Compositions and methods relating to IL27 receptor binding

Abstract

Provided herein are anti-IL27Rα V H H antibody and anti-gp130 V H IL27R-binding proteins, including H antibodies. TIFF2023536651000311.tif184170

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 061,562, filed August 5, 2020, U.S. Provisional Patent Application No. 63 / 078,745, filed September 15, 2020, and U.S. Provisional Patent Application No. 63 / 135,884, filed January 11, 2021, the disclosures of which are incorporated by reference in their entireties herein for all purposes. [Background technology]

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

[0003] IL-27 is a heterodimeric cytokine consisting of two non-covalently linked subunits, p28 and EBI3. The p28 subunit belongs to the four-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 Document 1)).

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

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

[0006] The other three domains are called fibronectin type III domains (Fn3). The sequence of each Fn3 domain is variable.

[0007] The gp130 receptor has six domains. The top domain D1 of gp130 binds to p28 of IL-27. D2 and D3 contribute little to IL-27 binding. The three juxtamembrane domains are Fn3 domains. The sequence of each Fn3 domain is variable.

[0008] The structure of IL-27R is not known, but the domain structure is. The structure of gp130 is known to be in complex with IL-6. Based on that structure, it is clear that the Fn3 domain does not contribute energetically to IL-27R complex formation. Rather, the structure of gp130 is such that domains 4 and 5 form a "C" at an 80% angle with each other. Certain residues in each of the "Toll" receptors 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 Document 3)).

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

[0011] Disclosure Summary The present disclosure provides compositions useful in cell receptor pairing to produce a desired effect useful in the treatment of disease in a mammalian subject.

[0012] Several advantages arise from the binding molecules described herein. IL-27, the natural ligand of IL-27R, brings gp130 and IL-27Ra into close proximity (i.e., by its simultaneous binding of IL-27). However, when IL-27 is used as a therapeutic substance in mammalian, particularly human subjects, it may also induce some harmful and undesirable effects by various mechanisms, including the presence of gp130 and IL-27Ra on other cell types, and the binding to gp130 and IL-27Ra on other cell types may also result in undesirable effects and / or undesirable signaling in cells expressing gp130 and IL-27Ra. The present disclosure is directed to methods and compositions that modulate the multiple effects of gp130 and IL-27Ra binding, 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 such that the binding molecules are full agonists. In some embodiments, the binding molecules described herein are designed such that the binding molecules are 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 undesired cell types. This can be achieved, for example, by binding to gp130 and IL-27Ra that has a different affinity compared to the affinity of IL-27 for gp130 and IL-27Ra, or by binding to gp130 and IL-27Ra that has a different E activity. maxThis can be achieved by the selection of binding molecules that cause a binding affinity to the IL-27 receptor relative to wild-type IL-27 binding. Because different cell types respond with different sensitivities to binding of a ligand to its cognate receptor, modulating the affinity of the dimeric ligand (or individual binding moieties thereof) for the IL-27 receptor relative to wild-type IL-27 binding can facilitate stimulation of desired activity while reducing undesired activity on non-target cells.

[0015] The present disclosure provides a bivalent binding molecule that is an agonist of the IL-27 receptor, comprising: a first single domain antibody (sdAb) that specifically binds to the extracellular domain of gp130 (the "anti-gp130 sdAb"); and a second single domain antibody that specifically binds to the extracellular domain of IL-27Ra (the "anti-IL-27Ra sdAb"); Including, the anti-gp130 sdAb and the anti-IL-27Ra sdAb are stably associated, and initial contacting of a cell expressing gp130 and IL-27Ra with an effective amount of the bivalent binding molecule results in dimerization of gp130 and IL-27Ra resulting in intracellular signaling characteristic of the IL-27 receptor when activated by its native cognate IL-27; The bivalent binding molecule is provided. In some embodiments, one or both of the sdAbs is a scFv. In some embodiments, one or both of the sdAbs is a VHH.

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

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

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

[0019] In some embodiments the first and second sdAb are provided as a single contiguous polypeptide which may optionally include a polypeptide linker intervening between the amino acid sequences of the first and second sdAb.

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

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

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

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

[0024] In some embodiments, the IL27R binding protein comprises a sequence 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, anti-IL27Rα V H The anti-gp130 VH antibody comprises a CDR1 having 0, 1, 2 or 3 amino acid changes relative to any one of SEQ ID NOs: 193-198; a CDR2 having 0, 1, 2 or 3 amino acid changes relative to any one of SEQ ID NOs: 199-204; and a CDR3 having 0, 1, 2 or 3 amino acid changes relative to any one of SEQ ID NOs: 205-210. HThe H antibody comprises a CDR1 having 0, 1, 2 or 3 amino acid changes relative to any one of SEQ ID NOs:211-217; a CDR2 having 0, 1, 2 or 3 amino acid changes relative to any one of SEQ ID NOs:218-224; and a CDR3 having 0, 1, 2 or 3 amino acid changes relative to any one of SEQ ID NOs:225-231.

[0026] In certain embodiments, the IL27R binding protein is an anti-IL27Rα V H CDR1, CDR2 and CDR3 in H antibody and anti-gp130 V H It includes CDR1, CDR2 and CDR3 in H antibody.

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

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

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

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

[0031] In certain embodiments, anti-IL27Rα V H H antibodies and anti-gp130 V H Each of the H antibodies comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identity to a sequence listed in a row of Table 3A.

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

[0033] In another aspect, the present disclosure provides an isolated nucleic acid encoding an IL27R binding protein described herein. In certain embodiments, the isolated nucleic acid comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity with any one of SEQ ID NOs: 109-192 or a sequence from Table 1B. The present disclosure also provides an expression vector comprising the nucleic acid. The present disclosure also provides an isolated host cell comprising the expression vector.

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

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

[0036] In some embodiments, the method further comprises administering one or more adjuvant agents 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 embodiments, the disease, disorder or condition is a viral infection, 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, cartilage inflammation, arthritis, rheumatoid arthritis, juvenile arthritis, juvenile rheumatoid arthritis, polyarticular juvenile rheumatoid arthritis, systemic juvenile rheumatoid arthritis, juvenile ankylosing spondylitis, juvenile enteropathic arthritis, juvenile reactive arthritis, juvenile Reiter's syndrome, SEA syndrome, juvenile dermatomyositis, juvenile psoriatic arthritis, juvenile scleroderma, juvenile systemic lupus erythematosus, juvenile vasculitis, small-articular rheumatoid arthritis, multi-related Rheumatoid arthritis, systemic rheumatoid arthritis, ankylosing spondylitis, enteropathic arthritis, reactive arthritis, Reiter's syndrome, SEA syndrome, psoriasis, psoriatic arthritis, dermatitis (eczema), exfoliative dermatitis or atopic dermatitis, pityriasis rubra pilaris, pityriasis rosea, parapsoriasis, pityriasis lichenoides, lichen planus, lichen nitidus, ichthyosa-like dermatitis, keratoderma, skin diseases, alopecia areata , pyoderma gangrenosum, vitiligo, pemphigoid, urticaria, porokeratosis, rheumatoid arthritis, seborrheic dermatitis, photodermatitis, seborrheic keratosis, senile keratosis, actinic keratosis, actin-induced keratosis, follicular keratosis, acne vulgaris, keloids, nevi, warts, condyloma or genital warts, and human papillomavirus (HPV) infection.

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

[0039] In particular, IL27R-binding proteins bind to CD8 + Binds to T cells and CD8 + Activates T cells. In some embodiments, the IL27R binding protein is CXCR5+ CD8 + Binds to T cells, CXCR5 + CD8 + IL27 activates T cells by activating memory-like CXCR5 T cells, e.g., during viral infection. + CD8 + It is known to be able to promote and maintain the rapid division of T cells. CXCR5 + CD8 + T cells maintain T cell responses during persistent infection or cancer and express CD8 after anti-PD1 treatment. + The IL27R binding proteins described herein can drive a proliferative burst of T cells, and thus are useful for maintaining and enhancing self-renewing T cells in chronic infections and neoplastic diseases such as cancer. [The present invention 1001] 1. An IL27 receptor (IL27R) binding protein that specifically binds to the L27Rα subunit (IL27Rα) and glycoprotein 130 subunit (gp130), upon binding to IL27Rα and gp130, causes multimerization of IL27Rα and gp130; and a single domain antibody (sdAb) that specifically binds to IL27Rα (anti-IL27Rα sdAb) and a sdAb that specifically binds to gp130 (anti-gp130 sdAb); The binding protein. [The present invention 1002] Anti-IL27Rα sdAb V H H antibody (anti-IL27Rα V H H antibody) and / or the anti-gp130 sdAb is V H H antibody (anti-gp130 V H 1001. The IL27R binding protein of the present invention, which is an H antibody. [The present invention 1003] The IL27R-binding protein of any of the present inventions 1001 to 1002, wherein the anti-IL27Rα sdAb and the anti-gp130 sdAb are linked together by a peptide linker. [The present invention 1004] 1003. An IL27R binding protein according to the invention, wherein the peptide linker comprises 1 to 50 amino acids. [The present invention 1005] The IL27R binding protein of the present invention 1004, wherein the peptide linker comprises the sequence GGGS (SEQ ID NO:108). [The present invention 1006] Anti-IL27Rα V H IL27R binding protein of any of claims 1002 to 1005, wherein the H antibody comprises a CDR1 having 0, 1, 2 or 3 amino acid changes relative to any one of the sequences of SEQ ID NOs: 193 to 198; a CDR2 having 0, 1, 2 or 3 amino acid changes relative to any one of the sequences of SEQ ID NOs: 199 to 204; and a CDR3 having 0, 1, 2 or 3 amino acid changes relative to any one of the sequences of SEQ ID NOs: 205 to 210. [The present invention 1007] anti-gp130v H IL27R binding protein of any of claims 1002 to 1006, wherein the H antibody comprises a CDR1 having 0, 1, 2 or 3 amino acid changes relative to any one of the sequences of SEQ ID NOs:211 to 217; a CDR2 having 0, 1, 2 or 3 amino acid changes relative to any one of the sequences of SEQ ID NOs:218 to 224; and a CDR3 having 0, 1, 2 or 3 amino acid changes relative to any one of the sequences of SEQ ID NOs:225 to 231. [The present invention 1008] IL27R binding protein of any of claims 1002 to 1005, comprising: a CDR1 having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to the sequence of CDR1 from a row of Table 1A, or having 0, 1, 2 or 3 amino acid changes, optionally conservative amino acid changes; a CDR2 having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to the sequence of CDR2 from the same row of Table 1A, or having 0, 1, 2 or 3 amino acid changes, optionally conservative amino acid changes; and a CDR3 having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to the sequence of CDR3 from the same row of Table 1A, or having 0, 1, 2 or 3 amino acid changes, optionally conservative amino acid changes. (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity, or a CDR3 having 0, 1, 2 or 3 amino acid changes, optionally conservative amino acid changes. H H antibody; and a CDR1 having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to the sequence of CDR4 from the same row of Table 1A, or having 0, 1, 2 or 3 amino acid changes, optionally conservative amino acid changes; a CDR2 having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to the sequence of CDR5 from the same row of Table 1A, or having 0, 1, 2 or 3 amino acid changes, optionally conservative amino acid changes; and a CDR6 having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity, or a CDR3 having 0, 1, 2 or 3 amino acid changes, optionally conservative amino acid changes. H H antibody. [The present invention 1009] IL27R binding protein of the present invention 1008 comprising a sequence at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identical to any one of the dual VHH dimer sequences shown in Table 1A. [The present invention 1010] Anti-IL27Rα V H CDR1, CDR2 and CDR3 in H antibody and anti-gp130 V H The IL27R-binding protein of any of claims 1002 to 1009, comprising CDR1, CDR2 and CDR3 in an H antibody. [The present invention 1011] Anti-gp130 V linked to the N-terminus of the linker H H antibody and anti-IL27Rα V linked to the C-terminus of the linker H The IL27R-binding protein of any one of claims 1001 to 1014, comprising an H antibody. [The present invention 1012] anti-gp130v H 1011. The IL27R binding protein of the present invention, wherein the H antibody comprises a sequence having at least 90% sequence identity with any one of SEQ ID NOs:232-237. [The present invention 1013] Anti-IL27Rα V H 1011. The IL27R binding protein of the present invention, wherein the H antibody comprises a sequence having at least 90% sequence identity with any one of SEQ ID NOs:238-244. [The present invention 1014] anti-gp130v H H antibody and anti-IL27Rα V H 1011 IL27R binding proteins of the invention, each of the H antibodies comprising a sequence having at least 90% identity to a sequence set forth in a row of Table 2A. [The present invention 1015] 1011. An IL27R binding protein of the present invention, comprising a sequence having at least 90% identity to any one of SEQ ID NOs: 1 to 42. [The present invention 1016] 1001. An IL27R binding protein of the present invention, comprising an anti-IL27Rα VHH antibody linked to the N-terminus of a linker and an anti-gp130 VHH antibody linked to the C-terminus of the linker. [The present invention 1017] Anti-IL27Rα V H 1016. An IL27R binding protein of the present invention, wherein the H antibody comprises a sequence having at least 90% sequence identity with any one of SEQ ID NOs:245-251. [The present invention 1018] anti-gp130v H 1016. An IL27R binding protein of the present invention, wherein the H antibody comprises a sequence having at least 90% sequence identity with any one of SEQ ID NOs:252-257. [The present invention 1019] Anti-IL27Rα V H H antibodies and anti-gp130 V H 1016 IL27R binding proteins of the invention, each of the H antibodies comprising a sequence having at least 90% identity to a sequence set forth in a row of Table 3A. [The present invention 1020] 1016. An IL27R binding protein of the present invention, comprising a sequence having at least 90% identity to any one of SEQ ID NOs: 43 to 84. [The present invention 1021] An isolated nucleic acid encoding an IL27R binding protein of any one of the present inventions 1001 to 1020. [The present invention 1022] 1021. An isolated nucleic acid of the present invention, comprising any one of SEQ ID NOs: 109-192 or a sequence having at least 90% sequence identity to a sequence in Table 1B. [The present invention 1023] An expression vector comprising the nucleic acid of the present invention. [The present invention 1024] An isolated host cell comprising a vector of the present invention. [The present invention 1025] A pharmaceutical composition comprising an IL27R binding protein of any of the present inventions 1001 to 1020 and a pharma- ceutically acceptable carrier. [The present invention 1026] A method of treating an autoimmune or inflammatory disease, disorder or condition, a neoplastic disease, or a viral infection in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an IL27R binding protein of any of the present inventions 1001-1020 or a pharmaceutical composition of the present invention 1025. [The present invention 1027] The method of claim 1026, further comprising administering one or more adjuvant agents selected from the group consisting of corticosteroids, Janus kinase inhibitors, calcineurin inhibitors, mTor inhibitors, IMDH inhibitors, biologics, vaccines and therapeutic antibodies. [The present invention 1028] The method of the present invention 1027, wherein the therapeutic antibody is an antibody that binds to a protein selected from the group consisting of BLyS, CD11a, CD20, CD25, CD3, CD52, IgEIL12 / IL23, IL17a, IL1β, IL4Rα, IL5, IL6R, integrin-α4β7, RANKL, TNFα, VEGF-A and VLA-4. [The present invention 1029] The disease, disorder or condition is selected from the group consisting of viral infection, Helicobacter pylori infection, 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, cartilage inflammation, arthritis, rheumatoid arthritis, juvenile arthritis, juvenile rheumatoid arthritis, polyarticular juvenile rheumatoid arthritis, systemic juvenile rheumatoid arthritis, juvenile ankylosing spondylitis, juvenile enteropathic arthritis, juvenile reactive arthritis, juvenile Reiter's syndrome, SEA syndrome, juvenile dermatomyositis, juvenile psoriatic arthritis, juvenile scleroderma, juvenile systemic lupus erythematosus, juvenile vasculitis, small joint rheumatoid arthritis, polyarticular rheumatoid arthritis, total Rheumatoid arthritis, ankylosing spondylitis, enteropathic arthritis, reactive arthritis, Reiter's syndrome, SEA syndrome, psoriasis, psoriatic arthritis, dermatitis (eczema), exfoliative dermatitis or atopic dermatitis, pityriasis rubra pilaris, pityriasis rosea, parapsoriasis, pityriasis lichenoides, lichen planus, lichen nitidus, ichthyosa-like dermatitis, keratoderma, skin disease, alopecia areata, pyoderma gangrenosum, vitiligo, pemphigoid , urticaria, porokeratosis, rheumatoid arthritis, seborrheic dermatitis, photodermatitis, seborrheic keratosis, senile keratosis, actinic keratosis, actin-induced keratosis, follicular keratosis, acne vulgaris, keloids, birthmarks, warts, including condyloma or condyloma acuminata, and human papillomavirus (HPV) infection. [Brief description of the drawings]

