These can be synthesized as fusion proteins. V has different binding activity and receptor targets. H H forms pairs with each other, and the bispecific V H H 2This allows for the creation of a binding protein that can be used to screen signaling in cells possessing one or both of the associated receptors.

[0113] In some embodiments, bispecificity V H H 2 This includes: CDR1 having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity with respect to the CDR1 sequence from the row in 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 with respect to the CDR2 sequence from the same row in Table 1A, or having 0, 1, 2, or 3 amino acid changes, optionally conservative amino acid changes; and CDR3 having at least 90% The first V contains a CDR3 having sequence identity of (for example, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) or having 0, 1, 2, or 3 amino acid changes, optionally conservative amino acid changes. H H antibody; and CDR1 has at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity with respect to the sequence of CDR4 from the same row in Table 1A, or has 0, 1, 2, or 3 amino acid changes, optionally conservative amino acid changes; CDR2 has at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity with respect to the sequence of CDR5 from the same row in Table 1A, or has 0, 1, 2, or 3 amino acid changes, optionally conservative amino acid changes; and CDR6 has at least 90% A second V containing a CDR3 having sequence identity of (for example, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) or having 0, 1, 2, or 3 amino acid changes, optionally conservative amino acid changes. H H antibody. In some embodiments, bispecificity V H H 2 This includes 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 double VHH dimer sequences shown in Table 1A.

[0114] In some embodiments, V described herein H H can be humanized to include the human framework domain. Humanized V H Examples of human germ cell lines that can be used to produce 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) TIFF2026090564000009.tif240167TIFF2026090564000010.tif241167TIFF2026090564000011.tif241167TIFF2026090564000012.tif240167TIFF2026090564000013.tif241167TIFF2026090564000014.tif236167TIFF2026090564000015.tif241167TIFF2026090564000016.tif236167TIFF2026090564000017.tif240167TIFF2026090564000018.tif241167TIFF2026090564000019.tif240167TIFF2026090564000020.tif240167TIFF2026090564000021.tif241167TIFF2026090564000022.tif236167TIFF2026090564000023.tif241167TIFF2026090564000024.tif236167TIFF2026090564000025.tif240167TIFF2026090564000026.tif241167TIFF2026090564000027.tif240167TIFF2026090564000028.tif241167TIFF2026090564000029.tif241167TIFF2026090564000030.tif236167TIFF2026090564000031.tif241167TIFF2026090564000032.tif236167TIFF2026090564000033.tif240167TIFF2026090564000034.tif241167TIFF2026090564000035.tif240167TIFF2026090564000036.tif241167TIFF2026090564000037.tif232167TIFF2026090564000038.tif240167TIFF2026090564000039.tif241167TIFF2026090564000040.tif240167TIFF2026090564000041.tif240167TIFF2026090564000042.tif241167TIFF2026090564000043.tif240167TIFF2026090564000044.tif241167TIFF2026090564000045.tif236167TIFF2026090564000046.tif241167TIFF2026090564000047.tif241167TIFF2026090564000048.tif236167TIFF2026090564000049.tif240167TIFF2026090564000050.tif241167TIFF2026090564000051.tif240167TIFF2026090564000052.tif241167TIFF2026090564000053.tif240167TIFF2026090564000054.tif241167TIFF2026090564000055.tif241167TIFF2026090564000056.tif236167TIFF2026090564000057.tif241167TIFF2026090564000058.tif241167TIFF2026090564000059.tif240167TIFF2026090564000060.tif241167TIFF2026090564000061.tif236167TIFF2026090564000062.tif241167TIFF2026090564000063.tif236167TIFF2026090564000064.tif240167TIFF2026090564000065.tif241167TIFF2026090564000066.tif241167TIFF2026090564000067.tif240167TIFF2026090564000068.tif241167TIFF2026090564000069.tif236167TIFF2026090564000070.tif241167TIFF2026090564000071.tif236167TIFF2026090564000072.tif240167TIFF2026090564000073.tif241167TIFF2026090564000074.tif240167TIFF2026090564000075.tif240167TIFF2026090564000076.tif241167TIFF2026090564000077.tif236167TIFF2026090564000078.tif241167TIFF2026090564000079.tif236167TIFF2026090564000080.tif240167TIFF2026090564000081.tif241167TIFF2026090564000082.tif240167TIFF2026090564000083.tif241167TIFF2026090564000084.tif241167TIFF2026090564000085.tif236167TIFF2026090564000086.tif241167TIFF2026090564000087.tif236167TIFF2026090564000088.tif240167TIFF2026090564000089.tif241167TIFF2026090564000090.tif240167TIFF2026090564000091.tif241167TIFF2026090564000092.tif232167TIFF2026090564000093.tif240167TIFF2026090564000094.tif241167TIFF2026090564000095.tif240167TIFF2026090564000096.tif240167TIFF2026090564000097.tif241167TIFF2026090564000098.tif240167TIFF2026090564000099.tif241167TIFF2026090564000100.tif236167TIFF2026090564000101.tif241167TIFF2026090564000102.tif241167TIFF2026090564000103.tif236167TIFF2026090564000104.tif240167TIFF2026090564000105.tif241167TIFF2026090564000106.tif240167TIFF2026090564000107.tif241167TIFF2026090564000108.tif240167TIFF2026090564000109.tif241167TIFF2026090564000110.tif241167TIFF2026090564000111.tif236167TIFF2026090564000112.tif241167TIFF2026090564000113.tif241167TIFF2026090564000114.tif240167TIFF2026090564000115.tif241167TIFF2026090564000116.tif236167TIFF2026090564000117.tif241167TIFF2026090564000118.tif236167TIFF2026090564000119.tif240167TIFF2026090564000120.tif241167TIFF2026090564000121.tif241167TIFF2026090564000122.tif240167TIFF2026090564000123.tif241167TIFF2026090564000124.tif236167TIFF2026090564000125.tif241167TIFF2026090564000126.tif236167TIFF2026090564000127.tif240167TIFF2026090564000128.tif241167TIFF2026090564000129.tif240167TIFF2026090564000130.tif240167TIFF2026090564000131.tif241167TIFF2026090564000132.tif236167TIFF2026090564000133.tif241167TIFF2026090564000134.tif236167TIFF2026090564000135.tif240167TIFF2026090564000136.tif241167TIFF2026090564000137.tif240167TIFF2026090564000138.tif241167TIFF2026090564000139.tif241167TIFF2026090564000140.tif236167TIFF2026090564000141.tif241167TIFF2026090564000142.tif236167TIFF2026090564000143.tif240167TIFF2026090564000144.tif241167TIFF2026090564000145.tif240167TIFF2026090564000146.tif241167TIFF2026090564000147.tif232167TIFF2026090564000148.tif240167TIFF2026090564000149.tif241167TIFF2026090564000150.tif240167TIFF2026090564000151.tif240167TIFF2026090564000152.tif241167TIFF2026090564000153.tif240167TIFF2026090564000154.tif241167TIFF2026090564000155.tif236167TIFF2026090564000156.tif241167TIFF2026090564000157.tif241167TIFF2026090564000158.tif236167TIFF2026090564000159.tif240167TIFF2026090564000160.tif241167TIFF2026090564000161.tif240167TIFF2026090564000162.tif241167TIFF2026090564000163.tif240167TIFF2026090564000164.tif241167TIFF2026090564000165.tif241167TIFF2026090564000166.tif236167TIFF2026090564000167.tif241167TIFF2026090564000168.tif241167TIFF2026090564000169.tif240167TIFF2026090564000170.tif241167TIFF2026090564000171.tif236167TIFF2026090564000172.tif241167TIFF2026090564000173.tif236167TIFF2026090564000174.tif240167TIFF2026090564000175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000209.tif241167TIFF2026090564000210.tif236167TIFF2026090564000211.tif241167TIFF2026 090564000212.tif241167TIFF2026090564000213.tif236167TIFF2026090564000214.tif240167TI FF2026090564000215.tif241167TIFF2026090564000216.tif240167TIFF2026090564000217.tif24 1167TIFF2026090564000218.tif240167TIFF2026090564000219.tif241167TIFF202609056400022 0.tif241167TIFF2026090564000221.tif236167TIFF2026090564000222.tif241167TIFF202609056 4000223.tif241167TIFF2026090564000224.tif240167TIFF2026090564000225.tif241167TIFF202 6090564000226.tif236167TIFF2026090564000227.tif241167TIFF2026090564000228.tif236167.

[0116] In some embodiments, the IL27R-binding proteins described herein (e.g., in Table 1A) are encoded by isolated nucleic acids that are substantially identical to any one of the sequences in Table 1B below. In some embodiments, the IL27R-binding proteins described herein (e.g., IL27R-binding proteins containing the sequences in Table 1A) are encoded by isolated nucleic acids containing sequences that have at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity with the sequences in Table 1B below.

[0117] (Table 1B) TIFF2026090564000229.tif186158TIFF2026090564000230.tif237158TIFF2026090564000231.tif236158TIFF2026090564000232.tif236158TIFF2026090564000233.tif237158TIFF2026090564000234.tif236158TIFF2026090564000235.tif237158TIFF2026090564000236.tif232158TIFF2026090564000237.tif236158TIFF2026090564000238.tif237158TIFF2026090564000239.tif236158TIFF2026090564000240.tif237158TIFF2026090564000241.tif236158TIFF2026090564000242.tif236158TIFF2026090564000243.tif237158TIFF2026090564000244.tif237158TIFF2026090564000245.tif232158TIFF2026090564000246.tif237158TIFF2026090564000247.tif237158TIFF2026090564000248.tif236158TIFF2026090564000249.tif237158TIFF2026090564000250.tif237158TIFF2026090564000251.tif236158TIFF2026090564000252.tif237158TIFF2026090564000253.tif237158TIFF2026090564000254.tif236158TIFF2026090564000255.tif237158TIFF2026090564000256.tif237158TIFF2026090564000257.tif236158TIFF2026090564000258.tif237158TIFF2026090564000259.tif232158TIFF2026090564000260.tif236158TIFF2026090564000261.tif237158TIFF2026090564000262.tif232158TIFF2026090564000263.tif237158TIFF2026090564000264.tif237158TIFF2026090564000265.tif236158TIFF2026090564000266.tif237158TIFF2026090564000267.tif236158TIFF2026090564000268.tif236158TIFF2026090564000269.tif237158TIFF2026090564000270.tif237158TIFF2026090564000271.tif237158TIFF2026090564000272.tif236158TIFF2026090564000273.tif236158TIFF2026090564000274.tif237158TIFF2026090564000275.tif237158TIFF2026090564000276.tif237158TIFF2026090564000277.tif232158TIFF2026090564000278.tif237158TIFF2026090564000279.tif237158TIFF2026090564000280.tif237158TIFF2026090564000281.tif232158TIFF2026090564000282.tif236158TIFF2026090564000283.tif237158TIFF2026090564000284.tif237158TIFF2026090564000285.tif237158TIFF2026090564000286.tif237158TIFF2026090564000287.tif236158TIFF2026090564000288.tif237158TIFF2026090564000289.tif237158TIFF2026090564000290.tif237158TIFF2026090564000291.tif237158TIFF2026090564000292.tif232158TIFF2026090564000293.tif237158TIFF2026090564000294.tif237158TIFF2026090564000295.tif237158TIFF2026090564000296.tif236158TIFF2026090564000297.tif237158TIFF2026090564000298.tif237158TIFF2026090564000299.tif236158TIFF2026090564000300.tif237158TIFF2026090564000301.tif237158TIFF2026090564000302.tif236158TIFF2026090564000303.tif237158TIFF2026090564000304.tif237158TIFF2026090564000305.tif236158TIFF2026090564000306.tif237158TIFF2026090564000307.tif237158TIFF2026090564000308.tif236158TIFF2026090564000309.tif237158TIFF2026090564000310.tif237158TIFF2026090564000311.tif236158TIFF2026090564000312.tif236158TIFF2026090564000313.tif237158TIFF2026090564000314.tif237158TIFF2026090564000315.tif232158TIFF2026090564000316.tif236158TIFF2026090564000317.tif237158TIFF2026090564000318.tif232158TIFF2026090564000319.tif236158TIFF2026090564000320.tif237158TIFF2026090564000321.tif237158TIFF2026090564000322.tif232158TIFF2026090564000323.tif236158TIFF2026090564000324.tif237158TIFF2026090564000325.tif237158TIFF2026090564000326.tif236158TIFF2026090564000327.tif236158TIFF2026090564000328.tif237158TIFF2026090564000329.tif237158TIFF2026090564000330.tif236158TIFF2026090564000331.tif237158TIFF2026090564000332.tif237158TIFF2026090564000333.tif236158TIFF2026090564000334.tif236158TIFF2026090564000335.tif237158TIFF2026090564000336.tif232158TIFF2026090564000337.tif237158TIFF2026090564000338.tif237158TIFF2026090564000339.tif236158TIFF2026090564000340.tif237158TIFF2026090564000341.tif237158TIFF2026090564000342.tif236158TIFF2026090564000343.tif237158TIFF2026090564000344.tif236158TIFF2026090564000345.tif236158TIFF2026090564000346.tif237158TIFF2026090564000347.tif232158TIFF2026090564000348.tif236158TIFF2026090564000349.tif237158TIFF2026090564000350.tif237158TIFF2026090564000351.tif232158TIFF2026090564000352.tif236158TIFF2026090564000353.tif237158TIFF2026090564000354.tif237158TIFF2026090564000355.tif232158TIFF2026090564000356.tif237158TIFF2026090564000357.tif237158TIFF2026090564000358.tif236158TIFF2026090564000359.tif237158TIFF2026090564000360.tif237158TIFF2026090564000361.tif236158TIFF2026090564000362.tif236158TIFF2026090564000363.tif2 37158TIFF2026090564000364.tif237158TIFF2026090564000365.tif236158TIFF2026090564000366.tif237158 TIFF2026090564000367.tif237158TIFF2026090564000368.tif236158TIFF2026090564000369.tif237158TIFF2 026090564000370.tif237158TIFF2026090564000371.tif236158TIFF2026090564000372.tif236158TIFF202609 0564000373.tif237158TIFF2026090564000374.tif237158TIFF2026090564000375.tif236158TIFF20260905640 00376.tif237158TIFF2026090564000377.tif237158TIFF2026090564000378.tif236158TIFF2026090564000379 .tif237158TIFF2026090564000380.tif232158TIFF2026090564000381.tif236158TIFF2026090564000382.tif2 37158TIFF2026090564000383.tif236158TIFF2026090564000384.tif236158TIFF2026090564000385.tif22158.

