Humanized IL-10 receptor-binding molecule and method of use

Abstract

This disclosure provides a humanized IL-10 agonist that binds to IL-10Rα and IL-10Rβ and comprises an IL-10Rα single-domain antibody and an IL-10Rβ single-domain antibody. TIFF2026521432000078.tif87140

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority to U.S. Provisional Patent Application No. 63 / 506,798, filed on 7 June 2023, and U.S. Provisional Patent Application No. 63 / 511,122, filed on 29 June 2023, the disclosures of these U.S. Provisional Applications being incorporated herein by reference in their entirety for all purposes. [Background technology]

[0002] Background of this disclosure Cytokines and growth factor ligands typically transmit signals through the multimerization of cell surface receptor subunits. In some cases, cytokines act as polyspecific (e.g., bispecific or trispecific) ligands that promote the association of such receptor subunits, bringing their intracellular domains into proximity and enabling intracellular signaling. Cytokines determine which receptor subunits will associate to form cytokine-receptor complexes. In this way, cytokines bridge individual receptor subunits to form receptor complexes that result in intracellular signaling.

[0003] One useful approach to cytokine engineering has been the use of single-domain antibodies (sdAbs), such as single variable-domain (sFv) antibodies and variable heavy-chain domain (VHH) antibodies. VHHs are sdAbs containing only the variable domain derived from heavy-chain antibodies derived from camelid animal species.

[0004] Therefore, in the art, there is a need to manipulate and generate single-domain molecules, such as single-domain antibodies (sdAbs) that independently bind to different receptors or receptor subunits, and dimers of sdAbs having two independent binding domains (i.e., two sdAbs with similar or different binding specificities), that have similar activity to their respective surrogate cytokines or cognitive cytokines, or that engage with cytokine receptor subunits by preferentially binding to a specific subunit (i.e., "biased" signaling), and that do not have independent off-target activity. [Overview of the project]

[0005] Summary of this disclosure This disclosure provides IL-10 agonist compounds and IL-10 agonist compositions comprising a humanized single-domain antibody (sdAb) (e.g., anti-IL-10Rα sdAb or VHH) conjugated to IL-10Rα, and a humanized single-domain antibody (sdAb) (e.g., anti-IL-10Rα sdAb or VHH) conjugated to IL-10Rβ. Such compositions are useful in the formation of cell-receptor pairs to produce desired effects that are helpful in treating diseases in mammalian subjects.

[0006] This disclosure provides an IL-10 agonist compound comprising at least a first domain (e.g., a first single-domain antibody polypeptide) that specifically binds to a first receptor subunit (e.g., IL-10Rα) and a second domain (e.g., a second single-domain antibody polypeptide) that specifically binds to a second receptor subunit (e.g., IL-10Rβ). In some embodiments, contact of the IL-10 agonist compound with cells expressing the first and second receptor subunits results in a functional association of the first and second receptor subunits, thereby triggering their interaction and resulting in downstream signaling. In some embodiments, the downstream signaling is different from the downstream signaling resulting from a native ligand binding to the native receptor subunit. In some embodiments, the first and second receptor subunits become close relatives in response to the binding of a cognitive ligand, which is referred to herein as a “native” cytokine receptor pair.

[0007] In some embodiments, the Disclosure provides an IL-10 agonist compound comprising a first domain (e.g., a first single-domain antibody polypeptide) that binds to IL-10Rα of the IL-10 receptor and a second domain (e.g., a second single-domain antibody polypeptide) that binds to IL-10Rβ of the IL-10 receptor. In some embodiments, contact of the IL-10 agonist compound with cells expressing IL-10Rα and IL-10Rβ of the IL-10 receptor results in functional association of IL-10Rα and IL-10Rβ, thereby resulting in functional dimerization of the receptor and downstream signaling.

[0008] In some embodiments, the present disclosure relates to an IL-10 agonist compound comprising a first single-domain antibody polypeptide conjugated to a second single-domain antibody polypeptide (e.g., covalently linked, covalently linked via a polypeptide linker or chemical linker, or stably associated via non-covalent linkage via an Fc construct, as described in more detail herein), wherein the first single-domain antibody polypeptide that specifically binds to the α subunit of the IL-10 receptor (IL 10Rα) comprises CDR1 containing an amino acid sequence selected from the group consisting of SEQ ID NO: 224-228, CDR2 containing an amino acid sequence selected from the group consisting of SEQ ID NO: 229-235, and CDR3 containing an amino acid sequence of SEQ ID NO: 236, and the second single-domain antibody polypeptide that specifically binds to the β subunit of the IL-10 receptor (IL 10Rβ) comprises CDR1, SEQ ID NO: 296 The present invention provides an IL-10 agonist compound comprising CDR2 containing an amino acid sequence selected from the group consisting of NO:297, and CDR3 containing an amino acid sequence selected from the group consisting of SEQ ID NO:298.

[0009] In some embodiments, the Disclosure provides an IL-10 agonist compound in which a first single-domain antibody and a second single-domain antibody are linked by a linker. In some embodiments, the Disclosure provides an IL-10 agonist compound comprising a single polypeptide chain, comprising a first single-domain antibody bound to IL-10Rα and a second single-domain antibody bound to IL-10Rβ, wherein the first single-domain antibody and the second single-domain antibody are optionally linked by a polypeptide linker, the polypeptide linker comprising 1 to 50, alternatively 1 to 30, alternatively 1 to 20, alternatively 1 to 15, alternatively 1 to 12, alternatively 1 to 10, alternatively 1 to 8, alternatively 1 to 6, alternatively 1 to 5, alternatively 1 to 4, alternatively 1 to 3, alternatively 1 to 2, and alternatively 1 amino acid.

[0010] In some embodiments, the disclosure provides an IL-10 agonist compound comprising a first single-domain antibody containing an amino acid sequence selected from the group consisting of SEQ ID NO: 25 to 48, and a second single-domain antibody containing an amino acid sequence selected from the group consisting of SEQ ID NO: 49 and SEQ ID NO: 50. In some embodiments, the disclosure provides an IL-10 agonist compound comprising a first single-domain antibody comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 25 to 48 and a second single-domain antibody comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 49 and SEQ ID NO: 50, wherein optionally, the first single-domain antibody and the second single-domain antibody are linked by a polypeptide linker comprising 1 to 50, alternatively 1 to 30, alternatively 1 to 20, alternatively 1 to 15, alternatively 1 to 12, alternatively 1 to 10, alternatively 1 to 8, alternatively 1 to 6, alternatively 1 to 5, alternatively 1 to 4, alternatively 1 to 3, alternatively 1 to 2, or alternatively 1 amino acid. In some embodiments, the disclosure provides an IL-10 agonist compound comprising a first single-domain antibody containing an amino acid sequence selected from the group consisting of SEQ ID NO: 25 to 48 and a second single-domain antibody containing an amino acid sequence selected from the group consisting of SEQ ID NO: 49 and SEQ ID NO: 50, wherein the first single-domain antibody and the second single-domain antibody are linked by a polypeptide linker selected from the group consisting of SEQ ID NO: 416 to 439.

[0011] In some embodiments, the disclosure provides an IL-10 agonist compound in which the C-terminus of a first single-domain antibody is conjugated to the N-terminus of a second single-domain antibody, and optionally, the C-terminus of the first single-domain antibody is conjugated to the N-terminus of the second single-domain antibody via a linker.

[0012] In some embodiments, the disclosure provides an IL-10 agonist compound in which the N-terminus of a first single-domain antibody is conjugated to the C-terminus of a second single-domain antibody, and optionally, the N-terminus of the first single-domain antibody is conjugated to the C-terminus of the second single-domain antibody via a linker. In some embodiments, the disclosure provides an IL-10 agonist compound comprising a first single-domain antibody that binds to the extracellular domain of IL-10Rα and a second single-domain antibody that binds to the extracellular domain of IL-10Rβ, wherein the N-terminus of the first single-domain antibody is conjugated to the C-terminus of the second single-domain antibody that binds to IL-10Rβ, and optionally, the N-terminus of the first single-domain antibody is conjugated to the C-terminus of the second single-domain antibody via a polypeptide linker containing 1 to 50, alternatively 1 to 30, alternatively 1 to 20, alternatively 1 to 15, alternatively 1 to 12, alternatively 1 to 10, alternatively 1 to 8, alternatively 1 to 6, alternatively 1 to 5, alternatively 1 to 4, alternatively 1 to 3, alternatively 1 to 2, or alternatively 1 amino acid. In some embodiments, the disclosure provides an IL-10 agonist compound comprising a first single-domain antibody that binds to the extracellular domain of IL-10Rβ and a second single-domain antibody that binds to the extracellular domain of IL-10Rα, wherein the N-terminus of the first single-domain antibody is conjugated to the C-terminus of the second single-domain antibody that binds to IL-10Rβ, and optionally, the N-terminus of the first single-domain antibody is conjugated to the C-terminus of the second single-domain antibody via a polypeptide linker containing 1 to 50, alternatively 1 to 30, alternatively 1 to 20, alternatively 1 to 15, alternatively 1 to 12, alternatively 1 to 10, alternatively 1 to 8, alternatively 1 to 6, alternatively 1 to 5, alternatively 1 to 4, alternatively 1 to 3, alternatively 1 to 2, or alternatively 1 amino acid.

[0013] In some embodiments, the disclosure provides IL-10 agonist compounds comprising polypeptides selected from the group consisting of SEQ ID NO: 1-24 and SEQ ID NO: 500-523.

[0014] In some embodiments, the disclosure provides IL-10 agonist compounds comprising polypeptides having at least 95% amino acid sequence identity to amino acid sequences selected from the group consisting of SEQ ID NO: 1-24 and SEQ ID NO: 500-523. In some embodiments, the disclosure provides IL-10 agonist compounds comprising polypeptides having at least 95%, alternatively at least 96%, alternatively at least 97%, alternatively at least 98%, alternatively at least 99%, or alternatively 100% amino acid sequence identity to amino acid sequences selected from the group consisting of SEQ ID NO: 1-24 and SEQ ID NO: 500-523. In some embodiments, the Disclosure provides IL-10 agonist compounds comprising a polypeptide containing at least 95%, alternatively at least 96%, alternatively at least 97%, alternatively at least 98%, alternatively at least 99%, or alternatively 100% amino acid sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 1-24 and SEQ ID NO: 500-523. In some embodiments, the Disclosure provides IL-10 agonist compounds comprising a polypeptide containing at least 95%, alternatively at least 96%, alternatively at least 97%, alternatively at least 98%, alternatively at least 99%, or alternatively 100% amino acid sequence identity to the amino acid (DR2463) of SEQ ID NO: 1. In some embodiments, the Disclosure provides IL-10 agonist compounds comprising a polypeptide containing at least 95%, alternatively at least 96%, alternatively at least 97%, alternatively at least 98%, alternatively at least 99%, or alternatively 100% amino acid sequence identity to the amino acid of SEQ ID NO:2 (DR2485). In some embodiments, the Disclosure provides IL-10 agonist compounds comprising a polypeptide containing at least 95%, alternatively at least 96%, alternatively at least 97%, alternatively at least 98%, alternatively at least 99%, or alternatively 100% amino acid sequence identity to the amino acid of SEQ ID NO:3 (DR2519).In some embodiments, the Disclosure provides IL-10 agonist compounds comprising a polypeptide having at least 95%, alternatively at least 96%, alternatively at least 97%, alternatively at least 98%, alternatively at least 99%, or alternatively 100% amino acid sequence identity with respect to the amino acid of SEQ ID NO:4 (DR2520).

[0015] In some embodiments, the Disclosure provides IL-10 agonist compounds comprising a polypeptide containing at least 95%, alternatively at least 96%, alternatively at least 97%, alternatively at least 98%, alternatively at least 99%, or alternatively 100% amino acid sequence identity to the amino acid of SEQ ID NO: 500 (DR2463, untagged). In some embodiments, the Disclosure provides IL-10 agonist compounds comprising a polypeptide containing at least 95%, alternatively at least 96%, alternatively at least 97%, alternatively at least 98%, alternatively at least 99%, or alternatively 100% amino acid sequence identity to the amino acid of SEQ ID NO: 501 (DR2485, untagged). In some embodiments, the Disclosure provides IL-10 agonist compounds comprising a polypeptide containing at least 95%, alternatively at least 96%, alternatively at least 97%, alternatively at least 98%, alternatively at least 99%, or alternatively 100% amino acid sequence identity to the amino acid of SEQ ID NO: 502 (DR2519, untagged). In some embodiments, the Disclosure provides IL-10 agonist compounds comprising a polypeptide containing at least 95%, alternatively at least 96%, alternatively at least 97%, alternatively at least 98%, alternatively at least 99%, or alternatively 100% amino acid sequence identity to the amino acid of SEQ ID NO: 503 (DR2520, untagged).

[0016] In some embodiments, this disclosure describes a first single-domain antibody polypeptide as follows: A second single-domain antibody polypeptide containing a combination of CDR1, CDR2, and CDR3 amino acid sequences selected from one of the rows in TIFF2026521432000002.tif95166 is given in the following table: The present invention provides an IL-10 agonist compound comprising a combination of CDR1, CDR2, and CDR3 amino acid sequences selected from one of the rows in TIFF2026521432000003.tif21166.

[0017] In some embodiments, the Disclosure relates to an IL-10 agonist compound comprising a first single-domain antibody polypeptide and a second single-domain antibody polypeptide, wherein the first single-domain antibody polypeptide is bound to the extracellular domain of IL-10Rα and comprises CDR1, CDR2, and CDR3, where, (a) CDR1 is a polypeptide containing the amino acid sequence of SEQ ID NO:224, CDR2 is a polypeptide containing the amino acid sequence of SEQ ID NO:229, and CDR3 is a polypeptide containing the amino acid sequence of SEQ ID NO:236, or CDR1 is a polypeptide containing the amino acid sequence of SEQ ID NO:224, CDR2 is a polypeptide containing the amino acid sequence of SEQ ID NO:230, and CDR3 is a polypeptide containing the amino acid sequence of SEQ ID NO:236, or CDR1 is a polypeptide containing the amino acid sequence of SEQ ID NO:224, CDR2 is a polypeptide containing the amino acid sequence of SEQ ID NO:231, and CDR3 is a polypeptide containing the amino acid sequence of SEQ ID NO:236. CDR1 is a polypeptide containing the amino acid sequence of SEQ ID NO:224, CDR2 is a polypeptide containing the amino acid sequence of SEQ ID NO:231, and CDR3 is a polypeptide containing the amino acid sequence of SEQ ID NO:236, or CDR1 is a polypeptide containing the amino acid sequence of SEQ ID NO:224, CDR2 is a polypeptide containing the amino acid sequence of SEQ ID NO:232, and CDR3 is a polypeptide containing the amino acid sequence of SEQ ID NO:236, or CDR1 is a polypeptide containing the amino acid sequence of SEQ ID NO:224, CDR2 is a polypeptide containing the amino acid sequence of SEQ ID NO:233, and CDR3 is a polypeptide containing the amino acid sequence of SEQ ID NO:236, or CDR1 is a polypeptide containing the amino acid sequence of SEQ ID NO:224, CDR2 is a polypeptide containing the amino acid sequence of SEQ ID NO:234, and CDR3 is a polypeptide containing the amino acid sequence of SEQ ID NO:236, or CDR1 is a polypeptide containing the amino acid sequence of SEQ ID NO:225, CDR2 is a polypeptide containing the amino acid sequence of SEQ ID NO:229, and CDR3 is a polypeptide containing the amino acid sequence of SEQ ID NO:236, or CDR1 is a polypeptide containing the amino acid sequence of SEQ ID NO:225, CDR2 is a polypeptide containing the amino acid sequence of SEQ ID NO:235, and CDR3 is a polypeptide containing the amino acid sequence of SEQ ID NO:236, or CDR1 is a polypeptide containing the amino acid sequence of SEQ ID NO:226, CDR2 is a polypeptide containing the amino acid sequence of SEQ ID NO:230, and CDR3 is a polypeptide containing the amino acid sequence of SEQ ID NO:236. CDR1 is a polypeptide containing the amino acid sequence of SEQ ID NO:228, CDR2 is a polypeptide containing the amino acid sequence of SEQ ID NO:229, and CDR3 is a polypeptide containing the amino acid sequence of SEQ ID NO:236, and / or (b) A second single-domain antibody polypeptide is bound to the extracellular domain of IL 10Rβ and comprises CDR1, CDR2 and CDR3, where, CDR1 is a polypeptide containing the amino acid sequence of SEQ ID NO:296, CDR2 is a polypeptide containing the amino acid sequence of SEQ ID NO:297, and CDR3 is a polypeptide containing the amino acid sequence of SEQ ID NO:298. Optionally, the first single-domain antibody polypeptide (IL-10Rα sdAb) that binds to the extracellular domain of IL-10Rα is at least 80% humanized compared to UniProt V3-23 (UniProt number P01764), and / or Optionally, a second single-domain antibody polypeptide (IL-10Rβ sdAb) that binds to IL-10Rβ is at least 89% humanized compared to Uniprot VH3-66 (UniProt A0A0C4DH42), and / or Furthermore, optionally, the N-terminus of the first single-domain antibody polypeptide is linked to the C-terminus of the second single-domain antibody polypeptide, or alternatively, the C-terminus of the first single-domain antibody polypeptide is linked to the N-terminus of the second single-domain antibody polypeptide via a polypeptide linker containing 1-50, alternatively 1-30, alternatively 1-20, alternatively 1-15, alternatively 1-12, alternatively 1-10, alternatively 1-8, alternatively 1-6, alternatively 1-5, alternatively 1-4, alternatively 1-3, alternatively 1-2, or alternatively 1 amino acid. We provide IL-10 agonist compounds.