[0040] [Figure 1] FIG. 1 of the accompanying drawings provides a schematic diagram of one embodiment of a bivalent binding molecule of the present disclosure comprising a first single domain antibody (1) and a second single domain antibody (3) and a linker (2), depicted as interacting with a cell membrane (10) bound heterodimeric receptor comprising 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), where on the bivalent binding molecule the intracellular domain (6) of the first receptor and the intracellular domain (9) of the second receptor are within a proximal distance (11). [Diagram 2]Figure 2 of the accompanying drawings provides schematic diagrams of two exemplary configurations of bivalent binding molecules of the present disclosure. Panel A provides a schematic diagram of an exemplary single polypeptide chain bivalent binding molecule comprising, in an amino to carboxy orientation, a first single domain antibody (1) and a second single domain antibody (3) and a linker (2). Panel B provides a schematic diagram of a bivalent binding 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 comprising a first subunit that is an Fc knob (13) and a second subunit that is an Fc hole (14), in which the bivalent binding molecule is covalently linked to the Fc domain subunits via an IgG hinge sequence (12). [Diagram 3]Figure 3 of the accompanying drawings provides schematic diagrams of two exemplary configurations of bivalent binding molecules of the present disclosure. Panel A provides a schematic diagram of an exemplary bivalent binding molecule construct comprising two bivalent binding molecules, each attached to a subunit of a knob-into-hole Fc domain, the construct comprising two polypeptide chains, the first polypeptide chain comprising, in the amino to carboxy orientation, 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), and the second polypeptide chain comprising, in the amino to carboxy orientation, 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), the first and second polypeptides being in stable association via knob-into-hole Fc domain interactions. Panel B provides a schematic diagram of an alternative arrangement of a bivalent binding molecule construct comprising two polypeptides, where the first polypeptide chain comprises, in amino to carboxy orientation, 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), and the second polypeptide chain comprises, in amino to carboxy orientation, 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), wherein the first and second polypeptides are in stable association via knob-into-hole Fc domain interactions. [Figure 4]Panel A of Figure 4 provides an alternative schematic diagram of a bivalent binding molecule configuration of the present disclosure in which the binding molecule comprises two polypeptides, one single domain antibody attached to each subunit of the knob-into-hole Fc domain, where the first polypeptide comprises, in amino to carboxy orientation, a first single domain antibody (1), an IgG hinge sequence (12) and an Fc knob subunit (13), and the second polypeptide comprises, in amino to carboxy orientation, a second single domain antibody (3), an IgG hinge sequence (12) and an Fc hole subunit (13), the first and second single domain antibodies being in stable association via knob-into-hole Fc domain interactions. Panel B of Figure 4 provides a schematic diagram of a binding molecule in which the binding domains are single domain antibodies linked via a transition metal coordination covalent complex. As shown, the binding molecule comprises two polypeptide subunits, a first subunit comprising a first single domain antibody (1) attached to a first chelating peptide (17) by a first linker (15), a second subunit comprising a second single domain antibody (3) attached to a second chelating peptide (18) by a second linker (16), and the first chelating peptide (17) and the second chelating peptide (18) form a coordinate covalent complex with a single transition metal ion ("M"), which may be in a kinetically labile oxidation state or a kinetically inactive oxidation state. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0041] Detailed Description of the Disclosure I. Introduction The present disclosure provides compositions useful in cell receptor pairing to produce a desired effect useful in the treatment of disease. In general, a binding protein is provided that includes a first domain that binds to IL27Rα and a second domain that binds to gp130, such that upon contact with a cell expressing IL27Rα and gp130, the binding protein causes 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 of IL27R, brings IL27Rα and gp130 into close proximity (i.e., by its simultaneous binding of IL27). However, when IL27 is used as a therapeutic substance in mammalian, particularly human subjects, it may also induce several harmful and undesirable effects by various mechanisms, including the presence of IL27Rα and gp130 on other cell types, and binding to IL27Rα and gp130 on other cell types may also result in undesirable effects and / or undesirable signaling in cells expressing IL27Rα and gp130. The present disclosure is directed to methods and compositions that modulate the multiple effects of IL27Rα and gp130 binding 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 such that the binding proteins provide maximum desired IL27 intracellular signaling from binding to IL27Rα and gp130 on desired cell types, while providing significantly less IL27 signaling on other undesired cell types. This can be achieved, for example, by binding to a binding protein that has a different affinity, or a different E-binding domain for IL27Rα and gp130, compared to the affinity of IL27 for IL27Rα and gp130. maxThis can be achieved by the selection of a binding protein that induces a binding activity. Different cell types respond with different sensitivities to the binding of a ligand to its cognate receptor, so modulating the affinity of the dimeric ligand (or its individual binding moieties) for the IL27 receptor compared to wild-type IL27 binding promotes the stimulation of desired activity while reducing undesired activity on non-target cells. Several methods are available for measuring downstream signaling activity. For example, in some embodiments, JAK / STAT signaling can be measured by the presence of phosphorylated receptor and / or phosphorylated STAT. In other embodiments, the expression of one or more downstream genes whose expression levels can be affected by the level of downstream signaling caused by the binding protein can also be measured.

[0044] Interleukin 27 (IL27) Structure: IL27 is a member of the IL-12 cytokine family. IL27 is a heterodimeric cytokine composed of two subunits: IL27A (also 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 that contains a 28 amino acid signal sequence that is post-translationally removed to yield a 215 amino acid mature protein. UniProtKB - Q8NEV9 (IL27A_HUMAN). The mature form of p28 (lacking the signal peptide) possesses the following amino acid sequence: TIFF0007665730000001.tif22128

[0045] Human IL27B (hIL27B) is expressed as a 229 amino acid preprotein that contains a 20 amino acid signal sequence that is post-translationally removed to yield a mature protein of 209 amino acids. UniProtKB - Q14213 (IL27B_HUMAN). The mature form of hIL27B (lacking the signal peptide) possesses the following amino acid sequence: TIFF0007665730000002.tif22128

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

[0047] Human IL27 receptor subunit alpha (hIL27RA) is expressed as a 636 amino acid preprotein that contains a 32 amino acid signal sequence that is post-translationally removed to yield a 604 amino acid mature protein. UniProtKB - Q6UWB1 (I27RA_HUMAN)

[0048] The mature form of hIL27RA (without the signal peptide) has the following amino acid sequence: TIFF0007665730000003.tif59128

[0049] The extracellular domain of hIL27RA (IL27RA-ECD) is a 484 amino acid polypeptide corresponding to amino acids 33 to 516 of the hIL27RA preprotein and possesses the following amino acid sequence: TIFF0007665730000004.tif50128

[0050] The human gp130 receptor subunit (hGP130) is also known as the IL6 receptor beta subunit. UniProtKB - P40189 (IL6RB_HUMAN. hGP130 is expressed as a 918 amino acid preprotein that contains a 22 amino acid signal sequence, which is removed post-translationally to yield a mature protein of 896 amino acids. The mature form of hGP130 possesses the following amino acid sequence: TIFF0007665730000005.tif87128

[0051] The extracellular domain of hGP130 (hGP130-ECD) is a 597 amino acid polypeptide corresponding to amino acids 23 to 619 of the hGP130 preprotein and possesses the following amino acid sequence: TIFF0007665730000006.tif59128

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

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

[0054] IL27 drives rapid clonal expansion of naive, but not memory, CD4 T cells. IL27 also potently synergizes with IL-12 to induce interferon-γ / IFN-γ production in naive CD4 T cells and binds to the cytokine receptor WSX-1 / TCCR. Another important role of IL-27 is its antitumor activity and antiangiogenic activity that activates the production of antiangiogenic chemokines.

[0055] Induction of IL10 IL-10-expressing Tr1 cells are induced by IL-27 through the transcription factor c-Maf, resulting in an anti-inflammatory response. The main activity of IL-10 is the suppression of inflammatory responses. STAT1 and STAT3 transcription factors, 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 below as well as throughout the specification. The definitions provided herein are non-limiting and should be read in light of the knowledge that would be known to one of ordinary skill in the art.

[0057] Before the present methods and compositions are described, it is to be understood that this invention is not limited to the particular methods or compositions described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing embodiments only, and is not intended to be limiting.

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

[0059] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs.Although any method and material similar or equivalent to the method and material described herein can be used in the practice or testing of this invention, some potential and preferred methods and materials will now be described.All publications mentioned herein are incorporated by reference to disclose and describe the method and / or material in the context in which they are cited.

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

[0061] Publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing contained herein should be construed as an admission that the present invention is not entitled to antedate such publications by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed.

[0062] Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Celsius (° C.), and pressure is at or near atmospheric. Standard abbreviations are used, including: bp = base pairs; kb = kilobase; pl = picoliter; s or sec = seconds; 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 = micromole; mM = millimole; M = mole; kDa = kilodalton; im = intramuscular; ip = intraperitoneal; SC or SQ = subcutaneous; QD = once daily; BID = twice daily; QW = weekly; QM = monthly; HPLC = high performance liquid chromatography; BW = Body weight; U = unit; ns = not statistically significant; PBS = phosphate-buffered saline; PCR = polymerase chain reaction; HSA = human serum albumin; MSA = mouse serum albumin; DMEM = Dulbecco's modified Eagle's medium; EDTA = ethylenediaminetetraacetic acid.

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

[0064] (Table) Amino acid abbreviations TIFF0007665730000007.tif97128

[0065] Standard methods in molecular biology are described in the scientific literature (see, e.g., Sambrook and Russell (2001) Molecular Cloning, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; and Ausubel, et al. (2001) Current Protocols in Molecular Biology, Vols. 1-4, John Wiley and Sons, Inc. New York, NY, which describe cloning and DNA mutagenesis in bacterial cells (Vol. 1), cloning in mammalian cells and yeast (Vol. 2), expression of glycoconjugates and proteins (Vol. 3), and bioinformatics (Vol. 4)). The scientific literature describes methods for protein purification, including immunoprecipitation, chromatography, electrophoresis, centrifugation, and crystallization, as well as chemical analysis, chemical modification, post-translational modification, production of fusion proteins, and glycosylation of proteins (see, e.g., Coligan, et al. (2000) Current Protocols in Protein Science, Vols. 1-2, John Wiley and Sons, Inc., NY).

[0066] Activate As used herein, the term "activate" is used in reference to a receptor or receptor complex to reflect a biological effect, either directly and / or by participation in a multicomponent signaling cascade, that results from the binding of an agonist ligand to the receptor in response to binding of the ligand.

[0067] Active:As used herein, the term "activity" is used with respect to a molecule to describe the properties of the molecule in relation to a test system (e.g., an assay) or the 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., alteration of cell membrane potential). Examples of such biological functions include, but are not limited to, the catalytic activity of a biological agent, the ability to stimulate intracellular signaling, the ability to regulate gene expression, cell proliferation, immune activity such as inflammatory response, etc. "Activity" is typically expressed as the level of biological activity per unit of the agent being tested, such as [catalytic activity] / [mg protein], [immune activity] / [mg protein], international unit (IU) of activity, [STAT5 phosphorylation] / [mg protein], [T cell proliferation] / [mg protein], plaque forming unit (pfu), etc. As used herein, the term "proliferative activity" refers to the activity of promoting cell proliferation and replication.

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

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

[0070] Agonist : As used herein, the term "agonist" refers to a first agent that specifically binds to a second agent ("target") and interacts with the target to cause or promote enhanced activation of the target. In some cases, an agonist is an activator of a receptor protein that modulates cell activation, enhances activation, sensitizes cells to activation by a second agent, or upregulates the expression of one or more genes, proteins, ligands, receptors, biological pathways that can lead to cell proliferation or pathways that result in cell death, such as by cell cycle arrest or apoptosis. In some embodiments, an agonist is an agent that binds to a receptor, modifies the state of the receptor, and results in a biological response. This response mimics the effect of the endogenous activator of the receptor. The term "agonist" includes partial agonists, full agonists, and superagonists. Agonists may be described as "full agonists" when such agonists produce substantially the complete biological response induced by the receptor under study (i.e., the response associated with the natural ligand / receptor binding interaction), or as partial agonists. In contrast to agonists, antagonists may specifically bind to a receptor but do not typically result in a signal cascade initiated by the receptor and may modify the action of an agonist at that receptor. Inverse agonists are agents that produce a pharmacological response opposite to that of an agonist. A "superagonist" is a type of agonist that can produce a maximal response greater than the endogenous agonist at the target receptor, and thus has greater than 100% activity of the natural ligand. A superagonist is typically a synthetic molecule that exhibits a response of 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 native form of the molecule when evaluated at similar concentrations in an equivalent assay.

[0071] Antagonists: As used herein, the term "antagonist" or "inhibitor" refers to a molecule that opposes the action of agonist.Antagonist prevents, reduces, inhibits, or neutralizes the activity of agonist, and antagonist can also prevent, inhibit, or reduce the constitutive activity of target, such as target receptor, even in the absence of specified agonist.Inhibitor is, for example, a molecule that reduces, blocks, prevents, delays activation, inactivates, desensitizes, or downregulates gene, protein, ligand, receptor, biological pathway, or cell.

[0072] antibody: As used herein, the term "antibody" refers collectively to: (a) glycosylated and non-glycosylated 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 C, which competes with the immunoglobulin from which it was derived for binding to the target molecule. H 2, F(ab')2, Fab, ScFv, V H , V LThe term "antibody" refers to immunoglobulin derivatives, including, but not limited to, F(ab'), ... The term "antibody" should not be construed as limited to any particular synthetic means, and includes not only naturally occurring antibodies isolatable from natural sources, but also engineered antibody molecules prepared by "recombinant" means, including antibodies isolated from transgenic animals that are transgenic for human immunoglobulin genes or hybridomas prepared therefrom, antibodies isolated from host cells transformed with a nucleic acid construct that results in the expression of the antibody, and antibodies isolated from combinatorial antibody libraries, including phage display libraries.

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

[0074] CDR:As used herein, the term "CDR" or "complementarity determining region" is intended to mean the non-contiguous antigen-binding sites found within the variable regions of both heavy and light chain immunoglobulin polypeptides. CDRs are 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) (also referred to herein as "Kabat 1991" or "Kabat"); Chothia, et al. (1987) J. Mol. Biol. 196:901-917 (also referred to herein as "Chothia"); and MacCallum, et al. (1996) J. Mol. Biol. 262:732-745, where these definitions include overlapping or subsets of amino acid residues when compared to each other. Nevertheless, application of either definition to refer to the CDRs of an antibody or grafted antibody or variants thereof is intended to be within the scope of the term as defined and used herein. The term "Chothia numbering" as used herein is recognized in the art and refers to a system of numbering amino acid residues based on the position of structural loop regions (Chothia et al. 1986, Science 233:755-758; Chothia & Lesk 1987, JMB 196:901-917; Chothia et al. 1992, JMB 227:799-817). For the purposes of 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 Kabat and Chothia numbering schemes.

[0075] Clonoplasm: Clonoplasm is defined as a collection of binding molecules derived from the same B cell precursor. The term "clonoplasm" is used herein to refer to a collection of antigen-binding molecules that belong to the same germline, have the same CDR3 length, and have 70% or more homology in CDR3 sequence.

