Compositions and methods related to receptor pairing

Abstract

The present application relates to compositions and methods related to receptor pairing. Provided herein are receptor binding proteins that bind to a native or non-native cytokine receptor pair to produce diversity of signal transduction beyond native receptor pairing.

Description

[0001] This application is a divisional application of Chinese Patent Application No. 202180068365.6.

[0002] Cross - reference to related patent applications

[0003] This application claims the priority benefits of U.S. Provisional Application No. 63 / 061,562, filed on August 5, 2020, U.S. Provisional Application No. 63 / 078,745, filed on September 15, 2020, and U.S. Provisional Application No. 63 / 135,884, filed on January 11, 2021, the disclosures of which are hereby incorporated by reference in their entireties for all purposes.

[0004] Sequence Listing

[0005] This application contains a sequence listing submitted electronically in ASCII format and hereby incorporated by reference in its entirety. The ASCII copy was created on September 15, 2021, named 106249 - 1263226_SL.txt, and is 725,661 bytes in size. Background Art

[0006] Cytokine and growth factor ligands typically signal through homodimeric or heterodimeric cell - surface receptors via Janus kinase (JAK / TYK) or receptor tyrosine kinase (RTK) - mediated trans - phosphorylation. However, the number of receptor - dimer pairings that occur in nature is limited to those driven by native ligands encoded within the genome.

[0007] In some cases, cytokines act as multispecific (e.g., bispecific or trispecific) ligands. The cytokine determines which receptors are included in the dimer by binding to the respective extracellular domains of two receptors. Thus, the cytokine serves to bridge or crosslink receptors in the signal transduction complex. Association of cytokine receptor domains or subunits results in activation of the intracellular JAK / STAT signal transduction pathway, which includes one or more of four Janus kinases (JAK1-3 and TYK2) (Ihle, Nature 377(6550):591-4, 1995; O’Shea and Plenge, Immunity 36(4):542-50, 2012) and several signal transducer and activator of transcription (STAT 1-6) proteins (Delgoffe, et al., Curr Opin Immunol. 23(5):632-8, 2011; Levy and Darnell, Nat Rev Mol Cell Biol. 3(9):651-62, 2002; Murray, J Immunol. 178(5):2623-9, 2007). Although cytokines typically bind specifically to the extracellular domains of cell surface receptors, the JAK / TYK / STAT signal transduction module is found in many combinations of endogenous cytokine receptor signal transduction complexes.

[0008] Given that the ligand determines the composition of receptor domains or subunits in the receptor complex and that the intracellular JAK / TYK and RTK enzymes are degenerate, the number of naturally occurring pairings of cytokine and growth factor receptor dimers represents only a small fraction of the total number of signaling-competent receptor pairings that the system theoretically permits. For example, the human genome encodes approximately forty different JAK / STAT cytokine receptors. In principle, approximately 1600 unique homodimeric and heterodimeric cytokine receptor pairs could be generated and have the potential to signal through different JAK / TYK / STAT combinations (Bazan, Proc Natl Acad Sci U S A. 87(18):6934-8, 1990; Huising et al., J Endocrinol. 189(1):1-25, 2006). However, as is currently known, the human genome encodes fewer than 50 different cytokine ligands (Bazan, Proc Natl Acad Sci U S A. 87(18):6934-8, 1990; Huising et al., J Endocrinol. 189(1):1-25, 2006), restricting the scope of cytokine receptor complex signal transduction to those that can be assembled by natural ligands. SUMMARY OF THE INVENTION

[0009] On the one hand, the present disclosure provides an IL12 receptor (IL12R) binding protein that specifically binds to IL12Rβ1 and IL12Rβ2, wherein the binding protein causes the multimerization of IL12Rβ1 and IL12Rβ2, and the multimerization causes the association of the intracellular domains of IL12Rβ2 and IL12Rβ1 and intracellular signal transduction, wherein the binding protein comprises a single-domain antibody (sdAb) that specifically binds to IL12Rβ1 (anti-IL12Rβ1 sdAb) and an sdAb that specifically binds to IL12Rβ2 (anti-IL12Rβ2 sdAb).

[0010] In some embodiments, the anti-IL12Rβ1 sdAb is a V H H antibody (anti-IL12Rβ1 V H H antibody) and / or the anti-IL12Rβ2 sdAb is a V H H antibody (anti-IL12Rβ2 V H H antibody). In some embodiments, the anti-IL12Rβ1 sdAb and the anti-IL12Rβ2 sdAb are joined directly or through a peptide linker. In some embodiments, the peptide linker comprises 1 to 50 amino acids. In some embodiments, the IL12R binding protein has a reduced E 最大 compared to IL12. In some embodiments, the IL12R binding protein has an increased E 最大 compared to IL12. In some embodiments, the IL12R binding protein has a similar potency compared to IL12.

[0011] On the other hand, the present invention provides a method of treating a neoplastic disease (such as cancer) in a subject in need thereof, the method comprising the step of administering to the subject an IL12R binding protein as described herein, wherein the IL12R binding protein binds to and activates natural killer, CD4 + T cells and / or CD8 + T cells. In some embodiments, the cancer is a solid tumor cancer.

[0012] On the other hand, the present invention provides an IL27 receptor (IL27R) binding protein that specifically binds to the IL27Rα subunit (IL27Rα) and the glycoprotein 130 subunit (gp130), wherein the binding protein causes the multimerization of IL27Rα and gp130, and the multimerization causes the association of the intracellular domains of gp130 and IL27Rα and intracellular signal transduction, wherein the binding protein comprises a single-domain antibody (sdAb) that specifically binds to IL27Rα (anti-IL27Rα sdAb) and an sdAb that specifically binds to gp130 (anti-gp130 sdAb).

[0013] In some embodiments, the anti-IL27Rα sdAb is a V H H antibody (anti-IL27Rα V H H antibody) and / or the anti-gp130 sdAb is a V H H antibody (anti-gp130 V H H antibody). In some embodiments, the anti-IL27Rα sdAb and the anti-gp130 sdAb are linked directly or via a peptide linker. In some embodiments, the peptide linker comprises 1 to 50 amino acids.

[0014] On the other hand, the present invention provides a method of treating a neoplastic disease (such as cancer) in a subject in need thereof, comprising administering to the subject an IL27R binding protein as described herein, wherein the IL27R binding protein binds to and activates CD8 + T cells, CD4 + T cells and / or regulatory T (Treg) cells. In some embodiments, the IL27R binding protein binds to and activates CD8 + T cells. In some embodiments, the IL27R binding protein binds to and activates CXCR5 + CD8 + T cells. In some embodiments, the cancer is a solid tumor cancer.

[0015] On the other hand, the present invention provides an IL10 receptor (IL10R) binding protein that specifically binds to the IL10Rα subunit (IL10Rα, also referred to herein as IL10R1) and IL10Rβ (also referred to herein as IL10R2), wherein the binding protein causes the multimerization of IL10Rα and IL10Rβ, and the multimerization causes the association of the intracellular domains of IL10Rβ and IL10Rα and intracellular signal transduction, wherein the binding protein comprises a single domain antibody (sdAb) that specifically binds to IL10Rα (anti-IL10Rα sdAb) and an sdAb that specifically binds to IL10Rβ (anti-IL10Rβ sdAb).

[0016] In some embodiments, the anti-IL10Rα sdAb is a V H H antibody (anti-IL10Rα V H H antibody) and / or the anti-IL10Rβ sdAb is a V H H antibody (anti-IL10Rβ V H H antibody). In some embodiments, the anti-IL10Rα sdAb and the anti-IL10Rβ sdAb are joined by a peptide linker. In some embodiments, the peptide linker comprises 1 to 50 amino acids.

[0017] In another aspect, the present disclosure provides a method of treating a neoplastic disease (such as cancer) in a subject in need thereof, comprising administering to the subject an IL10R binding protein as described herein, wherein the IL10R binding protein binds to and activates CD8 + T cells, CD4 + T cells, macrophages, and / or Treg cells. In some embodiments, the IL10R binding protein provides a longer therapeutic efficacy than pegylated IL10. In some embodiments, the cancer is a solid tumor cancer.

[0018] In other aspects, the IL10R binding proteins described herein can also be used to treat inflammatory diseases (such as Crohn's disease and ulcerative colitis), and autoimmune diseases (such as psoriasis, rheumatoid arthritis, and multiple sclerosis).

[0019] In another aspect, the present invention provides an interferon (IFN) lambda receptor (IFNλR) binding protein that specifically binds to the IL10Rβ and the alpha subunit of the IL28 receptor (IL28Rα) (IL28Rα), wherein the binding protein causes multimerization and downstream signal transduction of IL10Rβ and IL28Rα, and wherein the binding protein comprises a single domain antibody (sdAb) that specifically binds to IL10Rβ (anti-IL10Rβ sdAb) and an sdAb that specifically binds to IL28Rα (anti-IL28Rα sdAb).

[0020] In some embodiments, the anti-IL10Rβ sdAb is a V H H antibody (anti-IL10Rβ V H H antibody) and / or the anti-IL28Rα sdAb is a V H H antibody (anti-IL28Rα V H H antibody). In some embodiments, the anti-IL10Rβ sdAb and the anti-IL28Rα sdAb are joined directly or via a peptide linker. In some embodiments, the peptide linker comprises 1 to 50 amino acids.

[0021] In another aspect, the present invention characterizes a method of treating an infectious disease in a subject in need thereof, comprising administering to the subject an IFNλR binding protein as described herein, wherein the IFNλR binding protein binds to and activates macrophages, CD8 + T cells, CD4 + T cells, Treg cells, dendritic cells, and / or epithelial cells. In some embodiments, the IFNλR binding protein binds to and activates macrophages. In some embodiments, the infectious disease is influenza, hepatitis B, hepatitis C, or human immunodeficiency virus (HIV) infection.

[0022] On the other hand, the present invention provides a binding protein that specifically binds to IL10Rα and IL2Rγ, wherein the binding protein causes multimerization and downstream signal transduction of IL10Rα and IL2Rγ, and wherein the binding protein comprises a single domain antibody (sdAb) that specifically binds to IL10Rα (anti-IL10Rα sdAb) and an sdAb that specifically binds to IL2Rγ (anti-IL2Rγ sdAb).

[0023] In some embodiments, the anti-IL10Rα sdAb is a V H H antibody (anti-IL10Rα V H H antibody) and / or the anti-IL2Rγ sdAb is a V H H antibody (anti-IL2Rγ V H H antibody). In some embodiments, the anti-IL10Rα sdAb and the anti-IL2Rγ sdAb are joined directly or via a peptide linker. In some embodiments, the peptide linker comprises 1 to 50 amino acids.

[0024] On the other hand, the present invention provides a method of treating a neoplastic disease (such as cancer) in a subject in need thereof, comprising administering to the subject a binding protein that specifically binds to IL10Rα and IL2Rγ as described herein, wherein the binding protein binds to and activates CD8 + T cells and / or CD4 + T cells. In some embodiments, the method does not result in anemia.

[0025] On the other hand, the present invention provides a binding protein that specifically binds to a first receptor and a second receptor, wherein the first receptor is interferon gamma receptor 1 (IFNγR1) or IL28Rα, and the second receptor is preferentially expressed on myeloid cells and / or T cells, and wherein the binding protein causes multimerization of the first receptor and the second receptor and its downstream signal transduction, and wherein the binding protein comprises a single domain antibody (sdAb) that specifically binds to the first receptor and an sdAb that specifically binds to the second receptor.

[0026] In some embodiments, the sdAb that specifically binds to the first receptor is an anti-IFNγR1 V H H antibody. In some embodiments, the sdAb that specifically binds to the first receptor is an anti-IL28Rα V H H antibody. In some embodiments, the first receptor is IFNγR1 and the second receptor is IL2Rγ. In some embodiments, the first receptor is IL28Rα and the second receptor is IL2Rγ. In some embodiments, the sdAb that specifically binds to the first receptor and the sdAb that specifically binds to the second receptor are bound directly or via a peptide linker. In some embodiments, the peptide linker comprises 1 to 50 amino acids.

[0027] On the other hand, the present invention provides a method of treating a neoplastic disease (such as cancer) in a subject in need thereof, comprising administering to the subject a binding protein as described herein that binds to a first receptor (such as IFNγR1 or IL28Rα) and a second receptor (such as a receptor preferentially expressed on myeloid cells and / or T cells), wherein the binding protein binds to and activates myeloid cells and / or T cells. In some embodiments, the binding protein binds to and activates macrophages. In some embodiments, the binding protein binds to and activates CD8 + T cells and / or CD4 + T cells. Detailed embodiments

[0028] I. Introduction

[0029] The present invention provides compositions useful for the pairing of cellular receptors to produce desired effects useful for treating diseases. Generally, binding proteins are provided that at least comprise a first domain that binds to a first receptor and a second domain that binds to a second receptor such that upon contact with a cell expressing the first and second receptors, the binding protein causes a functional association of the first and second receptors, thereby initiating their interaction and resulting in downstream signal transduction. In some embodiments, the first and second receptors come into proximity in response to binding of certain cytokines and are referred to herein as "natural" cytokine receptor pairs. In other embodiments, the binding proteins described herein bind to two receptors that do not naturally interact by binding to a naturally occurring cytokine and are referred to herein as "unnatural" cytokine receptor pairs.

[0030] The binding proteins described herein have several advantages. In the case of natural cytokine receptor pairs, natural cytokines cause the natural cytokine receptor pairs to come into proximity (i.e., by their simultaneous binding to the cytokine). However, when some of these natural cytokines are used as therapeutic agents in mammalian (especially human) subjects, they may also trigger many adverse and undesirable effects through multiple mechanisms, including the presence of natural cytokine receptors on other cell types and the binding to those same receptor pairs on other cell types that may produce adverse effects or cause undesired signal transduction. The present invention relates to manipulating the multiple effects of cytokines such that the desired therapeutic signal transduction occurs, particularly in the desired cell or tissue subtypes, while minimizing undesired activity and / or intracellular signal transduction.

[0031] In some embodiments, the binding proteins described herein are designed such that the binding protein provides maximal desired signal transduction from a native cytokine receptor pair on the desired cell type, while signal transduction from the receptor is weaker on other undesired cell types, such that toxic effects caused by the other undesired cell types are reduced or there are no toxic effects. For example, this can be achieved by selecting a binding protein that has a different affinity compared to the affinity of the native cytokine pair for the same receptor or that elicits a different E 最大 of the target receptor. Since different cell types respond to ligand binding to its cognate receptor with different sensitivities, by modulating the affinity of the ligand for the receptor compared to native cytokine binding, stimulation of the desired activity is promoted while reducing undesired activity on non-target cells. To measure downstream signal transduction activity, a variety of methods can be used. For example, in some embodiments, JAK / STAT signal transduction can be measured by the presence of phosphorylated receptor and / or phosphorylated STAT. In other embodiments, the expression of one or more downstream genes can also be measured, the expression levels of which can be affected by the level of downstream signal transduction induced by the binding protein.

[0032] In other embodiments, the binding proteins described herein provide novel signal transduction, including but not limited to, by bringing two receptors that do not normally interact to a significant or measurable extent in nature into proximity, or by binding to a non-native cytokine receptor pair, to provide signal transduction in a specific target cell type. As an example of the latter, by binding to interferon gamma receptor 1 (IFNγR1) or IL28Rα and a second receptor that is uniquely or preferentially expressed on myeloid or T-cells, one of ordinary skill in the art can obtain the beneficial signal transduction elicited thereby, while by contacting the target cell with a binding protein that comprises a first domain that specifically binds to IFNγR1 or IL28Rα and a second domain that specifically binds to a receptor that is uniquely or preferentially expressed on myeloid or T-cells, binding to the same receptor (e.g., IFNγR1 or IL28Rα expressed on other cells of a human) is avoided or reduced, thereby restricting binding to other cells and targeting activation of IFNγR1 or IL28Rα by the binding protein that targets these target cells (myeloid or T-cells). The various receptor binding proteins described herein can be designed and customized to bind to a specific receptor or its domain or its subunit that is highly expressed on the cell surface of different cell types. By binding two separate receptors, these receptor binding proteins provide a method for selectively activating or inhibiting a specific cell type, which provides therapeutic and / or prophylactic activity for treating and / or preventing diseases, such as neoplastic diseases, such as cancer and infectious diseases.

[0033] II. Definitions

[0034] As used herein, the term "antibody" collectively refers 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) immunoglobulin derivatives, including but not limited to IgG(1-4)ΔC H 2. F(ab’) 2 , Fab, ScFv, V H , V L , tetravalent antibodies, trivalent antibodies, bispecific antibodies, dsFv, F(ab’) 3 , scFv-Fc, and (scFv) 2 , which compete with the immunoglobulin from which they are derived for binding to the target molecule. The term antibody is not limited to immunoglobulins from any particular mammalian species, including murine, human, equine, and camelid antibodies (e.g., human antibodies).