[0018] In certain embodiments, the Disclosure relates to an IL-10 agonist compound comprising a first single-domain antibody polypeptide and a second single-domain antibody polypeptide, wherein the first single-domain antibody polypeptide is bound to the extracellular domain of IL-10Rα and comprises CDR1, CDR2 and CDR3, where CDR1 is a polypeptide comprising the amino acid sequence of SEQ ID NO:224, CDR2 is a polypeptide comprising the amino acid sequence of SEQ ID NO:229, and CDR3 is a polypeptide comprising the amino acid sequence of SEQ ID NO:236, and optionally the first single-domain antibody polypeptide (IL-10Rα sdAb) bound to the extracellular domain of IL-10Rα is at least 80% humanized compared to UniProt V3-23 (UniProt number P01764), and (b) the second single-domain antibody polypeptide is bound to the extracellular domain of IL-10Rβ and comprises CDR1, CDR2 and CDR3, where CDR1 is SEQ ID The present invention provides an IL-10 agonist compound comprising a polypeptide containing the amino acid sequence NO:296, CDR2 being a polypeptide containing the amino acid sequence of SEQ ID NO:297, and CDR3 being a polypeptide containing the amino acid sequence of SEQ ID NO:298, and optionally a second single-domain antibody polypeptide (IL-10Rβ sdAb) that binds to IL-10Rβ being at least 89% humanized compared to Uniprot VH3-66 (UniProt A0A0C4DH42), and further optionally, the C-terminus of the first single-domain antibody polypeptide being conjugated to the N-terminus of the second single-domain antibody polypeptide via a polypeptide linker containing 1-50, alternatively 1-30, alternatively 1-20, alternatively 1-15, alternatively 1-12, alternatively 1-10, alternatively 1-8, alternatively 1-6, alternatively 1-5, alternatively 1-4, alternatively 1-3, alternatively 1-2, or alternatively 1 amino acid.

[0019] In certain embodiments, the Disclosure relates to an IL-10 agonist compound comprising a first single-domain antibody polypeptide and a second single-domain antibody polypeptide, wherein the first single-domain antibody polypeptide is bound to the extracellular domain of IL-10Rα and comprises CDR1, CDR2 and CDR3, where CDR1 is a polypeptide comprising the amino acid sequence of SEQ ID NO:224, CDR2 is a polypeptide comprising the amino acid sequence of SEQ ID NO:230, and CDR3 is a polypeptide comprising the amino acid sequence of SEQ ID NO:236, and optionally, the first single-domain antibody polypeptide (IL-10Rα sdAb) bound to the extracellular domain of IL-10Rα is at least 80% humanized compared to UniProt V3-23 (UniProt number P01764), and (b) the second single-domain antibody polypeptide is bound to the extracellular domain of IL-10Rβ and comprises CDR1, CDR2 and CDR3, where CDR1 is SEQ ID The present invention provides an IL-10 agonist compound comprising a polypeptide containing the amino acid sequence NO:296, CDR2 being a polypeptide containing the amino acid sequence of SEQ ID NO:297, and CDR3 being a polypeptide containing the amino acid sequence of SEQ ID NO:298, and optionally a second single-domain antibody polypeptide (IL-10Rβ sdAb) that binds to IL-10Rβ being at least 89% humanized compared to Uniprot VH3-66 (UniProt A0A0C4DH42), and further optionally, the C-terminus of the first single-domain antibody polypeptide being conjugated to the N-terminus of the second single-domain antibody polypeptide via a polypeptide linker containing 1-50, alternatively 1-30, alternatively 1-20, alternatively 1-15, alternatively 1-12, alternatively 1-10, alternatively 1-8, alternatively 1-6, alternatively 1-5, alternatively 1-4, alternatively 1-3, alternatively 1-2, or alternatively 1 amino acid.

[0020] In certain embodiments, the Disclosure relates to an IL-10 agonist compound comprising a first single-domain antibody polypeptide and a second single-domain antibody polypeptide, wherein the first single-domain antibody polypeptide is bound to the extracellular domain of IL-10Rα and comprises CDR1, CDR2 and CDR3, where CDR1 is a polypeptide comprising the amino acid sequence of SEQ ID NO:224, CDR2 is a polypeptide comprising the amino acid sequence of SEQ ID NO:231, and CDR3 is a polypeptide comprising the amino acid sequence of SEQ ID NO:236, and optionally the first single-domain antibody polypeptide (IL-10Rα sdAb) bound to the extracellular domain of IL-10Rα is at least 80% humanized compared to UniProt V3-23 (UniProt number P01764), and (b) the second single-domain antibody polypeptide is bound to the extracellular domain of IL-10Rβ and comprises CDR1, CDR2 and CDR3, where CDR1 is SEQ ID The present invention provides an IL-10 agonist compound comprising a polypeptide containing the amino acid sequence NO:296, CDR2 being a polypeptide containing the amino acid sequence SEQ ID NO:297, and CDR3 being a polypeptide containing the amino acid sequence SEQ ID NO:298, and optionally a second single-domain antibody polypeptide (IL-10Rβ sdAb) that binds to IL-10Rβ being at least 89% humanized compared to Uniprot VH3-66 (UniProt A0A0C4DH42), and further optionally, the C-terminus of the first single-domain antibody polypeptide being conjugated to the N-terminus of the second single-domain antibody polypeptide via a polypeptide linker containing 1-50, alternatively 1-30, alternatively 1-20, alternatively 1-15, alternatively 1-12, alternatively 1-10, alternatively 1-8, alternatively 1-6, alternatively 1-5, alternatively 1-4, alternatively 1-3, alternatively 1-2, or alternatively 1 amino acid.

[0021] In some embodiments, the Disclosure provides an IL-10 agonist compound comprising: a first single-domain antibody polypeptide comprising CDR1 containing the amino acid sequence of SEQ ID NO:224, CDR2 containing the amino acid sequence of SEQ ID NO:229, and CDR3 containing the amino acid sequence of SEQ ID NO:236; and a second single-domain antibody polypeptide comprising CDR1 containing the amino acid sequence of SEQ ID NO:296, CDR2 containing the amino acid sequence of SEQ ID NO:297, and CDR3 containing the amino acid sequence of SEQ ID NO:298.

[0022] In some embodiments, the Disclosure provides an IL-10 agonist compound comprising: a first single-domain antibody polypeptide comprising CDR1 containing the amino acid sequence of SEQ ID NO:224, CDR2 containing the amino acid sequence of SEQ ID NO:230, and CDR3 containing the amino acid sequence of SEQ ID NO:236; and a second single-domain antibody polypeptide comprising CDR1 containing the amino acid sequence of SEQ ID NO:296, CDR2 containing the amino acid sequence of SEQ ID NO:297, and CDR3 containing the amino acid sequence of SEQ ID NO:298.

[0023] In some embodiments, the Disclosure provides an IL-10 agonist compound comprising: a first single-domain antibody polypeptide comprising CDR1 containing the amino acid sequence of SEQ ID NO:224, CDR2 containing the amino acid sequence of SEQ ID NO:231, and CDR3 containing the amino acid sequence of SEQ ID NO:236; and a second single-domain antibody polypeptide comprising CDR1 containing the amino acid sequence of SEQ ID NO:296, CDR2 containing the amino acid sequence of SEQ ID NO:297, and CDR3 containing the amino acid sequence of SEQ ID NO:298.

[0024] In some embodiments, the disclosure provides IL-10 agonist compounds comprising polypeptides containing amino acid sequences selected from the group consisting of SEQ ID NO: 1-24 and SEQ ID NO: 500-523.

[0025] In some embodiments, the Disclosure provides an IL-10 agonist compound comprising a polypeptide containing the amino acid sequence (DR2463) of SEQ ID NO:1. In some embodiments, the Disclosure provides an IL-10 agonist compound comprising a polypeptide containing the amino acid sequence (DR2485) of SEQ ID NO:2. In some embodiments, the Disclosure provides an IL-10 agonist compound comprising a polypeptide containing the amino acid sequence (DR2519) of SEQ ID NO:3. In some embodiments, the Disclosure provides an IL-10 agonist compound comprising a polypeptide containing the amino acid sequence (DR2520) of SEQ ID NO:4.

[0026] In some embodiments, the disclosure provides an IL-10 agonist compound comprising a polypeptide containing the amino acid sequence (DR2463, untagged) of SEQ ID NO: 500. In some embodiments, the disclosure provides an IL-10 agonist compound comprising a polypeptide containing the amino acid sequence (DR2485, untagged) of SEQ ID NO: 501. In some embodiments, the disclosure provides an IL-10 agonist compound comprising a polypeptide containing the amino acid sequence (DR2519, untagged) of SEQ ID NO: 502. In some embodiments, the disclosure provides an IL-10 agonist compound comprising a polypeptide containing the amino acid sequence (DR2520, untagged) of SEQ ID NO: 4.

[0027] In some embodiments, this disclosure provides pharmaceutically acceptable formulations of the IL-10 agonist compounds disclosed herein.

[0028] In some embodiments, the Disclosure provides nucleic acid sequences encoding IL-10 agonist compounds disclosed herein. In some embodiments, the Disclosure provides recombinant vectors comprising such nucleic acids.

[0029] The IL-10 agonist compounds of this disclosure (e.g., single-domain antibody polypeptides that are IL-10 agonist compounds) are useful for treating or preventing diseases in mammalian subjects. In some embodiments, this disclosure provides a method for treating mammalian subjects suffering from autoimmune diseases, infectious diseases, or inflammatory diseases by administering a therapeutically effective dose of the IL-10 agonist compounds disclosed herein. In some embodiments, this disclosure provides a method for treating infectious diseases, including viral infections and chronic viral infections, in mammalian subjects by administering a therapeutically effective dose of the IL-10 agonist compounds disclosed herein. In some embodiments, this disclosure provides a method for treating mammalian subjects suffering from neoplastic diseases by administering a therapeutically effective dose of the IL-10 agonist compounds disclosed herein.

[0030] In another aspect, the Disclosure relates to a method for treating a neoplastic disease, such as cancer, in a subject that needs to be treated for a neoplastic disease, such as cancer, comprising administering to the subject an IL-10 agonist protein described herein, wherein the IL-10 agonist protein is CD8 + T cells, CD4 + The present invention provides a method for binding to and activating T cells, macrophages, and / or Treg cells. In some embodiments, the IL-10 agonist protein provides longer therapeutic efficacy than PEGylated IL-10. In some embodiments, the cancer is a solid tumor.

[0031] In some embodiments, the present disclosure provides means for inducing intracellular signaling in cells expressing IL-10Rα and IL-10Rβ, comprising (ii) a single-domain antibody polypeptide (IL-10Rα sdAb) conjugated to IL-10Rβ and being at least 89% humanized compared to UniProt VH3-66 (UniProt A0A0C4DH42), and a pharmaceutically acceptable carrier.

[0032] In some embodiments, the present disclosure provides compositions comprising IL-10 agonist compounds described herein, wherein the cells expressing IL-10Rα and IL-10Rβ are monocytes.

[0033] In some embodiments, the present disclosure provides compositions in which pSTAT3 signaling is induced by dimerizing the extracellular domain of the IL-10Rα subunit and the extracellular domain of the IL-10Rβ subunit of the IL-10 receptor (IL-10R) on a cell.

[0034] In some embodiments, the present disclosure provides compositions in which intracellular pSTAT-3 signaling in monocyte cells expressing IL10Rα and IL10R is superior to pSTAT-3 signaling in T cells expressing IL10Rα and IL10Rβ.

[0035] In some embodiments, the Disclosure provides compositions in which an IL-10 agonist compound (e.g., a single-domain antibody polypeptide that is an IL-10 agonist compound) includes a C-terminal amino acid modification that reduces immunogenicity caused by the existing antibody. In some embodiments, the first single-domain antibody polypeptide of the IL-10 agonist compound includes the C-terminal modification. In some embodiments, the second single-domain antibody polypeptide of the IL-10 agonist compound includes the C-terminal modification. In some embodiments, a C-terminal polypeptide selected from the first single-domain antibody polypeptide and the second single-domain antibody polypeptide includes the C-terminal modification.

[0036] In some embodiments, the Disclosure provides compositions comprising an IL-10 agonist compound (e.g., a single-domain antibody polypeptide that is an IL-10 agonist compound) with C-terminal amino acid modifications including an amino acid sequence selected from the group consisting of SEQ ID NO: 474 to 499.

[0037] In some embodiments, the disclosure provides compositions in which a compound is selected from the group consisting of SEQ ID NO: 121-135, 138-141, 144-155, 158-175, 179, and 182-195 and 199.

[0038] In another aspect, the present disclosure provides a method for treating mammalian subjects suffering from autoimmune diseases, infectious diseases, or inflammatory diseases by administering a therapeutically effective dose of an IL-10 agonist compound. In some embodiments, one sdAb of the binding molecule is scFv and the other sdAb is VHH.

[0039] In some embodiments, the first sdAb and the second sdAb are covalently bonded via chemical linkage.

[0040] In some embodiments, the first sdAb and the second sdAb are provided as a single, continuous polypeptide.

[0041] In some embodiments, the IL-10 agonist compound (e.g., a single-domain antibody polypeptide that is an IL-10 agonist compound) is provided as a single, continuous polypeptide.

[0042] This invention provides IL-10 agonist compounds, which are synthetic ligands for the IL-10 receptor.

[0043] In some embodiments, the present invention provides IL-10 agonist compounds comprising an IL-10Rα sdAb having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity compared to any one of SEQ ID NO: 25-48, or having 0, 1, 2, or 3 amino acid changes, optionally conservative amino acid changes. In some embodiments, the IL-10Rα sdAb includes a C-terminal amino acid modification that reduces immunogenicity caused by existing antibodies. In some embodiments, the IL-10Rα sdAb includes a C-terminal amino acid modification comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 475-497.

[0044] In some embodiments, the present invention provides IL-10 agonist compounds comprising IL-10Rβ sdAb having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity compared to either SEQ ID NO: 49 or SEQ ID NO: 50, or having 0, 1, 2, or 3 amino acid changes, optionally conservative amino acid changes. In some embodiments, the IL-10Rβ sdAb includes a C-terminal amino acid modification that reduces immunogenicity caused by existing antibodies. In some embodiments, the IL-10Rβ sdAb includes a C-terminal amino acid modification comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 475 to 497.

[0045] In some embodiments, the present invention provides an IL-10 agonist compound having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity with respect to any one of the IL-10 agonist compounds with SEQ ID NO: 1-24 and SEQ ID NO: 500-523 (e.g., a single-domain antibody polypeptide that is an IL-10 agonist compound). In some embodiments, the present invention provides an IL-10 agonist compound that is substantially identical to any one of the IL-10 agonist compounds with SEQ ID NO: 1-24 and SEQ ID NO: 500-523 (e.g., a single-domain antibody polypeptide that is an IL-10 agonist compound). In some embodiments, the present invention provides an IL-10 agonist compound that is identical in sequence to one of the IL-10 agonist compounds with SEQ ID NO: 1-24 and SEQ ID NO: 500-523 (e.g., a single-domain antibody polypeptide that is an IL-10 agonist compound). In some embodiments, the IL-10 agonist compound includes a C-terminal amino acid modification that reduces immunogenicity caused by the existing antibody. In some embodiments, the IL-10 agonist compound includes a C-terminal amino acid modification that includes an amino acid sequence selected from the group consisting of SEQ ID NO: 475-497.