[0076] Equivalent: As used herein, the term "equivalent" is used to describe the degree of difference between two measurements of an assessable quantitative or qualitative parameter. For example, if the difference between a first measurement of an assessable quantitative parameter and a second measurement of an assessable 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 that situation, the two measurements will be considered "equivalent". In some cases, a measurement can be considered "equivalent" if the difference between the other measurement is less than 30%, alternatively less than 25%, alternatively less than 20%, alternatively less than 15%, alternatively less than 10%, alternatively less than 7%, alternatively less than 5%, alternatively less than 4%, alternatively less than 3%, alternatively less than 2%, or even less than 1%. In certain embodiments, a measurement is equivalent to a reference standard if the difference between the reference standard and the first measurement 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 that is triggered by the interaction of two or more cell surface receptors that are brought into close proximity to one another.

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

[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) that is on the outside of the plasma membrane of a cell. The term "ECD" can include the extracytoplasmic portion of a transmembrane protein or the extracytoplasmic portion of a cell surface (or membrane-bound) protein.

[0080] identityAs used herein, the term "percent (%) sequence identity" or "substantially identical" used in the context of a nucleic acid or polypeptide refers to a sequence that has at least 50% sequence identity with a reference sequence. Alternatively, the percent sequence identity can be any integer between 50% and 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 as determined by BLAST using standard parameters, as described below. For sequence comparison, one sequence usually serves as a reference sequence to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are input into a computer, subsequence coordinates are designated as necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identity of the test sequence to the reference sequence based on the program parameters. The comparison window includes reference to any one segment of the number of contiguous positions, for example, a segment of at least 10 residues. In some embodiments, the comparison window has 10 to 600 residues, for example, 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 sequence can be compared to the reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. Suitable algorithms for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms described in Altschul et al. (1990) J. Mol. Biol. 215: 403-410 and Altschul, et al. (1977) Nucleic Acids Res. 25: 3389-3402, respectively. Software for performing BLAST analyses is publicly available through the website of the National Center for Biotechnology Information (NCBI).This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence that match or meet some positive threshold score T when aligned with words of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al., supra). These initial neighborhood word hits act as seeds to initiate searches to find longer HSPs that contain them. The word hits are then extended in both directions for each sequence as far as the total alignment score can be increased. For nucleotide sequences, the total score is calculated using the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0). For amino acid sequences, a scoring matrix is ​​used to calculate the total score. Extension of the word hits in each direction is stopped when the sum of the alignment scores drops from its maximum achieved value by an amount X; when the accumulation of the alignment of one or more negatively scoring residues causes the sum of the scores to fall below zero; or when the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) works similarly, but uses as defaults a word size (W) of 28, an expectation (E) of 10, M=1, N=-2, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a word size (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989)). The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, eg, Karlin & Altschul, Proc. Nat'l. Acad. Sci. USA 90:5873-5787 (1993)).One measure of similarity provided by the BLAST algorithm is the minimum sum probability (P(N)), which provides an indication of the probability that a match between two nucleotide or amino acid sequences would occur by chance. For example, an amino acid sequence may have a minimum sum probability of less than about 0.01, more preferably less than about 10, in the comparison of a test amino acid sequence with a reference amino acid sequence. -5 less than about 10 -20 If the sequence is less than 1, it is considered similar to the reference sequence.

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

[0082] Intracellular SignalingAs used herein, the terms "intracellular signaling" and "downstream signaling" are used interchangeably to refer to a cell signaling process triggered by the interaction of the intracellular domains (ICDs) of two or more cell surface receptors in close proximity to each other. In a receptor complex via the JAK / STAT pathway, the association of the ICDSs of the receptor subunits brings the JAK domains of the ICDs into close proximity, initiating a phosphorylation cascade in which STAT molecules are phosphorylated and translocate to the nucleus to associate with specific nucleic acid sequences, leading to the activation and expression of specific genes in the cell. The binding molecules of the present disclosure, when activated by its natural homolog IL27, provide intracellular signaling characteristic of the IL-27 receptor. Several methods are available for measuring downstream signaling activity. For example, in some embodiments, JAK / STAT signaling can be measured by the presence of phosphorylated receptor and / or phosphorylated STAT. In other embodiments, the expression of one or more downstream genes, whose expression levels can be affected by the level of downstream signaling 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 the receptor to which it binds, resulting in a change in the activity of the receptor. In one embodiment, the term "ligand" refers to a molecule that can act as an agonist or antagonist of a receptor, or a complex thereof. As used herein, the term "ligand" includes natural and synthetic ligands. "Ligand" also includes small molecules, such as peptide mimetics of cytokines and peptide mimetics of antibodies. A complex of a ligand and a receptor is referred to as a "ligand-receptor complex."

[0084] As used herein, the term "linker" refers to a link between two elements, e.g., protein domains. The linker can be a covalent bond or a peptide linker. The term "bond" refers to a chemical bond, e.g., an amide bond or a disulfide bond, or any type of bond resulting from a chemical reaction, e.g., chemical conjugation. The term "peptide linker" refers to an amino acid or polypeptide that can be used to link two protein domains to provide space and / or flexibility between the two protein domains.

[0085] Adjust: As used herein, the terms "modulate," "modulation," and the like refer to the ability of a test substance to affect, either positively or negatively, or directly or indirectly, a response in a biological system or a system that includes a biochemical pathway.

[0086] Polymerization As used herein, the term "multimerization" refers to the bringing into close proximity of two or more cell surface receptors, or domains or subunits thereof, such that the receptors, or domains or subunits thereof, can interact with each other to cause intracellular signaling.

[0087] N-terminus As used herein in the context of a polypeptide structure, the terms "N-terminus" (or "amino terminus") and "C-terminus" (or "carboxyl terminus") refer to the extreme amino and carboxyl termini of a polypeptide, respectively, whereas "N-terminal" and "C-terminal" refer to the relative positions towards the N-terminus and C-terminus, respectively, of the amino acid sequence of a polypeptide and can include the N- and C-terminal residues, respectively. The terms "directly N-terminal" or "directly C-terminal" are used to refer to the position of a first amino acid residue relative to a second amino acid residue when the first and second amino acid residues are covalently linked to provide a contiguous amino acid sequence.

[0088] nucleic acid The terms "nucleic acid", "nucleic acid molecule", "polynucleotide" and the like are used interchangeably herein to refer to a polymeric form of any length of nucleotides, either deoxyribonucleotides or ribonucleotides or analogs thereof. Non-limiting examples of polynucleotides include linear or circular nucleic acids, messenger RNA (mRNA), complementary DNA (cDNA), recombinant polynucleotides, vectors, probes, primers and the like.

[0089] Functionally linked The term "operably linked" is used herein to refer to the relationship between nucleic acid sequences encoding different functions that, when combined into a single nucleic acid sequence, provide a nucleic acid that, when introduced into a cell, can effect transcription and / or translation of the particular nucleic acid sequence in the cell. For example, DNA for a signal sequence is operably linked to DNA for a polypeptide if it is expressed as a preprotein involved in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation. Generally, "operably linked" means that the DNA sequences being linked are contiguous, and in the case of a secretory leader, contiguous and in reading phase. However, certain genetic elements, such as enhancers, need not be contiguous with respect to the sequences that produce 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 has only partial activation of the receptor compared to a full agonist. A partial agonist may exhibit both agonist and antagonist effects. For example, when both a full agonist and a partial agonist are present, the partial agonist acts as a competitive antagonist by competing with the full agonist for binding to the receptor, resulting in a net reduction in receptor activation compared to the contact of the receptor with the full agonist in the absence of the partial agonist. Clinically, partial agonists can be used to activate receptors to provide desired submaximal responses when insufficient amounts of endogenous ligand are present, or they can reduce overstimulation of the receptor when excess amounts of endogenous ligand are present. The maximum response (Emax) generated by a partial agonist is called its intrinsic activity, and can be expressed on a percentage scale where a full agonist would generate a 100% response. In some embodiments, the IL-27 binding molecule is an IL-27-induced E max Reduced E compared to max E max reflects the maximum response level in a cell type that can be obtained by a ligand (e.g., a binding molecule described herein or a natural cytokine (e.g., IL-27)). In some embodiments, the IL-27 binding molecules described herein inhibit the E1 activation induced by IL-27. max In another embodiment, the E of the IL-27 binding molecule described herein is at least 1% (e.g., 1% to 100%, 10% to 100%, 20% to 100%, 30% to 100%, 40% to 100%, 50% to 100%, 60% to 100%, 70% to 100%, 80% to 100%, 90% to 100%, 1% to 90%, 1% to 80%, 1% to 70%, 1% to 60%, 1% to 50%, 1% to 40%, 1% to 30%, 1% to 20%, or 1% to 10%). max is the E of the natural ligand IL-27 maxIn some embodiments, the E of an IL-27 binding molecule is increased by varying the linker length of the IL-27 binding molecule. max The IL-27 binding molecules can alter E. coli expression in the most desired cell types. max and reduced E in other cell types. max This can result in...

[0091] Polypeptides As used herein, the terms "polypeptide", "peptide" and "protein" are used interchangeably herein to refer to polymeric forms of amino acids of any length, which can include genetically encoded and non-genetically encoded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides with modified polypeptide backbones. These terms include fusion proteins, including, but not limited to, fusion proteins with heterologous amino acid sequences; fusion proteins with heterologous and homologous leader sequences; fusion proteins with or without an N-terminal methionine residue; fusion proteins with immunologically tagged proteins; fusion proteins of immunologically active proteins (e.g., antigenic diphtheria or tetanus toxin fragments), and the like.

[0092] As used herein, the terms "prevent", "preventing", "prevention", and the like refer to actions initiated on a subject prior to the onset of a disease, disorder, condition, or symptoms thereof, generally in relation to a subject predisposed to having a particular disease, disorder, or condition due to genetic, experiential, or environmental factors, such that the subject temporarily or permanently prevents, suppresses, inhibits, or reduces the risk of developing, or delays the onset of, a disease, disorder, condition, or other (e.g., as determined by the absence of clinical symptoms). In certain instances, the terms "prevent", "preventing", and "prevention" are also used to refer to the slowing of the progression of a disease, disorder, or condition from its current state to a more deleterious state.

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

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

[0095] Recombination: As used herein, the term "recombinant" is used as an adjective to refer to the way in which a polypeptide, a nucleic acid, or a cell has been modified using recombinant DNA technology. A recombinant protein is a protein produced using recombinant DNA technology and may be designated as such (e.g., rhIL2) using the abbreviation "r" in lowercase to indicate the way in which the protein was produced. Similarly, a cell is said to be a "recombinant cell" if it has been modified by the incorporation (e.g., transfection, transduction, infection) of an exogenous nucleic acid (e.g., ssDNA, dsDNA, ssRNA, dsRNA, mRNA, viral or non-viral vectors, plasmids, cosmids, etc.) using recombinant DNA technology. 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 (2d 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 a quantitative or qualitative change in an assessable biochemical or physiological parameter (e.g., concentration, density, adhesion, proliferation, activation, phosphorylation, migration, enzyme activity, gene expression level, gene expression rate, energy consumption rate, level or state of differentiation, where the change is correlated with activation, stimulation or treatment, or with internal mechanisms such as genetic programming. In certain contexts, terms such as "activation," "stimulation," and the like refer to cell activation that is regulated by external or environmental factors as well as internal mechanisms. In contrast, terms such as "inhibition," "downregulation," and the like refer to the opposite effect.

[0097] Single Domain Antibodies (sdAbs)The term "single domain antibody" or "sdAb" refers to an antibody that has a single (only one) monomeric variable antibody domain. An sdAb is capable of selectively binding to a specific antigen. The V domain, as further defined below, H The H antibody is an example of an sdAb.

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

[0099] Meeting in a stable mannerAs used herein, the term "stably associated" or "in stable association with" is used to refer to various means by which one molecule (e.g., a polypeptide) can be associated with another molecule over an extended period of time. The stable association of one molecule to another can be achieved by various means, including covalent bonds and non-covalent interactions. In some embodiments, the stable association of two molecules can be achieved by covalent bonds, such as peptide bonds. In other embodiments, the stable association of two molecules can be achieved 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 the sdAb of the bivalent binding molecule of the present disclosure can be achieved by non-covalent interactions. In one embodiment, non-covalent stable association of sdAbs of a bivalent binding molecule may 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 a first subunit of a heterodimeric receptor to the "Fc knob" monomer and a second sdAb that specifically binds to the extracellular domain of a second subunit of a heterodimeric receptor to the "Fc hole" monomer results in stable association of the first and second sdAbs. Knob-into-hole modifications are more fully described in Ridgway, et al. (1996) Protein Engineering 9(7):617-621, as well as U.S. Pat. No. 5,731,168, issued March 24, 1998, U.S. Pat. No. 7,642,228, issued January 5, 2010, U.S. Pat. No. 7,695,936, issued April 13, 2010, and U.S. Pat. No. 8,216,805, issued July 10, 2012.Knob-into-hole modification refers to a modification at the interface between two immunoglobulin heavy chains in the CH3 domain, in which: i) an amino acid residue in the CH3 domain of a first heavy chain is replaced by an amino acid residue with a larger side chain (e.g., tyrosine or tryptophan) to create a protrusion ("knob") from the surface; and ii) an amino acid residue in the CH3 domain of a second heavy chain is replaced by an amino acid residue with a smaller side chain (e.g., alanine or threonine), thereby generating a cavity ("hole") in the interface in the second CH3 domain, in 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" comprises the amino acid substitution T366W and optionally the amino acid substitution S354C in one of the antibody heavy chains, and the amino acid substitutions T366S, L368A, Y407V and optionally Y349C in the other antibody heavy chain. Additionally, the Fc domain may be modified by the introduction of cysteine ​​residues at positions S354 in one chain and Y349 in the other chain, resulting in a stabilizing disulfide bridge between the two antibody heavy chains in the Fc region (Carter, et al. (2001) Immunol Methods 248, 7-15). The knobs-into-holes format is used to facilitate expression of a first polypeptide (e.g., an IL27Rα-binding sdAb) on a first Fc monomer bearing a "knob" modification and a second polypeptide on a second Fc monomer carrying a "hole" modification to facilitate expression of a heterodimeric polypeptide conjugate.

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

[0101] In effect: As used herein, the term "substantially" refers to an amount, 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 reference amount, level, value, number, frequency, percentage, dimension, size, amount, weight or length. In one embodiment, "substantially the same" refers to an amount, level, value, number, frequency, percentage, dimension, size, amount, weight or length that produces about the same effect, e.g., a physiological effect, as the reference amount, level, value, number, frequency, percentage, dimension, size, amount, weight or length.

[0102] Suffering As used herein, the term "suffering" refers to a determination made by a physician about a subject that the subject requires or would benefit from treatment based on available information accepted in the art for the identification of a disease, disorder, or condition, including, but not limited to, x-rays, CT scans, conventional clinical diagnostic tests (e.g., blood counts), genomic data, protein expression data, immunohistochemistry. The term suffering is typically used in conjunction with a specific medical condition, such as "suffering from a neoplastic disease," to refer to a subject who has been diagnosed with the presence of a neoplasm.

[0103] Therapeutically Effective DoseAs used herein, the term "therapeutically effective amount" refers to administering an agent to a subject in a single dose, as part of a series of doses, alone, or as part of a pharmaceutical composition or treatment regimen, in an amount that can have any detectable positive effect on any symptom, aspect, or characteristic of a disease, disorder, or condition when administered to a subject. The therapeutically effective amount can be ascertained by measuring the relevant physiological effect, which can be adjusted in relation to the dosing regimen, as well as depending on diagnostic analysis such as the subject's condition. The parameters for evaluation to determine the therapeutically effective amount of an agent are determined by a physician using diagnostic criteria accepted in the art, including, but not limited to, signs such as age, weight, sex, general health, ECOG score, observable physiological parameters, blood levels, blood pressure, electrocardiogram, computed tomography, X-ray, and others. Alternatively or additionally, other parameters that are typically assessed in a clinical setting, such as body temperature, heart rate, normalization of blood chemistry, normalization of blood pressure, normalization of cholesterol levels, or any symptom, aspect, or characteristic of a disease, disorder, or condition, alteration of biomarker levels, increased survival time, increased progression-free survival, increased time to progression, increased time to treatment failure, increased event-free survival, increased time to next treatment, improved response rate, improved duration of response, and the like, may be monitored to determine whether a therapeutically effective amount of an agent has been administered to a subject, and these parameters are relied upon by clinicians in the art to assess improvement in a subject's condition in response to administration of the agent.