[0118] In some embodiments, bispecificity V H H 2 This includes: CDR1 having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to any one sequence of SEQ ID NO: 193-198, 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 any one sequence of SEQ ID NO: 199-204, or having 0, 1, 2, or 3 amino acid changes, optionally conservative amino acid changes; and at least 90% to any one sequence of SEQ ID NO: 205-210 Anti-gp130 V contains CDR3 having sequence identity of (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) or having 0, 1, 2, or 3 amino acid changes, optionally conservative amino acid changes. 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 any one sequence of SEQ ID NO:211~217, 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 any one sequence of SEQ ID NO:218~224, or having 0, 1, 2, or 3 amino acid changes, optionally conservative amino acid changes; and at least 90% to any one sequence of SEQ ID NO:225~231 Anti-IL27Rα V, containing CDR3 having sequence identity of (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) or having 0, 1, 2, or 3 amino acid changes, optionally conservative amino acid changes. H H antibody.

[0119] In a particular embodiment, the dual singularity V described herein H H 2 This includes anti-GP130 V compounds, CDR1, CDR2, and CDR3, as described in each row of Table 1 below. H Anti-IL27Rα V antibodies, including H antibody and CDR1, CDR2, and CDR3. H Contains H antibody. In some embodiments, anti-gp130 V H CDR1, CDR2, and CDR3 in H antibodies, as well as anti-IL27Rα V H In the H antibody, CDR1, CDR2, and CDR3 each independently contain at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity with respect to the sequences described 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, bispecificity V H H 2 It has an anti-GP130 V at its N-terminus. H H antibody and anti-IL27Rα V at the C-terminus H Contains H antibody. In some embodiments, bispecificity V H H 2 It has an anti-IL27Rα V at its N-terminus. H H antibody and anti-gp130 V at the C-terminus H Contains H antibody

[0121] (Table 1) TIFF2026090564000386.tif218163TIFF2026090564000387.tif218163TIFF2026090564000388.tif78163

[0122] Anti-GP130-Linker-Anti-Il27Rα V H H Bispecific V H H 2 This is the first V that binds to gp130 in the direction from the N-terminus to the C-terminus. H H (anti-gp130 VH (H antibody), linker, and second V that binds to IL27Rα H H (anti-IL27Rα V H It may contain (H antibody). In other words, the linker contains anti-gp130 V in the binding protein. H The C-terminus of H is linked to the anti-IL27Rα V in the binding protein. H The H is ligated to the N-terminus. In some embodiments, the purified peptide, for example, 6-histidine peptide ((His)6 (SEQ ID NO: 1531) or His-tag) is given bispecificity V H H 2 You may include it or not.

[0123] In a particular embodiment, the dual singularity V described herein H H 2 This is an anti-gp130 V containing a sequence that has at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity with one of the sequences SEQ ID NO:232~237. H Anti-IL27Rα V antibody; and an anti-IL27Rα V antibody containing a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity with one of the sequences SEQ ID NO:238-244. H Contains H antibody

[0124] In a particular embodiment, the dual singularity V described herein H H 2 This refers to the anti-GP130 V described in each row of Table 2A below or Table 1A above. H H antibody and anti-IL27Rα V H Contains H antibody. In some embodiments, in each row of Table 2A, anti-gp130 V H H antibody and anti-IL27Rα V HEach H antibody can independently contain 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, bispecificity V H H 2 This refers to the anti-GP130 V described in each row of Table 2A below. H H antibody and anti-IL27Rα V H A linker (e.g., a linker as described in Section IV) may be included between the H antibody and the antibody. In certain embodiments, the linker is GGGS (SEQ ID NO: 108) or (GGGS)n (SEQ ID NO: 1532), (GGS)nG (SEQ ID NO: 1533), or (GGGGS)n (SEQ ID NO: 1534), as described elsewhere in this specification. Anti-gp130 V H The H sequence is N-terminus relative to the linker, anti-IL27Rα V H The H sequence is C-terminus with respect to the linker. Examples of linkers are further described in Section IV below. Each V H The CDR sequence of H is underlined.

[0125] (Table 2A) TIFF2026090564000389.tif180150TIFF2026090564000390.tif225150TIFF2026090564000391.tif220150TIFF2026090564000392.tif249150

[0126] In a certain embodiment, bispecificity V H H 2 This includes a sequence that is substantially identical to one of the sequences with SEQ ID NO: 1 to 42. Such a bispecific V H H 2As shown in Table 2B below, it may have a sequence that has at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity with any one of the sequences SEQ ID NO: 1 to 42. In each of the sequences SEQ ID NO: 1 to 42, the linker GGGS (SEQ ID NO: 108) is shown in bold. Anti-gp130 V H The H sequence is N-terminus relative to the linker, anti-IL27Rα V H The H sequence is C-terminus with respect to the linker. Each V H The CDR sequence of H is underlined.

[0127] (Table 2B) TIFF2026090564000393.tif107150TIFF2026090564000394.tif224150TIFF20260905640 00395.tif224150TIFF2026090564000396.tif224150TIFF2026090564000397.tif135150

[0128] In some embodiments, the IL27R-binding proteins described herein (e.g., IL27R-binding proteins containing any one sequence of SEQ ID NO: 1 to 42) are encoded by isolated nucleic acids that are substantially identical to any one sequence of SEQ ID NO: 109 to 150, as shown in Table 2C below.

[0129] (Table 2C) TIFF2026090564000398.tif244150TIFF2026090564000399.tif232150TIFF20260905640 00400.tif232150TIFF2026090564000401.tif232150TIFF2026090564000402.tif228150 TIFF2026090564000403.tif224150TIFF2026090564000404.tif232150TIFF20260905640 00405.tif232150TIFF2026090564000406.tif232150TIFF2026090564000407.tif232150

[0130] Anti-IL27Rα V H H-Linker-AntiGP130 V H H Bispecific V H H 2 This is the first V that binds to IL27Rα in the direction from the N-terminus to the C-terminus. H H (anti-IL27Rα V H (H antibody), linker, and second V that binds to gp130 H H (anti-gp130 V H It may contain an anti-IL27Rα antibody in the binding protein. In other words, the linker contains an anti-IL27Rα antibody in the binding protein. H The C-terminus of H is connected to the anti-gp130 V in the binding protein. H The H is attached to the N-terminus. In some embodiments, the purified peptide, for example, 6-histidine peptide ((His)6 (SEQ ID NO: 1531) or His-tag) or Fc tag is used for bispecificity V H H 2 It can be included.

[0131] In a particular embodiment, the dual singularity V described herein H H 2This is an anti-IL27Rα V virus containing a sequence that has at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity with any one of the sequences with SEQ ID NO: 245-251. H Anti-gp130 V antibody; and an anti-gp130 V antibody containing a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity with one of the sequences SEQ ID NO:252-257. H Contains H antibody

[0132] In a particular embodiment, the dual singularity V described herein H H 2 This refers to the anti-IL27Rα V described in Table 3A below or in each row of Table A above. H H antibody and anti-gp130 V H Contains H antibody. In some embodiments, each row in Table 3A contains anti-IL27Rα V H H antibody and anti-gp130 V H Each H antibody can independently contain 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, bispecificity V H H 2 This refers to the anti-IL27Rα V described in each row of Table 3A below. H H antibody and anti-gp130 V H A linker (e.g., the linker described in Section IV) may be included between the H antibody and the anti-IL27Rα antibody. In certain embodiments, the linker is GGGS (SEQ ID NO: 108). H The H sequence is at the N-terminus relative to the linker, and is anti-gp130 V H The H sequence is C-terminus with respect to the linker. Examples of linkers are further described in Section IV below. Each V H The CDR sequence of H is underlined.

[0133] (Table 3A) TIFF2026090564000408.tif26150TIFF2026090564000409.tif220150TIFF20260905640 00410.tif225150TIFF2026090564000411.tif220150TIFF2026090564000412.tif188150

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

[0135] (Table 3B) TIFF2026090564000413.tif188150TIFF2026090564000414.tif228150TIFF2026090564000415.tif232150TIFF2026090564000416.tif212150

[0136] In some embodiments, the IL27R-binding proteins described herein (e.g., IL27R-binding proteins containing any one sequence of SEQ ID NO: 43-84) are encoded by isolated nucleic acids that are substantially identical to any one sequence of SEQ ID NO: 151-192, as shown in Table 3C below.