[0046] In one embodiment, the disclosure provides an IL-10Rα-binding molecule that preferentially activates T cells, such as CD8+ T cells, compared to monocytes. In one embodiment, the disclosure provides an IL-10Rα-binding molecule of formula (#1), wherein the affinity of IL-10Rα sdAb to the extracellular domain of IL-10Rα is higher than the affinity of IL-10Rβ sdAb to the extracellular domain of IL-10Rβ.

[0047] In some embodiments, the Disclosure provides IL-10 agonist compounds modified to provide an extended duration of action in vivo in mammalian subjects and pharmaceutically acceptable formulations thereof. In some embodiments, the Invention provides PEGylated IL-10 agonist compounds in which PEG is conjugated to an IL-10 agonist compound (e.g., a single-domain antibody polypeptide that is an IL-10 agonist compound). In some embodiments, PEGylation is performed at the C-terminus of IL-10R VHH2, and the PEG is a linear or branched PEG molecule having an average molecular weight of approximately 2,000 to approximately 80,000 daltons, alternatively approximately 2,000 to approximately 70,000 daltons, alternatively approximately 5,000 to approximately 50,000 daltons, alternatively approximately 10,000 to approximately 50,000 daltons, alternatively approximately 20,000 to approximately 50,000 daltons, alternatively approximately 30,000 to approximately 50,000 daltons, alternatively approximately 20,000 to approximately 40,000 daltons, or alternatively approximately 30,000 to approximately 40,000 daltons. In one embodiment of the present disclosure, the PEG is a 40kD branched PEG containing two 20kD arms.

[0048] This disclosure further provides pharmaceutically acceptable formulations of IL-10 agonist compounds for administration to mammalian subjects. This disclosure further provides pharmaceutically acceptable compositions for administration to mammalian subjects, the composition comprising a nucleic acid sequence encoding a polypeptide IL-10 agonist compound, a recombinant viral vector or nonviral vector encoding a polypeptide IL-10 agonist compound, or a recombinantly modified mammalian cell containing a nucleic acid sequence encoding a polypeptide IL-10 agonist compound, wherein in each case the nucleic acid sequence is functionally linked to one or more functional regulatory elements in the mammalian cell.

[0049] This disclosure provides nucleic acid sequences encoding polypeptide IL-10 agonist compounds. This disclosure further provides recombinant vectors comprising nucleic acid sequences encoding polypeptide IL-10 agonist compounds. This disclosure further provides recombinantly modified mammalian cells comprising nucleic acids encoding polypeptide IL-10 agonist compounds. This disclosure further provides methods for recombinant production, isolation, purification and characterization of polypeptide IL-10 agonist compounds and provides recombinant vectors comprising nucleic acid sequences encoding polypeptide IL-10 agonist compounds.

[0050] The disclosure also provides an expression vector comprising a nucleic acid encoding a bispecific IL-10 agonist compound functionally linked to one or more expression regulatory sequences. The disclosure also provides isolated host cells comprising an expression vector comprising a nucleic acid encoding an IL-10 agonist compound (e.g., a single-domain antibody polypeptide that is an IL-10 agonist compound) functionally linked to one or more expression regulatory sequences that are functional in host cells.

[0051] In another aspect, the present disclosure provides a pharmaceutical composition comprising an IL-10 agonist compound as described herein (e.g., a single-domain antibody polypeptide that is an IL-10 agonist compound) and a pharmaceutically acceptable carrier.

[0052] In another aspect, the Disclosure provides a method for treating an autoimmune or inflammatory disease, disorder, condition, or viral infection in a subject that requires treatment for such an autoimmune or inflammatory disease, disorder, or condition, or viral infection, comprising administering to the subject a therapeutically effective amount of an IL-10 agonist compound or a pharmaceutical composition described herein.

[0053] Several advantages can be obtained from the binding molecules described herein. IL-10, the natural ligand for the IL-10 receptor, causes IL-10Rα and IL-10Rβ to be in close proximity through their simultaneous binding to IL-10. However, when IL-10 is used as a therapeutic agent in mammalian subjects, particularly in human subjects, it can also trigger numerous adverse and undesirable effects through various mechanisms, including the presence of IL-10Rα and IL-10Rβ on other cell types, and the binding of IL-10Rα and IL-10Rβ on other cell types can result in undesirable effects and / or undesirable signaling in cells expressing IL-10Rα and IL-10Rβ. This disclosure relates to methods and compositions for modulating several undesirable adverse effects of IL-10 binding to IL-10Rα and IL-10Rβ such that desired therapeutic signaling occurs, particularly in desired cell or tissue subtypes, while minimizing undesirable activity and / or intracellular signaling.

[0054] In some embodiments, the IL-10 agonist compounds described herein (e.g., single-domain antibody polypeptides that are IL-10 agonist compounds) are partial agonists of the IL-10 receptor. In some embodiments, the binding molecules described herein are designed to be complete agonists. In some embodiments, the binding molecules described herein are designed to be superagonists.

[0055] In some embodiments, the binding molecule provides the maximum desired intracellular IL-10 signaling in desired cell types by binding to IL-10Rα and IL-10Rβ, while providing significantly lower IL-10 signaling in other undesirable cell types. This is because, for example, it has a different affinity for IL-10 compared to the affinity for IL-10 to IL-10Rα and IL-10Rβ, or a different affinity for IL-10Rα and IL-10Rβ. max This can be achieved by selecting the binding molecules that cause it.

[0056] Since different cell types respond with varying sensitivities to ligand binding to cognitive receptors, modulating the affinity of dimeric ligands (or their individual binding moieties) to the IL-10 receptor compared to wild-type IL-10 binding promotes the stimulation of desired activity while reducing undesirable activity in non-target cells. [Brief explanation of the drawing]

[0057] [Figure 1] This graph shows that in an LPS-induced monocyte secretion assay, the humanized IL-10Rα / IL-10Rβ VHH dimers DR1525 and DR2096 were not as potent as the parent molecule DR841 in terms of inhibition of IL1β production. [Figure 2] This graph shows that in an LPS-induced monocyte secretion assay, the humanized IL-10Rα / IL-10Rβ VHH dimers DR1525 and DR2096 were not as potent as the parent molecule DR841 in terms of inhibition of TNFα production. [Figure 3] This graph shows that in an LPS-induced monocyte secretion assay, the humanized IL-10Rα / IL-10Rβ VHH dimer DR2503 exhibits a titer comparable to that of the parent molecule DR841, as measured by inhibition of IL1β production. [Figure 4] This graph shows that in an LPS-induced monocyte secretion assay, the humanized IL-10Rα / IL-10Rβ VHH dimer DR2503 exhibits a titer comparable to that of the parent molecule DR841, as measured by inhibition of TNFα production. This graph also demonstrates the functionality of the IL10Rα / IL10Rβ VHH dimers DR2463 and DR2485. [Figure 5]Table 1 shows that the humanized IL10Rα / IL10Rβ VHH dimers DR2485 (SEQ ID NO: 2), DR2519 (SEQ ID NO: 3), and DR2520 (SEQ ID NO: 4) retain the ability to suppress the secretion of the pro-inflammatory cytokine IL-1β, similar to the unhumanized parental VHH dimer DR841 (Table 14; SEQ ID NO: 465). [Figure 6] Table 1 shows that the humanized IL10Rα / IL10Rβ VHH dimers DR2485 (SEQ ID NO: 2), DR2519 (SEQ ID NO: 3), and DR2520 (SEQ ID NO: 4) retain the ability to suppress the secretion of the pro-inflammatory cytokine TNFα, similar to the unhumanized parental VHH dimer DR841 (Table 14; SEQ ID NO: 465). [Figure 7] Table 1 shows that the humanized IL10Rα / IL10Rβ VHH dimers DR2485 (SEQ ID NO:2), DR2519 (SEQ ID NO:3), and DR2520 (SEQ ID NO:4) suppress the induction of IFN-γ in T cells. [Figure 8] Table 1 shows that the humanized IL10Rα / IL10Rβ VHH dimers DR2485 (SEQ ID NO:2), DR2519 (SEQ ID NO:3), and DR2520 (SEQ ID NO:4) suppress the production of granzyme B in T cells. [Modes for carrying out the invention]

[0058] Detailed description of the invention To facilitate understanding of this disclosure, certain terms and phrases are defined not only below but throughout this specification. The definitions provided herein are not limiting and should be read with consideration to the knowledge that a person skilled in the art would possess.

[0059] The present invention is not limited to the specific methods or compositions described, and should be understood to be such as they may vary. Furthermore, the technical terms used herein are intended only to describe specific embodiments, and such embodiments are not intended to be limiting.

[0060] Where a range of values ​​is provided, unless the context explicitly indicates otherwise, it is understood that each intermediate value between the upper and lower limits of this range, up to one-tenth of the lower limit, is also specifically disclosed. Any smaller range between any indicated value or intermediate value within the indicated range and any other indicated value or intermediate value within the indicated range is each included in the present invention. The upper and lower limits of these smaller ranges may independently be included in or excluded from the smaller range, and if there are any limits that are specifically excluded in the indicated range, each range in which one or both of these limits are included in the smaller range, or in which neither is included, is also included in the present invention. If the indicated range includes one or both of these limits, the range that excludes one or both of the limits that they include is also included in the present invention.

[0061] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art to which the present invention pertains. Any methods and substances similar to or equivalent to those described herein may be used in the practice or testing of the present invention, but several potential and preferred methods and substances are described herein. All publications referenced herein are incorporated herein by reference to disclose and describe the methods and / or substances in which the publications are cited in connection therewith.

[0062] It should be noted that, as used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural references unless the context otherwise explicitly indicates otherwise. For example, a reference to “cell” includes multiple such cells, and a reference to “peptide” includes one or more peptides and their equivalents, such as polypeptides known to those skilled in the art.

[0063] The publications discussed herein are provided only if their disclosures precede the filing date of this application. Nothing contained herein should be construed as acknowledging that the present invention is not granted prior rights to such publications by prior art. Furthermore, the publication dates presented may differ from the actual publication dates, which may need to be verified separately.

[0064] Unless otherwise specified, parts are parts by weight, molecular weight is the average molecular weight, temperature is degrees Celsius (°C), and pressure is atmospheric pressure or near atmospheric pressure. Standard abbreviations are used, including the following: bp = base pair; kb = kilobase; pl = picoliter; s or sec = second; min = minute; h or hr = hour; AA or aa = amino acid; kb = kilobase; nt = nucleotide; pg = picogram; ng = nanogram; μg = microgram; mg = milligram; g = gram; kg = kilogram; dl or dL = deciliter; μl or μL = microliter; ml or mL = milliliter; l or L = liter; μM = micromoles; mM = millimoles; M = mole; kDa = kilodalton; im = intramuscular; ip = intraperitoneal; SC or SQ = subcutaneous; QD = once daily; BID = twice daily; QW = once weekly; QM = once monthly; HPLC = high-performance liquid chromatography; BW = Body weight; U = unit; ns = not statistically significant; PBS = phosphate-buffered saline; PCR = polymerase chain reaction; HSA = human serum albumin; MSA = mouse serum albumin; DMEM = Dulbecco's modified Eagle medium; EDTA = ethylenediaminetetraacetic acid.

[0065] Throughout this disclosure, amino acids will be referred to according to either a one-letter code or a three-letter code. For the convenience of the reader, one-letter and three-letter amino acid codes are provided in Table 13.

[0066] Standard methods in molecular biology are described in scientific literature (see, for example, Sambrook and Russell. 2001. Molecular Cloning, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; and Ausubel et al. 2001. Current Protocols in Molecular Biology, Vols. 1-4, John Wiley and Sons, Inc. New York, NY, which describes cloning and DNA mutagenesis in bacterial cells (Vol. 1), cloning in mammalian cells and yeast (Vol. 2), expression of complex carbohydrates and proteins (Vol. 3), and bioinformatics (Vol. 4)). This scientific literature describes not only methods for protein purification, including immunoprecipitation, chromatography, electrophoresis, centrifugation, and crystallization, but also chemical analysis, chemical modification, post-translational modification, fusion protein production, and protein glycosylation (see, for example, Coligan et al. 2000. Current Protocols in Protein Science. Vols. 1-2, John Wiley and Sons, Inc., NY).

[0067] definition Unless otherwise specified, the following terms are intended to have the meanings set forth below. Other terms are defined elsewhere in the specification.

[0068] Activate As used herein, the term “activates” is used in reference to a receptor or receptor complex and reflects the biological effects resulting from the binding of an agonist ligand to a receptor in response to ligand binding, both directly and / or through involvement in a multi-component signaling cascade.

[0069] ActivationAs used herein, the term “activity” is used to describe, with respect to a molecule, its properties in relation to a test system (e.g., an assay), or its biological or chemical properties (e.g., the degree to which the molecule binds to another molecule), or the physical properties of a substance or cell (e.g., modification of the cell membrane potential). Examples of such biological functions include, but are not limited to, the catalytic activity of a biological agent, its ability to stimulate intracellular signaling, gene expression, cell proliferation, and its ability to modulate immune activity such as inflammatory responses. “Activity” is typically expressed as the level of bioactivity per unit of the agent being tested, such as [catalytic activity] / [mg protein], [immune activity] / [mg protein], international units of activity (IU), [STAT5 phosphorylation] / [mg protein], [T cell proliferation] / [mg protein], or plaque-forming units (pfu). As used herein, the term “proliferative activity” means the activity that promotes cell proliferation and replication.

[0070] Administer / Administer The terms “administer” and “administer” are used interchangeably herein to mean the act of contacting a subject, including, in vitro, in vivo, or ex vivo, with the active substance (e.g., orthologs, IL-10 orthologs, engineered cells expressing ortholog receptors, engineered cells expressing ortholog IL-10 receptors, CAR-T cells expressing ortholog IL-10 receptors, chemotherapeutic agents, antibodies, or pharmaceutical formulations comprising one or more of the above). Administration of the active substance may be achieved by any of the various methods approved in the Art, including, but not limited to, topical administration, intravascular injection (including intravenous or intra-arterial injection), intradermal injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, and inhalation. The term “administer” includes contact between the active substance and the cells, tissues, or organs, as well as contact between the active substance and the fluid in contact with the cells, tissues, or organs.

[0071] affinityAs used herein, the term "affinity" refers to the degree of specific binding of a first molecule (e.g., ligand) to a second molecule (e.g., receptor), and the dissociation rate constant k between the molecule and its target. off and the association rate constant k between the molecule and its target on The equilibrium dissociation constant K is the ratio of to D It is measured by [method].

[0072] AgonistAs used herein, the term “agonist” means a first active substance that specifically binds to a second active substance (“target”) and interacts with the target to cause or promote the enhancement of the target’s activation. In some cases, an agonist is an activator of a receptor protein that modulates, enhances, sensitizes a cell to activation by the second active substance, or upregulates one or more genes, proteins, ligands, receptors, biological pathways that may result in the expression of a gene, protein, ligand, receptor, cell proliferation, or cell death, such as cell cycle arrest or apoptosis. In some embodiments, an agonist is an active substance that binds to a receptor, modifies the state of the receptor, and results in a biological response that mimics the effect of the receptor’s endogenous activator. The term “agonist” includes partial agonists, full agonists, and superagonists. Agonists may be described as "full agonists" or "partial agonists" when such agonists produce a substantially complete biological response induced by the receptor under study (i.e., a response associated with the innate ligand / receptor binding interaction). In contrast to agonists, antagonists can bind specifically to a receptor but typically do not result in a signaling cascade initiated by the receptor, and can modify the action of the agonist at that receptor. Reverse agonists are agents that produce a pharmacological response opposite to that of the agonist. "Superagonists" are a type of agonist capable of producing a greater maximal response to a target receptor than the endogenous agonist, and therefore have greater activity than 100% of the innate ligand. A superagonist is typically a synthetic molecule that, when evaluated at similar concentrations in an equivalent assay, exhibits a response greater than 110%, alternatively greater than 120%, alternatively greater than 130%, alternatively greater than 140%, alternatively greater than 150%, alternatively greater than 160%, or alternatively greater than 170% in an evaluable quantitative or qualitative parameter of the molecule in its native form.

[0073] Antagonist: As used herein, the terms "antagonist" or "inhibitor" refer to a molecule that counteracts the action of an agonist. An antagonist prevents, reduces, inhibits, or neutralizes the activity of an agonist, and an antagonist can also prevent, inhibit, or reduce the constitutive activity of a target, such as a target receptor, even in the absence of a specific agonist. An inhibitor is, for example, a molecule that decreases, blocks, prevents, delays activation, inactivates, desensitizes, or downregulates a gene, protein, ligand, receptor, biological pathway, or cell.