[0104] TreatThe terms "treat", "treating", "treatment" and the like refer to an action initiated with respect to a subject after a disease, disorder, or condition, or a symptom thereof, has been diagnosed, observed, or otherwise in a subject, to temporarily or permanently eliminate, reduce, inhibit, alleviate, or ameliorate at least one of the underlying causes of, or at least one of the symptoms associated with, the disease, disorder, or condition afflicting the subject (e.g., administering a binding molecule described herein, or a pharmaceutical composition comprising same). Treatment includes an action taken with respect to a subject suffering from a disease, which action results in the inhibition of the disease in the subject (e.g., halting the progression of, or ameliorating one or more symptoms associated with, the disease, disorder, or condition).

[0105] VHH As used herein, "V H The term "H" refers to a type of sdAb that has a single monomeric heavy chain variable antibody domain. Such antibodies can be found or produced in camelid mammals (e.g., camels, llamas) that naturally lack light chains. H H was derived from immunization of camelids (including camels, llamas and alpacas) (see, e.g., Hamers-Casterman, et al. (1993) Nature 363:446-448), or V H Antibodies with a given specificity can be obtained by screening libraries (e.g., phage libraries) constructed with the H framework. Antibodies with a given specificity can be obtained by screening libraries (e.g., phage libraries) constructed with the V framework, obtained from immunization of cartilaginous fish, including but not limited to sharks. H In certain embodiments, the bispecific V polypeptide described herein may be derived from a non-mammalian source, such as H. H H 2 V in bond molecule H H is V H The equilibrium dissociation constant between H and the receptor is approximately 10, as determined, for example, by Scatchard analysis (Munsen, et al. 1980 Analyt. Biochem. 107:220-239).-6 Larger than M, alternatively about 10 -8 Larger than M, alternatively about 10 -10 Larger than M, alternatively about 10 -11 Larger than M, alternatively about 10 -10 Larger than M, about 10 -12 If M is greater than 1, 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. See, for example, Vincke, et al (2012) Chapter 8 in Methods in Molecular Biology , Walker, J. editor (Humana Press, Totowa NJ). Specific binding may be assessed using techniques known in the art, including but not limited to competitive ELISA, BIACORE® assays and / or KINEXA® assays. In some embodiments, the V H H can be humanized to include human framework regions. H Examples of human germline sequences that can be used to make 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 2and "VHH dimer" refers to a first and a second sdAb that are both VHHs and that bind to a first receptor, or a domain or subunit thereof. H H, and a second V that binds to a second receptor, or a domain or subunit thereof. H H.

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

[0108] III. IL27 Receptor Binding Proteins 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 a mammalian cell expressing both IL27Rα and gp130. In some embodiments, the IL27R binding protein is a bispecific V-type IL27R binding protein 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 a second V that binds to gp130 H H (anti-gp130 V H H antibody) and expressing IL27Rα and gp130, e.g., CD8 + T cells, CD4 + Bispecific V that upon binding to T cells and / or T regulatory (Treg) cells triggers dimerization of the two receptor subunits and downstream signaling H H 2It can be.

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

[0111] Exemplary V H H has a molecular weight of approximately 12-15 kDa, which is much smaller than that of conventional mammalian antibodies (150-160 kDa), which are composed of two heavy chains and two light chains. H H can be found or produced from mammals of the Camelidae family (e.g., camels, llamas, dromedaries, alpacas and guanacos) that do not naturally have light chains. 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 some embodiments, to prepare a binding protein, H H can be synthesized separately and then joined together by a linker. Alternatively, the bispecific V H H 2 can be synthesized as fusion proteins. V with different binding activities and receptor targets H Pairing of Hs to form bispecific V H H 2This binding protein can be screened for signaling in cells bearing one or both of the relevant receptors.

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

[0114] In some embodiments, the V H H can be humanized to include human framework regions. H Examples of human germline sequences that can be used to make 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) TIFF0007665730000008.tif240167TIFF0007665730000009.tif241167TIFF0007665730000010.tif241167TIFF0007665730000011.tif240167TIFF0007665730000012.tif241167TIFF0007665730000013.tif236167TIFF0007665730000014.tif241167TIFF0007665730000015.tif236167TIFF0007665730000016.tif240167TIFF0007665730000017.tif241167TIFF0007665730000018.tif240167TIFF0007665730000019.tif240167TIFF0007665730000020.tif241167TIFF0007665730000021.tif236167TIFF0007665730000022.tif241167TIFF0007665730000023.tif236167TIFF0007665730000024.tif240167TIFF0007665730000025.tif241167TIFF0007665730000026.tif240167TIFF0007665730000027.tif241167TIFF0007665730000028.tif241167TIFF0007665730000029.tif236167TIFF0007665730000030.tif241167TIFF0007665730000031.tif236167TIFF0007665730000032.tif240167TIFF0007665730000033.tif241167TIFF0007665730000034.tif240167TIFF0007665730000035.tif241167TIFF0007665730000036.tif232167TIFF0007665730000037.tif240167TIFF0007665730000038.tif241167TIFF0007665730000039.tif240167TIFF0007665730000040.tif240167TIFF0007665730000041.tif241167TIFF0007665730000042.tif240167TIFF0007665730000043.tif241167TIFF0007665730000044.tif236167TIFF0007665730000045.tif241167TIFF0007665730000046.tif241167TIFF0007665730000047.tif236167TIFF0007665730000048.tif240167TIFF0007665730000049.tif241167TIFF0007665730000050.tif240167TIFF0007665730000051.tif241167TIFF0007665730000052.tif240167TIFF0007665730000053.tif241167TIFF0007665730000054.tif241167TIFF0007665730000055.tif236167TIFF0007665730000056.tif241167TIFF0007665730000057.tif241167TIFF0007665730000058.tif240167TIFF0007665730000059.tif241167TIFF0007665730000060.tif236167TIFF0007665730000061.tif241167TIFF0007665730000062.tif236167TIFF0007665730000063.tif240167TIFF0007665730000064.tif241167TIFF0007665730000065.tif241167TIFF0007665730000066.tif240167TIFF0007665730000067.tif241167TIFF0007665730000068.tif236167TIFF0007665730000069.tif241167TIFF0007665730000070.tif236167TIFF0007665730000071.tif240167TIFF0007665730000072.tif241167TIFF0007665730000073.tif240167TIFF0007665730000074.tif240167TIFF0007665730000075.tif241167TIFF0007665730000076.tif236167TIFF0007665730000077.tif241167TIFF0007665730000078.tif236167TIFF0007665730000079.tif240167TIFF0007665730000080.tif241167TIFF0007665730000081.tif240167TIFF0007665730000082.tif241167TIFF0007665730000083.tif241167TIFF0007665730000084.tif236167TIFF0007665730000085.tif241167TIFF0007665730000086.tif236167TIFF0007665730000087.tif240167TIFF0007665730000088.tif241167TIFF0007665730000089.tif240167TIFF0007665730000090.tif241167TIFF0007665730000091.tif232167TIFF0007665730000092.tif240167TIFF0007665730000093.tif241167TIFF0007665730000094.tif240167TIFF0007665730000095.tif240167TIFF0007665730000096.tif241167TIFF0007665730000097.tif240167TIFF0007665730000098.tif241167TIFF0007665730000099.tif236167TIFF0007665730000100.tif241167TIFF0007665730000101.tif241167TIFF0007665730000102.tif236167TIFF0007665730000103.tif240167TIFF0007665730000104.tif241167TIFF0007665730000105.tif240167TIFF0007665730000106.tif241167TIFF0007665730000107.tif240167TIFF0007665730000108.tif241167TIFF0007665730000109.tif241167TIFF0007665730000110.tif236167TIFF0007665730000111.tif241167TIFF0007665730000112.tif241167TIFF0007665730000113.tif240167TIFF0007665730000114.tif241167TIFF0007665730000115.tif236167TIFF0007665730000116.tif241167TIFF0007665730000117.tif236167TIFF0007665730000118.tif240167TIFF0007665730000119.tif241167TIFF0007665730000120.tif241167TIFF0007665730000121.tif240167TIFF0007665730000122.tif241167TIFF0007665730000123.tif236167TIFF0007665730000124.tif241167TIFF0007665730000125.tif236167TIFF0007665730000126.tif240167TIFF0007665730000127.tif241167TIFF0007665730000128.tif240167TIFF0007665730000129.tif240167TIFF0007665730000130.tif241167TIFF0007665730000131.tif236167TIFF0007665730000132.tif241167TIFF0007665730000133.tif236167TIFF0007665730000134.tif240167TIFF0007665730000135.tif241167TIFF0007665730000136.tif240167TIFF0007665730000137.tif241167TIFF0007665730000138.tif241167TIFF0007665730000139.tif236167TIFF0007665730000140.tif241167TIFF0007665730000141.tif236167TIFF0007665730000142.tif240167TIFF0007665730000143.tif241167TIFF0007665730000144.tif240167TIFF0007665730000145.tif241167TIFF0007665730000146.tif232167TIFF0007665730000147.tif240167TIFF0007665730000148.tif241167TIFF0007665730000149.tif240167TIFF0007665730000150.tif240167TIFF0007665730000151.tif241167TIFF0007665730000152.tif240167TIFF0007665730000153.tif241167TIFF0007665730000154.tif236167TIFF0007665730000155.tif241167TIFF0007665730000156.tif241167TIFF0007665730000157.tif236167TIFF0007665730000158.tif240167TIFF0007665730000159.tif241167TIFF0007665730000160.tif240167TIFF0007665730000161.tif241167TIFF0007665730000162.tif240167TIFF0007665730000163.tif241167TIFF0007665730000164.tif241167TIFF0007665730000165.tif236167TIFF0007665730000166.tif241167TIFF0007665730000167.tif241167TIFF0007665730000168.tif240167TIFF0007665730000169.tif241167TIFF0007665730000170.tif236167TIFF0007665730000171.tif241167TIFF0007665730000172.tif236167TIFF0007665730000173.tif240167TIFF0007665730000174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000208.tif241167TIFF0007665730000209.tif236167TIFF0007665730000210.tif241167TIFF0007 665730000211.tif241167TIFF0007665730000212.tif236167TIFF0007665730000213.tif240167TI FF0007665730000214.tif241167TIFF0007665730000215.tif240167TIFF0007665730000216.tif24 1167TIFF0007665730000217.tif240167TIFF0007665730000218.tif241167TIFF000766573000021 9.tif241167TIFF0007665730000220.tif236167TIFF0007665730000221.tif241167TIFF000766573 0000222.tif241167TIFF0007665730000223.tif240167TIFF0007665730000224.tif241167TIFF000 7665730000225.tif236167TIFF0007665730000226.tif241167TIFF0007665730000227.tif236167.

[0116] In some embodiments, an IL27R binding protein described herein (e.g., in Table 1A) is encoded by an isolated nucleic acid that is substantially identical to any one of the sequences in Table 1B, below. In some embodiments, an IL27R binding protein described herein (e.g., an IL27R binding protein comprising a sequence in Table 1A) is encoded by an isolated nucleic acid that comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to a sequence in Table 1B, below.

[0117] (Table 1B) TIFF0007665730000228.tif186158TIFF0007665730000229.tif237158TIFF0007665730000230.tif236158TIFF0007665730000231.tif236158TIFF0007665730000232.tif237158TIFF0007665730000233.tif236158TIFF0007665730000234.tif237158TIFF0007665730000235.tif232158TIFF0007665730000236.tif236158TIFF0007665730000237.tif237158TIFF0007665730000238.tif236158TIFF0007665730000239.tif237158TIFF0007665730000240.tif236158TIFF0007665730000241.tif236158TIFF0007665730000242.tif237158TIFF0007665730000243.tif237158TIFF0007665730000244.tif232158TIFF0007665730000245.tif237158TIFF0007665730000246.tif237158TIFF0007665730000247.tif236158TIFF0007665730000248.tif237158TIFF0007665730000249.tif237158TIFF0007665730000250.tif236158TIFF0007665730000251.tif237158TIFF0007665730000252.tif237158TIFF0007665730000253.tif236158TIFF0007665730000254.tif237158TIFF0007665730000255.tif237158TIFF0007665730000256.tif236158TIFF0007665730000257.tif237158TIFF0007665730000258.tif232158TIFF0007665730000259.tif236158TIFF0007665730000260.tif237158TIFF0007665730000261.tif232158TIFF0007665730000262.tif237158TIFF0007665730000263.tif237158TIFF0007665730000264.tif236158TIFF0007665730000265.tif237158TIFF0007665730000266.tif236158TIFF0007665730000267.tif236158TIFF0007665730000268.tif237158TIFF0007665730000269.tif237158TIFF0007665730000270.tif237158TIFF0007665730000271.tif236158TIFF0007665730000272.tif236158TIFF0007665730000273.tif237158TIFF0007665730000274.tif237158TIFF0007665730000275.tif237158TIFF0007665730000276.tif232158TIFF0007665730000277.tif237158TIFF0007665730000278.tif237158TIFF0007665730000279.tif237158TIFF0007665730000280.tif232158TIFF0007665730000281.tif236158TIFF0007665730000282.tif237158TIFF0007665730000283.tif237158TIFF0007665730000284.tif237158TIFF0007665730000285.tif237158TIFF0007665730000286.tif236158TIFF0007665730000287.tif237158TIFF0007665730000288.tif237158TIFF0007665730000289.tif237158TIFF0007665730000290.tif237158TIFF0007665730000291.tif232158TIFF0007665730000292.tif237158TIFF0007665730000293.tif237158TIFF0007665730000294.tif237158TIFF0007665730000295.tif236158TIFF0007665730000296.tif237158TIFF0007665730000297.tif237158TIFF0007665730000298.tif236158TIFF0007665730000299.tif237158TIFF0007665730000300.tif237158TIFF0007665730000301.tif236158TIFF0007665730000302.tif237158TIFF0007665730000303.tif237158TIFF0007665730000304.tif236158TIFF0007665730000305.tif237158TIFF0007665730000306.tif237158TIFF0007665730000307.tif236158TIFF0007665730000308.tif237158TIFF0007665730000309.tif237158TIFF0007665730000310.tif236158TIFF0007665730000311.tif236158TIFF0007665730000312.tif237158TIFF0007665730000313.tif237158TIFF0007665730000314.tif232158TIFF0007665730000315.tif236158TIFF0007665730000316.tif237158TIFF0007665730000317.tif232158TIFF0007665730000318.tif236158TIFF0007665730000319.tif237158TIFF0007665730000320.tif237158TIFF0007665730000321.tif232158TIFF0007665730000322.tif236158TIFF0007665730000323.tif237158TIFF0007665730000324.tif237158TIFF0007665730000325.tif236158TIFF0007665730000326.tif236158TIFF0007665730000327.tif237158TIFF0007665730000328.tif237158TIFF0007665730000329.tif236158TIFF0007665730000330.tif237158TIFF0007665730000331.tif237158TIFF0007665730000332.tif236158TIFF0007665730000333.tif236158TIFF0007665730000334.tif237158TIFF0007665730000335.tif232158TIFF0007665730000336.tif237158TIFF0007665730000337.tif237158TIFF0007665730000338.tif236158TIFF0007665730000339.tif237158TIFF0007665730000340.tif237158TIFF0007665730000341.tif236158TIFF0007665730000342.tif237158TIFF0007665730000343.tif236158TIFF0007665730000344.tif236158TIFF0007665730000345.tif237158TIFF0007665730000346.tif232158TIFF0007665730000347.tif236158TIFF0007665730000348.tif237158TIFF0007665730000349.tif237158TIFF0007665730000350.tif232158TIFF0007665730000351.tif236158TIFF0007665730000352.tif237158TIFF0007665730000353.tif237158TIFF0007665730000354.tif232158TIFF0007665730000355.tif237158TIFF0007665730000356.tif237158TIFF0007665730000357.tif236158TIFF0007665730000358.tif237158TIFF0007665730000359.tif237158TIFF0007665730000360.tif236158TIFF0007665730000361.tif236158TIFF0007665730000362.tif2 37158TIFF0007665730000363.tif237158TIFF0007665730000364.tif236158TIFF0007665730000365.tif237158 TIFF0007665730000366.tif237158TIFF0007665730000367.tif236158TIFF0007665730000368.tif237158TIFF0 007665730000369.tif237158TIFF0007665730000370.tif236158TIFF0007665730000371.tif236158TIFF000766 5730000372.tif237158TIFF0007665730000373.tif237158TIFF0007665730000374.tif236158TIFF00076657300 00375.tif237158TIFF0007665730000376.tif237158TIFF0007665730000377.tif236158TIFF0007665730000378 .tif237158TIFF0007665730000379.tif232158TIFF0007665730000380.tif236158TIFF0007665730000381.tif2 37158TIFF0007665730000382.tif236158TIFF0007665730000383.tif236158TIFF0007665730000384.tif22158.