[0137] (Table 3C) TIFF2026090564000417.tif177150TIFF2026090564000418.tif232150TIFF2026090564000419.t if232150TIFF2026090564000420.tif232150TIFF2026090564000421.tif224150TIFF20260905640 00422.tif232150TIFF2026090564000423.tif232150TIFF2026090564000424.tif232150TIFF202 6090564000425.tif232150TIFF2026090564000426.tif232150TIFF2026090564000427.tif177150

[0138] Further IL27R-binding proteins Furthermore, additional IL27R-binding proteins include anti-IL27Rα V, as listed in Table 4 below. H In the H antibody, the three underlined CDR sequences and one of the SEQ ID NO:232-237 anti-gp130 V HThe H antibody may contain the three underlined CDR sequences. Further IL27R-binding proteins include anti-IL27Rα V H H antibody (for example, one of the sequences listed in Table 4 below) and anti-gp130 V H The antibody may contain an H antibody (e.g., one of SEQ ID NO: 232-237). In some embodiments, the binding protein may have an anti-IL27Rα V at its N-terminus. H H antibody and anti-gp130 V at the C-terminus H Contains H antibody. In some embodiments, the binding protein has anti-gp130 V at its N-terminus. H H antibody and anti-IL27Rα V at the C-terminus H Contains H antibody. In some embodiments, the binding protein is anti-IL27Rα V H H antibody and anti-gp130 V H The linker (e.g., one of SEQ ID NO: 85-108 (e.g., SEQ ID NO: 108)) is included between the H antibody and the linker. In a particular embodiment, the binding protein includes a purified tag, such as 6-histidine peptide (His)6 (SEQ ID NO: 1531) (His-tag).

[0139] (Table 4) TIFF2026090564000428.tif158150TIFF2026090564000429.tif64150

[0140] IL27R-binding proteins are anti-IL27Rα V, as described in each row of Table 5 below. H In the H antibody, the three underlined CDR sequences and anti-gp130 V H The H antibody may contain the three underlined CDR sequences. In some embodiments, the IL27R-binding protein is anti-IL27Rα V as described in each row of Table 5 below. H H antibody and anti-gp130 V H Contains H antibody. In some embodiments, the binding protein has anti-IL27Rα V at its N-terminus. H H antibody and anti-gp130 V at the C-terminusH It contains an H antibody. In some embodiments, the binding protein has an anti-gp130 V at the N-terminus H H antibody and an anti-IL27Rα V at the C-terminus H It contains an H antibody. In some embodiments, the binding protein is an anti-IL27Rα V H H antibody and an 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).

[0141] (Table 5) TIFF2026090564000430.tif60150TIFF2026090564000431.tif223150TIFF2026090564000432.tif223150TIFF2026090564000433.tif21150

[0142] Furthermore, the IL27R binding protein can contain a mouse anti-IL27Rα V H H antibody and a mouse anti-gp130 V H H antibody. In some cases, due to sequence or structural similarities between the extracellular domains of receptors from various mammalian species, immunization with an antigen derived from IL27Rα or gp130 of a first mammalian species may produce antibodies that specifically bind to receptors of one or more further mammalian species. Such antibodies are referred to as “cross-reactive.” For example, immunization of camelids with a human-derived antigen (e.g., hIL27Rα-ECD) may produce antibodies that cross-react to mouse and human receptors. The evaluation of the cross-reactivity of antibodies with respect to receptors from other mammalian species can be readily determined by those skilled in the art using methods for evaluating binding affinity and / or specific binding, such as flow cytometry or SPR, as described elsewhere in this specification. Consequently, the use of the terms “human IL27Rα VHH” or “hIL27Rα VHH” should not be understood as merely indicating that the species of IL27Rα antigen used for immunizing camelids from which VHH originates is human IL27Rα, but not as limiting the specific binding affinity of VHH to IL27Rα molecules of other mammalian species. Similarly, the use of the terms “mouse IL27Rα VHH” or “mIL27Rα VHH” should not be understood as limiting the specific binding affinity of VHH to IL27Rα molecules in other mammalian species, but simply indicating that the species of IL27Rα antigen used for immunization of camelid animals from which VHH originates is mouse IL27Rα. In some embodiments, the IL27R binding protein is a mouse anti-IL27Rα V described below H Three CDR sequences underlined in the H antibody sequence and a mouse anti-gp130 V described below H Three CDR sequences underlined in the H antibody sequence. In some embodiments, the IL27R binding protein is a mouse anti-IL27Rα V described below H H antibody and a mouse anti-gp130 V described below H H antibody. In some embodiments, the binding protein has a mouse anti-IL27Rα V at the N-terminus H H antibody and a mouse anti-gp130 V at the C-terminus HIt contains an H antibody. In some embodiments, the binding protein has a mouse anti-gp130 V at the N-terminus H H antibody and a mouse anti-IL27Rα V at the C-terminus H H antibody. In some embodiments, the binding protein is a mouse anti-IL27Rα V H H antibody and a mouse 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] Mouse anti-IL27Rα V H Examples of H antibody sequences: TIFF2026090564000434.tif188150TIFF2026090564000435.tif68150

[0144] Mouse anti-gp130 V H Examples of H antibody sequences: TIFF2026090564000436.tif146150TIFF2026090564000437.tif224150TIFF2026090564000438.tif228150TIFF2026090564000439.tif220150TIFF2026090564000440.tif68150

[0145] In some embodiments, the V H H described herein can be humanized to include a human framework region. Humanized V HExamples of human germ cell lines that can be used to produce 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 is triggered by IL27. max E reduced compared to max It has. E max This reflects the maximum response level in a cell type that can be obtained by a ligand (e.g., a binding molecule described herein or a native cytokine (e.g., IL27)). In some embodiments, the IL27R binding protein described herein is triggered by IL27. max It has at least 1% (for example, 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%). In other embodiments, the E of the IL27R-binding protein described herein. max E is the natural ligand IL27 max Larger than (for example, at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% larger). In some embodiments, the E of the IL27R-binding protein is increased by altering the linker length of the IL27R-binding protein. max It can change the IL27R binding protein in the most desired cell type. max This results in reduced E in other cell types.max It could lead to this.

[0147] IV. Linker As previously described, the binding domain of the binding protein of this disclosure is sequential (for example, the first V in the binding protein) H The second V in the binding protein is the C-terminal amino acid of H. H The binding domain of the binding protein may be linked to the N-terminal amino acid of H, or optionally linked by a linker. A linker is a link between two elements, for example, between protein domains. The bispecificity V described herein H H 2 In binding proteins, the linker consists of two Vs in the binding protein. H It is a linkage between H atoms. The linker can be a covalent bond or a peptide linker. In some embodiments, two V atoms in the binding protein H H is directly linked (i.e., via covalent bonds). Two V in the binding protein H The length of the linker between H is the length of the two Vs of the binding protein. H It can be used to regulate the proximity of H. By changing the length of the linker, the overall size and length of the binding protein can be adjusted to bind to a specific cell 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 close to 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 may contain 1 to 50 amino acids (e.g., 2 to 50, 5 to 50, 10 to 50, 15 to 50, 20 to 50, 25 to 50, 30 to 50, 35 to 50, 40 to 50, 45 to 50, 2 to 45, 2 to 40, 2 to 35, 2 to 30, 2 to 25, 2 to 20, 2 to 15, 2 to 10, 2 to 5 amino acids). The linker may also be a synthetic polymer, such as a chemical linker like a polyethylene glycol (PEG) polymer.

[0149] In some embodiments, the linker is the first V in the binding protein. H The C-terminus of H is connected to the second V in the binding protein. H It is attached to the N-terminus of H. In another embodiment, the linker is a second V in the binding protein. H The C-terminus of H is connected to the first V in the binding protein. H It is attached to the N-terminus of H.

[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 may contain motifs, such as multiple or repeating motifs, of GS, GGS, GGGGS (SEQ ID NO: 85), GGGGGS (SEQ ID NO: 86), GGSG (SEQ ID NO: 87), or SGGG (SEQ ID NO: 88). In certain embodiments, the peptide linker may contain 2 to 12 amino acids including the GS motif, for example TIFF2026090564000441.tif11155 may be included. In certain other embodiments, the peptide linker may include 3 to 12 amino acids containing the GGS motif, for example GGS, GGSGGS (SEQ ID NO:94), GGSGGSGGS (SEQ ID NO:95) and GGSGGSGGSGGS (SEQ ID NO:96). In yet another embodiment, the peptide linker may include 4 to 20 amino acids containing the GGSG (SEQ ID NO:87) motif, for example It may include TIFF2026090564000442.tif18150. In other embodiments, the peptide linker may include the motif of GGGGS (SEQ ID NO:85), for example, GGGGSGGGGS (SEQ ID NO:101) or GGGGSGGGGSGGGGS (SEQ ID NO:102).

[0151] Examples of movable linkers include glycine polymers (G)n, glycine-alanine polymers, alanine-serine polymers, glycine-serine polymers (for example, (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 Examples of mobile linkers include NO:1542), as well as combinations thereof, where m, n, and o are each independently at least 1 to 20 (e.g., selected from integers 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 mobile linkers. Glycine and glycine-serine polymers are relatively unstructured and can therefore function as neutral tethers between components. Examples of mobile linkers include, but are not limited to, GGSG (SEQ ID NO:87), GGSGG (SEQ ID NO:103), GGSSG (SEQ ID NO:104), GSGGG (SEQ ID NO:105), GGGSG (SEQ ID NO:106), and GSSSG (SEQ ID NO:107).

[0152] Further examples of mobile linkers include 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). Examples of movable 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 to extend the duration of action in vivo The binding proteins described herein can be modified to provide in vivo extension of lifespan and / or extension of duration of action in a target. In some embodiments, the binding proteins can be conjugated to a carrier molecule to provide desired pharmacological properties, such as extension of half-life. In some embodiments, the binding proteins can be covalently bound to the Fc domain of IgG, albumin, or other molecules, for example, by pegylation, glycosylation, etc., as known in the art, in order to extend their half-life.

[0154] In some embodiments, the binding protein is conjugated to the functional domain of an Fc-fusion chimeric polypeptide molecule. Fc-fusion conjugates have been shown to increase the systemic half-life of biologics, thus allowing biologic products to require less frequent administration. Fc binds to neonatal Fc receptors (FcRn) in endothelial cells lining blood vessels. Upon binding, the Fc-fusion molecule is protected from degradation and re-released into circulation, allowing it to circulate for a longer period. This Fc binding is thought to be the mechanism by which endogenous IgG retains its long plasma half-life. Recent Fc-fusion technologies annex 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 the preparation of Fc fusions can be a native or synthetic polypeptide homologous to the IgG C-terminal domain produced by the digestion of IgG with papain. IgG Fc has a molecular weight of approximately 50 kDa. The binding proteins described herein can conjugate the entire Fc region or a smaller portion that retains the ability to extend the cyclic half-life of the chimeric polypeptide in which it constitutes 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 harbor a heterologous polypeptide, which may be the same or different.

[0155] In some embodiments, when the binding proteins described herein are administered in the form of Fc fusions, particularly when the polypeptide chains conjugated to each subunit of the Fc dimer are different, the Fc fusions may be manipulated to have a "knob-into-hole modification." Knob-into-hole modifications are well described by Ridgway, et al. (1996) Protein Engineering 9(7):617-621 and U.S. Patent No. 5,731,168 issued on March 24, 1998. 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 with an amino acid residue having a larger side chain (e.g., tyrosine or tryptophan), creating a protrusion ("knob") from the surface; and ii) an amino acid residue in the CH3 domain of the second heavy chain is replaced with an amino acid residue having a smaller side chain (e.g., alanine or threonine), thereby creating a cavity ("hole") at the interface in the second CH3 domain, within which 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 amino acid substitution T366W and optionally amino acid substitution S354C in one antibody heavy chain, and amino acid substitution T366S, L368A, Y407V and optionally Y349C in the other antibody heavy chain. Furthermore, the Fc domain may be modified by introducing cysteine ​​residues at positions S354 and Y349, which results in a stabilizing disulfide crosslink between the two antibody heavy chains in the Fe region (Carter, et al. (2001) Immunol Methods 248, 7-15). The knob-into-hole configuration is used to promote the expression of a first polypeptide on a first Fc monomer having a "knob" modification to promote the expression of a heterodimer polypeptide conjugate, and a second polypeptide on a second Fc monomer having a "hole" modification.

[0156] In some embodiments, the binding protein may be conjugated with one or more water-soluble polymers. Examples of water-soluble polymers useful in the practice of this disclosure include polyethylene glycol (PEG), polypropylene glycol (PPG), polysaccharides (polyvinylpyrrolidone, copolymer 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 may be conjugated, or "PEGylated," with one or more polyethylene glycol molecules. While the method or site of PEG attachment to the binding protein may vary, in certain embodiments, PEGylation does not alter, or only minimally alters, the activity of the binding protein.