[0074] Antibody (Ab) : As used herein, the term "antibody" means any form of antibody (also known as immunoglobulin (Ig)) that exhibits the desired biological activity of binding to an antigen epitope as described herein. The term "antibody" includes, but is not limited to, polyclonal antibodies, monoclonal antibodies (including full-length monoclonal antibodies comprising two light chains and two heavy chains), multispecific antibodies (e.g., bispecific antibodies that bind to two or more antigens or antigen epitopes on a single antigen), fully human antibodies (huAb), humanized antibodies (hzAb), chimeric antibodies, single-chain variable fragment antibodies (scFv), single-domain antibodies (sdAb), variable heavy chain (VH) domain antibodies, diabodies (dAb), and antigen-binding fragments of heavy-chain-only antibodies (VHH) that contain the amino acid sequence of the variable region. As used herein, the term "antibody" includes (a) glycosylated and non-glycosylated immunoglobulins that specifically bind to a target molecule, such as an antigen (including, but not limited to, mammalian immunoglobulin classes IgG1, IgG2, IgG3, and IgG4), and (b) immunoglobulin derivatives that compete with the immunoglobulin from which they are derived for binding to the target molecule, such as, but not limited to, IgG(1-4) delta C H 2, F(ab')2, Fab, ScFv, V H 、V LThe term antibody collectively refers to tetrabodies, triabodies, diabodies, dsFv, F(ab')3, scFv-Fc, and (scFv)2. The term antibody is not limited to immunoglobulins derived from any specific mammalian species and includes antibodies from mouse, human, equid, camelid, and human (uman). The term antibody typically includes "heavy chain antibodies" and "VHH" obtained by immunization of camelids (including camels, llamas, and alpacas) as detailed in the definition of "VHH" below, for example, Hamers-Casterman et al. 1993. Nature. 363:446-448. Antibodies with a given specificity may also originate from non-mammalian sources, such as VHH obtained from immunization of cartilaginous fish (including, but not limited to, sharks). The term "antibody" encompasses not only antibodies that can be isolated from natural sources or from animals after immunization with an antigen, but also engineered antibodies, including monoclonal antibodies, bispecific antibodies, trispecific antibodies, chimeric antibodies, humanized antibodies, human antibodies, CDR-grafted antibodies, veneered antibodies, or deimmunized antibodies (for example, to remove B-cell epitopes and / or T-cell epitopes).

[0075] Human antibodies (hAb)The term "human antibody" includes not only antibodies obtained from humans, but also antibodies obtained from transgenic mammals that contain human immunoglobulin genes in such a way that, when stimulated with an antigen, the transgenic animal produces antibodies containing amino acid sequences specific to antibodies produced by humans. The term antibody includes both parental antibodies and their derivatives, such as affinity-mature antibodies, venated antibodies, CDR-grafted antibodies (including CDR-grafted VHH), humanized antibodies, camelized antibodies (in the case of non-camel-derived VHH), or binding molecules that contain the antibody binding domain (e.g., CDR) in a non-immunoglobulin scaffold. The term “antibody” is not limited to any specific synthetic means, but includes not only naturally occurring antibodies that can be isolated from natural sources, but also engineered antibody molecules prepared by “recombination” means, including antibodies isolated from transgenic animals that are transgenic for human immunoglobulin genes or hybridomas prepared therefrom, antibodies isolated from host cells transformed with nucleic acid constructs that result in antibody expression, antibodies isolated from combinatorial antibody libraries including phage display libraries, or chemically synthesized (e.g., solid-phase protein synthesis) antibodies. In one embodiment, “antibody” is a mammalian immunoglobulin. In some embodiments, an antibody is a “full-length antibody” that includes a variable domain and a constant domain that provide binding and effector functions. In some embodiments, a full-length antibody includes two light chains and two heavy chains, each light chain including a variable region and a constant region. In some embodiments, the term “full-length antibody” is used to refer to a conventional IgG immunoglobulin structure including two light chains and two heavy chains, where each light chain includes a variable region and a constant region that provide binding and effector functions. The term antibody encompasses antibody conjugates, which include modifications to prolong the duration of action, such as conjugation to fusion proteins or polymers (e.g., PEGylation), as detailed below.

[0076] Heavy chain (H)When used in relation to antibodies, the term "heavy chain" refers to a polypeptide chain that includes a variable region and a constant region that can be combined with a light chain. A heavy chain can be a human heavy chain sequence derived from a human heavy chain, or a humanized heavy chain created by introducing amino acid residue substitutions at specific positions in the amino acid sequence to produce a polypeptide with an amino acid sequence similar to that of a human polypeptide.

[0077] Steady-state region (C) The term "constant region" or "constant domain" refers to the carboxyl-terminal regions of the light and heavy chains that do not directly participate in antibody binding to antigen targets but exhibit various effector functions, such as interaction with Fc receptors. The amino acid sequence residues of the "constant region" or "constant domain" are generally more conserved (i.e., less variable) than other highly variable regions of immunoglobulins (including antigen-binding sites). The constant region includes the CH1, CH2, and CH3 regions of the heavy chain and the CL region of the light chain.

[0078] Variable region (V) The terms "variable region," "variable domain," "V region," or "V domain" refer to the variable light chain (VL) or variable heavy chain (VH) portion of an antibody located at the amino terminus of its light or heavy chain, which determines the binding specificity of each particular antibody to its specific antigen. The variable regions of the light chain (VL) and heavy chain (VH) can, together or individually, form a binding site (called a paratope) that binds to a target (called an antigen epitope). The binding region may consist of one or both of the VH and VL chains, for example, forming a bifunctional or bispecific antibody having two identical binding sites that bind to the same epitope or two different binding sites that bind to different epitopes.

[0079] Framework domain (FR)As used herein, the terms “framework region,” “framework,” or “FR” mean, when used in relation to an antibody, amino acid sequence regions and amino acid residues that have less variability compared to other amino acid residues in the variable region of the antibody and the constant region. Framework regions are adjacent to hypervariable regions (HVRs), and together they constitute the variable region of the antibody or antibody fragment. Hypervariable regions (HVRs) are also called complementarity-determining regions (CDRs), and these terms may be used interchangeably herein. Framework regions may also be defined as amino acid sequence residues that are not hypervariable amino acid residues or are not part of the amino acid sequence of a hypervariable region.

[0080] Complementarity Determination Area (CDR)The terms “complementarity-determining region” and “CDR” (also known as “hypervariable region” or “HVR” as described above) refer to segments of the variable region of an antibody that have amino acid residues that are more variable than other amino acid residues in the variable region and the constant region. As described above, the terms “complementarity-determining region” and “CDR” are synonymous with the term hypervariable region (HVR) and can be used interchangeably. The terms “complementarity-determining region” and “CDR” are commonly used in the art to describe amino acid residues in the antibody variable region (VL or VH) that are thought to mediate or participate in the interaction and binding between the antibody and the antigen epitope, or that have some structural and / or functional properties (i.e., are “complementary” to the amino acid residues on the epitope, or have amino acid residues that are physically or chemically “complementary”), however some CDR amino acid residues within the CDR region do not necessarily mediate or participate in the binding of the CDR region to the antigen epitope. Therefore, the use of the term CDR in this specification should not be interpreted as meaning that all amino acid residues of such a CDR are not necessarily physically or chemically complementary, nor should the term CDR be interpreted as meaning that the CDR amino acid residues of such a CDR constitute all of the amino acid residues that mediate or participate in the binding of the CDR region to the antigen epitope, since it is known that some amino acid residues outside the CDR region (i.e., amino acid residues in the framework region (FR)) may participate in or influence the binding of the antibody to the antigen epitope. Those skilled in the art will understand that, regardless of how the CDR sequence is defined, not all amino acid residues of the CDR sequence necessarily engage in antigen epitope contact, and that additional amino acid residues not assigned within the declared CDR region (i.e., amino acid residues in the FR1, FR2, FR3, or FR4 framework region) may also engage in antigen epitope contact or otherwise participate in target antigen epitope binding.Therefore, as used herein, the term CDR is interpreted to mean a region of an antibody's variable heavy chain (VH) or variable light chain (VL) region that is generally more variable (i.e., hypervariable) than the adjacent framework region (which is also "variable" relative to the antibody's constant region). As a result of such CDR variability, the "CDR" region is suitable for characterizing the chemical, physical, structural, and / or binding properties of the antibody (e.g., the antibody's structure, function, and / or binding properties), regardless of the actual or predicted structural, physical, chemical, or binding properties of that CDR region, and is therefore useful for distinguishing an antibody from others and establishing its novelty.

[0081] Single-domain antibodies such as scFv or VHH have different physical and chemical properties compared to standard antibodies, and therefore the CDR region of an sdAb may be defined differently from that of a standard antibody. The VL or VH chain of an sdAb typically contains three hypervariable regions (CDR1, CDR2, and CDR3) scattered among four framework regions (FR1, FR2, FR3, and FR4), forming a polypeptide sequence containing the linear polypeptide structure FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

[0082] The amino acid residues containing the CDR region of an antibody are generally defined according to the IMGT system using the Kabat numbering system. Conventional Kabat numbering systems for IgG molecules are found in, for example, Kabat et al. 1991. "Sequences of Proteins of Immunological Interest" (National Institutes of Health, Bethesda, Maryland, 5th edition, NIH publication number 91-3242), Kabat. 1978. Adv Prot Chem. 32:1-75, and Kabat et al. 1977. J Biol Chem. 252:6609-6616, Wu, TT and Kabat, EA. 1970. "Analysis of the sequences of the variable regions of Bence Jones proteins and myeloma light chains and their implications for antibody complementarity" J Exp Med. 132:211-250, and Kabat, EA and Wu, TT. 1991. "Identical V region amino acid sequences and segments of sequences in antibodies of different specificities. Relative contributions of VH and VL genes, minigenes, and complementarity determining regions to binding." "of antibody combining sites" is described in J Immunol. 147:1709-1719.More recently, a Kabat numbering scheme specifically for single-domain antibodies (sdAb) has been proposed in Chapter 14 of "Single-Domain Antibodies: Methods and Protocols" edited by Hussack G and Henry KA ("Methods in Molecular Biology" vol. 2446) (Humana, New York, NY 2022), and in Sulea, T. 2022, "Humanization of Camelid Single-Domain Antibodies," pp. 299-312.

[0083] The common numbering used in the Kabat numbering system for standard antibodies is described in Kabat et al. "Sequence of proteins of immunological interest." (US Public Health Service, NIH, Bethesda, Maryland, publication number 91). For VHH domains derived from camelids, the standard Kabat numbering scheme has been applied, as described by Riechmann and Muyldermans 2000. J Immunol Methods 240(1-2):185-195. In this numbering scheme, amino acid residues are numbered as follows (amino acid residue numbers in parentheses): FR1 (1-30), CDR1 (31-35B), FR2 (36-49), CDR2 (50-65), FR3 (66-92), CDR3 (93-102), and FR4 (103-113). It is known in the art that, in the case of VH polypeptides and VHH polypeptides derived from single-domain antibodies, the total number of amino acid residues in each CDR varies and may not exactly match the total number of amino acid residues indicated by a standard Kabat numbering scheme (i.e., one or more positions indicated by Kabat numbering may not be present in the actual sequence, or the actual sequence may contain more amino acid residues than the number allowed by Kabat numbering).

[0084] In the following paragraphs, we define the amino acid sequences of each of the three CDR regions of the sdAb variable region (including VHH and scFv) according to the Kabat antibody numbering scheme for sdAb.

[0085] CDR1 The term "CDR1" refers to an amino acid sequence that begins at amino acid residue 31 and ends at the Kabat amino acid residue immediately preceding amino acid residue 36. Since CDR1 may include amino acid insertions resulting from immunoglobulin VDJ gene segment recombination, CDR1 can alternatively be defined as an amino acid sequence that begins at the amino acid immediately following amino acid residue 30 and ends at the amino acid immediately preceding amino acid residue 36 (i.e., the amino acid sequence between amino acid residue 30, which is the last amino acid residue of FR1, and amino acid residue 36, which is the first amino acid residue of FR2).

[0086] CDR2 The term "CDR2" refers to an amino acid sequence that begins at amino acid residue 50 and ends at the amino acid residue immediately preceding amino acid residue 66. Alternatively, CDR2 can be defined as an amino acid sequence that begins at the amino acid residue immediately following amino acid residue 49 and ends at the amino acid residue immediately preceding amino acid residue 66 (i.e., the amino acid sequence between amino acid residue 49, which is the last amino acid residue of FR2, and amino acid residue 66, which is the first amino acid residue of FR3).

[0087] CDR3 The term "CDR3" refers to an amino acid sequence that begins at amino acid residue 93 and ends at amino acid residue 102. Since CDR3 may include amino acid insertions resulting from immunoglobulin VDJ gene segment recombination, CDR3 can alternatively be defined as an amino acid sequence that begins immediately after amino acid residue 92 and ends immediately before amino acid residue 103 (i.e., the amino acid sequence between amino acid residue 92, which is the last amino acid residue of FR3, and amino acid residue 103, which is the first amino acid residue of FR4).

[0088] Single-domain antibodyThe term "single-domain antibody" or "sdAb" refers to an antibody fragment consisting of a single monomeric variable antibody domain, which may contain one variable heavy chain domain (VH) of a heavy chain antibody or a common IgG molecule. sdAbs can selectively bind to specific antigens. A specific type of sdAb is the VHH molecule. Single-domain antibodies can be obtained by immunizing dromedary camels, camels, llamas, alpacas, or sharks with a desired antigen and then isolating the mRNA encoding the variable regions (VNAR and VHH) of the heavy chain antibody. Alternatively, sdAbs can also be produced from common mouse, rabbit, or human IgG, which has four strands. Humans may also produce sdAbs through the random generation of stop codons in the light chain. The term "single-domain antibody" or "sdAb" refers to an antibody that has a single (only one) monomeric variable antibody domain.

[0089] VHHAs used herein, the term "VHH" refers to a heavy-chain-only variable domain fragment obtained from, derived from, or induced from heavy-chain antibodies. A heavy-chain antibody is a functional antibody that has two heavy chains and no light chain. Heavy-chain antibodies are found in and can be obtained from camelid animals (e.g., camels and alpacas) that are members of the Camelidae family in biological classification. VHH antibodies were originally described as the antigen-binding immunoglobulin (variable) domain of a "heavy-chain antibody" (i.e., an "antibody lacking a light chain") (Hamers-Casterman et al., Nature 363:446-448 (1993)). The term "VHH domain" is used to distinguish these variable domains from the heavy chain variable domains (hereinafter referred to as "VH domains" or "VH") and the light chain variable domains (hereinafter referred to as "VL domains" or "VL") present in conventional four-chain antibodies.For further explanation of VHH, see Muyldermans' review (Reviews in Molec. Biotechnol. 74:277-302, (2001)), as well as the following patent applications mentioned as general background art: International Patent Publications WO9404678, WO9504079 and WO9634103 of Vrije Universiteit Brussel, WO9425591, WO9937681, WO0040968, WO0043507, WO0065057, WO0140310, WO0144301, EP1134231 and WO0248193 of Unilever, Vlaams Instituut voor WO9749805, WO0121817, WO03035694, WO03054016 and WO03055527 from Biotechnologie (VIB), WO03050531 from Algonomics NV and Ablynx NV, WO0190190 from the National Research Council of Canada, WO03025020 (EP1433793) from the Institute of Antibodies, and Ablynx See the international patent publications WO2004041867, WO2004041862, WO2004041865, WO2004041863, WO2004062551, WO2005044858, WO200640153, WO2006079372, WO2006122786, WO06122787, WO2006122825, WO2008101985, WO2008142164, and WO2015173325 by NV, as well as further published patent applications by Ablynx NV. See also the further prior art referenced in these applications, particularly the list of references mentioned on pages 41-43 of international application WO2006040153. These lists and references are incorporated herein by reference. Methods for obtaining VHH domains that bind to specific antigens or epitopes have been previously described, for example, in WO2006 / 040153 and WO2006 / 122786.As detailed in the above literature, camelid-derived VHH domains can be “humanized,” or “human-like,” by manipulation, for example, by replacing one or more amino acid residues in the amino acid sequence of the original VHH sequence with one or more amino acid residues present at corresponding positions in VH domains derived from conventional human four-chain antibodies. Humanized VHH domains may contain one or more fully human framework region sequences, and more specifically, may contain human framework region sequences derived from DP-29, DP-47, DP-51, or parts thereof (which may be combined with JH such as JH5). VHH CDRs can be transplanted into multiple types of binding proteins (e.g., antibodies), and the CDR retains binding. When a VHH CDR is grafted into a framework, it is manipulated to have potentially more favorable binding behavior. For example, VHH can be genetically linked to Fc domains, other VHHs, peptide tags, or toxins, and can be chemically conjugated to drugs, radionuclides, photosensitizers, and nanoparticles at specific sites. See Bannas et al. 2017. Front Immunol. 8:1603. In certain embodiments of the method, the binding protein is selected from single-chain antibodies (scFv), recombinant camelid heavy chain-only antibodies (VHH), shark heavy chain-only antibodies (VNAR), microproteins, darpin, anticarin, adonectin, aptamers, Sac7d derivatives (afitin, e.g., NANOFITIN, see Journal of Molecular Biology 383(5):1058-68, the contents of which are incorporated herein by reference), Fv, Fab, Fab', and F(ab')2. In some embodiments, the binding protein is a heterodimer, for example, the binding protein has a higher titer than its individual monomers. In alternative embodiments, the heteromultimeric neutralizing binding protein is a multimer, and the components of these multimers are associated by non-covalent or covalent bonds. VHH is a therapeutic protein derived from antibodies that possesses the unique structural and functional properties of naturally occurring heavy chain antibodies.VHH technology is based on fully functional antibodies derived from camelids that lack a light chain. These heavy-chain antibodies contain a single variable domain (VHH) and two constant domains (CH2 and CH3). The cloned and isolated VHH domain is a stable polypeptide with the antigen-binding ability of the original heavy-chain antibody. See U.S. Patent No. 5,840,526 by Castoran et al., issued November 24, 1998, and U.S. Patent No. 6,015,695 by Castoran et al., issued January 18, 2000. These patents are incorporated herein by reference in their entirety. VHH is marketed by Ablynx Inc. (Ghent, Belgium) under the trademark NANOBODIES®. Appropriate methods for preparing or isolating antibody fragments having the required binding specificity and affinity are described herein, including, for example, a method for selecting recombinant antibodies from a library by PCR (see U.S. Patent No. 5,455,030 by Ladner, published October 3, 1995, and U.S. Patent No. 7,745,587 by Devy et al., published June 29, 2010. These documents are incorporated herein by reference in their entirety).