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

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

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

[0121] (Table 1) TIFF0007665730000385.tif218163TIFF0007665730000386.tif218163TIFF0007665730000387.tif78163

[0122] Anti-GP130-Linker-Anti-Il27Rα V H H Bispecific V H H 2 From the N-terminus to the C-terminus, the first V H H (anti-gp130 VH H antibody), a linker, and a second V that binds to IL27Rα H H (anti-IL27Rα V H In other words, the linker can include an anti-gp130 VH antibody in the binding protein. H The C-terminus of H was linked to the anti-IL27Rα V H In some embodiments, the purified peptide is a 6-histidine peptide ((His)6 (SEQ ID NO:1531) or His-tag) to the bispecific V H H 2 may or may not be included in the

[0123] In certain embodiments, the bispecific V H H 2 The present invention relates to an anti-gp130 V antibody having 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 SEQ ID NOs: 232 to 237. H H antibodies; and anti-IL27Rα V antibodies comprising a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to any one of SEQ ID NOs: 238-244. H Contains H antibodies.

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

[0125] (Table 2A) TIFF0007665730000388.tif180150TIFF0007665730000389.tif225150TIFF0007665730000390.tif220150TIFF0007665730000391.tif249150

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

[0127] (Table 2B) TIFF0007665730000392.tif107150TIFF0007665730000393.tif224150TIFF00076657300 00394.tif224150TIFF0007665730000395.tif224150TIFF0007665730000396.tif135150

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

[0129] (Table 2C) TIFF0007665730000397.tif244150TIFF0007665730000398.tif232150TIFF00076657300 00399.tif232150TIFF0007665730000400.tif232150TIFF0007665730000401.tif228150 TIFF0007665730000402.tif224150TIFF0007665730000403.tif232150TIFF00076657300 00404.tif232150TIFF0007665730000405.tif232150TIFF0007665730000406.tif232150

[0130] Anti-IL27Rα V H H-Linker-Anti-GP130 V H H Bispecific V H H 2 From the N-terminus to the C-terminus, the first V H H (anti-IL27Rα V H H antibody), a linker, and a second V that binds to gp130. H H (anti-gp130 V H In other words, the linker can include an anti-IL27Rα V H antibody in the binding protein. H The C-terminus of H was linked to the anti-gp130 V H In some embodiments, the purified peptide is a 6-histidine peptide ((His)6 (SEQ ID NO:1531) or His-tag) or Fc-tag to the bispecific V H H 2 can be included in

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

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

[0133] (Table 3A) TIFF0007665730000407.tif26150TIFF0007665730000408.tif220150TIFF00076657300 00409.tif225150TIFF0007665730000410.tif220150TIFF0007665730000411.tif188150

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

[0135] (Table 3B) TIFF0007665730000412.tif188150TIFF0007665730000413.tif228150TIFF0007665730000414.tif232150TIFF0007665730000415.tif212150

[0136] In some embodiments, an IL27R binding protein described herein (e.g., an IL27R binding protein comprising the sequence of any one of SEQ ID NOs:43-84) is encoded by an isolated nucleic acid that is substantially identical to the sequence of any one of SEQ ID NOs:151-192, as set forth in Table 3C below. In some embodiments, an IL27R binding protein described herein (e.g., an IL27R binding protein comprising the sequence of any one of SEQ ID NOs:43-84) is encoded by an isolated nucleic acid that comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to the sequence of any one of SEQ ID NOs:151-192, as set forth in Table 3C below.

[0137] (Table 3C) TIFF0007665730000416.tif177150TIFF0007665730000417.tif232150TIFF0007665730000418.t if232150TIFF0007665730000419.tif232150TIFF0007665730000420.tif224150TIFF00076657300 00421.tif232150TIFF0007665730000422.tif232150TIFF0007665730000423.tif232150TIFF000 7665730000424.tif232150TIFF0007665730000425.tif232150TIFF0007665730000426.tif177150

[0138] Further IL27R-binding proteins Additionally, additional IL27R binding proteins include the anti-IL27Rα V H The three underlined CDR sequences in the H antibody and any one of SEQ ID NOs: 232 to 237 of the anti-gp130 V antibody HFurther IL27R binding proteins include the three underlined CDR sequences in the anti-IL27Rα VH antibody. H H antibody (e.g., any one of the sequences listed in Table 4 below) and anti-gp130 V H In some embodiments, the binding protein can include an anti-IL27Rα V H antibody (e.g., any one of SEQ ID NOs:232-237) at the N-terminus. H H antibody and C-terminal anti-gp130 V H In some embodiments, the binding protein comprises an anti-gp130 V H antibody at the N-terminus. H H antibody and C-terminal anti-IL27Rα V H In some embodiments, the binding protein comprises an anti-IL27Rα V H antibody. H H antibody and anti-gp130 V H A linker (e.g., any one of SEQ ID NOs:85 to 108 (e.g., SEQ ID NO:108)) is included between the H antibody and the H antibody. In certain embodiments, the binding protein is a 6-histidine peptide (His)6 (SEQ ID NO:1531) (His-tag).

[0139] (Table 4) TIFF0007665730000427.tif158150TIFF0007665730000428.tif64150

[0140] The IL27R binding proteins were selected from the group consisting of anti-IL27Rα V H The three CDR sequences underlined in the H antibody and the anti-gp130 V H In some embodiments, the IL27R binding protein can comprise the three underlined CDR sequences in the anti-IL27Rα VH antibody. H H antibodies and anti-gp130 V H In some embodiments, the binding protein comprises an anti-IL27Rα V H antibody at the N-terminus. H H antibody and C-terminal anti-gp130 VH In some embodiments, the binding protein comprises an anti-gp130 V H antibody at the N-terminus. H H antibody and C-terminal anti-IL27Rα V H In some embodiments, the binding protein comprises an anti-IL27Rα V H antibody. H H antibody and anti-gp130 V H A linker (e.g., any one of SEQ ID NOs:85 to 108 (e.g., SEQ ID NO:108)) is included between the H antibody and the H antibody. In certain embodiments, the binding protein is a 6-histidine peptide (His)6 (SEQ ID NO:1531) (His-tag).

[0141] (Table 5) TIFF0007665730000429.tif60150TIFF0007665730000430.tif223150TIFF0007665730000431.tif223150TIFF0007665730000432.tif21150

[0142] In addition, the IL27R binding protein was identified using mouse anti-IL27Rα V H H antibody and mouse anti-gp130 V H It may contain H antibodies. In some instances, 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 provide antibodies that specifically bind to the receptor of one or more additional mammalian species. Such antibodies are referred to as "cross-reactive." For example, immunization of a camelid with an antigen derived from a human (e.g., hIL27Rα-ECD) may generate antibodies that cross-react with mouse and human receptors. Assessment of the cross-reactivity of antibodies with respect to receptors derived from other mammalian species may be readily determined by one of skill in the art using methods for assessing binding affinity and / or specific binding described elsewhere herein, such as, for example, flow cytometry or SPR. As a result, the use of the term "human IL27Rα VHH" or "hIL27Rα VHH" merely indicates that the species of IL27Rα antigen used for immunization of the camelid from which the VHH is derived is human IL27Rα, but should not be understood as limiting with respect to the specific binding affinity of the VHH to the IL27Rα molecule of other mammalian species. Similarly, use of the term "mouse IL27Rα VHH" or "mIL27Rα VHH" simply indicates that the species of IL27Rα antigen used for immunization of the camelid from which the VHH is derived is mouse IL27Rα, but should not be understood as limiting with respect to the specific binding affinity of the VHH for IL27Rα molecules of other mammalian species. In some embodiments, the IL27R binding protein is a murine anti-IL27Rα V H The three CDR sequences underlined in the H antibody sequence and the mouse anti-gp130 V H In some embodiments, the IL27R binding protein can comprise the three CDR sequences underlined in the H antibody sequence. H H antibody and mouse anti-gp130 V as described below H In some embodiments, the binding protein comprises a murine anti-IL27Rα V H antibody at the N-terminus. H H antibody and C-terminal mouse anti-gp130 V HIn some embodiments, the binding protein comprises a mouse anti-gp130 V H antibody at the N-terminus. H H antibody and C-terminal mouse anti-IL27Rα V H In some embodiments, the binding protein comprises a mouse anti-IL27Rα V H antibody. H H antibody and mouse anti-gp130 V H A linker (e.g., any one of SEQ ID NOs:85 to 108 (e.g., SEQ ID NO:108)) is included between the H antibody and the H antibody. In certain embodiments, the binding protein is a 6-histidine peptide (His)6 (SEQ ID NO:1531) (His-tag).

[0143] Mouse anti-IL27Rα V H Example of H antibody sequence: TIFF0007665730000433.tif188150TIFF0007665730000434.tif68150

[0144] Mouse anti-gp130 V H Example of H antibody sequence: TIFF0007665730000435.tif146150TIFF0007665730000436.tif224150TIFF0007665730 000437.tif228150TIFF0007665730000438.tif220150TIFF0007665730000439.tif68150

[0145] In some embodiments, the V H H can be humanized to include human framework regions. HExamples of human germline sequences that can be used to make 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 an E1 induced by IL27. max Reduced E compared to max E max reflects the maximum response level in a cell type that can be obtained by a ligand (e.g., a binding molecule described herein or a natural cytokine (e.g., IL27)). In some embodiments, the IL27R binding proteins described herein inhibit the E1 activation induced by IL27. max In another embodiment, the E of the IL27R binding protein described herein is at least 1% (e.g., 1% to 100%, 10% to 100%, 20% to 100%, 30% to 100%, 40% to 100%, 50% to 100%, 60% to 100%, 70% to 100%, 80% to 100%, 90% to 100%, 1% to 90%, 1% to 80%, 1% to 70%, 1% to 60%, 1% to 50%, 1% to 40%, 1% to 30%, 1% to 20%, or 1% to 10%). max is the E of the natural ligand IL27 max In some embodiments, the E of the IL27R binding protein is increased by varying the linker length of the IL27R binding protein. max IL27R binding proteins can alter E. coli expression in the most desired cell types. max and reduced E in other cell types.max This can result in...

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

[0148] In some embodiments, the linker is a peptide linker. The peptide linker 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 be a chemical linker such as a synthetic polymer, for example, a polyethylene glycol (PEG) polymer.

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

[0150] Suitable peptide linkers are known in the art and include, for example, peptide linkers that contain flexible amino acid residues, such as glycine and serine. In certain embodiments, the peptide linker can include a motif, e.g., multiple or repeated 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 can include 2-12 amino acids including a GS motif, e.g., TIFF0007665730000440.tif11155. In certain other embodiments, the peptide linker can include 3-12 amino acids that include a GGS motif, such as GGS, GGSGGS (SEQ ID NO:94), GGSGGSGGS (SEQ ID NO:95) and GGSGGSGGSGGS (SEQ ID NO:96). In yet other embodiments, the peptide linker can include 4-20 amino acids that include a GGSG (SEQ ID NO:87) motif, such as TIFF0007665730000441.tif18150. In other embodiments, the peptide linker can include a GGGGS (SEQ ID NO:85) motif, e.g., GGGGSGGGGS (SEQ ID NO:101) or GGGSGGGGSGGGGS (SEQ ID NO:102).

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

[0152] Further examples of flexible 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=1-50, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 10-20, 20-30, 30-50. Exemplary flexible linkers include, but are not limited to, GGGS (SEQ ID NO:108), GGGGS (SEQ ID NO:85), GGSG (SEQ ID NO:87), GGSGG (SEQ ID NO:103), GSGSG (SEQ ID NO:104), GSGGG (SEQ ID NO:105), GGGSG (SEQ ID NO:106), and GSSSG (SEQ ID NO:107).

[0153] V. Modifications to Extend Duration of Action In Vivo The binding protein described herein can be modified to provide extended in vivo life span and / or extended duration of action in subjects.In some embodiments, the binding protein can be conjugated to carrier molecules to provide desired pharmacological properties, such as extended half-life.In some embodiments, the binding protein 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, to extend half-life.

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

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

[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 the present disclosure include polyethylene glycol (PEG), poly-propylene glycol (PPG), polysaccharides (polyvinylpyrrolidone, copolymers of ethylene glycol and propylene glycol, poly(oxyethylated polyols), polyolefin alcohols), polysaccharides), poly-alpha-hydroxy acids), polyvinyl alcohols (PVA), polyphosphazenes, polyoxazolines (POZ), poly(N-acryloylmorpholines), or combinations thereof.

[0157] In some embodiments, the binding protein may be conjugated with one or more polyethylene glycol molecules, or "PEGylated." Although the method or site of attachment of PEG to the binding protein can vary, in certain embodiments, PEGylation does not alter, or only minimally alters, the activity of the binding protein.

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

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

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

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

[0162] The present disclosure also contemplates compositions of conjugates in which the PEG has different n values, and thus various different PEGs are present in specific ratios. For example, some compositions contain a mixture of conjugates with n=1, 2, 3, and 4. In some compositions, the percentage of conjugates with n=1 is 18-25%, the percentage of conjugates with n=2 is 50-66%, the percentage of conjugates with n=3 is 12-16%, and the percentage of conjugates 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 fractions of conjugates, and then fractions containing, for example, conjugates with a desired number of PEGs attached are identified and purified free of unmodified protein sequences and conjugates with other numbers of PEGs attached.

[0163] PEG suitable for conjugation to polypeptide sequences is generally water soluble at room temperature and has the general formula R(O-CH-CH), where R is hydrogen or a protecting group such as an alkyl or alkanol group, and n is an integer between 1 and 1000. n It has OR. 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. Pat. No. 5,650,234), which react preferentially with lysine residues to form carbamate linkages, 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 a polypeptide through reductive amination.

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

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

[0167] In some embodiments, the PEGylation of binding protein is promoted by incorporating unnatural amino acids with unique side chains to promote site-specific PEGylation.The incorporation of unnatural amino acids into polypeptides to provide functional moieties for achieving site-specific PEGylation of such polypeptides is known in the art.See, for example, Ptacin et al., PCT International Application No. PCT / US2018 / 045257, filed August 3, 2018, and published February 7, 2019 under International Publication No. WO 2019 / 028419Al.

[0168] The PEG conjugated to the polypeptide sequence can be linear or branched. Branched PEG derivatives, "star PEGs" and multi-armed PEGs are contemplated by the present disclosure. In certain embodiments, the PEG useful in the practice of the present 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 ester (e.g., Sunbright® ME-100CS, Sunbright® ME-100AS, Sunbright® ME-100GS, Sunbright® ME-100HS, NOF), 20 kDa linear PEG-aldehyde (e.g., Sunbright® ME-200AL, NOF), 20 kDa linear PEG-NHS ester (e.g., Sunbright® ME-200CS, Sunbright® ME-200AS, Sunbright® ME-200GS, Sunbright® ME-200HS, NOF), 20 kDa 2-arm branched PEG-aldehyde, which comprises two 10 kDa linear PEG molecules (e.g., Sunbright® ME-200AL, NOF). GL2-200AL3, NOF), 20 kDa 2-arm branched PEG-NHS ester comprising two 10 kDA linear PEG molecules (e.g., Sunbright® GL2-200TS, Sunbright® GL200GS2, NOF), 40 kDa 2-arm branched PEG-aldehyde comprising two 20 kDA linear PEG molecules (e.g., Sunbright® GL2-400AL3), 40 kDa 2-arm branched PEG-NHS ester comprising two 20 kDA linear PEG molecules (e.g., Sunbright® GL2-400AL3, Sunbright® GL2-400GS2, NOF), linear 30 kDa These include PEG-aldehydes (e.g., Sunbright® ME-300AL) and linear 30 kDa PEG-NHS esters.