[0158] In some cases, when the VHH sequence described herein is used in the preparation of the IL27 binding molecule of this disclosure, the VHH has an N-terminal glutamine ("1Q") residue. The N-terminal glutamine residue has been observed to spontaneously cyclize, either under physiological conditions or in its vicinity, to form pyroglutamic acid (pE). (See, for example, Liu, et al (2011) J. Biol. Chem. 286(13): 11211-11217). In some embodiments, the formation of pyroglutamic acid complicates the N-terminal PEG conjugation, particularly when aldehyde chemistry is used for N-terminal PEGylation. Consequently, when PEGylating the IL27R binding molecule of this disclosure, particularly when aldehyde chemistry is used, the IL27Rα binding molecule having 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 molecule of the present disclosure comprises amino acid substitutions selected from groups Q1E and Q1D.

[0159] In some embodiments, selective PEGylation of the binding protein may be utilized, for example, by incorporating non-natural amino acids having side chains that facilitate selective PEG conjugation. Specific PEGylation sites can be selected so that PEGylation of the binding protein does not affect its binding to the target receptor.

[0160] In certain embodiments, an increase in half-life is greater than any decrease in biological activity. PEGs suitable for conjugation into polypeptide sequences are generally water-soluble at room temperature, with the general formula R(O-CH2-CH2) where R is a protecting group such as hydrogen or an alkyl or alkanol group, and n is an integer between 1 and 1000. n It has OR. If R is a protecting group, it generally has 1 to 8 carbon atoms. The PEG conjugated with the polypeptide sequence can be linear or branched. Branched PEG derivatives, "star PEGs," and multi-armed PEGs are considered in this disclosure.

[0161] The molecular weight of PEG used in this disclosure is not limited to any particular range. The PEG component of the binding protein may have a molecular weight 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 weight is about 5 kDa to about 10 kDa, about 5 kDa to about 15 kDa, about 5 kDa to about 20 kDa, about 10 kDa to about 15 kDa, about 10 kDa to about 20 kDa, about 10 kDa to about 25 kDa, or about 10 kDa to about 30 kDa. Linear or branched PEG molecules have molecular weights of approximately 2,000 to approximately 80,000 daltons, alternatively approximately 2,000 to approximately 70,000 daltons, alternatively approximately 5,000 to approximately 50,000 daltons, alternatively approximately 10,000 to approximately 50,000 daltons, alternatively approximately 20,000 to approximately 50,000 daltons, alternatively approximately 30,000 to approximately 50,000 daltons, alternatively approximately 20,000 to approximately 40,000 daltons, or alternatively approximately 30,000 to approximately 40,000 daltons. In one embodiment of this disclosure, PEG is a 40 kD branched PEG containing two 20 kD arms.

[0162] The present disclosure also contemplates conjugate compositions in which the PEG has different n values and thus different PEGs are present in specific 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 attached PEGs.

[0163] PEGs suitable for conjugation to polypeptide sequences are generally water-soluble at room temperature, where R is hydrogen or a protecting group such as an alkyl or alkanol group, and n is an integer from 1 to 1000, of the general formula R(O-CH2-CH2) n has O-R. 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 react preferentially 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 commonly occurs at the α-amino group at the N-terminus of a polypeptide, the epsilon-amino group at the side chain of a lysine residue, and the imidazole group at the side chain of a histidine residue. Since most recombinant polypeptides have a single α-amino group as well as several ε-amino and imidazole groups, numerous positional isomers can be generated depending on the chemical properties of the linker. General PEGylation strategies known in the art can be applied herein.

[0166] PEG can be conjugated to the binding protein of this disclosure by terminal reactive groups ("spacers") that mediate the binding between a free amino group or carboxyl group of one or more polypeptide sequences and polyethylene glycol. PEG having spacers that can be conjugated to a free amino group comprises N-hydroxysuccinilimide polyethylene glycol, which can be prepared by activating a succinate ester of polyethylene glycol with N-hydroxysuccinilimide.

[0167] In some embodiments, site-directed PEGylation of binding proteins is promoted by incorporating non-natural amino acids that support unique side chains. Incorporating non-natural amino acids into polypeptides to provide functional portions for achieving such site-directed PEGylation of polypeptides is known in the art. For example, see PCT international application number PCT / US2018 / 045257, filed on 3 August 2018 and published on 7 February 2019 as international publication number WO 2019 / 028419Al by Ptacin et al.

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

[0169] In some embodiments, linkers can be used to link binding proteins and PEG molecules. A suitable linker generally comprises a modified polypeptide sequence and a “mobile linker” of sufficient length to allow some movement between the linked components and molecules. Linker molecules are generally about 6–50 atomic lengths. Linker molecules may be, for example, arylacetylenes, ethylene glycol oligomers containing 2–10 monomer units, diamines, diacitors, amino acids, or combinations thereof. A suitable linker can be readily selected and can be any suitable length, e.g., 1 amino acid length (e.g., Gly), 2, 3, 4, 5, 6, 7, 8, 9, 10, 10–20, 20–30, 30–50, or over 50 amino acid lengths. Examples of mobile linkers are described in Section IV. Furthermore, multimers of these linker sequences (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 10-20, 20-30, or 30-50) may be linked together to provide a mobile linker that can be used to conjugate two molecules. Instead of polypeptide linkers, the linker can be a chemical linker, such as a PEG-aldehyde linker. In some embodiments, the binding protein is acetylated at its N-terminus by an enzymatic reaction with an N-terminal acetyltransferase and, for example, acetyl-CoA. Alternatively, or in addition to N-terminal acetylation, the binding protein may be acetylated at one or more lysine residues by an enzymatic reaction with, for example, a lysine acetyltransferase. See, for example, Choudhary et al. (2009) Science 325 (5942):834-840.

[0170] In other embodiments, the binding protein may be modified to include a further polypeptide sequence that functions as an antigenic tag, such as a FLAG sequence. The FLAG sequence is recognized by a biotinylated high-specificity anti-FLAG antibody as described herein (see, e.g., 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 known in the art (e.g., human serum albumin) to facilitate long-term in vivo exposure.

[0172] In some embodiments, the binding proteins of this disclosure (including fusion proteins of the binding proteins) are expressed as fusion proteins having one or more transition metal chelate polypeptide sequences. Incorporation of such transition metal chelate domains facilitates purified 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 chelate polypeptides useful in the practice of this disclosure are described in Smith et al., and Dobeli et al., U.S. Patent No. 5,320,663, issued May 10, 1995, the full teachings of which are incorporated herein by reference. Specific transition metal chelate polypeptides useful in the practice of this disclosure are peptides containing 3 to 6 consecutive histidine residues (SEQ ID NO: 1547), for example, 6-histidine peptide (His)6 (SEQ ID NO: 1531), often referred to in the art as "His tags."

[0173] The aforementioned fusion protein can be readily produced by recombinant DNA methods known in the art by constructing a recombinant vector in which the nucleic acid sequence encoding the binding protein is placed in frame at either the N-terminus or C-terminus of the binding protein, and the nucleic acid sequence encoding the fusion partner may optionally further include a nucleic acid sequence encoding a linker or spacer polypeptide in frame.

[0174] VI. Pharmaceutical Compositions The binding proteins of this disclosure may be administered to subjects in pharmaceutically acceptable dosage forms. Preferred formulations depend on the intended method of administration and therapeutic use. The pharmaceutically acceptable 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, e.g., human serum albumin, buffers, e.g., phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, e.g., protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulosic substances, and PEG. Supports of polypeptides in local or gel forms 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 pharmaceutically acceptable, non-toxic carriers, excipients, stabilizers, or diluents, which are defined as vehicles commonly used to formulate the pharmaceutical composition 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 doses and concentrations used and include: buffers, e.g., phosphates, citrates, and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (e.g., octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl, or benzyl alcohol; alkylparabens, e.g., methyl or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than approximately 10 residues) polypeptides; proteins, e.g., serum albumin, gelatin, or immunoglobulins; hydrophilic polymers, e.g., polyvinylpyrrolidone; amino acids, e.g., glycine, glutamine, asparagine, histidine, arginine, or lysine; This includes monosaccharides, disaccharides, and other sugars 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 used for in vivo administration are typically sterile. Sterilization of the compositions of this disclosure can be easily achieved by filtration through a sterile filtration membrane.

[0177] Typically, the compositions are prepared as injectable preparations, either as a liquid solution or a suspension; a solid form suitable for the solution or suspension of the liquid vehicle may also be prepared before injection. Preparations may also be emulsified or encapsulated in liposomes or microparticles such as polylactides, polyglycolides, or copolymers for enhanced adjuvant effects, as discussed above (Langer, Science 249: 1527, 1990 and Hanes, Advanced Drug Delivery Reviews 28: 97-119, 1997). The active ingredients of this disclosure may be administered in the form of accumulative injections or implantable preparations, which may be formulated in a manner that allows for sustained or pulsed release of the active ingredient. Pharmaceutical compositions are generally sterile, substantially isotonic, and formulated in full compliance with all Good Manufacturing Practice (GMP) regulations of the U.S. Food and Drug Administration.

[0178] The administration of binding proteins described herein may be achieved through any of the various methods approved in the art, including but not limited to local, intravascular injection (including intravenous or intra-arterial injection), intradermal injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, intracranial injection, intratumoral injection, intranodular injection, percutaneous, transmucosal, iontophoresis delivery, intralymphatic injection (Senti and Kundig (2009) Current Opinions in Allergy and Clinical Immunology 9(6):537-543), intragastric injection, intraprostatic injection, intracapsular injection (e.g., bladder), respiratory inhalers including nebulizers, intraocular injection, intraperitoneal injection, intrafocal injection, intraovarian injection, intracerebral injection or injection, intraventricular injection (ICVI), etc. In some embodiments, administration may include the administration of the binding protein itself (e.g., parenteral), as well as the administration of recombinant vectors (e.g., viral or nonviral vectors) to induce in situ expression of the binding protein in the subject. Alternatively, cells, such as those isolated from the target, can be recombinantly modified to express the binding protein of this disclosure.

[0179] The dosage of a pharmaceutical composition depends on factors such as the route of administration, the disease being treated, and the physical characteristics of the subject, including age, weight, and general health. Typically, the amount of binding protein contained in a single dose may be sufficient to effectively prevent, delay, or treat the disease without inducing significant toxicity. The pharmaceutical compositions disclosed herein are available in doses of 0.01 to 500 mg / kg (for example, 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). mg / kg, 400-500 mg / kg or 450-500 mg / kg) and, in a more specific embodiment, about 1-100 mg / kg (e.g., about 1-90 mg / kg, about 1-80 mg / kg, about 1-70 mg / kg, about 1-60 mg / kg, about 1-50 mg / kg, about 1-40 mg / kg, about 1-30 mg / kg, about 1-20 mg / kg, about 1-10 mg / kg, about 10-100 mg / kg, about 20-100 mg / kg, about 30-100 mg / kg, about 40-100 mg / kg, about 50-100 mg / kg, about 60-100 mg / kg, about 70-100 mg / kg, about 80-100 mg / kg or about 90-100 mg / kg) This may include doses of the binding protein described herein in the range of mg / kg.In some embodiments, the pharmaceutical compositions of this disclosure may include doses of the binding protein described herein in the range of 0.01 to 20 mg / kg (e.g., 0.01 to 15 mg / kg, 0.01 to 10 mg / kg, 0.01 to 8 mg / kg, 0.01 to 6 mg / kg, 0.01 to 4 mg / kg, 0.01 to 2 mg / kg, 0.01 to 1 mg / kg, 0.01 to 0.1 mg / kg, 0.01 to 0.05 mg / kg, 0.05 to 20 mg / kg, 0.1 to 20 mg / kg, 1 to 20 mg / kg, 2 to 20 mg / kg, 4 to 20 mg / kg, 6 to 20 mg / kg, 8 to 20 mg / kg, 10 to 20 mg / kg, 15 to 20 mg / kg). The dose may be adapted by a physician according to conventional factors such as the severity of the disease and different parameters of the subject.