[0090] Functional fragments of antibodies, such as chimeric antibodies, humanized antibodies, primate-like antibodies, venated antibodies, or single-chain antibody fragments, can also be manufactured. The aforementioned functional fragments or functional portions of antibodies include those that react with disease agents. For example, antibody fragments capable of binding to disease agents or parts thereof include, but are not limited to, scFv, Fab, VHH, Fv, Fab, Fab', and F(ab')2, and are encompassed by the present invention. Such fragments can be manufactured by enzymatic cleavage or recombination techniques. For example, papain cleavage or pepsin cleavage can be used to produce Fab fragments or F(ab')2 fragments, respectively. Antibody fragments are manufactured in various cleavage types using antibody genes in which one or more stop codons are introduced upstream of the native stop site. For example, a chimeric gene encoding an F(ab')2 heavy-chain peptide portion can be designed to include DNA sequences encoding the CH1 peptide domain and hinge region of the heavy-chain domain. Accordingly, the present invention encompasses polynucleic acids encoding IL-10 agonist compound (e.g., single-domain antibody polypeptides that are IL-10 agonist compounds) binding proteins as described herein. In certain embodiments, IL-10 agonist compound binding proteins are constructed as part of a multimeric protein and can ligate monomers or single binding regions (e.g., antibody fragments, microproteins, darpin, antikalin, adonectin, peptidomimetic molecules, aptamers, synthetic molecules, etc.). Any combination of binding protein type or binding region type can be ligated. In some embodiments, monomers or binding regions of a multimeric binding protein can be covalently linked. In other embodiments, monomer binding proteins can be modified, for example, by directly attaching them to another monomer binding protein (i.e., covalently bonding the C-terminus of one monomer to the N-terminus of the other) or indirectly (e.g., via linkers or spacers).In various embodiments, monomers are attached to or genetically fused to other monomers (for example, by recombinant proteins engineered to contain additional amino acid sequences that constitute the monomers). Thus, for example, the DNA encoding one monomer is joined to the DNA encoding a second monomer, with the reading frames aligned. Therefore, the DNA may contain additional nucleotides encoding additional amino acids between monomers, creating structurally indeterminate regions that separate the different monomers, thereby better promoting the free folding of each monomer into its active conformation or shape. Commercial techniques for protein fusion are used in various embodiments to attach monomers to the multimer-binding proteins of the present invention.

[0091] Fc The "Fc" region refers to an antibody fragment containing two heavy chain fragments, each containing the CH1 and CH2 domains of the antibody. These two heavy chain fragments are linked by two or more disulfide bonds and hydrophobic interactions of the CH3 domain. The Fc region is located in the C-terminal region of the immunoglobulin heavy chain and includes, for example, the native sequence Fc region, the recombinant Fc region, and the variant Fc region. While the boundary of the Fc region of the immunoglobulin heavy chain can vary, the human IgG heavy chain Fc region is often defined as extending from the amino acid residue at position Cys226 or Pro230 to its carboxyl terminus. The C-terminal lysine of the Fc region (residue 447 in the EU numbering system for the IgG molecule) may be removed, for example, during antibody production or purification, or by recombinant operation of the nucleic acid encoding the antibody heavy chain. Therefore, intact antibody compositions may include antibody populations from which all K447 residues have been removed, antibody populations from which no K447 residues have been removed, and antibody populations having a mixture of antibodies with and without K447 residues.

[0092] pieceThis disclosure also includes functional antigen-binding fragments and methods of using them. Where used herein, unless otherwise indicated, antibody “fragment” or antigen-binding “fragment” means an antigen-binding fragment of an antibody or a bispecific antibody, for example, an antibody fragment that retains the ability to specifically bind to an antigen to which a full-length antibody binds, for example, a fragment that retains one or more CDR regions. Examples of antigen-binding fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments, diabodies, linear antibodies, single-chain antibody molecules, for example scFv, and semi-bispecific molecules containing a heavy chain and a light chain of one antigen-binding arm. “Fab fragment” means an antibody fragment containing one light chain and the CH1 region and variable region of one heavy chain. The heavy chain of a Fab molecule cannot form a disulfide bond with another heavy chain molecule. “Fab fragment” can be a product of papain cleavage of an antibody. A "Fab' fragment" refers to an antibody fragment containing one light chain and a portion or fragment of one heavy chain that also contains the VH domain, the CH1 domain, and the region between the CH1 and CH2 domains, so that an interchain disulfide bond can be formed between the two heavy chains of the two Fab' fragments to form an F(ab')2 molecule. An "F(ab')2 fragment" refers to an antibody fragment containing two light chains and two heavy chains that contain a portion of the constant region between the CH1 and CH2 domains, so that an interchain disulfide bond is formed between these two heavy chains. Therefore, an F(ab')2 fragment consists of two Fab' fragments linked by a disulfide bond between the two heavy chains. An "F(ab')2 fragment" can be the product of pepsin cleavage of an antibody. An "Fv fragment" or "Fv region" refers to an antibody fragment that contains variable regions from both the heavy and light chains but lacks a constant region.

[0093] Single strand FvThe terms "single-chain Fv" antibody or "scFv" antibody refer to an antibody fragment that includes a VH domain and a VL domain, where these domains are present in a single polypeptide chain. Generally, an Fv polypeptide further includes a polypeptide linker between the VH and VL domains, enabling the scFv to adopt a structure desirable for antigen binding. For a review of scFv, see Pluckthun. 1994. Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds. Springer-Verlag, New York, pp. 269-315. See also International Publication No. WO 88 / 01649 and U.S. Patent Nos. 4,946,778 and 5,260,203. In one embodiment, the scFv includes a VH region, a peptide linker, and a VL region, from the N-terminus to the C-terminus. In another embodiment, scFv includes a VL region, a peptide linker, and a VH region, extending from the N-terminus to the C-terminus.

[0094] Fab A "Fab" consists of a heavy chain VH and CH1 region and a light chain VL and CL region, typically joined together by a disulfide bond, and possessing a single antigen-binding site. The VH, CH1, VL, and CL regions in the Fab can be arranged in various ways to confer the antigen-binding ability of this disclosure. For example, the VH and CH1 regions may be on one polypeptide, while the VL and CL regions may be on different polypeptides. Alternatively, the VH, CH1, VL, and CL regions may all be on the same polypeptide, in any different order.

[0095] DiabodyThis disclosure includes diabodies and methods of use thereof. As used herein, the term “diabody” or “diabodies” refers to a small antibody fragment having two antigen-binding sites, the fragment containing a heavy-chain variable domain (VH) (VH-VL or VL-VH) linked to a light-chain variable domain (VL) on the same polypeptide chain. By using a linker that is too short to allow pairing between two domains on the same chain, the domains are forced to pair with a complementary domain on another chain to create two antigen-binding sites. Diabodies are described in detail, for example, EP404097, WO 93 / 11161, and Holliger et al. 1993. Proc Natl Acad Sci USA 90:6444-6448. For a review of manipulated antibody variants, see Holliger and Hudson. 2005. Nat Biotechnol. 23:1126-1136. Domain antibodies, also known as "dAbs" (the terms "Domain Antibodies" and "dAbs" are trademarks used by the GlaxoSmithKline corporate group), are described in detail in Ward, ES et al. 1989. "Binding activities of a repertoire of single immunoglobulin variable domains secreted from Escherichia coli." Nature 341:544-546, Holt, LJ et al. 2003. "Domain antibodies: proteins for therapy." Trends in Biotechnology 21(11):484-490, and WO2003 / 002609. Domain antibodies essentially correspond to the VH or VL domains of non-camel mammalian (especially human) four-chain antibodies. To bind an epitope as a single antigen-binding domain, that is, without being paired with a VH domain or VL domain, specific selection of such antigen-binding properties is necessary, for example, by using a library of human single VH or VL domain sequences.Domain antibodies, such as VHH, have a molecular weight of approximately 13-16 kDa and, if derived from a complete human sequence, do not require humanization for purposes such as therapeutic use in humans. H Similar to the H domain, these are well expressed in prokaryotic expression systems, resulting in a significant reduction in overall manufacturing costs. Domain antibodies, along with the VHH domain, can be subjected to affinity maturation by introducing one or more modifications to the amino acid sequence of one or more CDRs. These modifications result in improved affinity of the resulting immunoglobulin single-chain variable domains to their respective antigens compared to their respective parent molecules. The affinity-matured immunoglobulin single variable domain molecule of the present invention can be prepared by methods known in the art, for example, as described in Marks et al. 1992. Biotechnology 10:779-783, or Barbas et al. 1994. Proc Nat Acad Sci. USA 91:3809-3813, Shier et al. 1995. Gene 169:147-155, Yelton et al. 1995. Immunol. 155:1994-2004, Jackson et al. 1995. J Immunol. 154(7):3310-9, and Hawkins et al. 1992. J Mol Biol. 226(3):889896, and KS Johnson and RE Hawkins. 1996. "Affinity maturation of antibodies using phage display," Oxford University Press.

[0096] bispecific antibodyThe “bispecific antibody” of this disclosure comprises an antigen-binding arm comprising a heavy-chain variable region and a light-chain variable region of either the antibody or its antigen-binding fragment described in the claims, and another antigen-binding arm that recognizes a different antigen. In one embodiment, the bispecific antibody is a heterodimer of an antigen-binding arm comprising a heavy-chain and a light-chain, and another antigen-binding arm comprising a heavy-chain and a light-chain that binds to a different antigen. These two antigen-binding arms associate to form a heterodimer via two heavy-chain constant regions having mutations in the CH3 region (see, for example, Figure X). The “multispecific antibody” comprises a bispecific antibody and further comprises an additional antigen-binding arm comprising a heavy-chain variable region and a light-chain variable region that targets at least one other antigen.

[0097] binding molecule As used herein, the term “binding molecule” refers to a bivalent molecule capable of binding to the extracellular domains of two cell surface receptors. In some embodiments, the binding molecule specifically binds to two different receptors (or their domains or subunits), resulting in the receptors (or domains or subunits) being kept in close proximity to each other, and consequently, the receptors (or domains or subunits), including their domains (e.g., intracellular domains), interact with each other to result in downstream signaling.

[0098] comparableAs used herein, the term “comparable” is used to describe the degree of difference between two measurements of an evaluable quantitative or qualitative parameter. For example, two measurements of an evaluable quantitative parameter and a second measurement of an evaluable parameter would be considered “comparable” if they do not deviate by a degree that a person skilled in the art would recognize as not producing a factually statistically significant difference between the two results under those circumstances. In some cases, measurements may be considered “comparable” if the deviation between one measurement and another is less than 30%, alternatively less than 25%, alternatively less than 20%, alternatively less than 15%, alternatively less than 10%, alternatively less than 7%, alternatively less than 5%, alternatively less than 4%, alternatively less than 3%, alternatively less than 2%, or alternatively less than 1%. In certain embodiments, a measurement is comparable to a reference standard if its deviation from that standard is less than 15%, alternatively less than 10%, or alternatively less than 5%. The term "comparable" is also used to describe the properties of chemical or biological entities that have similar or equivalent biological activity, function, or results.

[0099] conservative substitution As used herein, the terms “conservative amino acid substitution,” “conservative modified variant,” “conservative variant,” “functionally conserved variant,” or “conservative amino acid substitution” refer to the substitution of an amino acid in a protein with another amino acid having similar characteristics (e.g., charge, side chain size, hydrophobic / hydrophilicity, back chain conformation and stiffness) so that the modification can often be made without altering the biological activity of the protein. Those skilled in the art generally recognize that a single amino acid substitution in a non-essential region of a polypeptide does not substantially alter its biological activity (e.g., Watson et al. 1987. Molecular Biology of the Gene, 4 th(See ed. The Benjamin / Cummings Pub.Co. p.224). For example, amino acids in each of the following groups can be considered conserved 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. In addition, substitutions of structurally or functionally similar amino acids are less likely to disrupt biological activity. Exemplary conserved amino acid substitutions are shown in Table 1 herein. Typically, any modified antibody, bispecific antibody, or antigen-binding fragment of the present invention retains at least 10% of its binding activity (compared to the parent antibody) when its binding activity is expressed on a molar basis. Preferably, the antibody, bispecific antibody, or antigen-binding fragment of the present invention retains at least 20%, 50%, 70%, 80%, 90%, 95%, or 100% or more of the antigen-targeting affinity as a parent antibody. The antibody, bispecific antibody, or antigen-binding fragment of the present invention is also intended to contain conserved or non-conserved amino acid substitutions that do not substantially alter its biological activity.

[0100] isolatedThis disclosure provides isolated antibodies, antigen-binding fragments, and nucleotides. Where used herein, the term “isolated” means, when used in the context of “isolated nucleic acid molecule” or “isolated polynucleotide,” DNA or RNA of genomic, mRNA, cDNA, or any combination thereof, of synthetic origin, which is not related in whole or in part to any polynucleotides found in nature, or which is linked to a polynucleotide that is not linked in nature. The term “isolated” is also used in reference to a polypeptide of interest that, if it is naturally occurring, is in an environment different from the environment in which it can naturally occur. The term “isolated” encompasses polypeptides in a sample in which the polypeptide of interest is substantially concentrated and / or in a sample in which the polypeptide of interest is partially or substantially purified. If the polypeptide is not naturally occurring, the term “isolated” indicates that the polypeptide has been isolated from the environment in which it was synthesized, for example, isolated from a recombinant cell culture containing cells engineered to express the polypeptide, or isolated from a solution resulting from a solid-phase synthesis means. "Isolated" antibodies or bispecific antibodies or their antigen-binding fragments do not contain, at least partially, other biological molecules derived from the cells or cell cultures in which they are produced. Such biological molecules include nucleic acids, proteins, lipids, carbohydrates, or other materials such as cell debris and growth media. Isolated antibodies or antigen-binding fragments may also be in a state that does not contain, at least partially, components of the expression system, such as biological molecules derived from the host cell or its growth medium. In general, the term "isolated" is not intended to mean the complete absence of such biological molecules, or the absence of water, buffers, or salts, or components of a pharmaceutical formulation containing the antibody or fragment. For the purposes of this disclosure, it should be understood that "nucleic acid molecules containing" a particular nucleotide sequence does not include intact chromosomes.An isolated nucleic acid molecule "containing" a specified nucleic acid sequence may, in addition to the specified sequence, contain up to 10, and even up to 20, or more coding sequences of other proteins or parts or fragments thereof, or functionally linked regulatory sequences that control the expression of the coding region of the listed nucleic acid sequence, and / or vector sequences.