[0035] The term antibody also includes so-called "single-domain antibodies" or "sdAb" and "heavy-chain antibodies" or "V H H", which are further defined herein. V H H can be obtained from immunization of Camelidae (including camels, llamas, and alpacas) (see, e.g., Hamers-Casterman, et al. (1993) Nature 363:446-448) or by screening libraries constructed in a V H H framework (e.g., phage libraries). Antibodies with a given specificity can also be derived from non-mammalian sources, such as V H H obtained from immunization of cartilaginous fish (including but not limited to sharks). The term "antibody" includes antibodies isolated from natural sources or from animals immunized with an antigen, as well as engineered antibodies, including monoclonal antibodies, bispecific antibodies, trispecific, chimeric antibodies, humanized antibodies, human antibodies, CDR-grafted, veneered, or deimmunized (e.g., removing T cell epitopes) antibodies. The term "human antibody" includes antibodies obtained from humans, as well as antibodies obtained from transgenic mammals containing human immunoglobulin genes such that, upon antigen stimulation, the transgenic animal produces antibodies that contain the amino acid sequence characteristics of antibodies produced by humans.

[0036] The term includes the parent antibody and its derivatives, such as affinity matured, veneered, CDR-grafted, humanized, camelized (in the case of VHH), or binding molecules that contain the binding domain of an antibody (such as CDRs) in a non-immunoglobulin scaffold.

[0037] The term "antibody" should be interpreted as not being limited to any particular synthetic method, including naturally occurring antibodies that can be isolated from natural sources, and engineered antibody molecules prepared by "recombinant" means, including antibodies isolated from transgenic animals that transfer human immunoglobulin genes or hybridomas prepared therefrom, antibodies isolated from host cells transformed with nucleic acid constructs that lead to antibody expression, and antibodies isolated from combinatorial antibody libraries (including phage display libraries). In one embodiment, the "antibody" is a mammalian immunoglobulin. In some embodiments, the antibody is a "full-length antibody" that includes variable and constant domains that provide binding and effector functions.

[0038] The term antibody includes antibody conjugates, including those modified to prolong the duration of action, such as fusion proteins or conjugation to polymers (eg, PEGylation).

[0039] As used herein, the term "binding protein" refers to a protein that can bind to one or more cell surface receptors or their domains or subunits. In some embodiments, the binding protein specifically binds to two different receptors (or their domains or subunits), so that the receptors (or domains or subunits) remain close to each other, so that the receptors (or domains or subunits), including their domains (e.g., intracellular domains), interact and cause downstream signal transduction.

[0040] As used herein, the term "CDR" or "complementarity determining region" refers to the non-contiguous antigen binding sites found within the variable regions of heavy and light chain polypeptide immunoglobulin polypeptides. CDRs have been described by Kabat et al., J. Biol. Chem. 252, 6609-6616 (1977); Kabat et al., (1991) US Department of Health and Human Services, "Sequences of Proteins of Immunological Interest" (also referred to herein as Kabat 1991); Chothia et al., J. Mol. Biol. 196: 901-917 (1987) (also referred to herein as Chothia 1987); and MacCallum et al., J. Mol. Biol. 262: 732-745 (1996), where the definitions include overlaps or subsets of amino acid residues when compared to each other. However, the definition of CDRs referring to antibodies or grafted antibodies or variants thereof is intended to fall within the scope of the term defined or used herein. For the purposes of this disclosure, unless otherwise specifically stated, the positioning of CDR2 and 3 in the variable region of an antibody follows the Kabat numbering or simply "Kabat." The positioning of CDR1 in the variable region of an antibody follows a hybrid of the Kabat and Chothia numbering schemes.

[0041] As used herein, the term "conservative amino acid substitution" refers to an amino acid replacement in which a given amino acid is changed to a different amino acid having similar biochemical properties (e.g., charge, hydrophobicity, and size). For example, the amino acids in the following groups can be considered conservative amino acids of each other: (1) hydrophobic amino acids: alanine, isoleucine, leucine, tryptophan, phenylalanine, valine, proline, and glycine; (2) polar amino acids: glutamine, asparagine, histidine, serine, threonine, tyrosine, methionine, and cysteine; (3) basic amino acids: lysine and arginine; and (4) acidic amino acids: aspartic acid and glutamic acid.

[0042] As used herein, the term "interferon lambda receptor" or "IFNλR" refers to a heterodimeric receptor formed by the IL10Rβ receptor and the IL28 receptor alpha (IL28Rα) and bound by the ligand IFNλ. The subunit IL28Rα is also referred to as IFNLR1 (interferon lambda receptor 1). The human sequence of IL10Rβ is listed as UniProt ID NO. Q08334. The human sequence of IL28Rα is listed as UniProt ID NO. Q8IU57.

[0043] As used herein, the term "interferon gamma receptor 1" or "IFNγR1" refers to a subunit of the heterodimeric IFNγR formed by the subunit IFNγR1 and the subunit IFNγR2 and bound by the ligand IFNγ. The amino acid sequence of the human IFNγR1 polypeptide is known and is listed as UniProt ID NO. P15260.

[0044] As used herein, the term "interleukin 12 receptor" or "IL27R" refers to a heterodimeric receptor formed by the subunit IL12Rβ1 (IL12Rβ1) and the subunit IL12Rβ2 (IL12Rβ2) and bound by its cognate ligand IL12. The amino acid sequence of the human IL12Rβ1 is known and is listed as UniProt ID NO. P42701. The amino acid sequence of the human IL12Rβ2 is known and is listed as UniProt ID NO. Q99665.

[0045] As used herein, the term "interleukin 27 receptor" or "IL27R" refers to a heterodimeric receptor formed by the subunit IL27Rα (IL27Rα) and glycoprotein 130 (gp130) and bound by the ligand IL27. The human sequence of IL27Rα is listed as UniProt ID NO. Q6UWB1. The human sequence of gp130 is listed as UniProt ID NO. Q13514.

[0046] As used herein, the term "interleukin-10 receptor" or "IL10R" refers to a tetrameric receptor formed by two IL10Rα subunits (IL10Rα) and two IL10Rβ subunits (IL10Rβ) and bound by the ligand IL10. The amino acid sequence of human IL10Rα is listed as UniProt ID NO. Q13651. The amino acid sequence of human IL10Rβ is listed as UniProt ID NO. Q08334.

[0047] As used herein, the term "interleukin-2 receptor gamma" or "IL2Rγ" refers to the gamma subunit of the trimeric IL2R. IL2Rγ is also known as CD132. The amino acid sequence of human IL2Rγ is listed as UniProt ID NO. P31785.

[0048] As used herein, the term "linker" refers to a connection between two elements (such as protein domains). A linker can be a covalent bond or a peptide linker. The term "bond" refers to a chemical bond, such as an amide bond or a disulfide bond, or any kind of bond produced by a chemical reaction, such as chemical coupling. The term "peptide linker" refers to an amino acid or polypeptide that can be used to connect two protein domains to provide space and / or flexibility between the two protein domains.

[0049] As used herein, the term "oligomerization" refers to the proximity of two or more cell surface receptors or their domains or subunits to each other such that the receptors or their domains or subunits can interact and cause downstream signal transduction.

[0050] As used herein, the term "proximity" refers to the spatial proximity or physical distance between two cell surface receptors or their domains or subunits after the binding protein described herein binds to the two cell surface receptors or their domains or subunits. In some embodiments, after the binding protein binds to the cell surface receptor or its domain or subunit, the spatial proximity between the cell surface receptors or their domains or subunits can be, for example, less than about 500 angstroms, such as a distance of about 5 angstroms to about 500 angstroms. In some embodiments, the spatial proximity is less than about 5 angstroms, less than about 20 angstroms, less than about 50 angstroms, less than about 75 angstroms, less than about 100 angstroms, less than about 150 angstroms, less than about 250 angstroms, less than about 300 angstroms, less than about 350 angstroms, less than about 400 angstroms, less than about 450 angstroms, or less than about 500 angstroms. In some embodiments, the spatial proximity is less than about 100 angstroms. In some embodiments, the spatial proximity is less than about 50 angstroms. In some embodiments, the spatial proximity is less than about 20 angstroms. In some embodiments, the spatial proximity is less than about 10 angstroms. In some embodiments, the range of spatial proximity is about 10 to 100 angstroms, 50 to 150 angstroms, about 100 to 200 angstroms, about 150 to 250 angstroms, about 200 to 300 angstroms, about 250 to 350 angstroms, about 300 to 400 angstroms, about 350 to 450 angstroms, or about 400 to 500 angstroms. In some embodiments, the spatial proximity is less than about 250 angstroms, or less than about 200 angstroms, or less than about 150 angstroms, or less than about 120 angstroms, or less than about 100 angstroms, or less than about 80 angstroms, or less than about 70 angstroms, or less than about 50 angstroms.

[0051] As used herein, the term "downstream signaling" refers to the process of cell signaling resulting from the interaction of two or more cell surface receptors that are in proximity to each other.

[0052] As used herein, the term "percent (%) sequence identity" in the context of a nucleic acid or polypeptide refers to a sequence that has at least 50% sequence identity to a reference sequence. Additionally, the percent sequence identity can be any integer between 50% and 100%. In some embodiments, as determined by BLAST using standard parameters, the sequence has at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the reference sequence, as described below.

[0053] For sequence comparison, generally one sequence is used as a reference sequence against which a test sequence is compared. When using a sequence comparison algorithm, the test and reference sequences are input into a computer, and if desired, subsequence coordinates are specified, and sequence algorithm program parameters are specified. Default program parameters may be used, or alternative parameters may be specified. Then, the sequence comparison algorithm calculates the percent sequence identity of the test sequence relative to the reference sequence based on the program parameters.

[0054] A comparison window includes reference to a segment of any one of a plurality of contiguous positions, such as a segment of at least 10 residues. In some embodiments, the comparison window has from 10 to 600 residues, such as from about 10 to about 30 residues, from about 10 to about 20 residues, from about 50 to about 200 residues, or from about 100 to about 150 residues, where the sequences may be aligned with the reference sequence at the same number of contiguous positions after the two sequences are optimally aligned.

[0055] The algorithms suitable for determining percent sequence identity and percent 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. Software for performing BLAST analyses is publicly available from the National Center for Biotechnology Information (NCBI) website. This algorithm involves: first identifying high-scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence that, when aligned with words of the same length in a database sequence, can match or satisfy some positive threshold score T. T is referred to as the neighborhood word-scoring threshold (Altschul et al., supra). These initial neighborhood word hits are used as seeds to initiate a search to find longer HSPs that contain them. Then, the word hit is extended in both directions along each sequence until the cumulative alignment score is increased. For nucleotide sequences, the cumulative score is calculated using the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for a mismatched residue; always <0). For amino acid sequences, the cumulative score is calculated using a scoring matrix. Extension of the word hit in each direction is terminated when: the cumulative alignment score drops by X from its maximum achieved value; the cumulative score becomes zero or less due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The default values used by the BLASTN program (for nucleotide sequences) are as follows: word length (W) 28, expectation value (E) 10, M = 1, N = -2, and comparison of both strands. For amino acid sequences, the default values used by the BLASTP program are: word length (W) 3, expectation value (E) 10, and BLOSUM62 scoring matrix (see Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989)).

[0056] The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin and Altschul, Proc. Nat’l. Acad. Sci. USA 90:5873-5787 (1993)). One similarity measure provided by the BLAST algorithm is the sum probability and (P(N)), which indicates the probability that a match occurs by chance between two nucleotide or amino acid sequences. For example, if the sum probability and in a comparison of a test amino acid sequence with a reference amino acid sequence is less than about 0.01, more preferably less than about 10 -5 , and most preferably less than about 10 -20, the amino acid sequence is considered similar to the reference sequence.

[0057] As used herein, the term "single-domain antibody" or "sdAb" refers to an antibody having a single monomeric variable antibody domain. The sdAb is capable of selectively binding to a specific antigen. Further defined below, VH antibody is an example of sdAb. H VH antibody is an example of sdAb.

[0058] The term "specifically binds" as used herein refers to the degree of selectivity or affinity of one molecule for binding to another molecule. In the context of a binding pair (such as the binding protein / receptor, ligand / receptor, antibody / antigen, antibody / ligand, antibody / receptor binding pairs described herein), when the first molecule of the binding pair does not bind to other components present in the sample in significant amounts, the first molecule of the binding pair is said to specifically bind to the second molecule of the binding pair. When the affinity of the first molecule of the binding pair for the second molecule is at least 2-fold greater, or at least 5-fold greater, or at least 10-fold greater, or at least 20-fold greater, or at least 100-fold greater than the affinity of the first molecule for other components present in the sample, the first molecule of the binding pair is said to specifically bind to the second molecule of the binding pair.

[0059] In certain embodiments, if the equilibrium dissociation constant between VH and the receptor is greater than about 10 H M, or greater than about 10 6 M, or greater than about 10 8 M, or greater than about 10 10 M, or greater than about 10 11 M, or greater than about 10 10 M, greater than about 10 12 M, as determined, for example, by Scatchard analysis, then the VH in the bispecific VH H binding protein binds to the receptor (e.g., the first or second receptor of a natural or non-natural receptor pair) (Munsen, et al. 1980 Analyt. Biochem. 107:220-239). Specific binding can be evaluated using techniques known in the art, including but not limited to competitive ELISA, 2 assay and / or H assay. assay and / or assay.

[0060] As used herein, the terms "subject", "recipient", "individual", or "patient" refer to any mammalian subject in need of diagnosis, treatment, or therapy, particularly a human. These terms may be used interchangeably herein. A "mammal" for therapeutic purposes refers to any animal classified as a mammal, including humans, domestic and farm animals, non-human primates, and zoo, sports, or pet animals such as dogs, horses, cats, cows, sheep, goats, pigs, etc. In some embodiments, the mammal is a human.

[0061] The terms "treatment", "therapy", "treatment process", etc. refer to a course of action (e.g., administering a binding protein or a pharmaceutical composition comprising the same as described herein) or the like initiated after diagnosing, observing a disease, disorder, or condition or its symptoms in a subject, so as to temporarily or permanently eliminate, reduce, inhibit, alleviate, or improve at least one underlying cause of such disease, disorder, or condition troubling the subject, or at least one symptom associated with such disease, disorder, or condition. Treatment includes a course of action taken on a subject suffering from a disease, wherein the course of action results in the inhibition of the subject's disease (e.g., preventing the development of the disease, disorder, or condition or improving one or more symptoms associated therewith).

[0062] The terms "prevention", "prevention of", etc. as used herein refer to a course of action initiated before the occurrence of a subject's disease, disorder, condition, or its symptoms, such that the risk of the subject suffering from a certain disease, disorder, condition, or similar disease is temporarily or permanently prevented, alleviated, inhibited, or reduced (e.g., determined by the absence of clinical symptoms), or the onset thereof is postponed, generally in cases where the subject is predisposed to a certain specific disease, disorder, or condition due to genetic, experiential, or environmental factors. In certain cases, the terms "prevention", "prevention of" are also used to refer to slowing down the progression of a disease, disorder, or condition from its current state to a more harmful state.

[0063] As used herein, the term "V H H" is a single-domain antibody (sdAb) with a single monomeric heavy-chain variable antibody domain. Such an antibody can be found or produced in camelid mammals (such as camels, llamas), which are naturally lacking a light chain.

[0064] As used herein, the term "V H H 2 " refers to two V H H joined by a linker means (e.g., a covalent bond or a peptide linker). "Bispecific V H H 2 " refers to a V H H having a first V H H that binds to a first receptor or its domain or its subunit and a second V H H that binds to a second receptor or its domain or its subunit. 2 ​

[0065] III. Compositions and Methods

[0066] The present invention describes a variety of receptor-binding proteins that bind to natural cytokine receptor pairs or their domains or subunits, or non-natural cytokine receptor pairs or their domains or subunits to generate signal transduction diversity not seen in natural receptor pairings. A variety of receptor-binding proteins can be screened for binding to receptor pairs or their domains or subunits and signal transduction in therapeutically relevant cell types.

[0067] Receptor-binding proteins that bind to natural receptor pairs

[0068] IL12 receptor-binding protein

[0069] The IL12 receptor (IL12R) comprises the subunits IL12Rβ1 and IL12Rβ2. Provided herein are IL12R-binding proteins that specifically bind to IL12Rβ1 and IL12Rβ2. In some embodiments, the IL12R-binding protein binds to mammalian cells expressing both IL12Rβ1 and IL12Rβ2. In some embodiments, the IL12R-binding protein can be a bispecific V H H 2 . In other embodiments, the IL12R-binding protein can comprise a first domain (which is V H H) and a second domain (which can be a fragment of IL12 or, for example, an scFv).

[0070] The IL12R-binding protein can be a bispecific V H H having a first V H H (anti-IL12Rβ1 V H H antibody) that binds to IL12Rβ1 and a second V H H (anti-IL12Rβ2 V H H 2 antibody) that binds to IL12Rβ2, and when bound to cells expressing IL12Rβ1 and IL12Rβ2, such as natural killer or T cells (e.g., CD4 + T cells and / or CD8 + T cells), results in dimerization of the two receptor subunits and downstream signal transduction.

[0071] A linker can be used to join the anti-IL12Rβ1 V H H antibody and the anti-IL12Rβ2 V HH antibody. For example, the linker can simply be a covalent bond or a peptide linker. The peptide linker can comprise 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). Conjugating anti-IL12Rβ1V H H antibody and anti-IL12Rβ2V H The peptide linker of the H antibody can be a flexible glycine-serine linker. The linker can also be a chemical linker, such as a synthetic polymer, such as a polyethylene glycol (PEG) polymer.

[0072] anti-IL12Rβ1V H The H antibody can have a sequence with at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to any one of the sequences in SEQ ID NO: 105 - 111.