[0180] Pharmaceutical compositions containing binding proteins described herein may be administered to subjects in need, for example, once or multiple times (e.g., 1 to 10 times or more) daily, weekly, monthly, twice a year, annually, or as medically required. Dosages may be provided in single-dose or multi-dose regimens. The timing between doses may decrease as the medical condition improves and increase as the patient's health deteriorates. The course of treatment may be a single dose or a multi-dose regimen over a period of time. In some embodiments, a single dose is used. In some embodiments, two or more divided doses are used, 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 in each dose. A multi-day medication protocol over a certain period can be provided by a person skilled in the art (e.g., a physician) by monitoring the administration, taking into account the patient's response to the treatment, including side effects of the treatment and their adjustment, as discussed above.

[0181] VII. Indications immune disease This disclosure further provides a method for treating a subject suffering from a disease, disorder, or condition by administering a therapeutically effective dose of the IL27R-binding protein of this disclosure (or nucleic acid encoding the IL27R-binding protein, including a recombinant virus encoding the IL27R-binding protein). Disorders suitable for treatment with the IL27R-binding protein of this disclosure (including pharmaceutically acceptable formulations comprising the IL27R-binding protein and / or nucleic acid molecule encoding such IL27R-binding protein, including a recombinant virus encoding such IL27R-binding protein) include viral infections (e.g., AIDS, influenza, chronic HCV, chronic viral hepatitis B, C, or D), and Helicobacter pylori. 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, paraneoplastic autoimmune diseases, chondritis, arthritis, rheumatoid arthritis, juvenile arthritis, polyarticular juvenile rheumatoid arthritis, systemic juvenile rheumatoid arthritis, juvenile ankylosing spondylitis, juvenile enteroarthritis, juvenile reactive arthritis, juvenile Reiter's syndrome, SEA syndrome (seronegativity enthesopathy arthropathy). This includes inflammatory or autoimmune diseases, including but not limited to rheumatoid arthritis, juvenile dermatomyositis, juvenile psoriatic arthritis, juvenile scleroderma, juvenile systemic lupus erythematosus, juvenile vasculitis, small-joint rheumatoid arthritis, polyarticular rheumatoid arthritis, systemic rheumatoid arthritis, ankylosing spondylitis, enteritis arthritis, reactive arthritis, Reiter's syndrome, and SEA syndrome (seronegative enthesopathy, arthropathy syndrome).

[0182] Other examples of proliferative and / or differentiation disorders suitable for treatment with the IL27R-binding proteins of this disclosure (including recombinant viruses encoding such IL27R-binding proteins, and pharmaceutically acceptable formulations containing IL27R-binding proteins and / or nucleic acid molecules encoding such IL27R-binding proteins) include, but are not limited to, skin disorders. Skin disorders may involve abnormal activity of cells or groups of cells in the dermis, epidermis, or subcortex, or abnormalities at the dermal-epidermal junction. For example, skin disorders may involve 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 epidermis, such as the basal layer (stratum germinativum), spinous layer, granular layer, slender layer, or stratum corneum. In other embodiments, the disorder may be accompanied by abnormal activity of dermal cells in the dermis, such as the papillary or reticular layer, e.g., dermal endothelium, fibroblasts, and immune cells (e.g., mast cells or macrophages).

[0183] Examples of skin disorders include psoriasis, psoriatic arthritis, dermatitis (eczema), e.g., exfoliative dermatitis or atopic dermatitis, pityriasis rubra pilaris, pityriasis rosea, parapsoriasis, pityriasis lichenoid, lichen planus, lichen patella, ichthyosis-like dermatitis, keratoderma, skin diseases, alopecia areata, pyoderma gangrenosum, vitiligo, bullous pemphigoid (e.g., ocular scarring pemphigoid or bullous pemphigoid), urticaria, porokeratosis, and hyperproliferation of epithelial-associated cells lining the joint capsule. These include rheumatoid arthritis with inflammation, dermatitis such as seborrheic dermatitis and photodermatitis, keratosis such as seborrheic keratosis, senile keratosis, actinic keratosis, photo-induced keratosis and follicular keratosis, acne vulgaris, keloids and prevention of keloid formation, human papillomavirus (HPV) infections such as nevi, warts, condyloma or genital warts, and genital warts, leukoplakia, lichen planus, and keratitis. Skin disorders can be dermatitis, such as atopic dermatitis or allergic dermatitis, or psoriasis.

[0184] The compositions of this disclosure (including pharmaceutically acceptable formulations comprising a recombinant virus encoding such an IL27R-binding protein and / or a nucleic acid molecule encoding such an IL27R-binding protein) may also be administered to patients who have (or may have) psoriasis or a psoriatic disorder. The term “psoriasis” is intended to have its medical meaning, namely, having a disease that primarily affects the skin, causing raised, thickened, scaly, and ulcerative lesions. These lesions are typically well-defined erythematous papules covered with overlapping, shiny scales. The scales are usually silvery or slightly opaque. Nail involvement frequently occurs, resulting in pitting, detachment, thickening, and discoloration of the nails. Psoriasis may also be associated with arthritis, which can be debilitating. Overgrowth of keratinocytes, along with inflammation of the epidermis and decreased keratinocyte differentiation, is a major feature of psoriatic epidermal hyperplasia. Several mechanisms have been proposed to explain the keratinocyte hyperproliferation that characterizes psoriasis. Furthermore, abnormalities in cellular immunity are also thought to be involved in the pathogenesis of psoriasis. Examples of psoriatic disorders include chronic constant psoriasis, plaque psoriasis, moderate to severe plaque psoriasis, plaque psoriasis, exanthematous psoriasis, erythrodermic psoriasis, generalized pustular psoriasis, annular pustular psoriasis, or focal pustular psoriasis.

[0185] Combinations of IL27R-binding protein with additional therapeutic agents for autoimmune diseases: This disclosure provides the use of the IL27R-binding protein of this disclosure in combination with one or more additional active agents ("adjunct agents") in the treatment of autoimmune diseases. Where used herein, the term "adjunct agent" includes agents that can be administered or introduced separately, for example, agents separately formulated for another administration that can be administered or introduced in combination with the IL27R-binding protein (for example, may be provided in a kit) and / or therapeutics.

[0186] As used herein, the term “in combination with” means, when used in relation to the administration of multiple active agents to a subject, the administration of a first active agent and at least one additional (i.e., a second, third, fourth, fifth, etc.) active agent to the subject. For the purposes of the present invention, one active agent (e.g., IL27R-binding protein) is considered to be administered in combination with a second active agent (e.g., a therapeutic autoimmune antibody such as Humira®) if the biological effect resulting from the administration of the first active agent persists in the subject at the time of administration of the second active agent, and as a result, the therapeutic effects of the first active agent and the therapeutic effects of the second active agent overlap. For example, a therapeutic antibody may be administered every two weeks by IV infusion (e.g., adalimumab in the treatment of Crohn's disease), but the IL27R-binding protein of this disclosure may be administered more frequently, for example, daily, by IV, or weekly. However, while the administration of a first active agent (e.g., etanercept) provides a long-term therapeutic effect, and the administration of a second active agent (e.g., IL27R-binding protein) provides its therapeutic effect, the therapeutic effect of the first active agent is sustained, and as a result, even if the first active agent was administered at a time significantly separated from the administration time of the second active agent (e.g., several days or weeks), it is considered that the second and first active agents are administered together. In one embodiment, an active agent is considered to be administered together with a second active agent if the first and second active agents are administered simultaneously (within 30 minutes of each other), concurrently or sequentially. In some embodiments, if the first active substance and the second active substance are administered to each other within about 24 hours, preferably within about 12 hours, preferably within about 6 hours, preferably within about 2 hours, or preferably within about 30 minutes, the first active substance is considered to be administered "concurrently" with the second active substance. It should also be understood that the term "in combination" applies to situations in which the first active substance and the second active substance 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 active agent is administered before one or more other active agents, the IL27R-binding protein and co-active agents are administered or applied sequentially. In other embodiments, for example, if two or more active agents are administered simultaneously or approximately simultaneously, the IL27R-binding protein and co-active agents are administered simultaneously; the two or more active agents may be present in two or more separate formulations or combined as a single formulation (i.e., a co-formulation). Whether the active 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 adjunct is one or more adjuncts selected from the group consisting of corticosteroids (including but not limited to prednisone, budesonide, and prednirisone), 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), biologics such as abatacept (Orencia®) or etanercept (Enbrel®), and therapeutic antibodies. Examples of therapeutic antibodies that may be administered as adjuvants in combination with the IL27R binding protein of this disclosure in the treatment of autoimmune diseases include anti-CD25 antibodies (e.g., daclizumab and basiliximab), anti-VLA-4 antibodies (e.g., natalizumab), anti-CD52 antibodies (e.g., alemtuzumab), anti-CD20 antibodies (e.g., rituximab, ocrelizumab), anti-TNF antibodies (e.g., infliximab and adalimumab), anti-IL6R antibodies (e.g., tocilizumab), and anti-TNFα antibodies (e.g., adalimumab (Humi Examples of anti-IL antibodies include, but are not limited to, LA®, golimumab and infliximab, anti-integrin-α4β7 antibodies (e.g., vedolizumab), anti-IL17a antibodies (e.g., brodalumab or secukinumab), anti-IL4Rα antibodies (e.g., dupilumab), anti-RANKL antibodies, IL6R antibodies, anti-IL1β antibodies (e.g., canakinumab), anti-CD11a antibodies (e.g., efalizumab), anti-CD3 antibodies (e.g., muramonab), anti-IL5 antibodies (e.g., mepolizumab, reslizumab), anti-BLyS antibodies (e.g., belimumab), and anti-IL12 / IL23 antibodies (e.g., ustekinumab).

[0188] Many therapeutic antibodies have been approved for clinical use in the treatment of autoimmune diseases. Table 4 provides examples of antibodies approved by the U.S. Food and Drug Administration (FDA) for use in the treatment of autoimmune diseases in subjects suffering from autoimmune diseases, which may be administered as adjuvants in combination with the IL27R-binding protein (and optionally additional adjuvants) of this disclosure for the treatment of the indicated autoimmune diseases.

[0189] (Table 4) TIFF2026090564000443.tif207159

[0190] Treatment of neoplasms This disclosure provides a method for using IL-27R-binding molecules in the treatment of subjects suffering from neoplastic disease disorders or conditions by administering therapeutically effective doses of IL-27R-binding molecules (or nucleic acids encoding IL-27R-binding molecules, including recombinant vectors encoding IL-27R-binding molecules, as well as eukaryotic and prokaryotic cells modified to express IL-27R-binding molecules) as described herein.

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

[0192] Examples of benign neoplasms suitable for treatment using the compositions and methods of this 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 this disclosure include, but are not limited to, hyperplasia, atypia, metaplasia, and dysplasia. Examples of malignant neoplasms suitable for treatment using the compositions and methods of this disclosure include, but are not limited to, cancer (cancer arising from epithelial tissue such as skin or tissue covering internal organs), leukemia, lymphoma, and sarcomas, which typically originate from bone fat, 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), and hyperproliferative vascular diseases including scar formation, intimal smooth muscle cell hyperplasia, restenosis, and vascular occlusion.