[0101] control array The term "regulatory sequence" refers to a DNA sequence necessary for the expression of a functionally linked coding sequence in a particular host organism. Regulatory sequences suitable for prokaryotes include, for example, promoters, optionally operator sequences, and ribosome binding sites. Eukaryotic cells are known to use promoters, polyadenylation signals, and enhancers.

[0102] Functionally linkedThe term “functionally linked” is used herein to refer to a relationship between molecules arranged in a construct, typically polypeptides or nucleic acids, such that the function of each component molecule is preserved, but the functional linkage can result in a positive or negative modulation of the activity of the individual components of the construct. For example, the functional linkage of polyethylene glycol (PEG) molecules to a wild-type protein may result in a construct in which the biological activity of the protein is reduced compared to the wild-type molecule, but the two are nevertheless considered functionally linked. When the term “functionally linked” is applied to a relationship between multiple nucleic acid sequences encoding different functions, those multiple nucleic acid sequences, when combined as a single nucleic acid molecule, provide a nucleic acid that, for example, when introduced into a cell using recombination techniques, has the ability to result in the transcription and / or translation of a particular nucleic acid sequence in the cell. For example, a nucleic acid sequence encoding a signal sequence can be considered functionally linked to the polypeptide-coding DNA if it results in the expression of a preprotein, thereby promoting the secretion of the polypeptide; a promoter or enhancer can be considered functionally linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site can be considered functionally linked to a coding sequence if it is positioned to facilitate translation. Generally, the term "functionally linked" in relation to nucleic acid molecules means that the linked nucleic acid sequences are contiguous, and in the case of a molecule's secretion leader or associated subdomain, they are contiguous and their read phases are aligned. However, certain gene elements, such as enhancers, may function at a distance and may not need to be contiguous with respect to the sequences in which they exert their effect, but they can still be considered functionally linked.

[0103] cellWhere used herein, the terms “cell,” “cell line,” and “cell culture” are interchangeable, and these terms encompass offspring. Therefore, the terms “transformed” and “transformed cell” encompass both the primary target cell and any cultures derived therefrom, regardless of passage number. It is also understood that, due to intentional or accidental mutations, not all offspring will have strictly identical DNA contents. Mutant offspring possessing the same function or biological activity as those selected in the initially transformed cells are included. Where a clear designation is intended, it will be evident from the context.

[0104] ~ derived from As used herein, the term "derived from" in the context of an amino acid sequence or polynucleotide sequence (e.g., an amino acid sequence "derived from") means that a polypeptide or nucleic acid has a sequence based on a sequence of a reference polypeptide or nucleic acid (e.g., a naturally occurring polypeptide or coding nucleic acid), and is not limited to the source or how the protein or nucleic acid was made. For example, the term "derived from" includes homologs or variants of a reference amino acid or DNA sequence.

[0105] Effective concentration Where used herein, the term “effective concentration” or its abbreviation “EC” is used interchangeably to refer to the concentration of an active substance (e.g., anti-IL-10Rα-IL-10Rβ VHH dimer IL2 mutein) sufficient to produce a change in a given parameter in the test system. The abbreviation “E” refers to the magnitude of a given biological effect observed in the test system when the test system is exposed to the test substance. The abbreviation “EC” is used when the magnitude of the response is expressed as a factor of the concentration of the test substance ("C"). In the context of biological systems, the term Emax refers to the maximum magnitude of a given biological effect observed in response to the saturation concentration of the activated test substance. When the abbreviation EC is followed by a subscript (e.g., EC 40 , EC 50(e.g., the subscript indicates the percentage of the biological effect observed at that concentration relative to Emax. For example, a concentration of such a test substance sufficient to result in the induction of such a measurable biological parameter in a test system is 30% of the maximum level of the measurable biological parameter in response to the test substance. 30 It is said that "EC 100 The term "effective concentration" is used to mean the effective concentration of such an active substance that results in the maximum (100%) response of a measurable parameter in response to the active substance. Similarly, EC 50 The term "saturation concentration" (commonly used in the field of pharmacodynamics) refers to the concentration of an active substance sufficient to result in a maximum half-volume (50%) change in a measurable parameter. The term "saturation concentration" refers to the maximum possible amount of a test substance that can be dissolved in a standard volume of a particular solvent (e.g., water) under standard temperature and pressure conditions. In pharmacodynamics, the saturation concentration of a drug is typically used to mean a drug concentration sufficient to occupy all available receptors, and EC 50 This is the drug concentration required to produce the maximum half-dose effect. The EC of a specific effective concentration of a test substance may be omitted for certain parameters and test systems.

[0106] enriched As used herein, the term “enriched” means a sample that has been unnaturally manipulated so that the species of interest (e.g., molecule or cell) is present at a higher concentration (e.g., at least 3 times higher, alternatively at least 5 times higher, alternatively at least 10 times higher, alternatively at least 50 times higher, alternatively at least 100 times higher, or alternatively at least 1000 times higher) than the concentration of the species in the starting sample, e.g., a biological sample (e.g., a sample in which the molecule is naturally present or present after administration); or (b) a sample in which the molecule is present at a higher concentration than the environment in which the molecule was created (e.g., recombinantly modified bacteria or mammalian cells).

[0107] Extracellular domainAs used herein, the term “extracellular domain” or its abbreviation “ECD” refers to the portion of a cell surface protein (e.g., a cell surface receptor) located outside the plasma membrane of a cell. The term “ECD” may include the extracellular portion of a transmembrane protein or the extracellular portion of a cell surface (or membrane-bound protein).

[0108] Percent IdentityWhere used herein, the terms “percent (%) sequence identity” or “substantially identical” in the context of nucleic acids or polypeptides mean a sequence that has at least 50% sequence identity with a reference sequence. Alternatively, percent sequence identity can be any integer from 50% to 100%. In some embodiments, a sequence has at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with a reference sequence when determined by BLAST using standard parameters, as described below. In sequence comparison, typically one sequence acts as the reference sequence, and the test sequence is compared against it. When using a sequence comparison algorithm, the test sequence and reference sequence are entered into a computer, the coordinates of subsequences are specified as needed, and the program parameters for the sequence algorithm are specified. Default program parameters may be used, or different parameters may be specified. The sequence comparison algorithm then calculates the percentage sequence identity of the test sequence to the reference sequence based on program parameters. The comparison window includes a reference to any one segment of any number of consecutive positions, e.g., a segment of at least 10 residues. In some embodiments, the comparison window may be 10 to 600 residues, e.g., about 10 to about 30 residues, about 10 to about 20 residues, about 50 to about 200 residues, or about 100 to about 150 residues, within which the two sequences may be optimally aligned before being compared to the reference sequence at the same number of consecutive positions. Suitable algorithms for determining the percentage of sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms described in Altschul et al. 1990. J Mol Biol. 215:403-410 and Altschul et al. 1977. Nucleic Acids Res. 25:3389-3402, respectively. Software for performing BLAST analysis is publicly available through the website of the National Center for Biotechnology Information (NCBI).This algorithm first identifies high-scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence that match when aligned with words of the same length in the database sequence or satisfy a certain positive threshold score T, where T is called the adjacent word score threshold (Altschul et al., see above). This initial adjacent word hit acts as a seed to initiate a search for longer HSPs containing them. Then, word hits are extended in both directions for each sequence as long as the sum of the alignment scores can be increased. For nucleotide sequences, the sum of scores is calculated using parameters M (reward score for matching residue pairs; always > 0) and N (penalty score for mismatched residues; always < 0). For amino acid sequences, a scoring matrix is ​​used to calculate the sum of scores. The extension of word hits in each direction is stopped when the sum of the alignment scores decreases by amount X from its maximum achieved value; when the sum of the scores becomes zero or less due to the accumulation of alignments of one or more negative-scoring residues; or when the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of alignment. The BLASTN program (for nucleotide sequences) functions similarly, but uses a word size (W) of 28, an expected value (E) of 10, M=1, N=-2, and comparison of both strands as defaults. For amino acid sequences, the BLASTP program uses a word size (W) of 3, an expected value (E) of 10, and the BLOSUM62 scoring matrix as defaults (see Henikoff & Henikoff. 1989. Proc. Natl Acad Sci. USA 89:10915.). The BLAST algorithm also performs statistical analysis of the similarity between two sequences (see, for example, Karlin & Altschul. 1993. Proc Nat'l Acad Sci. USA 90:5873-5787).One measure of similarity provided by the BLAST algorithm is the minimum sum probability (P(N)), which provides an indicator of the probability that a match between two nucleotide or amino acid sequences occurs by chance. For example, for amino acid sequences, the minimum sum probability in the comparison between a test amino acid sequence and a reference amino acid sequence is less than about 0.01, more preferably about 10. -5 Less than, most preferably about 10 -20 If it is less than [a certain value], it is considered similar to the reference array.

[0109] human percentageIn relation to the degree to which the amino acid sequence of an IL-10 agonist compound is modified to be more human-like, the terms “human percentage,” “percent humanization,” or “humanized” refer to the percentage of amino acid residues in the target polypeptide sequence that are identical to a closely corresponding naturally occurring human polypeptide reference sequence, such as a similar human germline sequence. For example, as used herein with respect to polypeptide sequences such as sdAb, scFv, or VHH sequences, “human percentage” refers to the percentage of amino acid residues in the sdAb, scFv, or VHH amino acid sequence that are identical to a closely corresponding human germline reference sequence. There are many commonly used human germline sequences that can be selected as reference sequences for determining the “percent humanization.” The "humanization percentage" of the anti-IL-10RαVHH antibody sequences disclosed herein is the percentage of anti-IL-10RαVHH amino acid residues that are identical to the amino acid sequence of the closely corresponding germline sequence V3-23 (UniProt immunoglobulin heavy chain variable sequence 3-23, entry number P01764) (i.e., the ratio of the number of identical amino acid residues to the total number of amino acid residues). The "humanization percentage" of the anti-IL-10Rβ VHH sequences disclosed herein is the percentage of anti-IL-10RαVHH amino acid residues that are identical to the amino acid sequence of the closely corresponding germline sequence VH3-66 (UniProt immunoglobulin heavy chain variable sequence 3-66, entry number A0A0C4DH42) (i.e., the ratio of the number of identical amino acid residues to the total number of amino acid residues). For the purpose of calculating the humanization percentage, the V segment of a selected human germline sequence (V3-23 or VH3-66 above) is used as the reference germline sequence by comparison with the reference germline sequence of the IL-10Rα and IL-10Rβ humanization framework regions, including CDR1 and CDR2.

[0110] Intracellular signal transductionAs used herein, the terms “intracellular signaling” and “downstream signaling” are used interchangeably to refer to cellular signaling processes triggered by the interaction of two or more intracellular domains (ICDs) of cell surface receptors that are in close proximity to each other. In receptor complexes via the JAK / STAT pathway, the association of ICDS of receptor subunits brings the JAK domains of the ICDs into close proximity, causing the STAT molecule to be phosphorylated and translocate to the nucleus, where it associates with a specific nucleic acid sequence, initiating a phosphorylation cascade that results in the activation and expression of a specific gene within the cell. The IL-10 agonist compounds of this disclosure (e.g., single-domain antibody polypeptides of IL-10 agonist compounds), when activated by their native congener IL-10, provide intracellular signaling characteristic of the IL-10 receptor. Several methods are available to measure downstream signaling activity. For example, in some embodiments, JAK / STAT signaling can be measured by the presence of phosphorylated receptors and / or phosphorylated STAT. In other embodiments, the expression of one or more downstream genes, whose expression levels may be influenced by the level of downstream signaling triggered by binding molecules, can also be measured.

[0111] In sufficient quantity to elicit a responseWhere used herein, the phrase “in sufficient quantity to produce a response” is used to mean an amount of the test substance sufficient to provide a detectable change in the level of an indicator measured before (e.g., at baseline level) and after the application of the test substance to the test system. In some embodiments, the test system is a cell, tissue, or organism. In some embodiments, the test system is an in vitro test system, e.g., a fluorescence assay. In some embodiments, the test system is an in vivo system involving the measurement of changes in the level of parameters of a cell, tissue, or organism that reflect the biological function of the cell, tissue, or organism before and after the application of the test substance to the cell, tissue, or organism. In some embodiments, the indicator reflects the biological function or developmental state of a cell evaluated in the assay in response to the administration of a certain amount of the test substance. In some embodiments, the test system involves the measurement of changes in the level of an indicator of a cell, tissue, or organism that reflects the biological state of the cell, tissue, or organism before and after the application of one or more test substances to the cell, tissue, or organism. The term “in sufficient quantity to produce a response” may be sufficient to be a therapeutically effective dose, but may be more or less than a therapeutically effective dose.

[0112] Treatment is required Where used herein, the term “requiring treatment” refers to a determination made by a physician or other caregiver regarding a subject that the subject requires treatment or would potentially benefit from treatment. This determination is based on a variety of factors within the scope of the physician's or caregiver's expertise.

[0113] LigandAs used herein, the term “ligand” means a molecule that exhibits specific binding to a receptor and causes a change in the biological activity of the receptor to which it binds, resulting in a change in the activity of the receptor to which it binds. In one embodiment, the term “ligand” means a molecule or complex thereof that can act as an agonist or antagonist of a receptor. As used herein, the term “ligand” includes both natural and synthetic ligands. “Ligands” also include small molecules, such as peptide mimetic molecules of cytokines and peptide mimetic molecules of antibodies. A ligand-receptor complex is referred to as a “ligand-receptor complex.”

[0114] Joined As used herein, the term “conjugated” means that two elements, for example, two protein domains, are conjugated together or otherwise in a stable associated state with one another. Two proteins may be “conjugated” directly, with the C-terminus of one protein domain covalently bonded to the N-terminus of the second protein domain. Alternatively, two proteins may be “conjugated” using a linker polypeptide or other chemical linker compound.

[0115] Linker As used herein, the term “linker” refers to a link between two elements, such as protein domains. A linker may be a covalent bond or a peptide linker. The term “bond” refers to any type of bond created by a chemical bond, such as an amide bond or a disulfide bond, or by a chemical reaction, such as a chemical conjugation. The term “peptide linker” refers to an amino acid or polypeptide that can be used to link two protein domains to provide space and / or flexibility between them.

[0116] ModulateAs used herein, terms such as “modulate” and “modulation” refer to the ability of a test agent to directly or indirectly produce a positive or negative response in a system or biochemical pathway, including biological systems. The term modulator encompasses both agonists (including partial agonists, full agonists, and superagonists) and antagonists.

[0117] Multimerization As used herein, the term "multimerization" refers to bringing two or more cell surface receptors or their domains or subunits into close proximity so that the receptors or their domains or subunits can interact with each other to trigger intracellular signaling.

[0118] N-terminus and C-terminus Where used herein in relation to the structure of a polypeptide, “N-terminus” (or “amino-terminus”) and “C-terminus” (or “carboxyl-terminus”) refer to the outermost (last) amino-terminus and carboxyl-terminus of the polypeptide, respectively, while the terms “N-terminal” and “C-terminal” refer to the relative positions in the amino acid sequence of the polypeptide that are near or toward the N-terminus and C-terminus, respectively, and may include residues at or near the N-terminus and C-terminus, respectively. “Immediately N-terminal” refers to the relative position of a first amino acid residue in a consecutive polypeptide sequence relative to a second amino acid residue, where the first and second amino acid residues are covalently bonded, resulting in a consecutive amino acid sequence, with the first amino acid being closer to the N-terminus of the polypeptide. “Immediately C-terminal” refers to the relative position of a first amino acid residue in a consecutive polypeptide sequence relative to a second amino acid residue, where the first amino acid is closer to the C-terminus of the polypeptide.

[0119] neoplastic diseaseWhere used herein, the term “neoplastic disease” refers to a disorder or condition in an object resulting from excessive cell proliferation or unregulated (or dysregulated) cell replication, as will be discussed in more detail below. The term “neoplastic disease” refers to a disorder resulting from the presence of a neoplasm in an object. Neoplasms may be classified as (1) benign, (2) premalignant (or “precancerous”), and (3) malignant (or “cancerous”). The term “neoplastic disease” includes neoplasm-related diseases, disorders, and conditions that are directly or indirectly associated with neoplastic disease, such as angiogenesis and precancerous conditions, e.g., dysplasia or smoldering multiple myeloma. Examples of benign disorders resulting from dysregulated cell replication include hypertrophic scars, e.g., keloid scars.