[0073] anti-IL12Rβ2V H The H antibody can have a sequence with at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to any one of the sequences in SEQ ID NO: 58 - 63.

[0074] anti-IL12Rβ2V H The H antibody can have a sequence with at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to any one of the sequences in SEQ ID NO: 112 - 117.

[0075] In some embodiments, the IL12 receptor binding protein described herein can have anti-IL12Rβ1V H H, linker and anti-IL12Rβ2V H H, as listed in Table 1 below.

[0076]

[0077]

[0078]

[0079]

[0080] In some embodiments, E caused by IL12最大 In contrast, the IL12R binding protein has a reduced E 最大 . E 最大 reflects the maximum response level in cell types obtainable by a ligand (e.g., a binding protein or a native cytokine (e.g., IL12) as described herein). In some embodiments, the IL12R binding protein described herein has an E 最大 that is at least 1% of that caused by IL12 (e.g., between 1% and 100%, between 10% and 100%, between 20% and 100%, between 30% and 100%, between 40% and 100%, between 50% and 100%, between 60% and 100%, between 70% and 100%, between 80% and 100%, between 90% and 100%, between 1% and 90%, between 1% and 80%, between 1% and 70%, between 1% and 60%, between 1% and 50%, between 1% and 40%, between 1% and 30%, between 1% and 20%, or between 1% and 10%). In some embodiments, by varying the linker length of the IL12R binding protein, the E 最大 of the IL12R binding protein can be altered. The IL12R binding protein can elicit an E + in the cell types most in need (e.g., CD8 最大 T cells) and reduce E 最大 in other cell types (e.g., natural killer cells). In some embodiments, the E 最大 in natural killer cells caused by the IL12R binding protein described herein is between 1% and 100% of the E + in T cells (e.g., CD8 最大 T cells) caused by the IL12R binding protein (e.g., between 10% and 100%, between 20% and 100%, between 30% and 100%, between 40% and 100%, between 50% and 100%, between 60% and 100%, between 70% and 100%, between 80% and 100%, between 90% and 100%, between 1% and 90%, between 1% and 80%, between 1% and 70%, between 1% and 60%, between 1% and 50%, between 1% and 40%, between 1% and 30%, between 1% and 20%, or between 1% and 10%). In other embodiments, the E 最大 of the IL12R binding protein described herein is greater (e.g., at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% or 50% greater) than the E 最大 of the native ligand IL12.

[0081] The IL12R binding proteins described herein can be used to treat neoplastic diseases, such as cancer (e.g., solid tumor cancers (e.g., non-small cell lung cancer (NSCLC), renal cell carcinoma (RCC), or melanoma)), in a subject in need thereof. The IL12R binding proteins bind to and activate natural killer, CD4 + T cells and / or CD8 + T cells. The IL12R binding proteins can trigger different levels of downstream signal transduction in different cell types. For example, by varying the length of the linker between the anti-IL12Rβ1V H H antibody and the anti-IL12Rβ2V H H antibody in the IL12R binding protein, the IL12R binding protein can cause a higher level of downstream signal transduction in a desired cell type compared to an undesired cell type. In some embodiments, for example by varying the linker length, the IL12R binding protein can produce a higher level of downstream signal transduction in T cells (e.g., CD8 + T cells) compared to the level of downstream signal transduction in natural killer cells, a cell type that expresses both the IL12Rβ1 and IL12Rβ2 receptors but can be toxic when activated too strongly. In other embodiments, different anti-IL12Rβ1V H H antibodies with different binding affinities and different anti-IL12Rβ2V H H antibodies with different binding affinities can be combined to prepare different IL12R binding proteins. In addition, the orientation of the two antibodies in the binding protein can also be varied to prepare different binding proteins (i.e., anti-IL12Rβ1V H H antibody-linker-anti-IL12Rβ2V H H antibody, or anti-IL12Rβ2V H H antibody-linker-anti-IL12Rβ1V H H antibody). Different IL12R binding proteins can be screened to find the ideal binding protein that causes a higher level of downstream signal transduction in the desired cell type compared to an undesired cell type. In some embodiments, the IL12R binding protein can be a partial agonist that has different activities on different cell types (e.g., T cells relative to natural killer cells). For example, selective activation of T cells relative to natural killer cells is required to avoid the toxicity associated with IL12-activated natural killer cells. In some embodiments, the IL12R binding protein is a partial agonist, wherein the partial agonist selectively activates T cells relative to NK cells. In some embodiments, T cells (e.g., CD8 +The downstream signal transduction level in T cells) is at least 1.1, 1.5, 2, 3, 5, or 10 times the downstream signal transduction level in natural killer cells.

[0082] IL27 receptor binding protein

[0083] The IL27 receptor (IL27R) comprises the IL27Rα subunit (IL27Rα) and the glycoprotein 130 subunit (gp130). Provided herein are IL27R binding proteins that specifically bind to IL27Rα and gp130. In some embodiments, the IL27R binding protein binds to mammalian cells that express both IL27Rα and gp130. In some embodiments, the IL27R binding protein can be a bispecific V H H 2 . In other embodiments, the IL27R binding protein can comprise a first domain (which is V H H) and a second domain (which can be a fragment of IL27 or, for example, an scFv).

[0084] The IL27R binding protein can be a bispecific V with a first V H H that binds to IL27Rα (anti-IL27Rα V H H antibody) and a second V H H that binds to gp130 (anti-gp130 V H H antibody) H H 2 , and when bound to cells that express IL27Rα and gp130 (such as CD8 + T cells, CD4 + T cells and / or T regulatory (Treg) cells), results in dimerization of the two receptor subunits and downstream signal transduction.

[0085] Linkers can be used to conjugate the anti-IL27Rα V H H antibody and the anti-gp130 V H H antibody. For example, the linker can simply be a covalent bond or a peptide linker. The peptide linker can comprise 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 peptide linker that conjugates the anti-IL27Rα V H H antibody and the anti-gp130 V H H antibody can be a flexible glycine-serine linker. The linker can also be a chemical linker, such as a synthetic polymer, such as a polyethylene glycol (PEG) polymer.

[0086] Anti-IL27Rα V H The H antibody can have a sequence with at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity relative to any one of the sequences of SEQ ID NO: 70-75.

[0087] Anti-IL27Rα V H The H antibody can have a sequence with at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity relative to any one of the sequences of SEQ ID NO: 125-130.

[0088] Anti-gp130 V H The H antibody can have a sequence with at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity relative to any one of the sequences of SEQ ID NO: 24-29.

[0089] Anti-gp130 V H The H antibody can have a sequence with at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity relative to any one of the sequences of SEQ ID NO: 83-89.

[0090] In some embodiments, compared to E induced by IL27 最大 the IL27R binding protein has a reduced E 最大 . E 最大 reflects the maximum response level in cell types obtainable by a ligand (e.g., a binding protein or a native cytokine (e.g., IL27) described herein). In some embodiments, the IL27R binding protein described herein has an E induced by IL27 最大 of at least 1% (e.g., between 1% and 100%, between 10% and 100%, between 20% and 100%, between 30% and 100%, between 40% and 100%, between 50% and 100%, between 60% and 100%, between 70% and 100%, between 80% and 100%, between 90% and 100%, between 1% and 90%, between 1% and 80%, between 1% and 70%, between 1% and 60%, between 1% and 50%, between 1% and 40%, between 1% and 30%, between 1% and 20%, or between 1% and 10%). In other embodiments, the E of the IL27R binding molecule described herein最大 greater than (e.g., at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% greater) than that of the native ligand IL27 最大 . In some embodiments, by varying the linker length of the IL27R-binding protein, the E of the IL27R-binding protein can be altered 最大 . The IL27R-binding protein can elicit E in the cell types where it is most needed 最大 and reduce E in other cell types 最大 .

[0091] The IL27R-binding proteins described herein can be used to treat neoplastic diseases, such as cancer (e.g., solid tumor cancers; e.g., non-small cell lung cancer (NSCLC), renal cell carcinoma (RCC), or melanoma) and / or infectious diseases (e.g., bacterial infections and viral infections (e.g., viral infections caused by hepatitis C virus (HCV), human papillomavirus (HPV), or human immunodeficiency virus (HIV)) in a subject in need thereof. The IL27R-binding protein binds to and activates CD8 + T cells, CD4 + T cells, and / or T regulatory (Treg) cells. The IL27R-binding protein can trigger different levels of downstream signal transduction in different cell types. For example, by varying the length of the linker between the anti-IL27Rα V H H antibody and the anti-gp130 V H H antibody in the IL27R-binding protein, the IL27R-binding protein can elicit a higher level of downstream signal transduction in the desired cell types compared to the undesired cell types. In some embodiments, by varying the linker length, the IL27R-binding protein can elicit a higher level of downstream signal transduction in T cells (e.g., CD8 + T cells) compared to the level of downstream signal transduction in other cells. In other embodiments, different anti-IL27Rα V H H antibodies with different binding affinities and different anti-gp130 V H H antibodies with different binding affinities can be combined to prepare different IL27R-binding proteins. In addition, the orientation of the two antibodies in the binding protein can also be altered to prepare 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 HH antibody). Different IL27R-binding proteins can be screened to find an ideal binding protein that elicits a higher level of downstream signal transduction in the desired cell type compared to an undesired cell type. In some embodiments, the level of downstream signal transduction in T cells (e.g., CD8 + T cells) is at least 1.1, 1.5, 2, 3, 5, or 10 times the level of downstream signal transduction in other cells.

[0092] Specifically, the IL27R-binding protein binds to and activates CD8 + T cells. In some embodiments, the IL27R-binding protein binds to and activates CXCR5 + CD8 + T cells. It is well known that, for example, during viral infection, IL27 can promote and maintain the rapid proliferation of memory-like CXCR5 + CD8 + T cells. CXCR5 + CD8 + T cells can maintain the T cell response during persistent infection or cancer and drive a proliferation burst of CD8 + T cells after anti-PD1 treatment. Thus, the IL27R-binding proteins described herein contribute to maintaining and enhancing self-renewing T cells in chronic infections and neoplastic diseases such as cancer.

[0093] IL10 receptor-binding protein

[0094] The IL10 receptor (IL10R) comprises an IL10Rα subunit (IL10Rα) and an IL10Rβ subunit (IL10Rβ). Provided herein are IL10R-binding proteins that specifically bind to IL10Rα and IL10Rβ. In some embodiments, the IL10R-binding protein binds to mammalian cells that express both IL10Rα and IL10Rβ. In some embodiments, the IL10R-binding protein can be a bispecific V H H 2 as described below. In other embodiments, the IL10R-binding protein can comprise a first domain (which is a V H H) and a second domain (which can be a fragment of IL10 or, for example, an scFv).

[0095] The IL10R-binding protein can be a bispecific V H H with a first V H H that binds to IL10Rα (an anti-IL10Rα V H H antibody) and a second V H H that binds to IL10Rβ (an anti-IL10Rβ V H H antibody) 2, and when combined with cells expressing IL10Rα and IL10Rβ, such as T cells (e.g., CD8 + T cells or CD4 + T cells), macrophages, and / or Treg cells, it results in dimerization of the two receptor subunits and downstream signal transduction.

[0096] Linkers can be used to conjugate anti-IL10RαV H H antibodies and anti-IL10RβV H H antibodies. For example, the linker can simply be a covalent bond or a peptide linker. The peptide linker can 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 peptide linker that conjugates anti-IL10RαV H H antibodies and anti-IL10RβV H H antibodies can be a flexible glycine-serine linker. The linker can also be a chemical linker, such as a synthetic polymer, such as a polyethylene glycol (PEG) polymer.

[0097] Anti-IL10RαV H H antibodies can have a sequence with at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to any one of the sequences in SEQ ID NO: 44 - 50.

[0098] Anti-IL10RαV HThe H antibody can have a sequence comprising: having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity relative to the sequence of any one of SEQ ID NO: 388, 391, 394, 397, 400, 403 and 406, or having 0, 1, 2 or 3 amino acid changes, optionally conservative amino acid changes, in CDR1; having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity relative to the sequence of any one of SEQ ID NO: 389, 392, 395, 398, 401, 404 and 407, or having 0, 1, 2 or 3 amino acid changes, optionally conservative amino acid changes, in CDR2; and having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity relative to the sequence of any one of SEQ ID NO: 390, 393, 396, 399, 402, 405 and 408, or having 0, 1, 2 or 3 amino acid changes, optionally conservative amino acid changes, in CDR3.

[0099] Anti-IL10RβV H The H antibody can have a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity relative to the sequence of any one of SEQ ID NO: 51-57.

[0100] Anti-IL10RβV HThe H antibody can have a sequence comprising: 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 NO: 409, 412, 415, 418, 421, 424 and 427, or having 0, 1, 2 or 3 amino acid changes, optionally conservative amino acid changes in CDR1; 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 NO: 410, 413, 416, 419, 422, 425 and 428, or having 0, 1, 2 or 3 amino acid changes, optionally conservative amino acid changes in CDR2; and 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 NO: 411, 414, 417, 420, 423, 426 and 429, or having 0, 1, 2 or 3 amino acid changes, optionally conservative amino acid changes in CDR3.

[0101] Anti-IL10RβV H The H antibody can have 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 NO: 99 - 104.

[0102] In some embodiments, compared to E caused by IL10 最大 the IL10R binding protein has a reduced E 最大 . E 最大 reflects the maximum response level in cell types obtainable by a ligand (e.g., a binding protein or a native cytokine (e.g., IL10) as described herein). In some embodiments, the IL10R binding protein described herein has an E caused by IL10 最大at least 1% (e.g., between 1% and 100%, between 10% and 100%, between 20% and 100%, between 30% and 100%, between 40% and 100%, between 50% and 100%, between 60% and 100%, between 70% and 100%, between 80% and 100%, between 90% and 100%, between 1% and 90%, between 1% and 80%, between 1% and 70%, between 1% and 60%, between 1% and 50%, between 1% and 40%, between 1% and 30%, between 1% and 20%, or between 1% and 10%). In some embodiments, by varying the linker length of the IL10R-binding protein, the E of the IL10R-binding protein can be altered 最大 . The IL10R-binding protein can elicit E in the cell type most in need (e.g., CD8 + T cells) and reduce E in other cell types (e.g., macrophages 最大 . In some embodiments, the E in macrophages caused by the IL10R-binding proteins described herein 最大 is 1% to 100% (e.g., between 10% and 100%, between 20% and 100%, between 30% and 100%, between 40% and 100%, between 50% and 100%, between 60% and 100%, between 70% and 100%, between 80% and 100%, between 90% and 100%, between 1% and 90%, between 1% and 80%, between 1% and 70%, between 1% and 60%, between 1% and 50%, between 1% and 40%, between 1% and 30%, between 1% and 20%, or between 1% and 10%) of the E in T cells (e.g., CD8 最大 T cells) caused by the IL10R-binding protein. In other embodiments, the E of the IL10R-binding proteins described herein + is greater than (e.g., at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% or 50% greater) the E of the native ligand IL10 最大 . 最大 . 最大 .

[0103] In some embodiments, the present invention provides examples of IL10 receptor-binding proteins that comprise anti-IL10RαV H H, an optional linker, and anti-IL10Rβ2V H H. In some embodiments, the N-terminal V H H of the IL-10 binding molecule is anti-IL10RαV H H and the C-terminal V H H of the IL-10 receptor-binding protein is anti-IL10RβV H H, optionally comprising VH A linker between the Hs. In some embodiments, the N-terminal V of the IL-10 receptor-binding protein H Hs is anti-IL10Rβ V H Hs and the C-terminal V of the IL-10 receptor-binding protein H Hs is anti-IL10Rα V H Hs, optionally comprising V H A linker between the Hs. In some embodiments, the IL-10 receptor-binding protein can provide a purification handle, such as but not limited to an Ala-Ser-His-His-His-His-His-His (“ASH6”, SEQ ID NO: 430) purification handle, to facilitate purification of the receptor-binding protein by chelating peptide immobilized metal affinity chromatography (“CP-IMAC, as described in U.S. Patent No. 4,569,794).

[0104] A series of 98 IL10 receptor-binding proteins were prepared substantially according to Examples 1-4 herein, which comprise anti-IL10Rα V H Hs, a linker, and anti-IL10Rβ2 V H Hs, as well as an ASH6 (SEQ ID NO: 430) purification handle (SEQ ID NOs: 192-289), and the IL-10 activity was evaluated substantially according to Examples 5 and 6 herein. The arrangements of the V H Hs, linker, and purification handle elements of these 98 IL-10 receptor-binding proteins are provided in Table 2 below.

[0105]

[0106]

[0107]

[0108]

[0109]

[0110] As provided in more detail in Example 3 herein, the nucleic acid sequences encoding SEQ ID Nos: 192-289 were synthesized as SEQ ID Nos: 290-387 respectively, inserted into a recombinant expression vector and expressed and purified in HEK293 cells in 24-well plate format, substantially according to Example 4. Substantially according to Examples 5 and 6 herein, the activity of the supernatants of the IL-10 receptor-binding proteins containing SEQ ID Nos: 192-298 was evaluated using unstimulated and wild-type human IL-10 as controls. The results of these experiments are provided in Table 3 below.