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

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

[0171] In some embodiments, the binding proteins are expressed as fusion proteins with albumin molecules known in the art (eg, human serum albumin) to facilitate long-term exposure in vivo.

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

[0173] The above fusion proteins may be readily produced by recombinant DNA methods according to techniques known in the art by constructing a recombinant vector containing a nucleic acid sequence encoding the binding protein, either at the N-terminus or C-terminus of the binding protein, in frame with a nucleic acid sequence encoding a fusion partner, which may optionally further comprise a nucleic acid sequence encoding a linker or spacer polypeptide in frame.

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

[0175] The pharmaceutical composition may contain a pharma- ceutically acceptable, non-toxic carrier, excipient, stabilizer or diluent, which are defined as vehicles commonly used to formulate pharmaceutical compositions for administration to animals or humans. The diluent is selected so as not to affect the biological activity of the combination. Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed and include, for example, buffers such as phosphates, citrates, and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (e.g., octadecyidimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl, or benzyl alcohol; alkyl parabens, such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone; amino acids, such as glycine, glutamine, asparagine, histidine, arginine, or lysine; Monosaccharides, disaccharides, and other sugars including glucose, mannose, or dextrins; 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 non-ionic surfactants, such as TWEEN™, PLURONICS™, or polyethylene glycol (PEG).

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

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

[0178] Administration of the binding proteins described herein may be accomplished through any of a variety of art-recognized methods, including, but not limited to, topical, intravascular injection (including intravenous or intraarterial infusion), intradermal injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, intracranial injection, intratumoral injection, intranodal injection, transdermal, transmucosal, iontophoretic 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 system inhalers including nebulizers, intraocular injection, intraperitoneal injection, intralesional injection, intraovarian injection, intracerebral infusion or injection, intraventricular injection (ICVI), and the like. In some embodiments, administration includes administration of the binding protein itself (e.g., parenterally), as well as administration of a recombinant vector (e.g., viral or non-viral vector) to cause in situ expression of the binding protein in a subject. Alternatively, cells, such as cells isolated from a subject, can be recombinantly modified to express a binding protein of the disclosure.

[0179] The dosage of the pharmaceutical composition will depend on factors including the route of administration, the disease being treated, and the physical characteristics of the subject, such as age, weight, general health, etc. Typically, the amount of binding protein contained within a single dose will be that amount that effectively prevents, delays or treats the disease without inducing significant toxicity. The pharmaceutical compositions of the present disclosure may be administered in a concentration of 0.01-500 mg / kg (e.g., 0.01-450 mg, 0.01-400 mg, 0.01-350 mg, 0.01-300 mg, 0.01-250 mg, 0.01-200 mg, 0.01-150 mg, 0.01-100 mg, 0.01-50 mg, 0.01-10 mg, 0.01-1 mg, 0.1-500 mg / kg, 1-500 mg / kg, 5-500 mg / kg, 10-500 mg / kg, 50-500 mg / kg, 100-500 mg / kg, 150-500 mg / kg, 200-500 mg / kg, 250-500 mg / kg, 300-500 mg / kg, 350-500 mg / kg, 400-400 mg / kg, 450-500 mg / kg, 500-500 mg / kg, 600-650 mg / kg, 700-750 mg / kg, 800-850 mg / kg, 900-950 mg / kg, 1000-1000 mg / kg, 1500-1000 mg / kg, 2000-500 mg / kg, 2500-500 mg / kg, 3000-500 mg / kg, 3500-500 mg / kg, 4000-4000 mg / kg, 5000-500 mg / kg, 6000-6500 mg / kg, 7000-7500 mg / kg, 8000-8500 mg / kg, 90 mg / kg, 400 to 500 mg / kg, or 450 to 500 mg / kg), and in a more specific embodiment, about 1 to about 100 mg / kg (e.g., about 1 to about 90 mg / kg, about 1 to about 80 mg / kg, about 1 to about 70 mg / kg, about 1 to about 60 mg / kg, about 1 to about 50 mg / kg, about 1 to about 40 mg / kg, about 1 to about 30 mg / kg, about 1 to about 20 mg / kg, about 1 to about 10 mg / kg, about 10 to about 100 mg / kg, about 20 to about 100 mg / kg, about 30 to about 100 mg / kg, about 40 to about 100 mg / kg, about 50 to about 100 mg / kg, about 60 to about 100 mg / kg, about 70 to about 100 mg / kg, about 80 to about 100 mg / kg, or about 90 to about 100 The dosages may include doses of the binding proteins described herein in the range of 1 mg / kg.In some embodiments, the pharmaceutical composition of the present disclosure may comprise a dosage of the binding protein described herein in the range of 0.01-20 mg / kg (e.g., 0.01-15 mg / kg, 0.01-10 mg / kg, 0.01-8 mg / kg, 0.01-6 mg / kg, 0.01-4 mg / kg, 0.01-2 mg / kg, 0.01-1 mg / kg, 0.01-0.1 mg / kg, 0.01-0.05 mg / kg, 0.05-20 mg / kg, 0.1-20 mg / kg, 1-20 mg / kg, 2-20 mg / kg, 4-20 mg / kg, 6-20 mg / kg, 8-20 mg / kg, 10-20 mg / kg, 15-20 mg / kg). The dosage may be adapted by the physician according to conventional factors such as the extent of the disease and different parameters of the subject.

[0180] A pharmaceutical composition containing a binding protein described herein can be administered to a subject in need thereof, for example, one or more times (e.g., 1-10 or more times) daily, weekly, monthly, twice yearly, annually, or as medically necessary. Dosages can be provided in either single or multiple dose regimens. The timing between doses can decrease as the medical condition improves or increase as the patient's health condition declines. A course of treatment can be a single dose or multiple doses over a period of time. In some embodiments, a single dose is used. In some embodiments, two or more split 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 split dosing protocols can be the same or different in each administration. A multi-day dosing protocol over a period of time can be provided by one of skill in the art (e.g., a physician) monitoring administration taking into account the subject's response to treatment, including side effects of the treatment and adjustments thereto, as discussed above.

[0181] VII. Indications immune disease The present disclosure further provides methods of treating a subject suffering from a disease disorder or condition by administration of a therapeutically effective amount of an IL27R binding protein of the present disclosure (or a nucleic acid encoding an IL27R binding protein, including a recombinant virus encoding an IL27R binding protein). Disorders amenable to treatment with an IL27R binding protein of the present disclosure (including pharma- ceutically acceptable formulations comprising an IL27R binding protein and / or nucleic acid molecules encoding them, including recombinant viruses encoding such IL27R binding proteins) include viral infections (e.g., AIDS, influenza, chronic HCV, chronic viral hepatitis B, C, or D), 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, cartilage inflammation, arthritis, rheumatoid arthritis, juvenile arthritis, juvenile rheumatoid arthritis, polyarticular juvenile rheumatoid arthritis, systemic juvenile rheumatoid arthritis, juvenile ankylosing spondylitis, juvenile enteropathic arthritis, juvenile reactive arthritis, juvenile Reiter's syndrome, SEA syndrome (seronegative enthesopathy arthropathy) Inflammatory or autoimmune diseases, including, but not limited to, juvenile dermatomyositis, juvenile psoriatic arthritis, juvenile scleroderma, juvenile systemic lupus erythematosus, juvenile vasculitis, small-articular rheumatoid arthritis, polyarticular rheumatoid arthritis, systemic rheumatoid arthritis, ankylosing spondylitis, enteropathic arthritis, reactive arthritis, Reiter's syndrome, and SEA syndrome (Seronegativity, Enthesopathy, Arthropathy Syndrome).

[0182] Other examples of proliferative and / or differentiative disorders suitable for treatment with the IL27R binding proteins of the present disclosure (including pharma- ceutically acceptable formulations containing IL27R binding proteins and / or nucleic acid molecules encoding them, including recombinant viruses encoding such IL27R binding proteins) include, but are not limited to, skin disorders. Skin disorders can involve abnormal activity of cells or groups of cells in the dermis, epidermis, or hypodermis, or abnormalities at the dermal-epidermal junction. For example, skin disorders can 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 epidermal layers, such as the basal layer (stratum germinativum), stratum spinosum, stratum granular, stratum lucidum, or stratum corneum. In other embodiments, the disorder may involve abnormal activity of dermal cells, such as dermal endothelium, fibroblasts, immune cells (eg, mast cells or macrophages), found in the dermal layers, such as the papillary or reticular layers.

[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 lichenoidis, lichen planus, lichen nitidus, ichthyosa-like dermatitis, keratoderma, skin disease, alopecia areata, pyoderma gangrenosum, vitiligo, pemphigoid (e.g., ophthalmic cicatricial pemphigoid or bullous pemphigoid), urticaria, porokeratosis, hyperproliferation of epithelium-associated cells lining the joint capsule, and These include rheumatoid arthritis with inflammation and inflammation, dermatitis such as seborrheic dermatitis and sunburn, keratosis such as seborrheic keratosis, senile keratosis, actinic keratosis, actin-induced keratosis and follicular keratosis, acne vulgaris, keloids and prophylaxis against keloid formation, human papillomavirus (HPV) infections such as birthmarks, warts, condyloma or condyloma acuminata, and genital warts, leukoplakia, lichen planus, and keratitis. The skin disorder can be dermatitis, for example atopic dermatitis or allergic dermatitis, or psoriasis.

[0184] The compositions of the present disclosure (including pharma- ceutically acceptable formulations containing IL27R binding proteins and / or nucleic acid molecules encoding them, including recombinant viruses encoding such IL27R binding proteins) can also be administered to patients suffering from (or susceptible to) psoriasis or psoriatic disorders. The term "psoriasis" is intended to have its medical meaning, i.e., a disease that primarily affects the skin and produces raised, thickened, scaly, non-blemished lesions. The lesions are usually sharply demarcated erythematous papules covered with overlapping shiny scales. The scales are usually silvery or slightly opaque. Nail involvement is frequent, resulting in pitting, peeling, thickening and discoloration of the nails. Psoriasis can also be accompanied by arthritis, which can be disabling. Hyperproliferation of keratinocytes, along with epidermal inflammation and reduced differentiation of keratinocytes, is a key feature of psoriatic epidermal hyperplasia. Several mechanisms have been invoked to explain the keratinocyte hyperproliferation that characterizes psoriasis. Abnormalities in cell-mediated immunity are also thought to play a role in the pathogenesis of psoriasis. Examples of psoriatic disorders include chronic constant psoriasis, plaque psoriasis, moderate to severe plaque psoriasis, plaque psoriasis, eruptive psoriasis, erythrodermic psoriasis, generalized pustular psoriasis, annular pustular psoriasis, or localized pustular psoriasis.

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

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

[0187] In some embodiments, the adjunctive agent is one or more agents selected from the group consisting of corticosteroids (including but not limited to prednisone, budesonide, prednisone), 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 abatcept (Orencia®) or etanercept (Enbrel®), and therapeutic antibodies. Examples of therapeutic antibodies that may be administered as adjunctive agents in combination with the IL27R binding proteins of the present disclosure in treating 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), anti-TNFα antibodies (e.g., adalimumab (humectin), and anti-IL6R antibodies (e.g., humectin). non-limiting examples of antibodies that may be used include, but are not limited to, anti-IL12 / IL23 antibodies (e.g., ustekinumab), anti-IL5 antibodies (e.g., mepolizumab, reslizumab), 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 for autoimmune diseases. Examples of antibodies approved by the U.S. Food and Drug Administration (FDA) for use in treating autoimmune diseases in subjects suffering from autoimmune diseases that may be administered as adjunctive agents in combination with the IL27R binding proteins of the present disclosure (and optionally additional adjunctive agents) for treatment of the indicated autoimmune diseases are provided in Table 4.

[0189] (Table 4) TIFF0007665730000442.tif207159

[0190] Treatment of Neoplastic Disease The present disclosure provides methods of use of IL-27R binding molecules in treating a subject suffering from a neoplastic disease disorder or condition by administration of a therapeutically effective amount of the IL-27R binding molecules described herein (or nucleic acids encoding the IL-27R binding molecules, including recombinant vectors encoding the IL-27R binding molecules, and eukaryotic and prokaryotic cells modified to express the IL-27R binding molecules).

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

[0192] Examples of benign neoplasms suitable for treatment using the compositions and methods of the present disclosure include, but are not limited to, adenoma, fibroma, hemangioma, and lipoma. Examples of pre-malignant neoplasms suitable for treatment using the compositions and methods of the present 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 the present disclosure include, but are not limited to, carcinomas (cancers that originate from epithelial tissues such as skin or tissues that cover internal organs), leukemia, lymphoma, and sarcomas that typically originate from bone, fat, muscle, blood vessels, or connective tissue. The term neoplasm also includes virus-induced neoplasms such as warts and hyperproliferative vascular diseases, including EBV-induced diseases (i.e., infectious mononucleosis), scar formation, intimal smooth muscle cell hyperplasia, restenosis, vascular occlusion, etc.

[0193] The term "neoplastic disease" includes cancers characterized by solid and non-solid tumors, including, but not limited to, breast cancer, sarcoma (including, but not limited to, osteosarcoma and angiosarcoma and fibrosarcoma), leukemia, lymphoma, genitourinary cancer (including, but not limited to, ovarian, urethral, ​​bladder, and prostate cancer), gastrointestinal cancer (including, but not limited to, colon, esophageal and gastric cancer), lung cancer, myeloma, pancreatic cancer, liver cancer, kidney cancer, endocrine cancer, skin cancer, and brain or central nervous system (CNS) and peripheral nervous system tumors, including gliomas and neuroblastomas, astrocytomas, myelodysplastic disorders, malignant or benign, cervical intraepithelial neoplasia, intestinal polyposis, oral leukoplakia, histiocytosis, hyperproliferative scars including keloid scars, hemangiomas, hyperproliferative arterial stenosis, psoriasis, inflammatory arthritis, hyperkeratosis and papular desquamative rashes including arthritis.

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

[0195] As used herein, the term "hematopoietic neoplastic disorder" refers to a neoplastic disease involving hyperplastic / neoplastic cells arising from hematopoietic origin, e.g., myeloid, lymphoid or erythroid lineages, or precursor cells thereof.

[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 leukemia of ill-defined lineage. Exemplary myeloid disorders suitable for treatment according to the present disclosure include, but are not limited to, acute promyelocytic leukemia (APML), acute myeloid leukemia (AML), and chronic myeloid leukemia (CML).

[0197] Lymphoid neoplasms include, but are not limited to, precursor lymphoid neoplasms, mature B-cell neoplasms, mature T-cell neoplasms, Hodgkin's lymphoma, and immunodeficiency-associated lymphoproliferative disorders. Exemplary lymphoid disorders suitable for treatment according to the present 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 Waldenstrom's hypergammaglobulinemia (WM).

[0198] In some cases, hematopoietic neoplastic disorders result from poorly differentiated acute leukemias (e.g., erythroblastic leukemia and acute megakaryoblastic leukemia). As used herein, the term "hematopoietic neoplastic disorders" refers to malignant lymphomas, including, but not limited to, non-Hodgkin's 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] A determination of whether a subject is "suffering from a neoplastic disease" refers to a determination made by a physician about a subject based on available information accepted in the art for identifying a disease, disorder, or condition, including, but not limited to, x-rays, CT scans, conventional clinical diagnostic tests (e.g., blood counts, etc.), genomic data, protein expression data, immunohistochemistry, that the subject requires or would benefit from treatment.