[0120] nucleic acid The terms “nucleic acid,” “nucleic acid molecule,” “polynucleotide,” “nucleotide,” and the abbreviation “nt,” etc., are used interchangeably herein and refer to polymeric forms of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof. Non-exclusive examples of polynucleotides include linear and cyclic nucleic acids, messenger RNA (mRNA), complementary DNA (cDNA), recombinant polynucleotides, vectors, probes, primers, and the like.

[0121] Partial agonistAs used herein, the term “partial agonist” refers to a molecule that specifically binds to and activates a given receptor, but only partially activates the receptor compared to a full agonist. Partial agonists may exhibit both agonist and antagonist effects. For example, in the presence of both a full agonist and a partial agonist, the partial agonist acts as a competitive antagonist by competing with the full agonist for binding to the receptor, resulting in a net reduction in receptor activation compared to contact between the receptor and the full agonist in the absence of the partial agonist. Clinically, partial agonists can be used to activate a receptor in the presence of an insufficient amount of endogenous ligand to produce a desired submaximal response, or they can reduce receptor overstimulation in the presence of an excess amount of endogenous ligand. The maximum response (Emax) produced by a partial agonist is called its endogenous activity and may be expressed on a percentage scale compared to the 100% response produced by a full agonist. In some embodiments, an IL-10 agonist compound (e.g., a single-domain antibody polypeptide of an IL-10 agonist compound) is used to counteract the effects of IL-10. max E reduced compared to max It has. E max This reflects the maximum response level in a cell type that can be obtained by a ligand (e.g., a binding molecule described herein or a native cytokine (e.g., IL-10)). In some embodiments, the IL-10 agonist compounds described herein (e.g., a single-domain antibody polypeptide of an IL-10 agonist compound) are used to evoke E induced by IL-10. maxIt has at least 1% (for example, 1%-100%, 10%-100%, 20%-100%, 30%-100%, 40%-100%, 50%-100%, 60%-100%, 70%-100%, 80%-100%, 90%-100%, 1%-90%, 1%-80%, 1%-70%, 1%-60%, 1%-50%, 1%-40%, 1%-30%, 1%-20%, or 1%-10%). In other embodiments, the E of an IL-10 agonist compound as described herein (e.g., a single-domain antibody polypeptide of an IL-10 agonist compound) max E is the natural ligand IL-10 max Larger than (for example, at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% larger). In some embodiments, the linker length of the IL-10 agonist compound (e.g., a single-domain antibody polypeptide of the IL-10 agonist compound) is changed by altering the linker length of the IL-10 agonist compound (e.g., a single-domain antibody polypeptide of the IL-10 agonist compound). max This can alter the IL-10 agonist compound (e.g., a single-domain antibody polypeptide of an IL-10 agonist compound) in the most desired cell type. max This results in reduced E in other cell types. max It could lead to this.

[0122] Polypeptide:As used herein, the terms “polypeptide,” “peptide,” and “protein” are interchangeable to mean polymeric forms of amino acids of any length, which may include genetically encoded amino acids and genetically unencoded amino acids or amino acid sequences, chemically or biochemically modified or derivatized amino acids, and polypeptides having a modified polypeptide backbone. The term polypeptide includes, but is not limited to, fusion proteins having heterologous amino acid sequences; fusion proteins having heterologous and homologous leader sequences; fusion proteins having or not having an N-terminal methionine residue; fusion proteins with amino acid sequences that facilitate purification as chelate peptides; fusion proteins with immunologically tagged proteins; and fusion proteins containing peptides with immunologically active polypeptide fragments (e.g., antigenic diphtheria or tetanus toxin or toxoid fragments).

[0123] Prevent As used herein, the terms “prevent,” “prevent,” and “prevention” generally refer to actions initiated with respect to an object predisposed to a particular disease, disorder, or condition due to genetic, empirical, or environmental factors, to temporarily or permanently prevent, suppress, inhibit, or reduce the risk of the object developing the disease, disorder, condition, or other (determined, for example, by the absence of clinical symptoms), or to delay their onset. In certain specific cases, the terms “prevent,” “prevent,” and “prevention” may also be used to mean slowing the progression of a disease, disorder, or condition from its current state to a more harmful state.

[0124] Close proximityAs used herein, the term “proximity” means the spatial proximity or physical distance between two cell surface receptors, or their domains or subunits, after the binding molecule described herein has bound to those two cell surface receptors, or their domains or subunits. In some embodiments, the spatial proximity between cell surface receptors, or their domains or subunits, after the binding molecule has bound to those cell surface receptors, or their domains or subunits, may be, for example, less than about 500 angstroms, for example, a distance of about 5 angstroms to about 500 angstroms. In some embodiments, spatial proximity is less than approximately 5 angstroms, less than approximately 20 angstroms, less than approximately 50 angstroms, less than approximately 75 angstroms, less than approximately 100 angstroms, less than approximately 150 angstroms, less than approximately 250 angstroms, less than approximately 300 angstroms, less than approximately 350 angstroms, less than approximately 400 angstroms, less than approximately 450 angstroms, or less than approximately 500 angstroms. In some embodiments, spatial proximity is less than approximately 100 angstroms. In some embodiments, spatial proximity is less than approximately 50 angstroms. In some embodiments, spatial proximity is less than approximately 20 angstroms. In some embodiments, spatial proximity is less than approximately 10 angstroms. In some embodiments, spatial proximity extends to approximately 10–100 angstroms, approximately 50–150 angstroms, approximately 100–200 angstroms, approximately 150–250 angstroms, approximately 200–300 angstroms, approximately 250–350 angstroms, approximately 300–400 angstroms, approximately 350–450 angstroms, or approximately 400–500 angstroms. In some embodiments, spatial proximity is less than approximately 250 angstroms, alternatively less than approximately 200 angstroms, alternatively less than approximately 150 angstroms, alternatively less than approximately 120 angstroms, alternatively less than approximately 100 angstroms, alternatively less than approximately 80 angstroms, alternatively less than approximately 70 angstroms, or alternatively less than approximately 50 angstroms.

[0125] receptor As used herein, the term “receptor” means a polypeptide having a ligand-specific binding domain, the binding of which results in a change in at least one biological property of the polypeptide. In some embodiments, the receptor is a “soluble” receptor, not associated with the cell surface. In some embodiments, the receptor is a cell surface receptor comprising an extracellular domain (ECD) and a membrane-associated domain that fixes the ECD to the cell surface. In some embodiments of cell surface receptors, the receptor is a transmembrane polypeptide comprising an intracellular domain (ICD) and an extracellular domain (ECD) linked by a transmembrane domain, typically called a transmembrane domain. The binding of a ligand to a receptor results in a conformational change in the receptor, resulting in a measurable biological effect. In some cases, where the receptor is a transmembrane polypeptide comprising an ECD, TM, and ICD, the binding of the ligand to the ECD results in a measurable intracellular biological effect mediated by one or more domains of the ICD in response to the ligand's binding to the ECD. In some embodiments, the receptor is a component of a multicomponent complex for promoting intracellular signaling. For example, a ligand can bind to a cell surface molecule that is not associated with any intracellular signaling pathway in isolation, but once the ligand binds, it promotes the formation of a multimeric complex that leads to intracellular signaling.

[0126] RecombinantWhere used herein, the term “recombinant” is used as an adjective to describe a polypeptide, nucleic acid, or a method by which a cell has been modified using recombinant DNA technology. Recombinant proteins are proteins produced using recombinant DNA technology and may be denoted as such (e.g., rhIL-10) using the lowercase letter “r” to indicate the method by which the protein was produced. Similarly, if a cell has been modified using recombinant DNA technology by the incorporation of an exogenous nucleic acid (e.g., ssDNA, dsDNA, ssRNA, dsRNA, mRNA, viral or nonviral vector, plasmid, cosmid, etc.) (e.g., transfection, transduction, infection), then the cell is called a “recombinant cell.” Techniques and protocols for recombinant DNA technology are well known in the art, such as those found in Sambrook et al. 1989. Molecular Cloning: A Laboratory Manual (2nd ed., Cold Spring Harbor Laboratory Press, Plainview, NY) and other standard molecular biology laboratory manuals.

[0127] responseFor example, the term “response” of a cell, tissue, organ, or organism encompasses quantitative or qualitative changes in evaluable biochemical or physiological parameters (e.g., concentration, density, adhesion, proliferation, activation, phosphorylation, migration, enzyme activity, gene expression level, gene expression rate, energy expenditure rate, level or state of differentiation) that correlate with activation, stimulation, or treatment, contact with exogenous agents, or internal mechanisms, such as gene programming. In certain contexts, terms such as “activation” and “stimulation” refer to cellular activation regulated by external or environmental factors in addition to internal mechanisms; while terms such as “inhibition” and “downregulation” refer to the opposite effect. “Response” may be evaluated in vitro, for example, through the use of assay systems, surface plasmon resonance, enzyme activity, mass spectrometry, and amino acid or protein sequencing techniques. The “response” may be assessed in vivo quantitatively by evaluation of objective physiological parameters, such as body temperature, body weight, tumor volume, blood pressure, X-ray or other imaging techniques, or qualitatively through changes in reported subjective sensations of well-being, depression, agitation, or pain. In some embodiments, the level of CD3-activated primary human T cell proliferation may be assessed in a bioluminescence assay that generates a luminescence signal proportional to the amount of ATP present, directly proportional to the number of viable cells present in the culture, as described in Crouch et al. 1993. J Immunol Methods 160:81-8, or by using commercially available assays, such as the CellTiter-Glo® 2.0 Cell Viability Assay or CellTiter-Glo® 3D Cell Viability kits, commercially available from Promega Corporation, Madison WI 53711, as catalog numbers G9241 and G9681, substantially in accordance with the instructions provided by the producer.In some embodiments, the level of T cell activation in response to administration of a test substance may be determined by a described flow cytometry method, such that it is determined by the level of STAT (e.g., STAT1, STAT3, STAT5) phosphorylation according to methods well known in the art.

[0128] Significantly reduced binding As used herein, the term “significantly reduced binding” is used in reference to a variant of a first molecule that exhibits a significant reduction in affinity to a second molecule (e.g., a receptor) compared to the parental form of the first molecule (e.g., a ligand). With respect to an antibody variant, an antibody variant is said to “exhibit significantly reduced binding” if the variant binds to the native form of the receptor with an affinity of less than 20%, alternatively less than 10%, alternatively less than 8%, alternatively less than 6%, alternatively less than 4%, alternatively less than 2%, alternatively less than 1%, or alternatively less than 0.5% of the affinity of the parental antibody from which the variant originated. Similarly, with respect to variant ligands, a variant ligand exhibits "significantly reduced binding" when it binds to the receptor with an affinity of less than 20%, alternatively less than 10%, alternatively less than 8%, alternatively less than 6%, alternatively less than 4%, alternatively less than 2%, alternatively less than 1%, or alternatively less than 0.5% of that of the parent ligand from which the variant ligand originates. Similarly, with respect to variant receptors, a variant ligand exhibits "significantly reduced binding" when it binds to the variant receptor with an affinity of less than 20%, alternatively less than 10%, alternatively less than 8%, alternatively less than 6%, alternatively less than 4%, alternatively less than 2%, alternatively less than 1%, or alternatively less than 0.5% of that of the parent receptor from which the variant receptor originates.

[0129] Specific bindingAs used herein, the term “specifically binds” refers to the degree of affinity that a first molecule exhibits with respect to a second molecule. In the context of binding pairs (e.g., ligand / receptor, antibody / antigen), a first molecule of a binding pair is said to bind specifically to a second molecule of the binding pair if the first molecule of the binding pair does not bind in significant amounts to other components present in the sample. A first molecule of a binding pair is said to bind specifically to a second molecule of the binding pair if its affinity for the second molecule is at least twice, alternatively at least five times, alternatively at least ten times, alternatively at least twenty times, or alternatively at least one hundred times higher than its affinity for other components present in the sample. In certain embodiments, when the first molecule of a binding pair is an antibody, for example, when determined by scatchard analysis (Munsen. et al. 1980. Analyt Biochem. 107:220-239), the equilibrium dissociation constant between the antibody and the antigen (or the antigenic determinant (epitope) of a protein, antigen, ligand, or receptor) is approximately 10 -6 Higher than M, alternatively about 10 -8 Higher than M, alternatively about 10 -10 Higher than M, alternatively about 10 -11 Higher than M, about 10 -12 When M is higher, the antibody specifically binds to the antigen. In one embodiment, dissociation occurs at approximately 10 5 Higher than M, alternatively about 10 6 Higher than M, alternatively about 10 7 Higher than M, alternatively about 10 8 Higher than M, alternatively about 10 9 Higher than M, alternatively about 10 10 Higher than M, or alternatively around 10 11When M is higher than M, the ligand binds specifically to the receptor. Specific binding may be evaluated using techniques known in the art, including, but not limited to, competitive ELISA assays, radioactive ligand binding assays (e.g., saturated binding, scatchard plots, nonlinear curve fitting programs, and competitive binding assays); non-radioactive ligand binding assays (e.g., fluorescence polarization (FP), fluorescence resonance energy transfer (FRET); liquid-phase ligand binding assays (e.g., real-time polymerase chain reaction (RT-qPCR), and immunoprecipitation); and solid-phase ligand binding assays (e.g., multi-well plate assays, on-bead ligand binding assays, on-column ligand binding assays, and filter assays)), as well as surface plasmon resonance assays using commercially available instruments, e.g., Biacore 8+, Biacore S200, and Biacore T200 (GE Healthcare Bio-Sciences, 100 Results Way, Marlborough MA 01752) (see, e.g., Drescher et al. 2009. Methods Mol Biol. 493:323-343). In some embodiments, the disclosure provides molecules that specifically bind to IL-10R (e.g., IL-10R-binding sdAb). When used herein, the binding affinity of an IL-10 agonist compound to IL-10R may be determined and / or quantified by surface plasmon resonance ("SPR"). In evaluating the binding affinity of an IL-10 agonist compound to IL-10Rα or IL-10Rβ, either member of the binding pair may be immobilized, or the other element of the binding pair may be provided in the mobile phase.In some embodiments, the sensor chip on which the protein of interest is immobilized is conjugated with a substance to facilitate the binding of the protein of interest, which is, for example, a nitrilotriacetate (NTA) derivatized surface plasmon resonance sensor chip (e.g., Sensor Chip NTA, available from Cytiva Global Life Science Solutions USA LLC, Marlborough MA, catalog number BR100407), an anti-His tag antibody (e.g., anti-histidine CM5 chip, commercially available from Cytiva, Marlborough MA), protein A, or biotin. Consequently, to evaluate binding, it is often necessary to modify the protein to provide binding to the substance conjugated on the surface of the chip. For example, one member of a binding pair is evaluated by incorporating a chelated peptide containing a polyhistidine sequence (e.g., 6xHis or 8xHis) for retention of NTA on the conjugated chip. In some embodiments, the binding molecule may be immobilized on the chip, and the receptor subunit (or its ECD fragment) is present in the mobile phase. Alternatively, the receptor subunit (or its ECD fragment) may be immobilized on a chip, and the binding molecule may be provided in the mobile phase. In either situation, it should be noted that modifications of certain proteins for immobilization on a coated SPR chip may interfere with the binding properties of one or both components of the binding pair to be evaluated by SPR. In such cases, it may be necessary to switch the mobile and binding elements of the binding pair, or to use a chip with a binder that promotes non-interference with the conjugation of the protein to be evaluated. Alternatively, when evaluating the binding affinity of a binding molecule to a receptor subunit using SPR, the binding molecule may be derivatized by C-terminal addition of a polyHis sequence (e.g., 6xHis or 8xHis) and immobilized on an NTA derivatization sensor chip, and the receptor subunit for which binding affinity is being evaluated is provided in the mobile phase. Means for incorporating polyHis sequences into the C-terminus of binding molecules produced by recombinant DNA technology are well known to those skilled in the art of biotechnology.

[0130] recipient, subject, individual, or patient The terms “recipient,” “individual,” “subject,” and “patient” are used interchangeably herein and refer to any mammalian subject, in particular humans, to whom diagnosis, treatment, or therapy is desired. “Mammal” for treatment purposes refers to any animal classified as a mammal, including humans, livestock and farm animals, as well as zoo, sporting, or companion animals, such as dogs, horses, cats, cattle, sheep, goats, and pigs.

[0131] In essence pure As used herein, the term “substantially pure” means that the components of the composition constitute about 50% more, alternatively about 60% more, alternatively about 70% more, alternatively about 80% more, alternatively about 90% more, and alternatively about 95% more of the total content of the composition. “Substantially pure” protein constitutes about 50% more, alternatively about 60% more, alternatively about 70% more, alternatively about 80% more, alternatively about 90% more, and alternatively about 95% more of the total content of the composition.