[0111]

[0112]

[0113]

[0114]

[0115]

[0116]

[0117]

[0118]

[0119]

[0120] As can be seen from the data provided above, in the IL-10 activity assay, the IL-10 receptor binding protein showed significant IL-10 activity (Example 4). Specifically, based on the absorbance readings, the IL-10 activity was classified as low (higher than unstimulated and A 630 <1), medium (A 630 1-1.5) and high (A 630 >1.5). From the above data, 11 IL10R binding proteins showed high activity (SEQ ID No: 194, 209, 210, 211, 213, 218, 226, 233, 238, 244 and 250), 4 had medium activity (SEQ ID No: 203, 205, 207 and 269) and 8 VHHs had low activity (SEQ ID No: 212, 217, 219, 224, 227, 237, 239 and 249). In some embodiments, the present invention provides an IL10R binding protein, wherein the IL10R binding protein comprises a first anti-IL10R sdAb joined to a second anti-IL10R sdAb from amino to carboxy via a linker, according to Table 4 below:

[0121]

[0122]

[0123] and wherein the IL10R binding protein further optionally comprises a linker selected from SEQ ID No: 1-23.

[0124] The IL10R binding proteins described herein can be used to treat neoplastic diseases, such as cancer (e.g., solid tumor cancers; e.g., non-small cell lung cancer (NSCLC), renal cell carcinoma (RCC), or melanoma)) in a subject in need thereof. The IL10R binding proteins bind to and activate CD8 + T cells, CD4 + T cells, macrophages, and / or Treg cells. In some embodiments, the IL10R binding proteins described herein can provide longer therapeutic efficacy (e.g., lower effective doses, lower toxicity) than wild-type or pegylated IL10. The IL10R binding proteins can trigger different levels of downstream signal transduction in different cell types. For example, by varying the length of the linker between the anti-IL10RαV H H antibody and the anti-IL10RβV H H antibody in the IL10R binding protein, the IL10R binding protein can cause a higher level of downstream signal transduction in a desired cell type compared to an undesired cell type. In some embodiments, the IL10R binding protein is a partial agonist that selectively activates T cells (e.g., CD8 + T cells) rather than macrophages. In some embodiments, the activated T cells have upregulated IFNγ. In some embodiments, as a partial agonist, the IL10R binding protein can inhibit autoimmune inflammatory diseases (such as ulcerative colitis and Crohn's disease). In some embodiments, by varying the linker length, the IL10R binding protein can produce a higher level of downstream signal transduction in T cells (e.g., CD8 + T cells) compared to the level of downstream signal transduction in macrophages, a cell type that expresses both the IL10Rα and IL10Rβ receptors but can cause anemia when activated too strongly. When the downstream signal transduction in macrophages is activated to a high level, these activated macrophages can eliminate senescent red blood cells, resulting in anemia. The IL10R binding protein can cause a higher level of downstream signal transduction in T cells (e.g., CD8 + T cells) compared to the level of downstream signal transduction in macrophages, thereby avoiding anemia. In other embodiments, different anti-IL10RαV H H antibodies with different binding affinities and different anti-IL10RβV H H antibodies with different binding affinities can be combined to prepare different IL10R binding proteins. In addition, the orientation of the two antibodies in the binding protein can also be varied to prepare different binding proteins (i.e., anti-IL10RαV H H antibody-linker-anti-IL10RβV H H antibody, or anti-IL10RβV HH Antibody-Linker-Anti-IL10RαV H H antibody). Different IL10R binding proteins can be screened to find an ideal binding protein that causes higher levels of downstream signaling in desired cell types compared to undesired cell types. In some embodiments, T cells (e.g., CD8 + T cells) is at least 1.1, 1.5, 2, 3, 5, or 10 times greater than the level of downstream signaling in macrophages.

[0125] IFNλ receptor binding protein

[0126] Interferon (IFN) λ receptor (IFNλR) includes IL10Rβ and IL28 receptor (IL28R) α subunit (IL28Rα). Provided herein are IFNλR binding proteins that specifically bind to IL10Rβ and IL28Rα. In some embodiments, the IFNλR binding protein binds to mammalian cells expressing both IL10Rβ and IL28Rα. In some embodiments, the IFNλR binding protein can be a bispecific V as described below. H H 2 In other embodiments, the IFNλR binding protein may comprise a first domain (which is V H H) and a second domain (which may be a fragment of IFNλ or, for example, a scFv).

[0127] The IFNλR binding protein may have a first V binding to IL10Rβ H H(anti-IL10RβV H H antibody) and a second V H H (anti-IL28RαV H H antibody) H H 2 , and when bound to cells expressing IL10Rβ and IL28R, such as macrophages, T cells (e.g., CD8 + T cells or CD4 + T cells), Treg cells, dendritic cells and / or epithelial cells, leading to dimerization of the two receptor subunits and downstream signaling.

[0128] Linkers can be used to conjugate anti-IL10RβV H H antibody and anti-IL28RαV HH antibody. For example, the linker can simply be a covalent bond or a peptide linker. The peptide linker can 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). Conjugate anti-IL10RβV H H antibody and anti-IL28RαV H The peptide linker of the H antibody can be a flexible glycine-serine linker. The linker can also be a chemical linker, such as a synthetic polymer, such as a polyethylene glycol (PEG) polymer.

[0129] anti-IL10RβV H The H antibody can have a sequence with at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity relative to any one of the sequences of SEQ ID NO: 51-57.

[0130] anti-IL10RβV H The H antibody can have a sequence with at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity relative to any one of the sequences of SEQ ID NO: 99-104.

[0131] anti-IL28RαV H The H antibody can have a sequence with at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity relative to any one of the sequences of SEQ ID NO: 76-82.

[0132] In some embodiments, compared to the E induced by IFNλ 最大 the IFNλR binding protein has a reduced E 最大 . E 最大 reflects the maximum response level in cell types that can be obtained by a ligand (e.g., a binding protein or a native cytokine (e.g., IFNλ) as described herein). In some embodiments, the IFNλR binding protein described herein has an E induced by IFNλ 最大at least 1% (e.g., between 1% and 100%, between 10% and 100%, between 20% and 100%, between 30% and 100%, between 40% and 100%, between 50% and 100%, between 60% and 100%, between 70% and 100%, between 80% and 100%, between 90% and 100%, between 1% and 90%, between 1% and 80%, between 1% and 70%, between 1% and 60%, between 1% and 50%, between 1% and 40%, between 1% and 30%, between 1% and 20%, or between 1% and 10%). In other embodiments, the E of the IFNλR-binding protein described herein 最大 is greater (e.g., at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% or 50% greater) than the E of the native ligand IFNλ 最大 . In some embodiments, by varying the linker length of the IFNλR-binding protein, the E of the IFNλR-binding protein can be altered 最大 . The IFNλR-binding protein can elicit E 最大 in the cell type most in need (e.g., macrophages), and reduce E max in other cell types.

[0133] The IFNλR-binding protein of the present invention can be used to treat infectious diseases in a subject in need thereof. The IFNλR-binding protein binds to and activates macrophages, CD8 + T cells, CD4 + T cells, Treg cells, dendritic cells, and / or epithelial cells. In particular, the IFNλR-binding protein binds to and activates macrophages. Examples of infectious diseases include, but are not limited to, influenza, hepatitis B, hepatitis C, and human immunodeficiency virus (HIV) infection. In some embodiments, the IFNλR-binding protein can protect Kupffer cells in the liver from infectious diseases. The IFNλR-binding protein can elicit different levels of downstream signal transduction in different cell types. For example, by varying the length of the linker between the anti-IL10RβV H H antibody and the anti-IL28RαV H H antibody in the IFNλR-binding protein, the IFNλR-binding protein can elicit a higher level of downstream signal transduction in a desired cell type (e.g., macrophages) compared to an undesired cell type. In some embodiments, by varying the linker length, the IFNλR-binding protein can produce a regulation of downstream signal transduction in macrophages compared to the level of downstream signal transduction in other cell types. In other embodiments, different anti-IL10RβV H H antibodies with different binding affinities and different anti-IL28RαV HH antibodies can be combined to prepare different IFNλR-binding proteins. In addition, the orientation of the two antibodies in the binding protein can be altered to prepare different binding proteins (i.e., anti-IL10RβV H H antibody-linker-anti-IL28RαV H H antibody, or anti-IL28RαV H H antibody-linker-anti-IL10RβV H H antibody). Different IFNλR-binding proteins can be screened to find an optimal binding protein that elicits a higher level of downstream signal transduction in a desired cell type compared to an undesired cell type. In some embodiments, the level of downstream signal transduction in macrophages is at least 1.1, 1.5, 2, 3, 5, or 10 times the level of downstream signal transduction in other cell types.

[0134] IL23 receptor-binding protein

[0135] The IL23 receptor (IL23R) comprises the IL12Rβ1 subunit (IL12Rβ1) and the IL23R subunit. Provided herein are IL23R-binding proteins that specifically bind to IL12Rβ1 and IL23R. In some embodiments, the IL23R-binding protein binds to mammalian cells that express both IL12Rβ1 and IL23R. In some embodiments, the IL23R-binding protein can be a bispecific V H H 2 as described below. In other embodiments, the IL23R-binding protein can comprise a first domain that is a V H H and a second domain that can be a fragment of IL23 or, for example, a scFv.

[0136] The IL23R-binding protein can be a bispecific V H H that has a first V H H (anti-IL12Rβ1V H H antibody) that binds to IL12Rβ1 and a second V H H (anti-IL23R V H H 2 and, when bound to cells that express IL12Rβ1 and IL23R, such as T cells (e.g., CD8 + T cells or CD4 + T cells), macrophages, and / or Treg cells, results in dimerization of the two receptor subunits and downstream signal transduction.

[0137] A linker can be used to join the anti-IL12Rβ1V H H antibody and the anti-IL23R V HH antibody. For example, the linker can simply be a covalent bond or a peptide linker. The peptide linker can 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). Conjugate anti-IL12Rβ1V H H antibody and anti-IL23R V H The peptide linker of the H antibody can be a flexible glycine-serine linker. The linker can also be a chemical linker, such as a synthetic polymer, such as a polyethylene glycol (PEG) polymer.

[0138] anti-IL12Rβ1V H The H antibody can have a sequence with at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to any one of the sequences in SEQ ID NO: 105-111.

[0139] anti-IL23R V H The H antibody can have a sequence with at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to any one of the sequences in SEQ ID NO: 64-69.

[0140] anti-IL23R V H The H antibody can have a sequence with at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to any one of the sequences in SEQ ID NO: 118-124.

[0141] In some embodiments, compared to the E caused by IL23 最大 the IL23R binding protein has a reduced E 最大 . E 最大 reflects the maximum response level in cell types that can be obtained through a ligand (e.g., a binding protein or a native cytokine (e.g., IL23) as described herein). In some embodiments, the IL23R binding protein described herein has an E caused by IL23 最大at least 1% (e.g., between 1% and 100%, between 10% and 100%, between 20% and 100%, between 30% and 100%, between 40% and 100%, between 50% and 100%, between 60% and 100%, between 70% and 100%, between 80% and 100%, between 90% and 100%, between 1% and 90%, between 1% and 80%, between 1% and 70%, between 1% and 60%, between 1% and 50%, between 1% and 40%, between 1% and 30%, between 1% and 20%, or between 1% and 10%). In some embodiments, by varying the linker length of the IL23R binding protein, the E of the IL23R binding protein can be altered 最大 . The IL23R binding protein can elicit E + in the cell type where it is most needed (e.g., CD8 最大 T cells), and reduce E 最大 in other cell types (e.g., macrophages). In some embodiments, the E 最大 in macrophages elicited by the IL23R binding protein described herein is 1% to 100% (e.g., between 10% and 100%, between 20% and 100%, between 30% and 100%, between 40% and 100%, between 50% and 100%, between 60% and 100%, between 70% and 100%, between 80% and 100%, between 90% and 100%, between 1% and 90%, between 1% and 80%, between 1% and 70%, between 1% and 60%, between 1% and 50%, between 1% and 40%, between 1% and 30%, between 1% and 20%, or between 1% and 10%) of the E + in T cells (e.g., CD8 最大 T cells) elicited by the IL23R binding protein. In other embodiments, the E 最大 of the IL23R binding protein described herein is greater (e.g., at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% or 50% greater) than the E 最大 of the native ligand IL23.

[0142] The IL23R binding protein described herein can be used for wound healing. In particular, the IL23R binding protein described herein plays an important role in initiating wound healing, such as the healing of the skin keratinocyte layer. The IL23R binding protein binds to and activates CD8 + T cells, CD4 + T cells, macrophages, and / or Treg cells. The IL23R binding protein can elicit different levels of downstream signal transduction in different cell types. For example, by varying the anti-IL12Rβ1V in the IL23R binding protein HH antibody and anti-IL23R V H The length of the linker between the H antibody and the anti-IL23R V H can result in a higher level of downstream signal transduction in the desired cell type compared to the undesired cell type. In some embodiments, the IL23R binding protein can be a partial agonist that selectively activates T cells (e.g., CD8 + T cells) rather than macrophages. In other embodiments, different anti-IL12Rβ1 V + H H antibodies with different binding affinities and different anti-IL23R V H H H antibodies with different binding affinities can be combined to prepare different IL23R binding proteins. Additionally, the orientation of the two antibodies in the binding protein can be altered to prepare different binding proteins (i.e., anti-IL12Rβ1 V H H H antibody-linker-anti-IL23R V H H H antibody, or anti-IL23R V H H H antibody-linker-anti-IL12Rβ1 V H H H antibody). Different IL23R binding proteins can be screened to find the ideal binding protein that causes a higher level of downstream signal transduction in the desired cell type compared to the undesired cell type. In some embodiments, the level of downstream signal transduction in T cells (e.g., CD8 + T cells) is at least 1.1, 1.5, 2, 3, 5, or 10 times the level of downstream signal transduction in macrophages.

[0143] IL2 receptor binding protein

[0144] The IL2 receptor (IL2R) includes the CD25 subunit (CD25; also known as the IL2Rα subunit), the CD122 subunit (CD122; also known as the IL2Rβ subunit), and the CD132 subunit (CD132; also known as the IL2Rγ subunit). IL2R binding proteins that specifically bind to CD122 and CD132 are provided herein. In some embodiments, the IL2R binding protein binds to mammalian cells that express both CD122 and CD132. In some embodiments, the IL2R binding protein can be a bispecific V H H H 2 as described below. In other embodiments, the IL2R binding protein can comprise a first domain (which is a V 2 H H) and a second domain (which can be a fragment of IL2 or, for example, an scFv).

[0145] The IL2R binding protein can be a bispecific V H H H with a first V H HH antibody) and a second V that binds to CD132 H H (anti-CD132 V H H antibody) of bispecific V H H 2 and, when bound to cells expressing CD122 and CD132, such as T cells (e.g., CD8 + T cells or CD4 + T cells), macrophages, and / or Treg cells, results in dimerization of the two receptor subunits and downstream signal transduction.

[0146] The linker can be used to conjugate the anti-CD122 V H H antibody and the anti-CD132 V H H antibody. For example, the linker can simply be a covalent bond or a peptide linker. The peptide linker can 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 peptide linker that conjugates the anti-CD122 V H H antibody and the anti-CD132 V H H antibody can be a flexible glycine-serine linker. The linker can also be a chemical linker, such as a synthetic polymer, such as a polyethylene glycol (PEG) polymer.

[0147] The anti-CD122 V H H antibody can have a sequence with at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to any one of the sequences in SEQ ID NO: 30-37.

[0148] The anti-CD122 V H H antibody can have a sequence with at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to any one of the sequences in SEQ ID NO: 90 and 91.

[0149] The anti-CD132 V H H antibody can have a sequence with at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to any one of the sequences in SEQ ID NO: 38-43.

[0150] The anti-CD132 V HThe H antibody can have a sequence with at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to any one of the sequences in SEQ ID NO: 92 - 98.

[0151] In some embodiments, compared with that caused by IL2 最大 the IL2R binding protein has a reduced 最大 . 最大 reflects the maximum response level in cell types obtainable by a ligand (e.g., the binding proteins or native cytokines (e.g., IL2) described herein). In some embodiments, the IL2R binding protein described herein has an 最大 of at least 1% (e.g., between 1% and 100%, between 10% and 100%, between 20% and 100%, between 30% and 100%, between 40% and 100%, between 50% and 100%, between 60% and 100%, between 70% and 100%, between 80% and 100%, between 90% and 100%, between 1% and 90%, between 1% and 80%, between 1% and 70%, between 1% and 60%, between 1% and 50%, between 1% and 40%, between 1% and 30%, between 1% and 20%, or between 1% and 10%) of that caused by IL2. In some embodiments, by changing the linker length of the IL2R binding protein, the 最大 of the IL2R binding protein can be changed. The IL2R binding protein can cause an + in the cell type most in need (e.g., CD8 最大 T cells) and reduce the 最大 in other cell types (e.g., macrophages). In some embodiments, the 最大 in macrophages caused by the IL2R binding protein described herein is 1% to 100% (e.g., between 10% and 100%, between 20% and 100%, between 30% and 100%, between 40% and 100%, between 50% and 100%, between 60% and 100%, between 70% and 100%, between 80% and 100%, between 90% and 100%, between 1% and 90%, between 1% and 80%, between 1% and 70%, between 1% and 60%, between 1% and 50%, between 1% and 40%, between 1% and 30%, between 1% and 20%, or between 1% and 10%) of the + in T cells (e.g., CD8 最大 T cells) caused by the IL2R binding protein. In other embodiments, the 最大Greater than (e.g., at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% or 50% greater) than the natural ligand IL2 for E 最大 .