[0193] The term “neoplasmic disease” includes cancers characterized by solid and non-solid tumors, including but not limited to breast cancer, sarcomas (including, but not limited to, osteosarcoma, angiosarcoma, and fibrosarcoma), leukemia, lymphoma, 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 cancer, skin cancer, and tumors of the brain or central nervous system (CNS) and peripheral nervous system, including gliomas and neuroblastomas, astrocytomas, myelodysplastic disorders, malignant or benign tumors, cervical carcinoma in situ, intestinal polyposis, oral leukoplakia, histiocytosis, hyperproliferative scars including keloid scars, hemangiomas, hyperproliferative arterial stenosis, psoriasis, inflammatory arthritis, hyperkeratosis including arthritis, and papular desquamation.

[0194] The term neoplasm includes cancer. The term "cancer" refers to malignant diseases of epithelial or endocrine tissue, including respiratory cancers, digestive cancers, genitourinary cancers, testicular cancers, breast cancers, prostate cancers, endocrine cancers, and melanomas. The term neoplasm also includes adenocarcinoma. "Adenocarcinoma" refers to cancer that originates from glandular tissue or forms glandular structures that are recognizable by tumor cells.

[0195] As used herein, the term “hematopoietic neoplasm” means a neoplasm of hematopoietic origin, such as from bone marrow, lymphoid or erythrocyte lineage, or its progenitor cells, that involves hyperplasia / 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 neoplasms, and acute leukemias of unclear differentiation lineage. Exemplary myeloid disorders suitable for treatment according to this 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, progenitor lymphoid neoplasms, mature B-cell neoplasms, mature T-cell neoplasms, Hodgkin lymphoma, and immunodeficiency-associated lymphoproliferative disorders. Exemplary lymphoid disorders suitable for treatment according to this disclosure include, but are not limited to, 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 hypergammaglobulinemia (WM).

[0198] In some cases, hematopoietic neoplasm disorders result from poorly differentiated acute leukemias (e.g., erythroblastic leukemia and acute megakaryoblastic leukemia). As used herein, the term “hematopoietic neoplasm 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 (LGF), Hodgkin’s disease, and Reed-Sternberg disease.

[0199] The determination of whether a subject has a "neoplasmic disease" refers to a physician's assessment of a subject based on accepted and available information in the field for identifying a disease, disorder, or condition, including but not limited to X-rays, CT scans, conventional clinical diagnostic tests (e.g., blood counts), genomic data, protein expression data, and immunohistochemistry, that the subject requires or would benefit from treatment.

[0200] Evaluation of antineoplasm efficacy: Determining the effectiveness of the methods of this disclosure in the treatment of cancer generally relates to achieving one or more parameters approved in the art, such as reduction of lesions, particularly reduction of metastatic lesions, reduction of metastasis, reduction of tumor volume, improvement of ECOG score, and others. Determining the response to treatment can be assessed through the measurement of biomarkers that can provide useful, reproducible information in any aspect of IL-27R conjugate molecular therapy, including the presence and extent of the subject's response to such therapy and the presence and extent of adverse effects caused by such therapy. Examples, but not limited to, include: increased IFNγ, as well as upregulation of granzyme A, granzyme B, and perforin; increased number and enhanced function of CD8+ T cells; increased IFNγ, increased ICOS expression on CD8+ T cells, IL-10 expression T Reg This includes an increase in cells. Treatment response may be characterized by improvement on conventional measures of clinical efficacy, which may include not only complete response (CR), partial response (PR), and stable disease (SD) as defined by RECIST, 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 demonstrate efficacy in the treatment of neoplasms in mammalian subjects (e.g., humans).

[0201] Maintaining serum concentration: In some aspects of the present invention, the Disclosure provides methods and compositions for the treatment and / or prevention of neoplasms, disorders or conditions by administering a therapeutically effective dose of an IL-27R-binding molecule, wherein the serum concentration of the IL-27R-binding molecule is maintained at a therapeutically effective concentration or greater than a therapeutically effective concentration with respect to such an IL-27R-binding molecule for most of the period (i.e., longer than about 50% of the period, alternatively longer than about 60%, alternatively longer than about 70%, alternatively longer than about 80%, alternatively longer than about 90%) (for example, 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] Combinations of IL-27R binding molecules and therapeutic adjuvants: This disclosure provides methods for using the IL-27R conjugate molecule of this disclosure in combination with one or more further active agents ("adjunct agents"). Such further combinations are interchangeably referred to as "adjunct combinations" or "adjunct combination therapies," and therapeutic agents used in combination with the IL-27R conjugate molecule of this disclosure are referred to as "adjunct agents." As used herein, the term "adjunct agent" includes agents that can be administered or introduced separately, e.g., agents separately formulated for another administration that can be administered or introduced in combination with the IL-27R conjugate molecule (e.g., may be provided in a kit) and / or therapies.

[0203] Chemotherapy agents: In some embodiments, the adjunct agent is a chemotherapeutic agent. In some embodiments, the adjunct agent is a "cocktail" of multiple chemotherapeutic agents. In some embodiments, the chemotherapeutic agents or cocktail are administered in combination with one or more physical methods (e.g., radiotherapy). The term "chemotherapeutic agent" includes alkylating agents, e.g., thiotepa and cyclophosphamide; alkyl sulfonates, e.g., busulfan, improsulfan and piposulfan; aziridines, e.g., benzodepa, carbocon, metsuredepa and uredepa; ethyleneimines and methylamelamines, including altoretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide and trimethylolmelamine; nitrogen mustards, e.g., chlorambucil, chlornafadin, chlorophosphamide, estramustine, ifosfamide, mechloretamine, mechloretamine oxide hydrochloride, melphalan, nobuenvicin, fenesterine, prednimustine, trophosphamide, uracil mustard; and nitrosoureas, e.g., carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine. Antibiotics such as acrasinomycin, actinomycin, anthramycin, azaserin, bleomycin A2, kactinomycin, calichemycin, carabicin, caminomycin, cardinophilin, chromomycin, dactinomycin, daunorubicin and its derivatives, demethoxy-daunomycin, 11-deoxydaunorubicin, 13-deoxydaunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, esorubicin, idarubicin, and other mitomycins, marcelomycin, mitomycin C, N-methylmitomycin C, etc. Mycophenolic acid, nogaramycin, olibomycin, peplomycin, potfiromycin, puromycin, queramycin, rhodorubicin, streptonigrin, streptozocin, tubercidine, ubenimex, dinostatin, zolubicin; antimetabolites, such as methotrexate and 5-fluorouracil (5-FU);Folic acid analogs, e.g., denopterin, methotrexate, pteropterin, trimethrexate, dideazatetrahydrofolate, and folinic acid; purine analogs, e.g., fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs, e.g., ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxlysine, 5-FU; androgens, e.g., carsterone, dromostanolone propionate, epithiostanol, mepitiostane, testolactone; anti-adrenal drugs, e.g., aminoglutethimide, mitotane, trilostane; folic acid supplements, e.g., flolinic acid; acegraton; aldofsphamide glycoside; aminolevulinic acid; amsacrine; bestrabusil; bisantren; Edatrexate; Defofamine; Demecolsin; Diadiquan; Eflornithine; Erliptinium acetate; Etoglucide; Gallium nitrate; Hydroxyurea; Lentinan; Ronidamin; Mitoguazone; Mitoxantrone; Mopidamol; Nitracrine; Pentostatin; Fenamet; Pirarubicin; Podophyllic acid; 2-Ethylhydrazide; Procarbazine; Lazoxane; Schizophyllan; Spirogermanium; Tenuazonic acid; Triadicone; 2,2',2"-Trichlorotriethylamine; Urethane; Vindesine; Dacarbazine; Mannomustine; Mitobronitol; Mitractol; Pipobroman; Gacitosine; Arabinoside (Ara-C); Cyclophosphamide; Thiotepa; Taxoids, e.g., paclitaxel, nab-paclitaxel, and doxetaxel; chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum and platinum-coordinate compounds, e.g., cisplatin, oxaplatin, and carboplatin; vinblastine; etoposide (VP-16); ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; navelbine; novantrone; teniposide; daunomycin; aminopterin; xeloda; ibandronate;CPT11; topoisomerase inhibitors; difluoromethylornithine (DMFO); retinoic acid; esperamicin; capecitabine; taxanes, e.g., paclitaxel, docetaxel, carbaditaxel; carminomycin, adriamycin, e.g., 4'-epiaadriamycin, 4-adriamycin-14-benzoate, adriamycin-14-octanoate, adriamycin-14-naphthalene acetate; colchicine and any pharmaceutically acceptable salts, acids, or derivatives of the above, but not limited to these.

[0204] The term “chemotherapeutic agents” also includes anti-estrogen agents, such as tamoxifen, raloxifen, aromatase inhibitory 4(5)-imidazole, 4-hydroxytamoxifen, trioxyfen, keoxyfen, onapristone, and toremifene; as well as anti-androgen agents, such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; and anti-hormonal 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 above.

[0205] In some embodiments, adjuvants include, but are 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 experienced clinicians in the art, as practiced in known chemotherapy regimens: cytokines or cytokine antagonists, e.g., IL-12, INFα, or anti-epithelial growth factor receptors, irinotecan; tetrahydrofolate antimetabolites, e.g., pemetrexed; antibodies against tumor antigens, monoclonal antibody-toxin complexes, T-cell adjuvants, bone marrow grafts, or antigen-presenting cells (e.g., dendritic cell therapy), antitumor vaccines, replicable viruses, signaling inhibitors (e.g., Gleevec® or Herceptin). One or more chemotherapeutic or biological agents identified in the art as useful for treating neoplasms, including but not limited to one or more combinations of the above, including immunomodulatory factors for achieving additive or synergistic suppression of tumor growth (registered trademark), nonsteroidal anti-inflammatory drugs (NSAIDs), cyclooxygenase-2 (COX-2) inhibitors, steroids, TNF antagonists (e.g., Remicade (registered trademark) and Enbrel (registered trademark)), interferon-β1a (Avonex (registered trademark)), and interferon-β1b (Betaseron (registered trademark)), for achieving additive or synergistic suppression of tumor growth.

[0206] In some embodiments, IL-27R-binding molecules are administered in combination with BRAF / MEK inhibitors, kinase inhibitors such as sunitinib, PARP inhibitors such as olaparib, EGFR inhibitors such as osimertinib (Ahn, et al. (2016) J Thorac Oncol 11:S115), IDO inhibitors such as epacadostat, and oncolytic viruses such as tarimozine-laharpalepbec (T-VEC).

[0207] Antitumor antigen antibody therapeutic substances as adjunct agents In some embodiments, the “adjunct agent” is a therapeutic antibody (including, but not limited to, bispecific T cell-inducing antibodies (BITEs), biaffinity retargeting (DART) constructs, and trispecific killer enforcer (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, ibiritumomab, 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 It is an antibody that binds to at least one tumor antigen selected from the group consisting of VEGF (e.g., bevacizumab), VEGFR, VEGFR2 (e.g., ramucirumab), PDGFRα (e.g., ofatumumab (olartumumab)), EGFR (e.g., cetuximab, panitumumab, and nesitumumab), ERBB2 (e.g., trastuzumab), ERBB3, MET, IGF1R, EPHA3, TRAIL R1, TRAIL R2, RANKL RAP, tenascin, integrin αVβ3, and integrin α4β1.

[0209] Examples of FDA-approved antibody therapeutics that can be used as adjuvant agents for the treatment of neoplasms include those provided in the table below.

[0210] (Table) Approved antineoplasmic disease antibodies and indications TIFF2026090564000444.tif85150TIFF2026090564000445.tif171150

[0211] physical method In some embodiments, the adjuvant is one or more non-pharmacological means (e.g., local or total body radiotherapy or surgery). As an example, the disclosure considers a treatment regimen in which the radiation phase is preceded or followed by treatment using a treatment regimen comprising an IL-27R-binding molecule and one or more adjuvants. In some embodiments, the disclosure further considers the use of the IL-27R-binding molecule in combination with surgery (e.g., tumor resection). In some embodiments, the disclosure further considers the use of the IL-27R-binding molecule in combination with bone marrow transplantation, peripheral blood stem cell transplantation or other types of transplantation therapy.