[0200] Assessment of Anti-Neoplastic Efficacy: Determining the effectiveness of the disclosed method in treating cancer generally relates to the achievement of one or more parameters recognized in the art, such as reduction in lesions, particularly metastatic lesions, reduction in metastasis, reduction in tumor volume, improvement in ECOG score, and others. Determining response to treatment can be assessed through the measurement of biomarkers that can provide reproducible information useful in any aspect of IL-27R binding molecule therapy, including the presence and degree of subject response to such therapy and the presence and degree of adverse effects caused by such therapy. By way of example and not of limitation, biomarkers include increased IFNγ, and 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 cells, and IL-27R binding molecule upregulation. Reg Treatment response may be characterized by improvements in traditional measures of clinical efficacy, such as complete response (CR), partial response (PR), and stable disease (SD) for target lesions, including complete response (CR), incomplete response / stable disease (SD) as defined by RECIST, as well as immune-related complete response (irCR), immune-related partial response (irPR), and immune-related stable disease (irSD) as defined by immune-related response criteria (irRC), which are considered by those skilled in the art to evidence efficacy in treating neoplastic disease in mammalian (e.g., human) subjects.

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

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

[0203] Chemotherapeutic 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 agent or cocktail is administered in combination with one or more physical methods (e.g., radiation therapy). The term "chemotherapeutic agent" includes alkylating agents such as thiotepa and cyclophosphamide; alkylsulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodepa, carboquone, meturedepa and uredepa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide and trimethylolmelamine; nitrogen mustards such as chlorambucil, chlornaphazine, chlorophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, nobuenbiquine, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosoureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine; Antibiotics, such as aclacinomycin, actinomycin, anthramycin, azaserine, bleomycins such as bleomycin A2, cactinomycin, calicheamicin, carabicin, caminomycin, carzinophilin, chromomycin, dactinomycin, daunorubicin and its derivatives such as demethoxy-daunomycin, 11-deoxydaunorubicin, 13-deoxydaunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, esorubicin, idarubicin, mitomycins such as marcellomycin, mitomycin C, N-methylmitomycin C; Mycophenolic acid, nogalamycin, olivomycin, peplomycin, potfiromycin, puromycin, queramycin, lodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; antimetabolites such as methotrexate and 5-fluorouracil (5-FU);Folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate, dideazatetrahydrofolic acid, and folinic acid; purine analogues such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogues such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, 5-FU; androgens such as calsterone, dromostanolone propionate, epithiostanol, mepitiostane, testolactone; anti-adrenal drugs such as aminoglutethimide, mitotane, trilostane; folic acid supplements such as floric acid; aceglatone; aldophosphamide glycosides; aminolevulinic acid; amsacrine; bestravcil; bisantrene; Edatrexate; Defofamine; Demecolcine; Diaziquone; Eflornithine; Elliptinium acetate; Etoglucide; Gallium nitrate; Hydroxyurea; Lentinan; Lonidamine; Mitoguazone; Mitoxantrone; Mopidamol; Nitracrine; Pentostatin; Fenamet; Pirarubicin; Podophyllic acid; 2-Ethylhydrazide; Procarbazine; Razoxane; Sizofiran; Spirogermanium; Tenuazonic acid; Triazicon; 2,2',2"-Trichlorotriethylamine; Urethane; Vindesine; Dacarbazine; Mannomustine; Mitobronitol; Mitolactol; Pipobroman; Gacytosine; Arabinoside (Ara-C); Cyclophosphamide; Thiotepa; Taxoids such as paclitaxel, nab-paclitaxel and doxetaxel; chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum and platinum complexes such as 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 such as paclitaxel, docetaxel, carbazitaxel; carminomycin, adriamycin such as 4'-epidariamycin, 4-adriamycin-14-benzoate, adriamycin-14-octanoate, adriamycin-14-naphthalene acetate; colchicine as well as pharmaceutically acceptable salts, acids, or derivatives of any of the above;

[0204] The term "chemotherapeutic agent" also includes anti-hormonal agents that act to regulate or inhibit the action of hormones on tumors, such as anti-estrogens, including, for example, tamoxifen, raloxifene, aromatase-inhibiting 4(5)-imidazoles, 4-hydroxytamoxifen, trioxyphene, keoxifene, onapristone, and toremifene; and anti-androgens, such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; and pharmaceutically acceptable salts, acids, or derivatives of any of the above.

[0205] In some embodiments, the adjunctive agent is a cytokine or cytokine antagonist, such as IL-12, INFα, or anti-epidermal growth factor receptor, irinotecan, as practiced in known chemotherapy treatment regimens, including, but not limited to, TAC, FOLFOX, TPC, FEC, ADE, FOLFOX-6, EPOCH, CHOP, CMF, CVP, BEP, OFF, FLOX, CVD, TC, FOLFIRI, PCV, FOLFOXIRI, ICE-V, XELOX, and others readily recognized by a skilled clinician in the art; a tetrahydrofolate anti-metabolite, such as pemetrexed; an antibody against a tumor antigen, a monoclonal antibody-toxin conjugate, a T cell adjuvant, a bone marrow graft, or an antigen presenting cell (e.g., dendritic cell therapy), an anti-tumor vaccine, a replication competent virus, a signal transduction inhibitor (e.g., Gleevec® or Herceptin), (R)) or one or more chemical or biological agents identified in the art as being useful in the treatment of neoplastic diseases, including, but not limited to, immunomodulators, nonsteroidal anti-inflammatory drugs (NSAIDs), cyclooxygenase-2 (COX-2) inhibitors, steroids, TNF antagonists (e.g., Remicade (R) and Enbrel (R)), interferon-β1a (Avonex (R)), and interferon-β1b (Betaseron (R)), as well as combinations of one or more of the foregoing to achieve additive or synergistic inhibition of tumor growth.

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

[0207] Anti-tumor antigen antibody therapeutic agents as adjunct agents In some embodiments, the "auxiliary agent" is a therapeutic antibody (including bispecific and trispecific antibodies that bind to one or more tumor-associated antigens, including but not limited to bispecific T cell engaging antibodies (BITEs), dual affinity retargeting (DART) constructs, and trispecific killer engager (TriKE) constructs).

[0208] In some embodiments, the therapeutic antibody is selected from the group consisting of 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 , VEGF (e.g., bevacizumab), VEGFR, VEGFR2 (e.g., ramucirumab), PDGFRα (e.g., ofatumumab (olartumumab)), EGFR (e.g., cetuximab, panitumumab, and necitumumab), ERBB2 (e.g., trastuzumab), ERBB3, MET, IGF1R, EPHA3, TRAIL R1, TRAIL R2, RANKL RAP, tenascin, integrin αVβ3, and integrin α4β1.

[0209] Examples of antibody therapeutics that are FDA approved and can be used as adjunctive agents for use in the treatment of neoplastic diseases include those provided in the table below.

[0210] (Table) Approved anti-neoplastic antibodies and indications TIFF0007665730000443.tif85150TIFF0007665730000444.tif171150

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

[0212] Combination with immune checkpoint modulators: In some embodiments, the "co-agent" is an immune checkpoint regulator for treating and / or preventing neoplastic disease in a subject, as well as diseases, disorders or conditions associated with neoplastic disease. The term "immune checkpoint pathway" refers to a biological response triggered by the binding of a first molecule (e.g., a protein such as PD1) expressed on an antigen-presenting cell (APC) to a second molecule (e.g., a protein such as PDL1) expressed on an immune cell (e.g., T cell), which regulates the immune response either through stimulation (e.g., upregulation of T cell activity) or inhibition (e.g., downregulation of T cell activity). Molecules involved in the formation of binding pairs that regulate the immune response are generally referred to as "immune checkpoints". Such biological responses regulated by immune checkpoint pathways are mediated by intracellular signaling pathways that lead 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 first cell surface expressed molecule to a second 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 immune responses.

[0213] As used herein, the term "immune checkpoint pathway regulator" refers to a molecule that inhibits or stimulates the activity of immune checkpoint pathway in biological systems, including immune-competent mammals.An immune checkpoint pathway regulator may exert its effect 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 may exert its effect on upstream and / or downstream reactions in immune checkpoint pathways.For example, an immune checkpoint pathway regulator may regulate the activity of SHP2, a tyrosine phosphatase involved in PD-1 and CTLA-4 signal transduction. The term "immune checkpoint pathway regulator" encompasses both immune checkpoint pathway regulators that can at least partially downregulate the function of an inhibitory immune checkpoint (referred to herein as "immune checkpoint pathway inhibitors" or "immune checkpoint pathway antagonists") and immune checkpoint pathway regulators that can at least partially upregulate the function of a stimulatory immune checkpoint (referred to herein as "immune checkpoint pathway effectors" or "immune checkpoint pathway agonists").

[0214] Immune checkpoint regulators include, but are not limited to, immune checkpoint antagonists (e.g., antagonist antibodies) that bind to T cell inhibitory receptors, including, but not limited to, PD1 (also referred to 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 regulators are agonists that trigger checkpoint pathways that result in the 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, agonistic antibodies that bind to T cell activation receptors such as ICOS (e.g., JTX-2011, Jounce Therapeutics), OX40 (e.g., MEDI6383, Medinum), CD27 (e.g., varlilumab, 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, Medinum; INCAGN1876, Agenus).

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

[0216] In one embodiment, the immune checkpoint pathway modulator is a negative immune checkpoint pathway antagonist that inhibits the binding of PD1 to PDL1 and / or PDL2 ("PD1 pathway inhibitor"). The term PD1 pathway inhibitor includes monoclonal antibodies that interfere with the binding of PD1 to PDL1 and / or PDL2. Examples of commercially available PD1 pathway inhibitors useful as adjunctive agents in the treatment of neoplastic diseases include antibodies that interfere with the binding of PD1 to PDL1 and / or PDL2, including, but not limited to, nivolumab (Opdivo®, BMS-936558, MDX1106, commercially available from BristolMyers Squibb, Princeton NJ), pembrolizumab (Keytruda® MK-3475, lambrolizumab, commercially available from Merck and Company, Kenilworth NJ), and atezolizumab (Tecentriq®, Genentech / Roche, South San Francisco Calif.). Additional PD1 pathway inhibitor antibodies are in clinical development, 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). Additional antibody PD1 pathway inhibitors are described in U.S. Patent No. 8,217,149, issued July 10, 2012 (Genentech, Inc.); U.S. Patent No. 8,168,757, issued May 1, 2012 (Merck Sharp and Dohme Corp.), U.S. Patent No. 8,008,449, issued August 30, 2011 (Medarex), and U.S. Patent No. 7,943,743, issued 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 the PD-L2 IgG2a fusion protein AMP-224, the PDL2 fusion protein AMP-514, Amplimmune and Glaxo SmithKline), aptamers (Wang, et al. (2018) 145:125-130), peptide PD1 pathway inhibitors (Sasikumar, et al., U.S. Patent No. 9,422,339 issued August 23, 2016 and Sasilkumar, et al., U.S. Patent No. 8,907,053 issued December 9, 2014), small molecules (CA-170, AUPM-170, Aurigene / Curis; Sasikumar, et al., 1,2,4-oxadiazole and thiadiazole compounds as immunomodulators (PCT / IB2016 / 051266 filed March 7, 2016, published September 15, 2016 as WO2016142833A1) and Sasikumar, et al PCT / IB2016 / 051343 filed March 9, 2016, published as WO2016142886A2), BMS-1166 and Chupak LS and Zheng X. (2015) WO 2015 / 034820 A1, EP3041822 B1 granted August 9, 2017; WO2015034820 A1; and Chupak, et al. 2015) WO 2015 / 160641 A2. WO 2015 / 160641 A2, Currently in clinical development according to Chupak, et al. Sharpe, et al. WO 2011082400 A2 published July 7, 2011; U.S. Patent No. 7,488,802 issued February 10, 2009.

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

[0219] In some embodiments, the IL-27R binding molecule is administered in combination with an antagonist of a negative immune checkpoint pathway (a "TIM3 pathway inhibitor") that inhibits the ability of TIM3 to bind to a TIM3-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, published September 8, 2016 (F. Hoffman-LaRoche); Karunsky, U.S. Patent No. 9,631,026, issued April 27, 2017; Karunsky, Sabatos-Peyton et al., U.S. Patent No. 8,841,418, issued September 23, 2014; U.S. Patent No. 9,605,070; Takayanagi et al., U.S. Patent No. 8,552,156, issued October 8, 2013.

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

[0221] In some embodiments, the IL-27R binding molecule is administered in combination with an A2aR inhibitor. A2aR inhibits the activation of CD4+ T cells by Reg A2aR inhibits T cell responses by stimulating cells to develop. 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, which is a ligand for A2aR. In addition, loss of A2aR is associated with enhanced, sometimes pathological, inflammatory responses to infection. Inhibition of A2aR can be caused by administration of molecules such as antibodies that block the binding of adenosine, or by adenosine analogs. Such agents can be used in combination with IL-27R binding molecules for use in treating disorders such as cancer and Parkinson's disease.

[0222] In some embodiments, IL-27R binding molecules are administered in combination with an inhibitor of IDO (indoleamine 2,3-dioxygenase). IDO downregulates immune responses mediated via the oxidation of tryptophan, resulting in the inhibition of T cell activation and the induction of T cell apoptosis, creating an environment in which tumor-specific cytotoxic T lymphocytes are functionally inactive or can no longer attack the cancer cells of the subject. Indoximod (NewLink Genetics) is an IDO inhibitor that is being evaluated in metastatic breast cancer.

[0223] As discussed above, the present invention provides methods for treating neoplastic diseases (e.g., cancer) in a mammalian subject by administration of an IL-27R binding molecule in combination with an agent that modulates at least one immune checkpoint pathway, including immune checkpoint pathway regulators that modulate two, three, or more immune checkpoint pathways.

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

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

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

[0227] Adjunctive Agent Cellular Therapeutic Agents and Methods: In some embodiments, the method of the present disclosure may include a combination of administration of IL-27R binding molecules and auxiliary agents in the form of cell therapy for treating neoplastic disease, autoimmune disease, or inflammatory disease. Examples of cell therapy that can be applied to be used in combination with the method 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 (TIL), engineered Treg cells. Since engineered T cell products are usually activated ex vivo before their administration to a subject, thus providing upregulated levels of CD25, cell products including such activated engineered T cell types can be applied to further support the administration of CD25-biased IL-27R binding molecules described herein.

[0228] In some embodiments of the method of the present disclosure, the auxiliary agent is "chimeric antigen receptor T cell" and "CAR-T cell", which are used interchangeably to refer to T cell that has been recombinantly modified to express chimeric antigen receptor. As used herein, the terms "chimeric antigen receptor" and "CAR" are used interchangeably to refer to a chimeric polypeptide that includes multiple functional domains arranged in sequence from amino-terminus to carboxy-terminus: (a) antigen binding domain (ABD), (b) transmembrane domain (TD); and (c) one or more cytoplasmic signaling domains (CSD), where the aforementioned domains may be optionally linked by one or more spacer domains. CAR may also further include a signal peptide sequence that is routinely removed during post-translational processing and presentation of CAR on the cell surface of cells transformed with an expression vector that includes a nucleic acid sequence encoding CAR. CARs useful for carrying out the present invention are prepared according to principles well known in the art. See, e.g., Eshhaar et al., U.S. Patent No. 7,741,465 B1, issued June 22, 2010; Sadelain, et al. (2013) Cancer Discovery 3(4):388-398; Jensen and Riddell (2015) Current Opinions in Immunology 33:9-15; Gross, et al. (1989) PNAS(USA) 86(24):10024-10028; Curran, et al. (2012) J Gene Med 14(6):405-15. Examples of commercially available CAR-T cell products that can be modified to incorporate the orthogonal receptors of the invention include axicabtagene ciloreucel (sold as Yescarta® by Gilead Pharmaceuticals) and tisagenlecleucel (sold as Kymriah® by Novartis).In some embodiments, the CAR-T carries a CAR that specifically binds to a cell surface molecule associated with a tumor cell 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.

[0229] physical method : In some embodiments, the adjunct agent is an anti-neoplastic physical method, including, but not limited to, radiation therapy, cryotherapy, hyperthermia, surgery, laser ablation, and proton beam therapy.