[0152] The IL2R-binding proteins described herein can be used to treat neoplastic diseases, such as cancer (e.g., solid tumor cancers, such as non-small cell lung cancer (NSCLC), renal cell carcinoma (RCC), melanoma, kidney cancer or lung cancer) in a subject in need thereof. The IL2R-binding protein binds to and activates CD8 + T cells, CD4 + T cells, macrophages and / or Treg cells. The IL2R-binding protein can elicit different levels of downstream signal transduction in different cell types. For example, by altering the length of the linker between the anti-CD122 V H H antibody and the anti-CD132 V H H antibody in the IL2R-binding protein, the IL2R-binding protein can cause a higher level of downstream signal transduction in a desired cell type compared to an undesired cell type. In some embodiments, the IL2R-binding protein can be a partial agonist that selectively activates T cells (e.g., CD8 + T cells) rather than macrophages. In some embodiments, as a partial agonist the IL2R-binding protein can inhibit autoimmune inflammatory diseases (such as lupus, type 2 diabetes, ulcerative colitis and Crohn's disease). In some embodiments, by altering the linker length, the IL2R-binding protein can cause a higher level of downstream signal transduction in T cells (e.g., CD8 + T cells) compared to the level of downstream signal transduction in other cell types. In other embodiments, different anti-CD122 V H H antibodies with different binding affinities and different anti-CD132 V H H antibodies with different binding affinities can be combined to prepare different IL2R-binding proteins. In addition, the orientation of the two antibodies in the binding protein can also be altered to prepare different binding proteins (i.e., anti-CD122 V H H antibody-linker-anti-CD132 V H H antibody, or anti-CD132 V H H antibody-linker-anti-CD122V H H antibody). Different IL2R-binding proteins can be screened to find an ideal binding protein that causes a higher level of downstream signal transduction in a desired cell type compared to an undesired cell type. In some embodiments, T cells (e.g., CD8 +The downstream signal transduction level in T cells is at least 1.1, 1.5, 2, 3, 5 or 10 times that of other cell types.

[0153] IL22 receptor binding protein

[0154] The IL22 receptor (IL22R) comprises an IL22R1 subunit (IL22R1) and an IL10Rβ subunit (IL10Rβ). Although IL10Rβ is expressed on a variety of cells, particularly immune cells including monocytes, T cells, B cells and NK cells, in contrast, expression of the IL22R1 subunit of the IL22 receptor complex is mainly observed in non-immune tissues, including skin, small intestine, liver, colon, lung, kidney and pancreas, see, e.g., Wolk, et al. (2004) Immunity 21(2):241-254. Provided herein are IL22R binding proteins that specifically bind to IL22R1 and IL10Rβ. In some embodiments, the IL22R binding protein binds to mammalian cells expressing both IL22R1 and IL10Rβ. In some embodiments, the IL22R binding protein can be a bispecific V H H 2 . In other embodiments, the IL22R binding protein can comprise a first domain (which is a V H H) and a second domain (which can be a fragment of IL22 or, e.g., an scFv).

[0155] The IL22R binding protein can be a bispecific V H H having a first V H H that binds to IL22R1 (anti-IL22R1 V H H antibody) and a second V H H that binds to IL10Rβ (anti-IL10Rβ V H H) 2 , and when bound to cells expressing IL22R1 and IL10Rβ (such as epithelial cells), results in dimerization of the two receptor subunits and downstream signal transduction. IL22R is expressed on tissue cells and is absent on immune cells. IL22R1 is expressed almost exclusively on cells of non-hematopoietic origin, such as epithelial cells, renal tubular cells and pancreatic duct cells.

[0156] Linkers can be used to conjugate the anti-IL22R1 V H H antibody and the anti-IL10Rβ V HH antibody. For example, the linker can simply be a covalent bond or a peptide linker. The peptide linker can comprise from 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). Conjugate anti-IL22R1 V H H antibody and anti-IL10Rβ V H The peptide linker of the H antibody can be a flexible glycine-serine linker. The linker can also be a chemical linker, such as a synthetic polymer, such as a polyethylene glycol (PEG) polymer.

[0157] anti-IL10Rβ V H The H antibody can have a sequence with at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity relative to any one of the sequences of SEQ ID NO: 51-57.

[0158] anti-IL10Rβ V H The H antibody can have a sequence with at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity relative to any one of the sequences of SEQ ID NO: 99-104.

[0159] In some embodiments, compared to E caused by IL22 最大 the IL22R binding protein has a reduced E 最大 . E 最大 reflects the maximum response level in cell types obtainable by a ligand (e.g., a binding protein or a native cytokine (e.g., IL22) as described herein). In some embodiments, the IL22R binding protein described herein has an E caused by IL22 最大 of at least 1% (e.g., between 1% and 100%, between 10% and 100%, between 20% and 100%, between 30% and 100%, between 40% and 100%, between 50% and 100%, between 60% and 100%, between 70% and 100%, between 80% and 100%, between 90% and 100%, between 1% and 90%, between 1% and 80%, between 1% and 70%, between 1% and 60%, between 1% and 50%, between 1% and 40%, between 1% and 30%, between 1% and 20%, or between 1% and 10%). In some embodiments, by changing the linker length of the IL22R binding protein, the E of the IL22R binding protein can be changed 最大 .

[0160] The IL22R binding protein can elicit E in the cell types that most need it (such as epithelial cells, tumor cells expressing IL22R1). 最大 and reduce E in other cell types 最大 . In some embodiments, the E in macrophages elicited by the IL22R binding protein described herein 最大 is 1% to 100% of the E in epithelial cells elicited by the IL22R binding protein (for example, between 10% and 100%, between 20% and 100%, between 30% and 100%, between 40% and 100%, between 50% and 100%, between 60% and 100%, between 70% and 100%, between 80% and 100%, between 90% and 100%, between 1% and 90%, between 1% and 80%, between 1% and 70%, between 1% and 60%, between 1% and 50%, between 1% and 40%, between 1% and 30%, between 1% and 20%, or between 1% and 10%). In other embodiments, the E of the IL22R binding protein described herein 最大 最大 is greater (for example, at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% greater) than the E of the native ligand IL22 最大 .

[0161] The biological activity of IL22 is regulated by a specific endogenous antagonist, the IL22 binding protein (IL22BP), which is thought to be a soluble neutralizing decoy receptor for IL22. Since wild-type IL22 has a higher affinity for IL22BP than the IL22 receptor complex, it is thought that IL22BP controls the biological activity of IL22 in vivo. In one embodiment, compared to IL22BP, the IL22R binding protein of the present invention can provide preferential binding to the IL22 receptor complex, thereby avoiding endogenous antagonism and modulating the IL22 activity stemming from the presence of endogenous IL22BP. In some embodiments, the IL22R binding protein described herein exhibits 1% to 100% of the affinity of the native ligand IL22 for IL22BP (for example, between 10% and 100%, between 20% and 100%, between 30% and 100%, between 40% and 100%, between 50% and 100%, between 60% and 100%, between 70% and 100%, between 80% and 100%, between 90% and 100%, between 1% and 90%, between 1% and 80%, between 1% and 70%, between 1% and 60%, between 1% and 50%, between 1% and 40%, between 1% and 30%, between 1% and 20%, or between 1% and 10%).

[0162] ​The IL22R-binding proteins described herein can be used to treat neoplastic diseases, such as cancer (e.g., solid tumor cancers, such as non-small cell lung cancer (NSCLC), renal cell carcinoma (RCC), melanoma, renal cancer or lung cancer)) in a subject in need thereof. The IL22R-binding protein binds to and activates epithelial cells. The IL22R-binding protein can initiate different levels of downstream signal transduction in target cells. For example, by altering the length of the linker between the anti-IL22R1 V H H antibody and the anti-IL10RβV H H antibody, the IL22R-binding protein can cause different (e.g., higher or lower) levels of downstream signal transduction in desired cell types compared to undesired cell types. In some embodiments, the IL22R-binding protein can be a partial agonist that selectively activates epithelial cells. In some embodiments, the IL22R-binding protein as a partial agonist can be used to treat or prevent diseases such as psoriasis, graft-versus-host disease, inflammatory diseases of the lung and airway, such as pulmonary fibrosis, ventilator-induced lung injury, neoplastic diseases (e.g., tumors expressing IL22R1), liver fibrosis, diseases associated with liver injury, such as alcoholic (acute or chronic) steatosis, pancreatitis, lupus, type 2 diabetes, ulcerative colitis, and Crohn's disease. In some embodiments, by altering the linker length, the IL22R-binding protein can cause a higher level of downstream signal transduction in epithelial cells compared to the level of downstream signal transduction in other cell types. In other embodiments, different anti-IL22R1 V H H antibodies with different binding affinities and different anti-IL10RβV H H antibodies with different binding affinities can be combined to prepare different IL22R-binding proteins. In addition, the orientation of the two antibodies in the binding protein can also be altered to prepare different binding proteins (i.e., anti-IL22R1 V H H antibody-linker-anti-IL10RβV H H antibody, or anti-IL10RβV H H antibody-linker-anti-IL22R1 V H H antibody). Different IL22R-binding proteins can be screened to find the ideal binding protein that causes a higher level of downstream signal transduction in the desired cell type compared to undesired cell types. In some embodiments, the level of downstream signal transduction in the target cell is at least 1.1, 1.5, 2, 3, 5, or 10 times the level of downstream signal transduction in other cell types or cells from different tissues.

[0163] Receptor-binding proteins that bind to non-native receptor pairs

[0164] Receptor-binding proteins that bind IL10Rα and IL2Rγ

[0165] The present disclosure provides binding proteins that specifically bind to IL10Rα and IL2Rγ. In some embodiments, the binding protein binds to mammalian cells that express both IL10Rα and IL2Rγ. In some embodiments, the binding protein is a bispecific VH that specifically binds to the extracellular domain of IL10Rα H (anti-IL10Rα VH H antibody) and a second VH that specifically binds to the extracellular domain of IL2Rγ H (anti-IL2Rγ VH H antibody), and when bound to cells that express both IL10Rα and IL2Rγ, such as T cells (e.g., CD8 H T cells and / or CD4 2 T cells), causes dimerization of the two receptor subunits and downstream signal transduction. In some embodiments, the binding protein that specifically binds to IL10Rα and IL2Rγ can be a bispecific VH + as described below. In other embodiments, the binding protein can comprise a first domain (which is a VH + ) and a second domain (which can be a fragment of IL10Rα or IL2Rγ or, for example, an scFv). H 2 H

[0166] A linker can be used to join the anti-IL10Rα VH H antibody and the anti-IL2Rγ VH H antibody. For example, the linker can simply be a covalent bond or a peptide linker. The peptide linker can comprise from 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 peptide linker that joins the anti-IL10Rα VH H antibody and the anti-IL2Rγ VH H antibody can be a flexible glycine-serine linker. The linker can also be a chemical linker, such as a synthetic polymer, such as a polyethylene glycol (PEG) polymer.

[0167] anti-IL10Rα VH H ​​​The H antibody can have a sequence with at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity relative to any one of the sequences of SEQ ID NO: 44 - 50.

[0168] Anti - IL2RγV H The H antibody can have a sequence with at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity relative to any one of the sequences of SEQ ID NO: 38 - 43.

[0169] Anti - IL2RγV H The H antibody can have a sequence with at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity relative to any one of the sequences of SEQ ID NO: 92 - 98.

[0170] In some embodiments, compared with E of IL10 最大 the binding protein that specifically binds to IL10Rα and IL2Rγ has a reduced E 最大 . E 最大 reflects the maximum response level in cell types that can be obtained by a ligand (e.g., the binding proteins described herein or native cytokines (e.g., IL10)). In some embodiments, the binding protein that specifically binds to IL10Rα and IL2Rγ described herein has 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%) of E caused by IL10. In some embodiments, by changing the linker length of the binding protein that specifically binds to IL10Rα and IL2Rγ, the E of the binding protein can be changed 最大 . The binding protein can generate E 最大 in the cell type CD8 + T cells where it is most needed. In some embodiments, the E 最大 in CD8 + T cells caused by the binding protein that specifically binds to IL10Rα and IL2Rγ is 最大 for the E 最大 in other T cells caused by the binding proteinfrom 1% to 100% (e.g., between 10% and 100%, between 20% and 100%, between 30% and 100%, between 40% and 100%, between 50% and 100%, between 60% and 100%, between 70% and 100%, between 80% and 100%, between 90% and 100%, between 1% and 90%, between 1% and 80%, between 1% and 70%, between 1% and 60%, between 1% and 50%, between 1% and 40%, between 1% and 30%, between 1% and 20%, or between 1% and 10%). In other embodiments, E of a binding protein that specifically binds to IL10Rα and IL2Rγ 最大 greater than (e.g., at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% greater) the E of the native ligand 最大 .

[0171] The binding proteins that bind to IL10Rα and IL2Rγ described herein can be used to treat diseases in a subject in need thereof, including but not limited to 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). The binding protein binds to and activates CD8 + T cells and / or CD4 + T cells. In certain embodiments, the method does not result in anemia. It is known that IL10 is active on macrophages and T cells. In some embodiments, the methods provided herein using the binding proteins of the invention that bind to IL10Rα and IL2Rγ result in selective activation of T cells relative to macrophage activation. Selective activation of T cells relative to macrophages is beneficial because IL10-activated macrophages can phagocytose senescent red blood cells, which presents as anemia in patients receiving IL10. The binding proteins described herein provide selective massive activation of T cells while providing minimal activation of macrophages, thereby producing a molecule with lower side effects (such as anemia) relative to the native IL10 ligand. Other problems and toxicities associated with IL10 activation are described in, for example, Fioranelli and Grazia, J Integr Cardiol 1(1):2-6, 2014. These problems can be avoided by using the binding proteins of the invention that specifically bind to IL10Rα and IL2Rγ.

[0172] In some embodiments, the binding proteins that bind IL10Rα and IL2Rγ can initiate different levels of downstream signaling in different cell types. For example, by varying the length of the linker between the anti-IL10RαV H H antibody and the anti-IL2RγV H H antibody in the binding protein, relative to other T cells, CD8+ Downstream signaling of the binding protein in T cells. In other embodiments, different anti-IL10Rα V H H antibodies with different binding affinities and different anti-IL2Rγ V H H antibodies can be combined to prepare different binding proteins. Additionally, the orientation of the two antibodies in the binding protein can be altered to prepare different binding proteins (i.e., anti-IL10Rα V H H antibody-linker-anti-IL2Rγ V H H antibody, or anti-IL2Rγ V H H antibody-linker-anti-IL10Rα V H H antibody). Different binding proteins can be screened to find the desired binding protein that elicits a higher level of downstream signaling in the desired cell type compared to undesired cell types. In some embodiments, the level of downstream signaling in CD8 + T cells is at least 1.1, 1.5, 2, 3, 5, or 10 times the level of downstream signaling in other T cells.

[0173] A receptor-binding protein that binds IFNγR1 or IL28Rα and myeloid cells and / or T cells

[0174] Also provided herein is a binding protein that specifically binds a first receptor and a second receptor, wherein the first receptor is interferon gamma receptor 1 (IFNγR1) or IL28Rα, and the second receptor is preferentially expressed on myeloid cells and / or T cells. In some embodiments, the binding protein binds to mammalian cells that express both the first receptor and the second receptor. For example, if the binding protein binds to IFNγR1 as the first receptor and IL2Rγ as the second receptor expressed on T cells, the binding protein can selectively initiate downstream signaling in T cells. In some embodiments, the binding protein can be a bispecific V H H 2 . In other embodiments, the binding protein can comprise a first domain (which is V H H) and a second domain (which can be a fragment of IFNγR1 or IL28Rα or, for example, an scFv).

[0175] In one embodiment, the binding protein is a bispecific V H H that specifically binds a first V H H to the first receptor (e.g., an anti-IFNγR1 V H H antibody or an anti-IL28Rα V H H antibody) and a second V H H that specifically binds to the second receptor 2and when bound to cells expressing IFNγR1 or IL28Rα and cells expressing a second receptor (e.g., myeloid cells and / or T cells), results in dimerization of the two receptors and downstream signal transduction.

[0176] The linker can be used to join two V H H. For example, the linker can simply be a covalent bond or a peptide linker. The peptide linker can comprise from 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 peptide linker joining two V H H can be a flexible glycine-serine linker. The linker can also be a chemical linker, such as a synthetic polymer, such as a polyethylene glycol (PEG) polymer.

[0177] Anti-IL28Rα V H H antibodies can have a sequence with at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to any one of the sequences of SEQ ID NO: 76 - 82.

[0178] In certain embodiments of the binding proteins described herein, the binding protein binds to the first receptor IFNγR1 and the second receptor IL2Rγ. In certain embodiments, the binding protein can activate T cells and avoid activating macrophages. In other embodiments, different antibodies with different binding affinities for the first receptor and different antibodies with different binding affinities for the second receptor can be combined to prepare different binding proteins. Additionally, the orientation of the two antibodies in the binding protein can also be altered to prepare different binding proteins (i.e., V H H antibody against the first receptor - linker - V H H antibody against the second receptor, or V H H antibody against the second receptor - linker - V H H antibody against the first receptor). Different binding proteins can be screened to find the desired binding protein that causes a higher level of downstream signal transduction in the desired cell type compared to an undesired cell type. In some embodiments, the level of downstream signal transduction in T cells is at least 1.1, 1.5, 2, 3, 5 or 10 times the level of downstream signal transduction in macrophages.