[0212] Combination with immune checkpoint regulators: In some embodiments, “adjunctivitis” are immune checkpoint modifiers for treating and / or preventing not only neoplasms in a subject but also diseases, disorders, or conditions associated with neoplasms. The term “immune checkpoint pathway” refers to a biological response induced by the binding of a first molecule (e.g., a protein such as PD1) expressed on antigen-presenting cells (APCs) to a second molecule (e.g., a protein such as PDL1) expressed on immune cells (e.g., T cells), which modulates the immune response either by stimulating (e.g., upregulation of T cell activity) or inhibiting (e.g., downregulation of T cell activity). The molecules involved in the formation of binding pairs that modulate the immune response are generally called “immune checkpoints.” The biological responses modulated 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. Immune checkpoint pathways are generally triggered by the binding of a primary cell surface molecule to a secondary cell surface molecule associated with the immune checkpoint pathway (e.g., PD1 binding to PDL1, CTLA4 binding to CD28, etc.). Activation of immune checkpoint pathways can result in stimulation or inhibition of the immune response.

[0213] As used herein, the term “immune checkpoint pathway modulator” refers to a molecule that inhibits or stimulates the activity of an immune checkpoint pathway in a biological system, including an immune-eligible mammal. Immune checkpoint pathway modulators may exert their effects by binding to immune checkpoint proteins (such as immune checkpoint proteins expressed on the surface of antigen-presenting cells (APCs), such as cancer cells and / or immune T effector cells), or by influencing upstream and / or downstream reactions in the immune checkpoint pathway. For example, immune checkpoint pathway modulators may modulate the activity of SHP2, a tyrosine phosphatase involved in PD-1 and CTLA-4 signaling. The term “immune checkpoint pathway modulator” encompasses both immune checkpoint pathway modulators that can at least partially downregulate the function of inhibitory immune checkpoints (hereinafter referred to as “immune checkpoint pathway inhibitors” or “immune checkpoint pathway antagonists”) and immune checkpoint pathway modulators that can at least partially upregulate the function of stimulative immune checkpoints (hereinafter referred to as “immune checkpoint pathway effectors” or “immune checkpoint pathway agonists”).

[0214] Immune checkpoint modulators include, but are not limited to, immune checkpoint antagonists (e.g., antagonist antibodies) that bind to T cell inhibitory receptors, including, but are not limited to, 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, immune checkpoint modulators are agonists that induce checkpoint pathways resulting 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 ICOSL 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, but are not limited to, agonist antibodies that bind to T cell activating receptors such as ICOS (e.g., JTX-2011, Jounce Therapeutics), OX40 (e.g., MEDI6383, Medimmune), CD27 (e.g., valrirumab, 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).

[0215] Examples of 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 anticytotoxic T lymphocyte antigen 4 (CTLA4) pathway inhibitors.

[0216] In one embodiment, immune checkpoint pathway modulators are antagonists of the negative immune checkpoint pathway ("PD1 pathway inhibitors") that inhibit the binding of PD1 to PDL1 and / or PDL2. 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 adjunct agents in the treatment of neoplasms include, but are not limited to, nivolumab (Opdivo®, BMS-936558, MDX1106, BristolMyers Squibb, marketed by Princeton NJ), pembrolizumab (Keytruda®, MK-3475, lambrolizumab, marketed by Merck and Company, Kenilworth NJ), and atezolizumab (Tecentriq®, Genentech / Roche, South San Francisco CA), which are antibodies that interfere with the binding of PD1 to PDL1 and / or PDL2. Further PD1 pathway inhibitory antibodies, including but not limited to durvalumab (MEDI4736, Medimmune / AstraZeneca), pidilizumab (CT-011, CureTech), PDR001 (Novartis), BMS-936559 (MDX1105, BristolMyers 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 issued on July 10, 2012 (Genentech, Inc.); U.S. Patent No. 8,168,757 issued on May 1, 2012 (Merck Sharp and Dohme Corp.); U.S. Patent No. 8,008,449 issued on August 30, 2011 (Medarex); and U.S. Patent No. 7,943,743 issued on May 17, 2011 (Medarex, Inc.).

[0217] The term PD1 pathway inhibitor is not limited to antagonist antibodies. Non-antibody biological PD1 pathway inhibitors are also in clinical development, including PD-L2 IgG2a fusion protein AMP-224 and PD-L2 fusion protein AMP-514 (Amplimmune and Glaxo SmithKline), aptamers (Wang, et al. (2018) 145:125-130), peptide PD1 pathway inhibitors (Sasikumar, et al., U.S. Patent No. 9,422,339 issued August 23, 2016 and Sasikumar, et al., U.S. Patent No. 8,907,053 issued December 9, 2014), and 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 on September 15, 2016 as WO2016142833A1) 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; currently in clinical development under U.S. Patent No. 7,488,802 issued on February 10, 2009.

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

[0219] In some embodiments, the IL-27R-binding molecule is administered in combination with a negative immune checkpoint pathway antagonist ("TIM3 pathway inhibitor") that inhibits TIM3's ability to bind to its activating ligand. 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; and Takayanagi et al., U.S. Patent No. 8,552,156, issued October 8, 2013.

[0220] In some embodiments, IL-27R-binding molecules are administered in combination with inhibitors of both LAG3 and PD1, as blocking LAG3 and PD1 has been suggested to synergistically reduce 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 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, IL-27R-binding molecules are administered in combination with A2aR inhibitors. A2aR inhibits CD4+ T cells. Reg It inhibits the T cell response by stimulating cells in a developing direction. 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, A2aR deletion is associated with an enhanced, sometimes pathological, inflammatory response to infection. Inhibition of A2aR can be induced by administering molecules such as antibodies that block adenosine binding, or by adenosine analogs. Such agents can be used in combination with IL-27R-binding molecules for use in cancer and treatment disorders such as Parkinson's disease.

[0222] In some embodiments, IL-27R-binding molecules are administered in combination with IDO (indoleamine 2,3-dioxygenase) inhibitors. IDO downregulates the immune response mediated via tryptophan oxidation, resulting in inhibition of T cell activation and induction of T cell apoptosis, creating an environment in which tumor-specific cytotoxic T lymphocytes are functionally inactivated or no longer able to attack target 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 neoplasms (e.g., cancer) in mammals by administering an IL-27R-binding molecule in combination with an active agent that modulates at least one immune checkpoint pathway, which includes immune checkpoint pathway modulators that modulate two, three, or more immune checkpoint pathways.

[0224] In some embodiments, IL-27R-binding molecules are administered in combination with immune checkpoint modulators that modulate multiple immune checkpoint pathways. Multiple immune checkpoint pathways can be modulated by the administration of multifunctional molecules that act as modulators of multiple immune checkpoint pathways. Examples of such multiple immune checkpoint pathway modulators include, but are not limited to, bispecific or multispecific antibodies. Examples of multispecific antibodies capable of acting as modulators of multiple immune checkpoint pathways are known in the art. For example, U.S. Patent Application Publication 2013 / 0156774 describes bispecific and multispecific agents (e.g., antibodies) that target cells co-expressing PD1 and TIM3, as well as methods of use thereof. Furthermore, double blockade of BTLA and PD1 has been shown to enhance antitumor immunity (Pardoll, (April 2012) Nature Rev. Cancer 12:252-64). This disclosure considers the use of IL-27R-binding molecules in combination with immune checkpoint pathway modulators that target multiple immune checkpoint pathways, including but not limited to bispecific antibodies that bind to both PD1 and LAG3. Thus, antitumor immunity can be enhanced at multiple levels, and combination strategies can be generated considering various mechanistic considerations.

[0225] In some embodiments, IL-27R-binding molecules may be administered in combination with two, three, four, or more checkpoint pathway modulators. Such combinations may be advantageous in that the immune checkpoint pathways may 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 conventional chemotherapy such as tyrosine kinase inhibitors. In some cases, it may take more than six months after the initiation of treatment with immune checkpoint pathway inhibitors before objective characteristics of the therapeutic response are observed. Therefore, decisions regarding treatment with immune checkpoint pathway inhibitors in combination with the IL-27R binding molecules of this disclosure must often be made over a longer progression-free period than with conventional chemotherapy. The desired response can be any outcome considered favorable under these circumstances. In some embodiments, the desired response is prevention of disease, disability, or condition progression, while in other embodiments, the desired response is regression or stabilization of one or more characteristics of the disease, disability, or condition (e.g., reduction of tumor size). In yet another embodiment, the desired response is reduction or elimination of one or more adverse effects associated with one or more active agents of this combination.

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

[0228] In some embodiments of the methods of this disclosure, the adjutants are "chimeric antigen receptor T cells" and "CAR-T cells," which 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" are used interchangeably to refer to a chimeric polypeptide comprising a plurality of functional domains arranged in the sequence from the amino terminus to the carboxyl terminus: (a) an antigen-binding domain (ABD), (b) a transmembrane domain (TD); and (c) one or more cytoplasmic signaling domains (CSD), wherein the aforementioned domains may optionally be linked by one or more spacer domains. The CAR may also further comprise a signal peptide sequence that is conventionally removed during post-translational processing and presentation of the CAR on the cell surface of cells transformed with an expression vector containing the nucleic acid sequence encoding the CAR. CARs useful for carrying out the present invention are prepared according to principles well known in the art. For example, see Eshhaar et al., U.S. Patent No. 7,741,465 B1, published 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 receptor of the present invention include axicaptagene silolucel (marketed by Gilead Pharmaceuticals as Yescarta®) and tisagenlecleucel (marketed by Novartis as Kymriah®).In some embodiments, CAR-T possesses a CAR that specifically binds to cell surface molecules 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 are associated with tumor cells.

[0229] physical method : In some embodiments, the adjunct agents are antineoplastic physical methods including, but not limited to, radiotherapy, cryotherapy, hyperthermia, surgery, laser ablation, and proton beam therapy.

[0230] kit: This disclosure also considers pharmaceutical compositions, IL-27R-binding molecules, and kits comprising the pharmaceutical compositions. The kits generally take the form of a physical structure containing various components, such as those described below, and can be used, for example, in the practice of the methods described above. The kits may include a ready-to-use form of a pharmaceutical composition suitable for administration to a subject, or an IL-27R-binding molecule in a form requiring preparation before administration, e.g., thawing, reconstitution, or dilution. If the IL-27R-binding molecule is in a form that needs to be reconstituted by the user, the kit may also include a sterile container providing a reconstitution medium, such as a buffer, a pharmaceutically acceptable excipient, etc. The kits of this disclosure can be designed for conditions necessary to properly maintain the components contained therein (e.g., refrigeration or freezing). The kits may further include a label or accompanying document containing identification information about the components therein and instructions for their use. Each component of the kit may be contained in an individual container, and all the various containers may be placed in a single package. The label or accompanying document may include manufacturer information such as lot number and expiration date. Labels or accompanying instructions may, for example, be incorporated into a physical structure containing the components, contained separately within a physical structure, or affixed to the components of the kit (e.g., ampoules, syringes, or vials). Labels or accompanying instructions may be provided in physical form or in computer-readable media. In some embodiments, the actual instructions are not present in the kit, and conversely, the kit provides means for obtaining instructions via an internet site, including through 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) from a remote source in accordance with administrative regulations (e.g., HIPAA).

[0231] Any maximum numerical limitation described herein is intended to include all numerical limitations smaller than it, as if such smaller limitations were explicitly stated herein. Any minimum numerical limitation described herein includes all numerical limitations larger than it, as if such larger limitations were explicitly stated herein. Any numerical range described herein includes all numerical ranges narrower than it that fall within such a wider range, as if all such narrower ranges were explicitly stated herein.

[0232] None of the references cited herein constitute prior art. The consideration of the references presents the claims of their authors, and the inventors reserve the right to challenge the accuracy and validity of the cited references. While several sources, including articles in scientific journals, patent documents, and textbooks, are referenced herein, it will be evident that this reference does not constitute an acceptance that any of these documents form part of common general knowledge in the art.