[0230] kit: The present disclosure also contemplates pharmaceutical compositions, kits including IL-27R binding molecules and pharmaceutical compositions thereof. The kits are generally in the form of physical structures housing various components as described below, and can be utilized, for example, in practicing the methods described above. The kits can contain the IL-27R binding molecules in the form of pharmaceutical compositions suitable for administration to a subject, ready for use, or in a form that requires preparation, e.g., thawing, reconstitution or dilution, prior to administration. If the IL-27R binding molecules are in a form that requires reconstitution by the user, the kits can also include a sterile container providing a reconstitution medium, including buffers, pharma- ceutically acceptable excipients, and the like. The kits of the present disclosure can be designed for the conditions necessary to properly maintain the components contained therein (e.g., refrigeration or freezing). The kits may further contain a label or package insert that includes identifying information for the components therein and instructions for their use. Each component of the kit can be contained within an individual container, and the various containers can all be in one package. The label or package insert can include manufacturer information, such as lot number and expiration date. The label or package insert can be, for example, incorporated into a physical structure that contains the component, contained separately within a physical structure, or affixed to a component of the kit (e.g., an ampoule, a syringe, or a vial). The label or package insert can be provided in a physical form or in a computer readable medium. In some embodiments, the actual instructions are not present in the kit, but rather the kit provides a means for obtaining the instructions via an internet site, including by secure access from a remote source, for example, by providing a password (or a scannable code, such as a bar code or QR code, on the container of the IL-27R binding molecule or the kit containing it) in accordance with government regulations (e.g., HIPAA).

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

[0232] Any reference cited herein is not admitted to constitute prior art. The discussion of references presents what their authors assert, and the inventors reserve the right to challenge the accuracy and pertinence of the cited documents. Although several sources of information, including scientific journal articles, patent documents, and textbooks, are referenced herein; it will be clearly understood that this reference does not constitute an admission that any of these documents form part of the common general knowledge in the art.

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

[0234] In some embodiments, the adjunct agent is a vaccine. The IL27R binding proteins of the invention can be administered to a subject in combination with a vaccine as an adjuvant to enhance the immune response to the vaccine according to the teachings of U.S. Patent No. 5,800,819, issued Sep. 1, 1998 to Doyle et al. Examples of vaccines that may be combined with the IL27R binding proteins of the invention include HSV vaccines, Bordetella pertussis vaccines, Escherichia coli vaccines, pneumococcal vaccines, including multivalent pneumococcal vaccines such as Prevnar® 13, diphtheria, tetanus and whooping cough vaccines (including combination vaccines such as Pediatrix® and Pentacel®), chickenpox vaccines, Haemophilus influenzae type B vaccines, human papilloma virus vaccines such as Garasil®, polio vaccines, leptospirosis vaccines, combined respiratory vaccines, Moraxella vaccines, live attenuated or killed virus vaccine products such as bovine respiratory disease vaccine (RSV), Fluzone® and Quadravlent Fluzone. (registered trademark), feline leukemia vaccine, transmissible gastroenteritis vaccine, COVID-19 vaccine, and rabies vaccine.

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

[0236] Example 1 Camels were allowed to acclimate in the laboratory for at least 7 days prior to immunization. Antigen was diluted in 1x PBS (approximately 1 mg total antigen). Antigen quality was assessed by SDS-PAGE to ensure purity (e.g., >80%). First, 10 mL of CFA (followed by six doses of IFA) was added to the mortar, then 10 mL antigen in 1x PBS was slowly added to the mortar while grinding with a pestle. The antigen and CFA / IFA were ground until the components appeared milky and appeared difficult to disperse. Camels were injected subcutaneously at least six sites on their body with antigen emulsified in CFA, with approximately 2 mL injected at each site (10 mL total per camel). Injecting larger volumes at more sites resulted in stronger immune responses. Immunizations were performed seven times, one week apart (7 days). To avoid leakage of the emulsion, the needle was inserted into the subcutaneous space for 10–15 seconds after each injection. Alternatively, leakage was prevented by gently pulling the plunger of the syringe. Blood samples were taken 3 days after the seventh immunization.

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

[0238] To identify VHHs that bind to IL27Rα, biopanning of the phage library was performed. 96-well plates were coated with IL27Rα, and the phage library was incubated in each well to allow phage-expressed IL27Rα-reactive VHHs to bind to IL27Rα on the plate. Non-specifically bound phages were washed away, and specifically bound phages were isolated. After selection, the enriched phage library expressing IL27Rα-reactive VHHs was amplified in TG1 cells. The aforementioned biopanning process was repeated for 2–3 rounds to enrich the library of VHHs selective for IL27Rα. Once biopanning was completed, three individual phage clones of the 96-well plate were isolated to perform periplasmic extract ELISA (PE-ELISA) on antigen-coated plates to identify positive VHH binders. Briefly, 96-well plates were coated with antigen and PBS under the same conditions. The wells were then blocked for 1 h at 37 °C. Then, 100 μl of extracted antibody was added to each well and incubated for 1 hour. After that, 100 μl of HRP-conjugated anti-tag polyclonal antibody was added to each well and incubated at 37°C for 1 hour. The plate was developed with TMB substrate. The reaction was stopped by adding H2SO4. The absorbance at 450 nm was read in a microtiter plate reader, and antibodies with absorbance at least 3-fold higher in antigen-coated wells than in PBS-coated controls were 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. Binding proteins were purified essentially as follows: 96×4 mL of supernatant from 4×24-well blocks was re-arrayed into 4×96-well, 1 mL blocks using a Hamilton Star automation system. PhyNexus micropipette tips (Biotage, San Jose CA) holding 80 μL of Ni-Excel IMAC resin (Cytiva) were equilibrated with wash buffer: PBS pH 7.4, 30 mM imidazole. PhyNexus tips were immersed and pipetted 1 mL for 14 cycles across all 4×96-well blocks. PhyNexus tips were washed in 2×1 mL blocks holding wash buffer. PhyNexus tips were eluted in 3×0.36 mL blocks holding elution buffer: PBS pH 7.4, 400 mM imidazole. PhyNexus chips were regenerated in 3 x 1 mL blocks of 0.5 M sodium hydroxide.

[0240] Purified protein eluates were quantified using a Biacore® T200 essentially according to the following procedure: 10 μL of the initial 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 onto a CM5 series S chip previously functionalized with an anti-histidine capture antibody (Cytiva): injections were performed at 5 μL / min for 18 seconds. Capture levels were recorded after a 60 second buffer wash. To eliminate cell-to-cell surface variations, a known V HA standard curve of H concentrations (270, 90, 30, 10, 3.3, 1.1 μg / mL) was obtained. 96 captures were interpolated to the standard curve using a nonlinear model including specific and nonspecific one-site binding. The concentration of the first elution block varied from 12 to 452 μg / mL, corresponding to 4 to 149 μg. To confirm that the molecular weight of the eluate corresponded to the expected value (approximately 30 kDa), SDS-PAGE analysis of five randomly selected samples was performed.

[0241] Protein concentrations were normalized using a Hamilton Star automated system essentially following the procedure below: Concentration values ​​were imported into an Excel spreadsheet where pipetting volumes were calculated to perform a dilution to 50 μg / mL in 0.22 mL. The spreadsheet was imported into a dedicated Hamilton Star method that performed dilution pipetting using the first elution block and elution buffer as diluent. The final normalized plate was sterile filtered using a 0.22 μm filter plate (Corning).

[0242] Example 3 All experiments were performed on a Biacore T200 instrument (Cytiva) equipped with a Protein A chip in 10 mM Hepes, 150 mM NaCl, 0.05% (v / v) polysorbate 20 (PS20) and 3 mM EDTA (HBS-EP+ buffer). Mono-Fc VHH ligands were run at 5 μl / min for various times ranging from 18 to 300 s to reach the capture loads listed in the table below.

[0243] After ligand capture, injections of a two-fold dilution series of his-tagged cytokine receptors, typically including at least five concentrations between 1 μM and 1 nM, were performed in either high performance or single cycle kinetics mode. Surface regeneration was achieved by flowing 10 mM glycine-HCl, pH 1.5 (60 s, 50 μL / min). Buffer-subtracted sensograms 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 that fits the kinetic analysis. Calculated Rmax was generated using the formula Rmax = Loading (RU) × Ligand valence × (Molecular weight of analyte / Molecular weight of ligand). Surface activity was defined as the ratio of Rmax experimental / calculated. See the table below for sample information and experimental results. Anti-hGP130 Mono-Fc VHH (ligand) that binds to hGP130-his (Sino Biological, Cat. No. 10974) TIFF0007665730000445.tif69151Anti-hIL27Ra Mono-Fc VHH (ligand) that binds to hIL27Ra-his (Origene, Cat. No. TP307012) TIFF0007665730000446.tif61151 * Both the association and dissociation rate constants can be suppressed at Rmax > 100. If present, this effect is likely offset by the kinetic ratio, i.e., the affinity constant.

[0244] Example 4 Binding for all VHHs was confirmed by ELISA. One representative VHH from each clone was selected for further analysis by surface plasmon resonance using a Biacore T200, see below. TIFF0007665730000447.tif206150TIFF0007665730000448.tif154150

Claims

1. 1. An IL27 receptor (IL27R) binding protein that specifically binds to the IL27R alpha subunit (IL27Rα) and glycoprotein 130 subunit (gp130), contacting a cell expressing IL27Rα and gp130 with the binding protein to induce multimerization of IL27Rα and gp130; and the binding protein comprises a single domain antibody (sdAb) that specifically binds IL27Rα (anti-IL27Rα sdAb) and an sdAb that specifically binds gp130 (anti-gp130 sdAb); Anti-IL27Rα sdAb is anti-IL27Rα V H H antibody and the anti-gp130 sdAb is anti-gp130 V H H antibody, anti-gp130v H wherein the H antibody comprises a set of CDR1, CDR2, and CDR3, the set comprising: the amino acid sequence of SEQ ID NO:196, the amino acid sequence of SEQ ID NO:202, and the amino acid sequence of SEQ ID NO:208, respectively; the amino acid sequence of SEQ ID NO:193, the amino acid sequence of SEQ ID NO:199, and the amino acid sequence of SEQ ID NO:205, respectively; the amino acid sequence of SEQ ID NO:194, the amino acid sequence of SEQ ID NO:200, and the amino acid sequence of SEQ ID NO:206, respectively; the amino acid sequence of SEQ ID NO:195, the amino acid sequence of SEQ ID NO:201, and the amino acid sequence of SEQ ID NO:207, respectively; the amino acid sequence of SEQ ID NO:197, the amino acid sequence of SEQ ID NO:203, and the amino acid sequence of SEQ ID NO:209, respectively; and The amino acid sequence of SEQ ID NO:198, the amino acid sequence of SEQ ID NO:204, and the amino acid sequence of SEQ ID NO:210, respectively. and Anti-IL27Rα V H wherein the H antibody comprises a set of CDR1, CDR2, and CDR3, the set comprising: the amino acid sequence of SEQ ID NO:214, the amino acid sequence of SEQ ID NO:221, and the amino acid sequence of SEQ ID NO:228, respectively; the amino acid sequence of SEQ ID NO:211, the amino acid sequence of SEQ ID NO:218, and the amino acid sequence of SEQ ID NO:225, respectively; the amino acid sequence of SEQ ID NO:212, the amino acid sequence of SEQ ID NO:219, and the amino acid sequence of SEQ ID NO:226, respectively; the amino acid sequence of SEQ ID NO:213, the amino acid sequence of SEQ ID NO:220, and the amino acid sequence of SEQ ID NO:227, respectively; the amino acid sequence of SEQ ID NO:215, the amino acid sequence of SEQ ID NO:222, and the amino acid sequence of SEQ ID NO:229, respectively; the amino acid sequence of SEQ ID NO:216, the amino acid sequence of SEQ ID NO:223, and the amino acid sequence of SEQ ID NO:230, respectively; and The amino acid sequence of SEQ ID NO:217, the amino acid sequence of SEQ ID NO:224, and the amino acid sequence of SEQ ID NO:231, respectively. and a set of CDR1, CDR2, and CDR3 comprising: IL27R-binding proteins.

2. anti-gp130v H The IL27R binding protein of claim 1, wherein the H antibody comprises a set of CDR1, CDR2, and CDR3, and the CDR1, CDR2, and CDR3 comprise the amino acid sequence of SEQ ID NO:196, the amino acid sequence of SEQ ID NO:202, and the amino acid sequence of SEQ ID NO:208, respectively.

3. Anti-IL27Rα V H The IL27R binding protein of claim 2, wherein the H antibody comprises a set of CDR1, CDR2, and CDR3, and the CDR1, CDR2, and CDR3 comprise the amino acid sequence of SEQ ID NO:214, the amino acid sequence of SEQ ID NO:221, and the amino acid sequence of SEQ ID NO:228, respectively.

4. Anti-IL27Rα V H The IL27R binding protein of claim 2, wherein the H antibody comprises a set of CDR1, CDR2, and CDR3, and the CDR1, CDR2, and CDR3 comprise the amino acid sequence of SEQ ID NO:216, the amino acid sequence of SEQ ID NO:223, and the amino acid sequence of SEQ ID NO:230, respectively.

5. 1. An IL27 receptor (IL27R) binding protein that specifically binds to the IL27R alpha subunit (IL27Rα) and glycoprotein 130 subunit (gp130), The binding protein is upon binding to IL27Rα and gp130, causes multimerization of IL27Rα and gp130; and a single domain antibody (sdAb) that specifically binds to IL27Rα (anti-IL27Rα sdAb) and an sdAb that specifically binds to gp130 (anti-gp130 sdAb); Anti-IL27Rα sdAb is anti-IL27Rα V H H antibody and the anti-gp130 sdAb is anti-gp130 V H H antibody, The IL27 receptor (IL27R) binding protein comprises the amino acid sequence of SEQ ID NO:

64. IL27 receptor (IL27R) binding protein.

6. 1. An IL27 receptor (IL27R) binding protein that specifically binds to the IL27R alpha subunit (IL27Rα) and glycoprotein 130 subunit (gp130), The binding protein is upon binding to IL27Rα and gp130, causes multimerization of IL27Rα and gp130; and a single domain antibody (sdAb) that specifically binds to IL27Rα (anti-IL27Rα sdAb) and an sdAb that specifically binds to gp130 (anti-gp130 sdAb); Anti-IL27Rα sdAb is anti-IL27Rα V H H antibody, and the anti-gp130 sdAb is anti-gp130 V H H antibody, The IL27 receptor (IL27R) binding protein comprises the amino acid sequence of SEQ ID NO:

76. IL27 receptor (IL27R) binding protein.

7. The IL27R binding protein of claim 1, wherein the anti-IL27Rα sdAb and the anti-gp130 sdAb are linked via a polypeptide linker of 1 to 50 amino acids.

8. The IL27R binding protein of claim 1, comprising an amino acid sequence having at least 90% identity to any of the amino acid sequences of SEQ ID NOs:406, 430, 454, 478, 502, 526, 409, 433, 457, 481, 505, 529, 411, 435, 459, 483, 507, 531, 414, 438, 462, 486, 510, 534, 420, 444, 468, 492, 516, 540, 424, 448, 472, 496, 520, 544, 426, 450, 474, 498, 522, and 546.

9. The IL27R binding protein of claim 1, comprising any of the amino acid sequences of SEQ ID NOs:406, 430, 454, 478, 502, 526, 409, 433, 457, 481, 505, 529, 411, 435, 459, 483, 507, 531, 414, 438, 462, 486, 510, 534, 420, 444, 468, 492, 516, 540, 424, 448, 472, 496, 520, 544, 426, 450, 474, 498, 522, and 546.

10. The IL27R binding protein of claim 1, comprising the amino acid sequence of SEQ ID NO:486 or the amino acid sequence of SEQ ID NO:

496.

11. The IL27R binding protein of claim 1, comprising, in the amino-terminal to carboxy-terminal direction, an anti-IL27Rα sdAb and an anti-gp130 sdAb.

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