[0179] In certain embodiments of the binding proteins described herein, the binding protein binds to the first receptor IL28Rα and the second receptor IL2Rγ.

[0180] The binding proteins described herein can be used to treat neoplastic diseases, such as cancer (e.g., solid tumor cancers; e.g., non-small cell lung cancer (NSCLC), renal cell carcinoma (RCC), or melanoma)) in a subject in need thereof. In some embodiments, the binding protein binds to and activates myeloid cells and / or T cells. In certain embodiments, the binding protein binds to and activates macrophages. In certain embodiments, the binding protein binds to and activates CD8 + T cells and / or CD4 + T cells.

[0181] IV. Single-domain antibodies and V H H

[0182] A single-domain antibody (sdAb) is an antibody that contains a single monomeric variable antibody domain. Like a full-length antibody, it is capable of selectively binding to a specific antigen. The complementarity-determining regions (CDRs) of an sdAb are located within the single-domain polypeptide. Single-domain antibodies can be engineered from heavy-chain antibodies found in camelids, which are referred to as V H H. Cartilaginous fish also have heavy-chain antibodies (IgNAR, "immunoglobulin new antigen receptor"), from which single-domain antibodies referred to as V NAR can be obtained. The dimeric variable domains of human or murine consensus immunoglobulin G (IgG) can also be split into monomers to prepare sdAbs. Although most current research on sdAbs is based on heavy-chain variable domains, sdAbs derived from light chains have also been shown to specifically bind to targets, see, e.g., Moller et al., J Biol Chem. 285(49):38348–38361, 2010. In some embodiments, the sdAb consists of a single monomeric light-chain variable antibody domain.

[0183] An sdAb can be a heavy-chain antibody (V H H). V H H is an sdAb having a single monomeric heavy-chain variable antibody domain. Similar to conventional antibodies, V H H is capable of selectively binding to a specific antigen. The binding proteins described herein can comprise two V H H (e.g., V H H 2 ) joined by a linker (peptide linker). The binding protein can be a bispecific V H H 2 , which comprises a first V H H that binds to a first receptor or a domain or a subunit thereof and a second V H H that binds to a second receptor or a domain or a subunit thereof, wherein the two V H H are joined by a linker.

[0184] Exemplary VH H has a molecular weight of about 12 - 15 kDa, which is much smaller than that of traditional mammalian antibodies (150 - 160 kDa) composed of two heavy chains and two light chains. V H H can be found or produced in camelid mammals such as camels, llamas, dromedaries, alpacas, and vicuñas, which are naturally lacking in light chains. sdAb and V H Descriptions of H can be found in, for example, 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" ( Antibodies) (Basel) 8(1), 2018.

[0185] To prepare a bispecific V H H 2 binding protein, in some embodiments, two V H H can be synthesized separately and then joined together by a linker. Alternatively, the bispecific V H H 2 can be synthesized as a fusion protein. V H H with different binding activities and receptor targets can be paired to prepare a bispecific V H H 2 . The binding protein can be screened for signal transduction on cells carrying one or two related receptors.

[0186] V. Linker

[0187] As described above, the binding domains of the binding proteins of the present invention can be joined continuously (e.g., the C - terminal amino acid of the first V H H in the binding protein is joined to the N - terminal amino acid of the second V H H in the binding protein), or the binding domains of the binding protein can optionally be joined by a linker. A linker is a connection between two elements, such as a protein domain. In the bispecific V H H 2 binding proteins described herein, the linker is the connection between the two V H H in the binding protein. The linker can be a covalent bond or a peptide linker. In some embodiments, the two V H H in the binding protein are joined directly (i.e., by a covalent bond). The length of the linker between the two V H H in the binding protein can be used to adjust the two V HProximity. By varying the length of the linker, the overall size and length of the binding protein can be adjusted to bind to a specific cell receptor or its domain or its subunit. For example, if the binding protein is designed to bind to two receptors or their domains or subunits that are close to each other on the same cell, a short linker can be used. In another example, if the binding protein is designed to bind to two receptors or their domains or subunits located on two different cells, a long linker can be used.

[0188] In some embodiments, the linker is a peptide linker. The peptide linker can comprise from 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 can also be a chemical linker, such as a synthetic polymer, such as a polyethylene glycol (PEG) polymer.

[0189] In some embodiments, the linker joins the C-terminus of the first VH in the binding protein H to the N-terminus of the second VH in the binding protein. H In other embodiments, the linker joins the C-terminus of the second VH H in the binding protein to the N-terminus of the first VH H in the binding protein.

[0190] Suitable peptide linkers are known in the art and include, for example, peptide linkers containing flexible amino acid residues such as glycine and serine. In certain embodiments, the peptide linker may comprise a motif of GS, GGS, GGGGS (SEQ ID NO:1), GGGGGGGS (SEQ ID NO:2), GGSG (SEQ ID NO:3) or SGGG (SEQ ID NO:4), such as multiple or repeating motifs. In certain embodiments, the peptide linker may comprise 2 to 12 amino acids, which include the motif of GS, such as GS, GSGS (SEQ ID NO:5), GSGSGS (SEQ ID NO:6), GSGSGSGS (SEQ ID NO:191), GSGSGSGSGS (SEQ ID NO:7) or GSGSGSGSGSGS (SEQ ID NO:8). In certain other embodiments, the peptide linker may comprise 3 to 12 amino acids, which include the motif of GGS, such as GGS, GGSGGS (SEQ ID NO:9), GGSGGSGGS (SEQ ID NO:10) and GGSGGSGGSGGS (SEQ ID NO:11). In still other embodiments, the peptide linker may comprise 4 to 20 amino acids, which include the motif of GGSG (SEQ ID NO:3), such as GGSGGGSG (SEQ ID NO:12), GGSGGGSGGGSG (SEQ ID NO:13), GGSGGGSGGGSGGGSG (SEQ ID NO:14) or GGSGGGSGGGSGGGSGGGSG (SEQ ID NO:15). In other embodiments, the peptide linker may comprise the motif of GGGGS (SEQ ID NO:1), such as GGGGSGGGGS (SEQ ID NO:16) or GGGGSGGGGSGGGGS (SEQ ID NO:17).

[0191] VI. Modifications for Extended In Vivo Duration

[0192] The binding proteins described herein can be modified to provide an extended in vivo lifespan and / or an extended duration of action in a subject. In some embodiments, the binding protein can be conjugated to a carrier molecule to provide desired pharmacological properties, such as an extended half-life. In some embodiments, the binding protein can be covalently linked to the Fc domain of IgG, albumin or other molecules to extend its half-life, for example, by polyethylene glycolylation, glycosylation, etc. known in the art.

[0193] In some embodiments, the binding protein is conjugated to the functional domain of an Fc-fusion chimeric polypeptide molecule. Fc-fusion conjugates have been shown to increase the systemic half-life of biopharmaceuticals, such that biopharmaceutical products can be administered less frequently. The Fc binds to the neonatal Fc receptor (FcRn) that lines the endothelial cells of blood vessels, and upon binding, the Fc-fusion molecule is protected from degradation and is re-released into the circulation, allowing the molecule to remain in the circulation for a longer period of time. This Fc binding is thought to be the mechanism by which endogenous IgG maintains its long plasma half-life. Recent Fc-fusion technologies have linked a single copy of a biopharmaceutical to the Fc region of an antibody, optimizing the pharmacokinetic and pharmacodynamic properties of the biopharmaceutical compared to traditional Fc-fusion conjugates. The "Fc region" used to prepare the Fc-fusion can be a naturally occurring or synthetic polypeptide that is homologous to the IgG C-terminal domain produced by papain digestion of IgG. The molecular weight of IgG Fc is 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 circulatory half-life of the chimeric polypeptide as a part. Additionally, the full-length or fragment of the 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, which can be the same or different.

[0194] In some embodiments, when the binding proteins described herein are to be administered in the form of Fc fusions, particularly where the polypeptide chains conjugated to the respective subunits of the Fc dimer are different, the Fc fusions can be engineered to have a "knob-into-hole modification". The knob-into-hole modification is 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. The knob-into-hole modification refers to a modification at the interface between two immunoglobulin heavy chains in the CH3 domain, wherein: i) in the CH3 domain of the first heavy chain, an amino acid residue is replaced with an amino acid residue having a larger side chain (e.g., tyrosine or tryptophan), creating a protrusion ("knob") from the surface and ii) in the CH3 domain of the second heavy chain, an amino acid residue is replaced with an amino acid residue having a smaller side chain (such as alanine or threonine), creating a cavity ("hole") within the interface of the second CH3 domain, wherein the protruding side chain ("knob") of the first CH3 domain is received within the cavity in the second CH3 domain. In one embodiment, the "knob-into-hole modification" includes the amino acid substitutions T366W and optionally S354C in one 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 can be modified by introducing cysteine residues at positions S354 and Y349, thereby creating a stable disulfide bond between the two antibody heavy chains in the Fc region (Carter, et al. (2001) Immunol Methods 248, 7-15). The knob-into-hole format is used to facilitate the expression of the first polypeptide on the first Fc monomer having the "knob" modification and the second polypeptide on the second Fc monomer having the "hole" modification to promote the expression of the heterodimeric polypeptide conjugate.

[0195] In some embodiments, the binding protein can be conjugated to one or more water-soluble polymers. Examples of water-soluble polymers useful in the practice of the present invention include polyethylene glycol (PEG), polypropylene glycol (PPG), polysaccharides (copolymers of polyvinylpyrrolidone, ethylene glycol, and propylene glycol, poly(oxyethylated polyols), polyolefinic alcohols), polysaccharides), poly-α-hydroxy acids, polyvinyl alcohol (PVA), polyphosphazenes, polyoxazolines (POZ), poly(N-acrylmorpholine), or combinations thereof.

[0196] In some embodiments, the binding protein can be conjugated to one or more polyethylene glycol molecules or "PEGylated". Although the method or site of PEG attachment to the binding protein may vary, in certain embodiments PEGylation does not alter or only minimally alters the activity of the binding protein.

[0197] In some embodiments, selective PEGylation of the binding protein can be employed, for example, by incorporating unnatural amino acids with side chains to facilitate selective PEG conjugation. Specific PEGylation sites can be selected such that PEGylation of the binding protein does not affect its binding to the target receptor.

[0198] In certain embodiments, the increase in half-life is greater than any decrease in biological activity. PEGs suitable for conjugation to polypeptide sequences are generally water-soluble at room temperature and have the general formula R(O-CH 2 -CH 2 ) n O-R, where R is hydrogen or a protecting group, such as an alkyl or alkanol group, and n is an integer from 1 to 1000. When R is a protecting group, it generally has 1 to 8 carbons. The PEG conjugated to the polypeptide sequence can be linear or branched. The present invention contemplates branched PEG derivatives, "star-PEG", and multi-arm PEGs.

[0199] The molecular weight of the PEG used in the present invention is not limited to any particular range. The PEG moiety of the binding protein can have a molecular weight greater than about 5 kDa, greater than about 10 kDa, greater than about 15 kDa, greater than about 20 kDa, greater than about 30 kDa, greater than about 40 kDa, or greater than about 50 kDa. In some embodiments, the molecular weight is from 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 molecular weight of linear or branched PEG molecules ranges from about 2,000 to about 80,000 daltons, or about 2,000 to about 70,000 daltons, or about 5,000 to about 50,000 daltons, or about 10,000 to about 50,000 daltons, or about 20,000 to about 50,000 daltons, or about 30,000 to about 50,000 daltons, or about 20,000 to about 40,000 daltons, or about 30,000 to about 40,000 daltons. In one embodiment of the present disclosure, the PEG is a 40 kD branched PEG comprising two 20 kD arms.

[0200] The present invention also contemplates compositions of conjugates, where the PEG has different n values, and thus there are multiple different PEGs present at specific ratios. For example, some compositions contain mixtures of conjugates where n = 1, 2, 3, and 4. In some compositions, the percentage of the conjugate with n = 1 is 18 - 25%, the percentage of the conjugate with n = 2 is 50 - 66%, the percentage of the conjugate with n = 3 is 12 - 16%, and the percentage of the conjugate with n = 4 is up to 5%. Such compositions can be produced by reaction conditions and purification methods known in the art. Chromatography can be used to resolve fractions of the conjugate, and then fractions containing conjugates with, for example, the desired number of PEGs attached can be identified and purified from unmodified protein sequences and conjugates with other numbers of PEGs attached.

[0201] PEGs suitable for conjugation to polypeptide sequences are generally water-soluble at room temperature and have the general formula R(O-CH 2 -CH 2 ) n O-R, where R is hydrogen or a protecting group, such as an alkyl or alkanol group, and n is an integer from 1 - 1000. When R is a protecting group, it generally has 1 - 8 carbons.

[0202] Two widely used first-generation activated monomethoxy PEGs (mPEGs) are succinimidyl carbonate PEG (SC-PEG; see, e.g., Zalipsky, et al. (1992) Biotehnol. Appl. Biochem 15:100 - 114) and benzotriazole carbonate PEG (BTC-PEG; see, e.g., Dolence, et al. U.S. Patent No. 5,650,234), which preferably react with lysine residues to form carbamate linkages, but are also known to react with histidine and tyrosine residues. The N-terminal single site of a polypeptide is targeted using a PEG-aldehyde linker via reductive amination.

[0203] PEGylation often occurs at the α-amino group at the N-terminus of a polypeptide, the ε-amino group on the side chain of a lysine residue, and the imidazole group on the side chain of a histidine residue. Since most recombinant polypeptides have a single α-group and several ε-groups and imidazole groups, depending on the chemical nature of the linker, many positional isomers can be produced. General PEGylation strategies known in the art can be applied herein.

[0204] PEG can be attached to the binding proteins of the present invention through a terminal reactive group ("spacer"), which mediates the bonding between the free amino or carboxyl group of one or more polypeptide sequences and the polyethylene glycol. PEGs with a spacer that can bind to a free amino group include N-hydroxysuccinimide polyethylene glycol, which can be prepared by activating the succinate ester of polyethylene glycol with N-hydroxysuccinimide.

[0205] In some embodiments, PEGylation of binding proteins is facilitated by incorporating unnatural amino acids with unique side chains to promote site-specific PEGylation. Incorporating unnatural amino acids into polypeptides to provide functional moieties for 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, International Publication No. WO 2019 / 028419A1).

[0206] The PEG coupled to the polypeptide sequence can be linear or branched. The present invention contemplates branched PEG derivatives, "star-PEG" and multi-arm PEGs. Specific embodiments of PEG useful in the practice of the present invention include 10 kDa linear PEG-aldehyde (e.g., ME-100AL, NOF America Corporation, One North Broadway, White Plains, NY 10601 USA), 10 kDa linear PEG-NHS ester (e.g., ME-100CS, ME-100AS, ME-100GS, ME-100HS, NOF), 20 kDa linear PEG-aldehyde (e.g. ME-200AL, NOF, 20 kDa linear PEG-NHS ester (e.g., ME-200CS, ME-200AS, ME-200GS, ME-200HS, NOF), 20 kDa 2-arm branched PEG-aldehyde, the 20 kDa PEG-aldehyde, which comprises two 10 kDa linear PEG molecules (e.g., GL2-200AL3, NOF), 20 kDa 2-arm branched PEG-NHS ester, the 20 kDa PEG-NHS ester, which comprises two 10 kDa linear PEG molecules (e.g., GL2-200TS, GL200GS2, NOF), 40 kDa 2-arm branched PEG-aldehyde, the 40 kDa PEG-aldehyde, which comprises two 20 kDa linear PEG molecules (e.g., GL2-400AL3), 40 kDa 2-arm branched PEG-NHS ester, the 40 kDa PEG-NHS ester, which comprises two 20 kDa linear PEG molecules (e.g., GL2-400AL3, GL2-400GS2, NOF), linear 30 kDa PEG-aldehyde (e.g., ME-300AL) and linear 30 kDa PEG-NHS ester.

[0207] In some embodiments, a linker can be used to join a binding protein and a PEG molecule. Suitable linkers include "flexible linkers" which are typically long enough to allow some movement between the modified polypeptide sequence and the attached components and molecules. Linker molecules are generally about 6 - 50 atoms in length. Linker molecules can also be, for example, arylacetylenes, ethylene glycol oligomers containing 2 - 10 monomer units, diamines, diacids, amino acids, or combinations thereof. Suitable linkers can be readily selected and can be of any suitable length, such as 1 amino acid (e.g., Gly), 2, 3, 4, 5, 6, 7, 8, 9, 10, 10 - 20, 20 - 30, 30 - 50, or more than 50 amino acids.