[0233] The aforementioned antibodies, useful as adjuvants in the practice of the methods of this disclosure, may be administered alone or in the form of any antibody-drug conjugate (ADC) comprising the 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 may be administered to a subject in combination with a vaccine as an adjuvant to enhance the immune response to the vaccine, in accordance with the teachings of Doyle et al., U.S. Patent No. 5,800,819 issued September 1, 1998. 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 multivalent pneumococcal vaccines such as Prevnar® 13, diphtheria, tetanus and pertussis vaccines (including combination vaccines such as Pediatrix® and Pentacel®), varicella vaccine, Haemophilus influenzae type B vaccine, human papillomavirus vaccines such as Garasil®, polio vaccine, leptospirosis vaccine, respiratory combination vaccine, Moraxella vaccine, attenuated live or dead virus vaccine products such as bovine respiratory disease (RSV) vaccine, Fluzone® and Quadravlent Fluzone. Examples include multivalent human influenza vaccines (such as registered trademarks), feline leukemia vaccine, infectious gastroenteritis vaccine, COVID-19 vaccine, and rabies vaccine.

[0235] For prophylactic use, the pharmaceutical composition or medicine is administered to patients who are susceptible to or at risk of the disease in an amount sufficient to eliminate or reduce the risk of the disease, mitigate its severity, or delay the onset of the disease, including the biochemical, histological, and / or behavioral symptoms of the disease, its complications, and intermediate pathological phenotypes that appear during the onset of the disease. [Examples]

[0236] Example 1 Camels were acclimatized in the research facility for at least 7 days prior to 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 its purity (e.g., over 80%). For the first injection, 10 mL of CFA (followed by 6 injections of IFA) was added to a mortar, and then 10 mL of antigen in 1×PBS was slowly added to the mortar while grinding it with a pestle. The antigen and CFA / IFA appeared to be difficult to disperse and were ground until the components appeared milky white. Camels were subcutaneously injected with the antigen emulsified in CFA into at least 6 sites on their bodies, with approximately 2 mL injected into each site (total 10 mL per camel). A stronger immune response was produced by injecting more sites and larger amounts. Immunization was performed 7 times, once a week (7 days). To avoid leakage of the emulsion, the needle was inserted into the subcutaneous space for 10-15 seconds after each injection. Alternatively, gently pulling back the plunger of the syringe also prevented leakage. Blood samples were taken three days after the seventh immunization.

[0237] Three days after the final injection of the immunization protocol, 100 mL of blood was collected from camels. RNA was extracted from the blood and transcribed into cDNA. A reverse transcription sequence of approximately 900 bp encoding the VH-CH1-hinge-CH2-CH3 construct was isolated from a desired fragment of approximately 700 bp encoding the VHH-hinge-CH2-CH3 construct. The purified fragment of approximately 700 bp was amplified by nested PCR. The amplified sequence was digested with Pst1 and Not1. A Pst1 / Not1 digested fragment of approximately 400 bp was inserted into a Pst1 / Not1 digested pMECS phagemide vector so that the VHH-encoding sequence was in a frame with the DNA sequence encoding the HA / His sequence. The PCR-generated sequence and the pMECS phagemide vector were digested with Pst1 and Not1, and then ligated to pMECS / Nb recombinant. After ligation, the product was transformed into E. coli TG1 cells by electroporation. These transformants were enriched with growth medium and then transferred to 2YT + 2% glucose agar plates.

[0238] Biopanning of a phage library was performed to identify VHH that binds to IL27Rα. IL27Rα was coated onto a 96-well plate, and the phage library was incubated in each well to ensure that phage-expressed IL27Rα-reactive VHH bound 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 VHH was amplified in TG1 cells. The biopanning process described above was repeated 2-3 rounds to enrich the library of VHH selective for IL27Rα. After biopanning was complete, three individual phage clones from the 96-well plate were isolated to perform periplasmic extract ELISA (PE-ELISA) on an antigen-coated plate to identify positive VHH conjugates. In short, an antigen and PBS were coated onto a 96-well plate under the same conditions. The wells were then blocked at 37°C for 1 hour. Next, 100 μl of extracted antibody was added to each well and incubated for 1 hour. Then, 100 μl of HRP-conjugated anti-tagged polyclonal antibody was added to each well and incubated at 37°C for 1 hour. The plates were stained 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 whose absorbance in antigen-coated wells was at least 3 times higher than that of PBS-coated controls were identified as specific binding molecules and were subjected to sequence analysis.

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

[0240] The purified protein eluate was quantified using a Biacore® T200 according to essentially the following procedure. The first 10 μL of 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 tip pre-functionalized with an anti-histidine capture antibody (Cytiva): injection was performed at 5 μL / min for 18 seconds. Capture levels were recorded after 60 seconds of buffer washing. To eliminate intercellular surface variability, known V values ​​were recorded in each of the four Biacore tip flow cells. HStandard curves for H concentration (270, 90, 30, 10, 3.3, 1.1 μg / mL) were obtained. 96 captures were interpolated against the standard curves using a nonlinear model including specific and nonspecific single-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 was performed on five randomly selected samples to confirm that the molecular weight of the eluate corresponded to the expected value (approximately 30 kDa).

[0241] Protein concentrations were normalized using a Hamilton Star automated system, essentially following these steps: The concentration values ​​were imported into an Excel spreadsheet containing calculated pipetting volumes for dilution to 50 μg / mL in 0.22 mL. The spreadsheet was then imported into a dedicated Hamilton Star system for dilution pipetting using the initial elution block and elution buffer as diluents. Finally, the normalized plates were aseptically filtered using a 0.22 μm filter plate (Corning).

[0242] Example 3 All experiments were performed in 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). Mono-Fc VHH ligand was flowed at 5 μl / min for various durations ranging from 18 to 300 seconds until the capture loads listed in the table below were reached.

[0243] After ligand capture, injection of a 2-fold dilution series of his-tagged cytokine receptors, typically containing at least five concentrations between 1 μM and 1 nM, was performed in either high-performance mode or single-cycle kinetics mode. Surface regeneration was achieved by flowing 10 mM glycine-HCl, pH 1.5 (60 seconds, 50 μL / min). Sensograms with buffer subtracted were processed with Biacore T200 Evaluation Software and globally fitted to a 1:1 Langmuir binding model (bulk shift set to zero) to extract kinetic 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 formula Rmax = Load (RU) × Ligand valence × (Analyte molecular weight / Ligand molecular weight). Surface activity was defined as the ratio of experimental Rmax to calculated Rmax. 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) TIFF2026090564000446.tif69151hIL27Ra-his binding anti-hIL27Ra Mono-Fc VHH (ligand) (Origene, catalog number TP307012) TIFF2026090564000447.tif61151 * Both the binding rate constant and the dissociation rate constant can be suppressed when 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. For further analysis using surface plasmon resonance with Biacore T200, one representative VHH was selected from each clonal trait. See below. TIFF2026090564000448.tif206150TIFF2026090564000449.tif154150

Claims

1. An IL27 receptor (IL27R) binding protein that specifically binds to the L27Rα subunit (IL27Rα) and the glycoprotein 130 subunit (gp130), When binding to IL27Rα and gp130, it causes polymerization of IL27Rα and gp130, and This includes 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.

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

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

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

5. The IL27R-binding protein according to claim 4, wherein the peptide linker comprises the sequence 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 to any one sequence of SEQ ID NO: 193 to 198; CDR2 having 0, 1, 2, or 3 amino acid changes to any one sequence of SEQ ID NO: 199 to 204; and CDR3 having 0, 1, 2, or 3 amino acid changes to any one sequence of SEQ ID NO: 205 to 210.

7. anti-gp130v H The IL27R-binding protein according to any one of claims 2 to 6, wherein the H antibody comprises CDR1 having 0, 1, 2, or 3 amino acid changes to any one sequence of SEQ ID NO: 211 to 217; CDR2 having 0, 1, 2, or 3 amino acid changes to any one sequence of SEQ ID NO: 218 to 224; and CDR3 having 0, 1, 2, or 3 amino acid changes to any one sequence of SEQ ID NO: 225 to 231.

8. The IL27R-binding protein according to any one of claims 2 to 5, including the following: CDR1 having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity with respect to the CDR1 sequence from the row in 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 with respect to the CDR2 sequence from the same row in Table 1A, or having 0, 1, 2, or 3 amino acid changes, optionally conservative amino acid changes; and CDR3 having at least 90% The first V contains a CDR3 having sequence identity of (for example, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) or having 0, 1, 2, or 3 amino acid changes, optionally conservative amino acid changes. H H antibody; and CDR1 has at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity with respect to the sequence of CDR4 from the same row in Table 1A, or has 0, 1, 2, or 3 amino acid changes, optionally conservative amino acid changes; CDR2 has at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity with respect to the sequence of CDR5 from the same row in Table 1A, or has 0, 1, 2, or 3 amino acid changes, optionally conservative amino acid changes; and CDR6 has at least 90% A second V containing a CDR3 having sequence identity of (for example, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) or having 0, 1, 2, or 3 amino acid changes, optionally conservative amino acid changes. 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 double 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. Anti-GP130 V bonded to the N-terminus of the linker H H antibody and anti-IL27Rα V linked to the C-terminus of the linker H An IL27R-binding protein according to any one of claims 1 to 14, comprising an H antibody.

12. anti-gp130v 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 SEQ ID NO:232-237.

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 with SEQ ID NO: 238 to 244.

14. anti-gp130v 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 row of Table 2A.

15. The IL27R-binding protein according to claim 11, comprising a sequence having at least 90% identity with one of the sequences SEQ ID NO: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 SEQ ID NO: 245 to 251.

18. anti-gp130v 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 SEQ ID NO:252-257.

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 row of Table 3A.

20. The IL27R-binding protein according to claim 16, comprising a sequence having at least 90% identity with one of the sequences with SEQ ID NO: 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 one sequence from SEQ ID NO: 109 to 192 or a sequence having at least 90% sequence identity with a sequence in Table 1B.

23. An expression vector comprising the nucleic acid described in claim 21.

24. Isolated host cells comprising the vector according to claim 23.

25. A pharmaceutical composition comprising an 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, neoplasm or viral infection in a subject requiring treatment, comprising the step of administering a therapeutically effective amount of the IL27R-binding protein described in any one of claims 1 to 20 or the pharmaceutical composition described in claim 25 to a subject.

27. The method according to claim 26, further comprising the step of 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 aforementioned disease, disorder, or condition is caused by a viral infection, such as Helicobacter pylori. 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, chondritis, arthritis, rheumatoid arthritis, juvenile arthritis, juvenile rheumatoid arthritis, juvenile rheumatoid arthritis, polyarticular juvenile rheumatoid arthritis, systemic juvenile juvenile rheumatoid arthritis, juvenile ankylosing spondylitis, juvenile enteroarthritis, juvenile reactive arthritis, juvenile Reiter's syndrome, SEA syndrome, juvenile dermatomyositis, juvenile psoriatic arthritis, juvenile scleroderma, juvenile systemic lupus erythematosus, juvenile vasculitis, small-joint rheumatoid arthritis, polyarticular rheumatoid arthritis, all Rheumatoid arthritis, ankylosing spondylitis, enteritis 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 lichenoid, lichen planus, lichen patella, ichthyosis-like dermatitis, keratoderma, skin diseases, alopecia areata, pyoderma gangrenosum, vitiligo, bullous pemphigoid The method according to any one of claims 26 to 28, selected from urticaria, porokeratosis, rheumatoid arthritis, seborrheic dermatitis, photodermatitis, seborrheic keratosis, senile keratosis, actinic keratosis, photoinduced keratosis, follicular keratosis, acne vulgaris, keloids, nevi, warts, verruca including condyloma or genital warts, and human papillomavirus (HPV) infection.

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