[0208] Examples of flexible linkers include glycine polymers (G)n, glycine-alanine polymers, alanine-serine polymers, glycine-serine polymers (e.g., (GmSo)n (SEQ ID NO:431), (GSGGS)n (SEQ ID NO:432), (GmSoGm)n (SEQ ID NO:433), (GmSoGmSoGm)n (SEQ ID NO:434), (GSGGSm)n (SEQ ID NO:435), (GSGSmG)n (SEQ ID NO:436), and (GGGSm)n (SEQ ID NO:437) and combinations thereof, where m, n, and o are each independently selected from integers of at least 1 to 20, e.g., 1 - 18, 2 - 16, 3 - 14, 4 - 12, 5 - 10, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) and other flexible linkers. Glycine and glycine-serine polymers are relatively unstructured and thus can be used as neutral tethers between components. Examples of flexible linkers include, but are not limited to, GGSG (SEQ ID NO:3), GGSGG (SEQ ID NO:18), GSGSG (SEQ ID NO:19), GSGGG (SEQ ID NO:20), GGGSG (SEQ ID NO:21), and GSSSG (SEQ ID NO:22). Other examples of flexible linkers are described in Section V.

[0209] Other examples of flexible linkers include glycine polymers (G)n or glycine-serine polymers (e.g., (GS)n (SEQ ID NO:438), (GSGGS)n (SEQ ID NO:439), (GGGS)n (SEQ ID NO:440), and (GGGGS)n (SEQ ID NO:441), where n = 1 to 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:23), GGGGS (SEQ ID NO:1), GGSG (SEQ ID NO:3), GGSGG (SEQ ID NO:18), GSGSG (SEQ ID NO:19), GSGGG (SEQ ID NO:20), GGGSG (SEQ ID NO:21), and GSSSG (SEQ ID NO:22). Polymers of these linker sequences (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 10-20, 20-30, or 30-50) can be linked together to provide a flexible linker that can be used to couple two molecules. Alternatively, the polypeptide linker can be a chemical linker, e.g., a PEG-aldehyde linker. In some embodiments, the binding protein is acetylated at the N-terminus by an enzymatic reaction with an N-terminal acetyltransferase and, e.g., acetyl-CoA. In addition to or instead of N-terminal acetylation, the binding protein can be acetylated at one or more lysine residues, e.g., by an enzymatic reaction with a lysine acetyltransferase. See, e.g., Choudhary et al. (2009) Science 325(5942):834-840.

[0210] In other embodiments, the binding protein can be modified to include other polypeptide sequences that function as antigen tags, e.g., the FLAG sequence. As described herein, the FLAG sequence can be biotinylated and recognized by highly specific anti-FLAG antibodies (see, e.g., Blanar et al. (1992) Science 256:1014 and LeClair, et al. (1992) PNAS-USA 89:8145). In some embodiments, the binding protein further includes a C-terminal c-myc epitope tag.

[0211] In some embodiments, the binding protein is expressed as a fusion protein with an albumin molecule (e.g., human serum albumin), which is known in the art to promote extended in vivo exposure.

[0212] In some embodiments, the binding proteins of the invention (including fusion proteins comprising such binding proteins) are expressed as fusion proteins having one or more transition metal chelating polypeptide sequences. Incorporation of such transition metal chelating domains facilitates purification by immobilized metal affinity chromatography (IMAC), as described in U.S. Patent No. 4,569,794, issued February 11, 1986 to Smith et al. Examples of transition metal chelating polypeptides useful in the practice of the invention are described in Smith et al. (supra) and in U.S. Patent No. 5,320,663, issued May 10, 1995 to Dobeli et al., the entire teachings of which are incorporated herein by reference. A specific transition metal chelating polypeptide useful in the practice of the invention is a peptide (SEQ ID NO: 443) comprising 3 - 6 contiguous histidine residues, such as the 6 - histidine peptide (His) 6 (SEQ ID NO: 442), which is often referred to in the art as a "His - tag".

[0213] The above - mentioned fusion proteins can be readily produced by recombinant DNA methods by techniques known in the art, by constructing a recombinant vector that contains a nucleic acid sequence comprising a nucleic acid sequence encoding a binding protein in - frame with a nucleic acid sequence encoding an N - terminal or C - terminal fusion partner of the binding protein, which sequence may optionally further include a nucleic acid sequence encoding a linker or spacer polypeptide in - frame.

[0214] VII. Pharmaceutical Compositions

[0215] The binding proteins of the invention can be administered to a subject in a pharmaceutically acceptable dosage form. The preferred formulation depends on the intended mode of administration and therapeutic application. The pharmaceutical dosage forms of the binding proteins described herein include physiologically acceptable carriers which are themselves non - toxic and non - therapeutically active. Examples of such carriers include ion - exchange agents, alumina, aluminum stearate, lecithin, serum proteins such as human serum albumin, buffering substances such as phosphates, sucrose, 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. Topical or gel - based carriers for polypeptides include polysaccharides such as sodium carboxymethylcellulose or methylcellulose, polyvinylpyrrolidone, polyacrylates, polyoxyethylene - polyoxypropylene - block copolymers, PEG, polyamino acids, amino acid copolymers and lipid aggregates (such as oily droplets or liposomes).

[0216] The pharmaceutical composition may also include pharmaceutically acceptable, non-toxic carriers, excipients, stabilizers or diluents, which are defined as carriers commonly used in formulating pharmaceutical compositions for administration to animals or humans. The diluent is selected so as not to affect the biological activity of the combination. At the dosages and concentrations employed, the acceptable carriers, excipients or stabilizers are non-toxic to the recipient and include: buffers such as phosphate, citrate and other organic acid buffers; antioxidants, including ascorbic acid and methionine; preservatives (such as octadecyl dimethyl benzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl esters of p-hydroxybenzoic acid, such as methyl or propyl p-hydroxybenzoate; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); polypeptides of low molecular weight (less than about 10 residues); 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 dextrin; chelating agents, such as EDTA; sugars, such as sucrose, mannitol, trehalose or sorbitol; counterions forming salts, such as sodium; metal complexes (e.g., Zn-protein complexes); and / or nonionic surfactants such as TWEEN TM , PLURONICS TM or polyethylene glycol (PEG).

[0217] Preparations for in vivo administration are usually sterile. Sterilization of the compositions of the present invention can be readily achieved by filtration through sterile filtration membranes.

[0218] Generally, the compositions are prepared as injectable, liquid solutions or suspensions; solid forms suitable for solution or suspension in a liquid carrier 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 effects, as described above (Langer, Science 249:1527, 1990 and Hanes, Advanced Drug Delivery Reviews 28:97-119, 1997). The agents of the present invention can be administered in the form of depot injections or implantable formulations, which can be formulated to allow for sustained or pulsed release of the active ingredient. The pharmaceutical compositions are generally formulated to be sterile, substantially isotonic and in full compliance with all Good Manufacturing Practice (GMP) regulations of the U.S. Food and Drug Administration.

[0219] Administration of the binding protein described herein can be achieved by any of a variety of methods recognized in the art, including but not limited to topical, intravascular injection (including intravenous or intra-arterial infusion), intradermal injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, intracranial injection, intratumoral injection, intranodal injection, transdermal, transmucosal, iontophoretic delivery (Senti and Kundig (2009) Current Opinions in Allergy and Clinical Immunology 9(6):537-543), intralymphatic injection, intragastric infusion, intraprostatic injection, intravesical infusion (e.g., into the bladder), respiratory inhaler (including nebulizer), intraocular injection, intra-abdominal injection, intralesional injection, intraovarian injection, intracerebral infusion or injection, intraventricular injection (ICVI), etc. In some embodiments, Administration includes administration of the binding protein itself (e.g., parenterally), as well as administration of a recombinant vector (e.g., viral or non-viral vector) to effect in situ expression of the binding protein in a subject. Alternatively, cells, such as cells isolated from a subject, can be recombinantly modified to express the binding protein of the invention.

[0220] The dosage of the pharmaceutical composition depends on the route of administration, the disease to be treated, and the physical characteristics of the subject, such as factors like age, body weight, general health condition, etc. Typically, the amount of the binding protein contained in a single dose can be an amount effective for preventing, delaying, or treating the disease without causing significant toxicity. The dosage range of the binding protein described herein that the pharmaceutical composition of the present invention can contain is as follows: 0.01 to 500 mg / kg (for example, 0.01 to 450 mg, 0.01 to 400 mg, 0.01 to 350 mg, 0.01 to 300 mg, 0.01 to 250 mg, 0.01 to 200 mg, 0.01 to 150 mg, 0.01 to 100 mg, 0.01 to 50 mg, 0.01 to 10 mg, 0.01 to 1 mg, 0.1 to 500 mg / kg, 1 to 500 mg / kg, 5 to 500 mg / kg, 10 to 500 mg / kg, 50 to 500 mg / kg, 100 to 500 mg / kg, 150 to 500 mg / kg, 200 to 500 mg / kg, 250 to 500 mg / kg, 300 to 500 mg / kg, 350 to 500 mg / kg, 400 to 500 mg / kg, or 450 to 500 mg / kg), and, in more specific embodiments, about 1 to about 100 mg / kg (for example, 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 mg / kg). In some embodiments, the dosage range of the binding protein described herein that the pharmaceutical composition of the present invention can contain is as follows: 0.01 to 20 mg / kg (for example, 0.01 to 15 mg / kg, 0.01 to 10 mg / kg, 0.01 to 8 mg / kg, 0.01 to 6 mg / kg, 0.01 to 4 mg / kg, 0.01 to 2 mg / kg, 0.01 to 1 mg / kg, 0.01 to 0.1 mg / kg, 0.01 to 0.05 mg / kg, 0.05 to 20 mg / kg, 0.1 to 20 mg / kg, 1 to 20 mg / kg, 2 to 20 mg / kg, 4 to 20 mg / kg, 6 to 20 mg / kg, 8 to 20 mg / kg, 10 to 20 mg / kg, 15 to 20 mg / kg). A doctor can adjust the dosage according to conventional factors such as the degree of the disease and different parameters of the subject.

[0221] The pharmaceutical compositions containing the binding proteins described herein can be administered to a subject in need thereof once or multiple times (e.g., 1 - 10 times or more) daily, weekly, monthly, semi - annually, annually, or as medically required. The dosage can be provided in a single - dose or multi - dose regimen. The dosing interval can be decreased as the condition improves or increased as the patient's health deteriorates. The 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, it is administered in two or more divided doses over a period of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 21, 28, 30, 60, 90, 120, or 180 days. Each dose administered in such a divided - dosing regimen can be the same or different in each administration. The multi - day dosing regimen over a time period can be provided by a skilled artisan (such as a doctor), who monitors the administration, taking into account the subject's response to the treatment, including adverse reactions to the treatment and their modulation, as described above.

[0222] VIII. Indications

[0223] Neoplastic diseases

[0224] The present invention provides a method for treating a subject suffering from a neoplastic disease by administering a therapeutically effective amount of a binding protein as described herein (or a nucleic acid encoding the binding protein, including a recombinant vector encoding the binding protein).

[0225] The compositions and methods of the present invention are used to treat a subject suffering from a neoplastic disease, characterized by the presence of a tumor, including benign and malignant tumors, and neoplastic diseases. Examples of benign tumors suitable for treatment with the compositions and methods of the present invention include, but are not limited to, adenomas, fibromas, hemangiomas, and lipomas. Examples of pre - malignant tumors suitable for treatment with the compositions and methods of the present invention include, but are not limited to, hyperplasia, atypia, metaplasia, and dysplasia. Examples of malignant tumors suitable for treatment with the compositions and methods of the present invention include, but are not limited to, carcinomas (cancers originating from epithelial tissues, such as the skin or the tissues lining visceral organs), leukemias, lymphomas, and sarcomas (usually originating from bone, fat, muscle, blood vessels, or connective tissues). The term tumor also includes virus - induced tumors, such as warts and Epstein - Barr virus - induced diseases (i.e., infectious mononucleosis), scar formation, proliferative vascular diseases, including intimal smooth muscle cell hyperplasia, restenosis, and vascular occlusion, etc.

[0226] The term "neoplastic disease" includes cancers characterized by solid and non-solid tumors, including but not limited to breast cancer; sarcomas (including but not limited to osteosarcoma, angiosarcoma, and fibrosarcoma), leukemia, lymphoma, genitourinary cancers (including but not limited to ovarian cancer, urethral cancer, bladder cancer, and prostate cancer); gastrointestinal cancers (including but not limited to colorectal cancer, esophageal cancer, and gastric cancer); lung cancer; myeloma; pancreatic cancer; liver cancer; kidney cancer; endocrine cancer; skin cancer; and brain or central and peripheral nerve (CNS) system tumors, malignant or benign, including gliomas, neuroblastomas, astrocytomas, myelodysplastic syndromes; carcinoma in situ of the cervix; intestinal polyps; oral leukoplakia; histiocytose, hypertrophic scars, including keloids, hemangiomas; proliferative arterial stenosis, psoriasis, inflammatory arthritis; hyperkeratosis and papulosquamous eruptions, including arthropathy.

[0227] The term neoplastic disease includes carcinoma. The term "carcinoma" refers to a malignant tumor of epithelial or endocrine tissue, including carcinomas of the respiratory system, gastrointestinal system, genitourinary system, testicular cancer, breast cancer, prostate cancer, endocrine system cancer, and melanoma. The term neoplastic disease includes adenocarcinoma. "Adenocarcinoma" refers to a cancer that originates from glandular tissue or in which the tumor cells form recognizable glandular structures.

[0228] The term "hematopoietic neoplastic disorder" refers to a neoplastic disorder involving proliferating / neoplastic cells of hematopoietic origin, e.g., originating from the myeloid, lymphoid, or erythroid lineages, or their precursor cells. 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 precursor neoplasms, and acute leukemia of ambiguous lineage. Exemplary myeloid disorders suitable for treatment according to the present disclosure include, but are not limited to, acute promyelocytic leukemia (APML), acute myelogenous leukemia (AML), and chronic myelogenous leukemia (CML). Lymphoid neoplasms include, but are not limited to, precursor lymphoid neoplasms, mature B-cell neoplasms, mature T-cell neoplasms, Hodgkin lymphoma, and immunodeficiency-related lymphoproliferative disorders. Exemplary lymphoid diseases suitable for treatment according to the present disclosure include, but are not limited to, acute lymphoblastic leukemia (ALL), which includes B-lineage ALL and T-lineage ALL, chronic lymphocytic leukemia (CLL), prolymphocytic leukemia (PLL), hairy cell leukemia (HLL), and Waldenström macroglobulinemia (WM).

[0229] In some cases, hematopoietic neoplastic disorders arise from poorly differentiated acute leukemias such as erythroleukemia and acute megakaryocytic leukemia. As used herein, the term "hematopoietic neoplastic disorder" refers to malignant lymphomas, including but not limited to non-Hodgkin lymphoma and its variants, peripheral T-cell lymphoma, adult T-cell leukemia / lymphoma (ATL), cutaneous T-cell lymphoma (CTCL), large granular lymphocyte leukemia (LGL), Hodgkin disease, and Reed-Sternberg disease.

[0230] Determining whether a subject "has a neoplastic disease" refers to a determination by a physician, based on the available information in the art for identifying diseases, disorders, or conditions (including but not limited to X-rays, CT-scans, conventional laboratory diagnostic tests (e.g., blood cell counts, etc.), genomic data, protein expression data, immunohistochemistry), that the subject requires or would benefit from treatment.

[0231] The determination of the efficacy of the methods of the present invention in treating cancer generally relates to achieving one or more parameters recognized in the art, such as lesion reduction, particularly reduction of metastatic lesions, metastasis reduction, tumor volume reduction, improvement in ECOG score, and the like. Determining the response to treatment can be evaluated by measuring biomarkers that can provide reproducible information useful in any aspect of the conjugate protein treatment, including the presence and extent of the subject's response to such treatment and the presence and extent of untoward effects caused by such treatment. The response to treatment is characterized by an improvement in the conventional measures of clinical efficacy and can be, for example, complete remission (CR), partial remission (PR), stable disease (SD) with respect to target lesions, complete remission (CR), "incomplete remission / stable disease (SD), as defined by RECIST, and immune-related complete remission (irCR), immune-related partial remission (irPR), and immune-related stable disease (irSD), as defined by immune-related response criteria (irRC), which are considered by those skilled in the art as evidence of the efficacy of treating neoplastic diseases in mammalian (such as human) subjects.

[0232] Infectious diseases

[0233] The present invention provides methods of treating a subject having an infectious disease by administering a therapeutically effective amount of a conjugate protein as described herein (or a nucleic acid encoding a conjugate protein, including a recombinant vector encoding a conjugate protein).

[0234] In some embodiments, the infection is a chronic infection, i.e., an infection that has not been cleared by the host immune system for a period of up to 1 week, 2 weeks, etc.). In some cases, chronic infections involve the integration of pathogen genetic elements into the host genome, for example, retroviruses, lentiviruses, hepatitis B virus, etc. In other cases, chronic infections, such as certain intracellular bacteria or protozoan pathogens, are caused by pathogen cells within host cells. Additionally, in some embodiments, the infection is in a latent phase, such as herpesviruses or human papillomaviruses.

[0235] Viral pathogens of interest include, but are not limited to, retroviruses, hepadnaviruses, lentiviruses, and other pathogens such as HIV-1; HIV-2, HTLV, FIV, SIV, etc., and hepatitis A, B, C, D, and E viruses. In some embodiments, the methods of the invention involve diagnosing a patient as having an infection; or selecting a patient previously diagnosed as having an infection; treating the patient with a variant type III interferon treatment regimen, optionally in combination with other treatments; and monitoring the treatment efficacy of the patient. Monitoring can measure clinical indicators of the infection such as fever, white blood cell count, etc., and / or directly monitor the presence of the pathogen. Treatment can be combined with other active agents. Cytokines can also be included, such as interferon γ, tumor necrosis factor α, interleukin 12, etc. Antiviral agents such as acyclovir, ganciclovir, etc. can also be used for treatment. Subjects suspected of having an infection, including HCV infection, can be screened prior to treatment. In addition, subjects undergoing treatment can be tested to determine the activity and efficacy of the treatment. A significant improvement in one or more parameters indicates efficacy. Adjusting the dosing regimen and number of doses to provide the best benefit to the patient based on various factors (e.g., patient-dependent factors such as the severity of the disease, etc., the compound administered, etc.) is well within the skill of an ordinary healthcare provider (e.g., a clinician). For example, HCV infection in an individual can be detected and / or monitored by the presence of HCV RNA in the blood and / or the presence of anti-HCV antibodies in their serum. Other clinical signs and symptoms that can be used for diagnosis and / or monitoring of treatment include assessment of liver function and assessment of liver fibrosis (e.g., which may accompany chronic viral infection).

[0236] Subjects to whom the treatments described herein can be administered include naive individuals (e.g., individuals diagnosed with an infection but who have not previously received treatment) and individuals who have previously failed treatment ("treatment failure" patients). For HCV treatment, previous treatments include, for example, monotherapy with IFN-α (e.g., IFN-α and / or pegylated IFN-α) or combination treatment with IFN-α, where combination treatment can include administration of IFN-α and an antiviral agent such as ribavirin. Treatment failure patients include non-responders (i.e., individuals in whom previous HCV treatment has not significantly or sufficiently reduced HCV titers to provide a clinically significant response, e.g., previous IFN-α monotherapy, previous combination treatment with IFN-α and ribavirin, or previous combination treatment with pegylated IFN-α and ribavirin); and relapsers (i.e., individuals who have previously received HCV treatment (e.g., individuals who have previously received IFN-α monotherapy, individuals who have previously received combination treatment with IFN-α and ribavirin, or individuals who have previously received combination treatment with pegylated IFN-α and ribavirin), in whom HCV titers have been reduced to provide a clinically significant response, but in whom the reduced HCV titers have not been maintained due to subsequent increases in HCV titers).

[0237] Other subjects of interest for treatment disclosed herein include subjects that are "difficult to treat" due to the nature of HCV infection. "Difficult to treat" subjects are those 1) with a high titer HCV infection, which is typically defined as having at least about 10 5 , at least about 5×10 5 or at least about 10 6 HCV genome copies per milliliter of serum, 2) infected with an HCV genotype recognized in the art as being associated with treatment failure (e.g., HCV genotype 1, its subtypes (e.g., 1a, 1b, etc.), and its quasispecies) or 3) both.

[0238] In other embodiments, methods for treating or reducing primary or metastatic cancer are provided, the regimens comprising contacting a subject in need of treatment with a therapeutically effective amount or dose of an IFNλ synthase or IFNλ variant polypeptide. The effective dose for treating cancer depends on many different factors, including the mode of administration, the target site, the physiological state of the patient, whether the patient is human or animal, other drugs being taken, and whether the treatment is prophylactic or therapeutic. Typically, the patient is human, but non-human mammals can also be treated, e.g., companion animals such as dogs, cats, horses, etc., and laboratory mammals such as rabbits, mice, rats, etc. The therapeutic dose can be titrated to optimize safety and efficacy.

[0239] In prophylactic applications, relatively low doses can be administered at relatively infrequent intervals over a long period of time. Some patients continue treatment for the rest of their lives. In other therapeutic applications, relatively high doses may sometimes be required at relatively short intervals until the progression of the disease slows or terminates, preferably until the patient shows partial or complete improvement of the disease symptoms. Thereafter, a prophylactic regimen can be administered to the patient.

[0240] In other embodiments, the methods of the invention include treating, reducing or preventing tumor growth, tumor metastasis or tumor invasion of cancer, said cancer including carcinoma, blood cancer, melanoma, sarcoma, glioma, especially epithelial-derived cancers expressing IFNλR1 and IFNAR1 or IFNAR2, or IL-10Rβ and IFNAR1 or IFNAR2. In some embodiments, the responsiveness of cancer to the IFNλ synthetic factor is evaluated by determining whether the cancer expresses the cognate receptor activated by the synthetic factor (e.g., determining the expression of IFNλR1 and IFNAR1 or IFNAR2). Tissues known to express IFNλR1 include, for example, lung, heart, liver (hepatocytes), prostate, keratinocytes and melanocytes. Cancers responsive to IFNλ and the IFNλ synthetic factor may include but are not limited to melanoma, fibrosarcoma, hepatocellular carcinoma, bladder cancer, Burkitt lymphoma, colorectal cancer, glioblastoma, non-small cell lung cancer, esophageal cancer and osteosarcoma, etc.

[0241] For prophylactic applications, the pharmaceutical composition or medicament is administered to a patient susceptible to or at risk of a disease, its complications and intermediate pathological phenotypes occurring during the development of the disease, in an amount sufficient to eliminate or reduce the risk, reduce the severity or delay the onset of the disease (including the biochemical, histological and / or behavioral symptoms of the disease).

[0242] Examples

[0243] Example – V h H generation:

[0244] Before immunization, the camels were acclimated in the research institution for at least 7 days. The antigen was diluted with 1xPBS (total antigen amount about 1 mg). The quality of the antigen was evaluated by SDS-PAGE to ensure purity (e.g., >80%). For the first time, 10 mL of CFA (subsequently 6 times with IFA) was added to the mortar, then 10 mL of the antigen in 1×PBS was slowly added to the mortar and ground with a pestle. The antigen and CFA / IFA were ground until the component showed a milky white color and seemed difficult to disperse. The antigen emulsified in CFA was subcutaneously injected at at least six sites on the camel, about 2 mL at each site (10 mL in total for each camel). A stronger immune response can be generated by injecting at more sites and larger volumes. Immunization was performed weekly (7 days) for 7 times. After each injection, the needle was inserted into the subcutaneous space for 10 to 15 seconds to avoid leakage of the emulsion. Alternatively, gently pulling the syringe plunger can also prevent leakage. Blood samples were collected 3 days after the 7th immunization.

[0245] After immunization, a library was constructed. Briefly, RNA was extracted from the blood and transcribed into cDNA. V was obtained by two-step PCR HRegion H, with a fragment of approximately 400 bp. The PCR products and the vector of pMECS phagemid were digested with Pst I and Not I, and then ligated to the pMECS / Nb recombinant. After ligation, the product was transformed into Escherichia coli (E. coli) TG1 cells by electroporation. Then, the transformants were enriched in the growth medium and plated. Finally, the library size was estimated by counting the number of colonies.

[0246] After library construction, library biopanning was performed to screen for candidates against the antigen. The phage display technique was applied in this process. Positive colonies were identified by PE-ELISA.

[0247] Example 2. Generation of anti-hIL10R VHH

[0248] Camelids were immunized weekly with the extracellular domains of human IL10Rα (amino acids 22 - 235, UniProtKB Q13651, hIL-10Rα ecd ) and IL10Rβ (amino acids 20 - 220, UniProtKB Q08334, hIL-10Rβ ecd ) for 7 consecutive weeks, and PBMC were harvested on day 52. A phage display library was constructed and biopanned as described in Example 1 above. Fifty VHH sequences were obtained after selection on hIL10-R1, and 47 VHH sequences were obtained after selection on hIL10-R2. The sequences were clonotyped using germline assignment and CDR3 sequence similarity.

[0249] Example 3. Synthesis of DNA encoding synthetic factors

[0250] Seven unique anti-hIL-10Rα ecd sequences (SEQ ID No: 44 - 50) and seven unique anti-hIL-10Rβ ecd sequences (SEQ ID No: 51 - 57) were selected from each group, and DNA composed of one DNA encoding an IL-10Rα VHH and one DNA encoding an IL-10Rβ VHH, separated by a linker sequence of DNA encoding GGGS (SEQ ID NO: 23), was synthesized. The DNA was used for each possible VHH combination and in two orientations for a total of 98 7x7 x 2 = 98 VHH dimers. An Ala-Ser ("AS") linker was added to the 3' end of each DNA construct, followed by His-6 (SEQ ID NO: 442) DNA (ASH6, SEQ ID NO: 430). The codon-optimized DNA sequences encoding these constructs are provided as SEQ ID No: 290 - 237, and the orientation of their components is described in Table 2 of the above specification.

[0251] Example 4. Recombinant production and purification

[0252] The codon-optimized DNA insert (SEQ ID No: 290 - 237) was cloned into modified pcDNA3.4 (Genscript) for small-scale expression in HEK293 cells in 24-well plates. The supernatant cell IL2R-binding protein was purified essentially according to the following procedure. Using a Hamilton Star automated system, 96 x 4 mL supernatants in 4 x 24-well blocks were rearranged into 4 x 96-well, 1 mL blocks. PhyNexus micropipette tips (Biotage, San Jose CA) were used to aspirate 80 μL of Ni-Excel IMAC resin (Cytiva) equilibrated with wash buffer: PBS pH 7.4, 30 mM imidazole. The PhyNexus tips were dipped onto all 4 x 96-well blocks and cycled through 14 cycles of 1 mL pipetting.

[0253] The PhyNexus tips were washed in 2 x 1 mL blocks containing wash buffer. The PhyNexus tips were eluted in 3 x 0.36 mL blocks containing elution buffer: PBS pH 7.4, 400 mM imidazole. The PhyNexus tips were regenerated in 3 x 1 mL blocks of 0.5 M sodium hydroxide.

[0254] Essentially according to the following procedure, using the T200 to quantify the purified protein eluate. 10 μL of the first 96 x 0.36 mL eluate was transferred to a 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 pre-functionalized with an anti-histidine capture antibody (Cytiva): injected at 5 μL / min for 18 s. The capture level was recorded after 60 s of buffer wash. A standard curve of known VH H concentrations (270, 90, 30, 10, 3.3, 1.1 μg / mL) was obtained in each of the 4 Biacore chip flow cells to account for variability in the cell surface. The 96 captures were interpolated against the standard curve using a non-linear model including both specific and non-specific one-site binding. The concentrations in the first elution block varied between 12 and 452 μg / mL, corresponding to 4 - 149 μg. Five randomly selected samples were analyzed by SDS-PAGE to ensure that the molecular weight of the eluate was as expected (approx. 30 kDa).

[0255] Basically, according to the following procedure, the concentration of the protein was standardized using the Hamilton Star automated system. The concentration values were entered into an Excel spreadsheet, where the pipetting volumes were calculated to dilute to 50 μg / mL in 0.22 mL. This spreadsheet was entered into the Hamilton Star method, which was dedicated to performing dilution pipetting using the first elution block and elution buffer as diluents. The final standardized plate was sterile filtered using a 0.22 μm filter plate (Corning) and the materials for the following in vitro assays.

[0256] Example 5. IL10 activity assay:

[0257] HEK-Blue TM The IL-10 reporter cell line (Invivogen, San Diego, CA) was used to screen for IL10R1 / R2 VHH. HEK-Blue TM The IL-10 cells were generated by stable transfection of the human embryonic kidney HEK293 cell line with genes encoding the hIL-10Rα and β chains, human STAT3, and the STAT3-inducible SEAP (secreted embryonic alkaline phosphatase) reporter gene. IL-10 binds to its receptor on the surface of HEK-Blue TM IL-10 cells, triggering JAK1 / STAT3 signal transduction and subsequent SEAP production. Then, the signal was detected by quantifying the SEAP activity in the cell culture supernatant using the QUANTI-Blue TM development solution (Invivogen, San Diego, CA), and the absorbance value was measured at 630 nm by spectrophotometry. Since STAT3 is also involved in the signal transduction of cytokines such as IFN-α / β and IL-6, the HEK-Blue TM IL-10 cells were knocked out for the expression of hIFNAR2 and hIL-6R.

[0258] Example 6. Screening of SEQ ID NO: 192 - 289:

[0259] For screening IL10R1 / R2 VHH, HEK-Blue TMIL-10 cells were seeded at 50,000 cells per well in 96-well plates and treated with 25 nM or 100 nM protein (in triplicate) for 24 hours. Recombinant animal-free human IL-10 (Shenandoah Biotechnology, Inc., Warwick, PA, catalog number 100-83AF) was used as a positive control, and unstimulated cells were used as a negative control. Twenty-four hours after treatment, 20 μl of cell supernatant was transferred to flat-bottom 96-well plates and the assay was developed by adding 180 μl of QUANTI-Blue TM (Invitrogen) for 2 hours. Absorbance values at 630 nm were measured on a multilabel microplate reader (PerkinElmer, Inc., Waltham, MA). The results of the screening are listed in Table 3 of the specification.

[0260] Informal Sequence Listing

[0261]

[0262]

[0263]

[0264]

[0265]

[0266]

[0267]

[0268]

[0269]

[0270]

[0271]

[0272]

[0273]

[0274]

[0275]

[0276]

[0277]

[0278]

[0279]

[0280]

[0281]

[0282]

[0283]

[0284]

[0285]

[0286]

[0287]

[0288]

[0289]

[0290]

[0291]

[0292]

[0293]

[0294]

[0295]

[0296]

[0297]

[0298]

[0299]

[0300]

[0301]

[0302]

[0303]

[0304]

[0305]

[0306]

[0307]

[0308]

[0309]

[0310]

[0311]

[0312]

[0313]

[0314]

[0315]

[0316]

[0317]

[0318]

[0319]

[0320]

[0321]

[0322]

[0323]

[0324]

[0325]

[0326]

[0327]

[0328]

[0329]

[0330]

[0331]

[0332]

[0333]

[0334]

[0335]

[0336]

[0337]

[0338]

[0339]

[0340]

[0341]

[0342]

[0343]

[0344]

[0345]

[0346]

[0347]

[0348]

[0349]

[0350]

[0351]

[0352]

[0353]

[0354]

[0355]

[0356]

[0357]

[0358]

[0359]

[0360]

[0361]

[0362]

[0363]

[0364]

[0365]

[0366]

[0367]

[0368]

[0369]

[0370]

[0371]

[0372]

[0373]

[0374]

[0375]

[0376]

[0377]

[0378]

[0379]

[0380]

[0381]

[0382]

[0383]

[0384]

[0385]

[0386]

[0387]

[0388]

[0389]

[0390]

[0391]

[0392]

[0393]

[0394]

[0395]

[0396]

[0397]

[0398]

[0399]

[0400]

[0401]

[0402]

[0403]

[0404]

[0405]

[0406]

[0407]

[0408]

[0409]

[0410]

[0411]

[0412]

[0413]

[0414]

[0415]

[0416]

[0417]

[0418]

[0419]

[0420]

[0421]

[0422]

[0423]

[0424]

[0425]

[0426]

[0427]

[0428]

[0429]

[0430]

[0431]

[0432]

[0433] It should be understood that the embodiments described herein are for illustrative purposes only, and those skilled in the art should understand the various modifications or changes made accordingly, and they are included within the spirit and scope of the present application and the appended claims. The sequences of the sequence accession numbers cited herein are incorporated herein by reference.

Claims

1. An IL10 receptor (IL10R) binding protein that specifically binds to the IL10Rα subunit (IL10Rα) and IL10Rβ, wherein said binding protein causes the multimerization and downstream signal transduction of IL10Rα and IL10Rβ, and wherein said binding protein comprises a single domain antibody (sdAb) that specifically binds to IL10Rα (anti-IL10Rα sdAb) and an sdAb that specifically binds to IL10Rβ (anti-IL10Rβ sdAb).

2. The IL10R binding protein according to claim 1, wherein said anti-IL10Rα sdAb is a VHH antibody and / or said anti-IL10Rβ sdAb is a VHH antibody.

3. The IL10R binding protein according to claim 1, wherein said anti-IL10Rα sdAb and said anti-IL10Rβ sdAb are joined by a peptide linker.

4. The IL10R binding protein according to claim 3, wherein said peptide linker comprises 1 - 50 amino acids.

5. The IL10R binding protein according to claim 1, wherein said anti-IL10Rα sdAb is selected from the group consisting of SEQ ID NO:44 - 50, and said -IL10Rβ sdAb is selected from the group consisting of SEQ ID No:51 - 57.

6. The IL10R binding protein according to claim 5, wherein said anti-IL10Rα sdAb is joined to the anti-IL10Rβ sdAb by a linker selected from SEQ ID No:1 - 23.

7. The IL10R binding protein according to claim 5, wherein said IL10R binding protein comprises a first anti-IL10R sdAb joined to a second anti-IL10R sdAb by a linker from amino to carboxyl as follows: wherein said linker is selected from the group consisting of SEQ ID No:1 - 23.

8. The IL-10 receptor binding protein according to claim 5, which is selected from the group consisting of SEQ ID No:194, 209, 210, 211, 213, 218, 226, 233, 238, 244, 250, 203, 205, 207, 269, 212, 217, 219, 224, 227, 237, 239 and 249.

9. Use of the IL10R binding protein according to claim 1 in the preparation of a pharmaceutical composition for treating an inflammatory disease in a subject in need thereof.

10. The use according to claim 9, wherein said inflammatory disease is Crohn's disease, ulcerative colitis or an autoimmune disease.

11. The use according to claim 10, wherein said inflammatory disease is an autoimmune disease selected from psoriasis, rheumatoid arthritis or multiple sclerosis.

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