[0132] To suffer from ~ As used herein, the term “suffering from” refers to a decision made by a physician regarding a subject based on available information acceptable in the field of identifying a disease, disorder, or condition, including but not limited to X-ray, CT scan, conventional laboratory diagnostic tests (e.g., blood count), genomic data, protein expression data, and immunohistochemistry, that the subject requires or would benefit from treatment. The term “suffering from” is typically used in relation to a specific disease condition; for example, “suffering from a neoplastic disease” refers to a subject diagnosed with the presence of a neoplasm.

[0133] T cellsAs used herein, the terms "T cell" or "T-cell" are used in their conventional sense and refer to lymphocytes that differentiate in the thymus, possess specific cell surface antigen receptors, and control the initiation or suppression of cell-mediated and humoral immunity, as well as those that lyse antigen-carrying cells. In some embodiments, T cells include naive CD8 + T cells, cytotoxic CD8 + T cells, naive CD4 + T cells, helper T cells, for example T H 1. T H 2, T H 9, T H 11, T H 22, T FH ; regulatory T cells, e.g. T R 1. Tregs, inducible Tregs; memory T cells, such as central memory T cells, effector memory T cells, NKT cells, tumor-infiltrating lymphocytes (TILs), and engineered variants of such T cells, including but not limited to CAR-T cells, recombinantly modified TILs, and TCR-manipulated cells.

[0134] Therapeutic effective doseWhere used herein, the term “therapeutic dose” is used to refer to a single dose of an active substance administered to a subject that, when administered to the subject, can have any detectable positive effect on any symptom, aspect, or feature of a disease, disorder, or condition, either as part of a series of doses, alone, or as part of a pharmaceutical composition or therapeutic regimen. The therapeutic dose can be determined by measuring the relevant physiological effect, which may be adjusted in relation to the drug regimen and in accordance with diagnostic analysis such as the condition of the subject. Parameters for evaluation to determine the therapeutic dose of an active substance are determined by a physician using diagnostic criteria approved in the art, including but not limited to age, weight, sex, overall health status, ECOG score, observable physiological parameters, blood levels, blood pressure, electrocardiogram, computed tomography, radiography, and other indicators. Alternatively or additionally, other parameters typically evaluated in the clinical context, such as body temperature, heart rate, normalization of blood chemistry, normalization of blood pressure, normalization of cholesterol levels, or any symptoms, phases, or characteristics of the disease, disorder, or condition, modification of biomarker levels, increased survival, extended progression-free survival, extended time to progression, increased time to treatment failure, extended event-free survival, extended time to subsequent treatment, improved response rate, and improved duration of response, may be monitored to determine whether a therapeutically effective dose of the active ingredient was administered to the subject, and these parameters are relied upon by clinicians in the art to assess the improvement in the subject's condition in response to administration of the active ingredient.

[0135] regulatory T cells The terms “regulatory T cells” or “Treg cells” as used herein include, but are not limited to, effector T cells (Teff) and other T cells capable of suppressing the response of CD4 +This refers to a type of T cell. Treg cells are characterized by the expression of CD4, the α-subunit of the IL2 receptor (CD25), and the transcription factor forkheadbox P3 (FOXP3) (Sakaguchi. 2004. Annu Rev Immunol. 22: 531-62). + T cells, other than regulatory T cells, CD4 + This refers to T cells.

[0136] transmembrane domain The terms “transmembrane domain” or “TM” refer to a domain of a transmembrane polypeptide that is embedded in the cell membrane when the transmembrane polypeptide is associated with the cell membrane and is peptidyl-linked to the extracellular domain (ECD) and intracellular domain (ICD) of the transmembrane polypeptide. The transmembrane domain may be homogeneous (naturally associated) or heterogeneous (not naturally associated) with either or both of the extracellular and / or intracellular domains. In some embodiments, if the receptor is a chimeric receptor containing an intracellular domain derived from a first parent receptor and a second extracellular domain derived from a second different parent receptor, the transmembrane domain of the chimeric receptor is the transmembrane domain that is typically associated with either the ICD or ECD of the parent receptor from which the chimeric receptor derives. Alternatively, the transmembrane domain of a receptor may be an artificial amino acid sequence that spans the plasma membrane. In some embodiments, if the receptor is a chimeric receptor comprising an intracellular domain derived from a first parent receptor and a second extracellular domain derived from a second different parent receptor, the transmembrane domain of the chimeric receptor is a transmembrane domain that is typically associated with either the ICD or ECD of the parent receptor from which the chimeric receptor originates.

[0137] TreatTerms such as “to treat,” “to treat,” and “treatment” mean any action initiated with respect to a subject after a disease, disorder, or condition, or its symptoms, has been diagnosed, observed, or otherwise caused by such disease, disorder, or condition, or at least one of the symptoms associated with such disease, disorder, or condition, either temporarily or permanently, or by administering a binding molecule as described herein or a pharmaceutical composition containing such molecule. Treatment includes any action taken with respect to a subject suffering from a disease, such action resulting in an inhibition of the disease in the subject (e.g., halting the progression of the disease, disorder, or condition, or restoring one or more of the symptoms associated with it).

[0138] Wild type As used herein, the terms “wild-type,” “WT,” or “natural” are used to mean an amino acid or nucleotide sequence that is found in nature and has not been modified by human intervention.

[0139] Interleukin-10 receptor agonist compounds This disclosure provides IL-10 agonist compounds that are synthetic ligands for the IL-10 receptor. In some embodiments, the IL-10 agonist compound (e.g., a single-domain antibody polypeptide that is an IL-10 agonist compound) is a humanized IL-10 agonist compound that is a ligand for the human IL-10 receptor.

[0140] The IL-10 agonist compounds of this disclosure (e.g., single-domain antibody polypeptides that are IL-10 agonist compounds) comprise two or more single-domain antibodies (sdAbs) that selectively bind to the extracellular domains of the IL-10Rα receptor subunit and the IL-10Rβ receptor subunit. In one embodiment, the present disclosure provides an IL-10R receptor-binding molecule comprising an IL-10 receptor (IL-10R) agonist compound that binds to the IL-10R receptor, a first single-domain antibody (sdAb) ("IL-10Rα sdAb") that specifically binds to the extracellular domain of the IL-10Rα subunit of the IL-10 receptor, and a second single-domain antibody ("IL-10Rβ sdAb") that specifically binds to the extracellular domain of the IL-10Rβ subunit of the IL-10 receptor, wherein the first sdAb and the second sdAb are in a stable associated state (for example, conjugated by a linker such as a polypeptide linker as described herein, or non-covalently conjugated by the association of the IL-10Rα VHH-Fc polypeptide to the IL-10Rβ VHH-Fc polypeptide). In some embodiments, the IL-10Rα and IL-10Rβ subunits of the IL-10 receptor dimerize in response to contact with an IL-10 agonist compound. When cells expressing IL-10Rα and IL-10Rβ are contacted with an effective amount of an IL-10 agonist compound (e.g., a single-domain antibody polypeptide that is an IL-10 agonist compound), the intracellular domains of IL-10Rα and IL-10Rβ come into close proximity, resulting in intracellular signal transduction.

[0141] The IL-10 agonist compounds of this disclosure (e.g., single-domain antibody polypeptides that are IL-10 agonist compounds) are useful in the treatment or prevention of diseases in mammalian subjects. In some embodiments, this disclosure provides treatment or prevention of autoimmune diseases in mammalian subjects by administration of a therapeutically effective dose of the IL-10 agonist compound of this disclosure. In some embodiments, this disclosure provides treatment or prevention of infectious diseases, including viral infections and chronic viral infections, in mammalian subjects by administration of a therapeutically effective dose of the IL-10 agonist compound of this disclosure. In some embodiments, this disclosure provides treatment or prevention of neoplastic diseases in mammalian subjects by administration of a therapeutically effective dose of the IL-10 agonist compound of this disclosure. In some embodiments, this disclosure provides treatment or prevention of neoplastic, infectious, or autoimmune diseases in mammalian subjects by administration of a therapeutically effective dose of the IL-10 agonist compound of this disclosure in combination with one or more supplemental therapeutic agents.

[0142] In some embodiments, the disclosure provides IL-10 agonist compounds modified to provide an extended duration of action in vivo in mammalian subjects, and pharmaceutically acceptable formulations thereof.

[0143] This disclosure further provides pharmaceutically acceptable formulations of IL-10 agonist compounds for administration to mammalian subjects. This disclosure further provides pharmaceutically acceptable compositions for administration to mammalian subjects, the composition comprising a nucleic acid sequence encoding a polypeptide IL-10 agonist compound, a recombinant viral vector or nonviral vector encoding a polypeptide IL-10 agonist compound, or a recombinantly modified mammalian cell containing a nucleic acid sequence encoding a polypeptide IL-10 agonist compound, wherein in each case the nucleic acid sequence is functionally linked to one or more functional regulatory elements in the mammalian cell.

[0144] In some embodiments, the IL-10 agonist compounds of this disclosure (e.g., single-domain antibody polypeptides that are IL-10 agonist compounds) are polypeptides. This disclosure provides nucleic acid sequences encoding polypeptide IL-10 agonist compounds. This disclosure further provides recombinant vectors comprising nucleic acid sequences encoding polypeptide IL-10 agonist compounds. This disclosure further provides recombinantly modified mammalian cells comprising nucleic acids encoding polypeptide IL-10 agonist compounds. This disclosure further provides methods for recombinant production, isolation, purification, and characterization of polypeptide IL-10 agonist compounds.

[0145] The cognitive ligand for the IL-10 receptor (IL-10R) is the cytokine IL-10. The term IL-10 encompasses human and mouse (murine) (or mouse) IL-10. Human IL-10 (hIL-10) is a non-covalently linked homodimer protein containing two identical subunits. Each human IL-10 monomer is expressed as a 178-amino acid preprotein containing an 18-amino acid signal sequence, which is post-translationally removed to form a 160-amino acid mature protein. The canonical amino acid sequence of the mature human IL-10 protein is disclosed as UniProt Reference No. P22301. Mouse (or mouse (murine)) IL-10 (mIL-10) is a non-covalently linked homodimer protein containing two identical subunits. Each mouse IL-10 monomer is expressed as a 178-amino acid preprotein containing an 18-amino acid signal sequence, which is removed post-translation to a 160-amino acid mature protein. The canonical amino acid sequence of the mature mouse IL-10 protein without the signal sequence (corresponding to amino acids 19-178 of the preprotein) is disclosed as UniProt Reference No. P18893. IL-10 agonist compounds activate IL-10 signaling in cells expressing the IL-10 receptor. This disclosure provides IL-10 agonist compounds engineered to provide selective levels of intracellular signaling in cells expressing the IL-10 receptor. The present invention provides IL-10 agonist compounds engineered to generate intracellular signaling in specific cell types.

[0146] The IL-10 receptor is a heterodimer protein complex comprising the IL-10Rα subunit and the IL-10Rβ subunit. Interaction of IL-10 on the surface of mammalian cells expressing the IL-10Rα and IL-10Rβ subunits results in dimerization of IL-10Rα and IL-10Rβ and subsequent intracellular signaling. The intracellular signaling characteristic of IL-10-mediated dimerization of IL-10Rα and IL-10Rβ is the activation of the JAK / STAT pathway, particularly the phosphorylation of the STAT3 molecule, a component of the intracellular signaling pathway that, in combination with other components of the signaling pathway, results in modulation of gene expression. In some embodiments, the IL-10 receptor is the human IL-10 receptor. As used herein, the terms “IL-10 receptor” and “IL-10R” are interchangeable to refer to the heterodimer complex of IL-10R comprising the IL-10Rα and IL-10Rβ subunits.

[0147] The IL-10Rα component of the human IL-10 receptor is the human IL-10R (hIL-10Rα) protein. The canonical full-length hIL-10Rα protein is listed in the UniProt database, ID No. Q13651. The IL-10Rα component of the mouse IL-10 receptor is the mouse IL-10Rα (mIL-10Rα) protein. The canonical full-length mMIL-10Rα is listed in the UniProt database, ID No. Q61727.

[0148] The IL-10Rβ component of the human IL-10 receptor is the human IL-019R (hIL-10Rβ) protein. The canonical full-length hIL-10Rβ precursor is listed in the UniProt database, ID No. Q08334. The canonical mouse IL-10Rβ (mIL-10Rβ) is listed in the UniProt database, ID No. Q61190.

[0149] Single-domain antibody The IL-10 agonist compounds of this disclosure (e.g., single-domain antibody polypeptides that are IL-10 agonist compounds) comprise two or more single-domain antibodies. The term "single-domain antibody" (sdAb), as used herein, refers to an antibody fragment comprising a monomeric variable antibody domain that specifically binds to an antigen and can compete for binding with the parent antibody from which it originates. The term "single-domain antibody" encompasses scFv and VHH molecules. In some embodiments, one or both of the sdAb of the cytokine receptor-binding molecule are scFv. In some embodiments, one or both of the sdAb are VHH. In some embodiments, one or both of the sdAb are scFv.

[0150] The term single-domain antibody encompasses, but is not limited to, engineered sdAbs, including chimeric sdAbs, CDR-grafted sdAbs, and humanized sdAbs. In some embodiments, one or more of the sdAbs for incorporation into the IL-10 agonist compounds of this disclosure (e.g., single-domain antibody polypeptides that are IL-10 agonist compounds) are CDR-grafted. CDRs obtained from antibodies, heavy-chain antibodies, and sdAbs derived therefrom may be grafted onto alternative frameworks to generate CDR-grafted sdAbs, as described in Saerens et al. (2005) J. Mol Biol 352:597-607. Any framework region may be used with the CDRs described herein.

[0151] In some embodiments, one or more sdAbs for incorporation into an IL-10 agonist compound (e.g., a single-domain antibody polypeptide that is an IL-10 agonist compound) are chimeric sdAbs in which the CDR is derived from one species (e.g., camel) and the framework and / or constant region is derived from another species (e.g., human or mouse). In certain embodiments, the framework region is a human sequence or a humanized sequence. Thus, IL-10 agonist compounds containing one or more humanized sdAbs are considered to be within the scope of this disclosure.

[0152] In some embodiments, one or more of the cytokine receptor agonist compounds sdAbs of the present disclosure are VHHs. As used herein, the term "VHH" typically refers to a single-domain antibody derived from camelid antibodies obtained by immunization of camelids (including camels, llamas, and alpacas) (see, e.g., Hamers-Casterman et al. 1993 Nature 363:446-448). Single-domain antibodies can also be derived from non-mammalian sources, such as VHHs obtained from IgNAR antibodies resulting from immunization of cartilaginous fish (including, but not limited to, sharks), so VHHs are also called heavy-chain antibodies. A VHH is a type of single-domain antibody (sdAb) containing a single monomeric variable antibody domain. It can selectively bind to a specific antigen, like a full-length antibody.

[0153] The complementarity-determining region (CDR) of VHH is located within a single-domain polypeptide. VHH can be manipulated from heavy-chain antibodies found in camelids. Exemplary VHHs have a molecular weight of approximately 12–15 kDa, which is much smaller than traditional mammalian antibodies (150–160 kDa) composed of two heavy chains and two light chains. VHH can be found in camelid mammals that naturally lack light chains (e.g., camels, llamas, dromedaries, alpacas, and guanacos), or can be produced from these animals. Descriptions of sdAb and VHH can be found, for example, in De Greve et al. 2019. Curr Opin Biotechnol. 61:96-101, Ciccarese et al. 2019. Front Genet. 10:997, Chanier and Chames. 2019. Antibodies (Basel) 8(1), and De Vlieger et al. 2018. Antibodies (Basel) 8(1). CDRs derived from camelid VHH can be used to prepare CDR-grafted VHH that can be incorporated into IL-10 agonist compounds (e.g., single-domain antibody polypeptides that are IL-10 agonist compounds).

[0154] In some embodiments, the VHH for incorporation into the IL-10 agonist compound of this disclosure (e.g., a single-domain antibody polypeptide that is an IL-10 agonist compound) is a humanized VHH containing a human framework region or a framework region into which additional amino acid residues common to the human framework region are incorporated. Human framework regions useful in the preparation of humanized VHHs include, but are not limited to, VH3-23 (e.g., UniProt ID:P01764), VH3-74 (e.g., UniProt ID:A0A0B4J1X5), VH3-66 (e.g., UniProt ID:A0A0C4DH42), VH3-30 (e.g., UniProt ID:P01768), VH3-11 (e.g., UniProt ID:P01762), and VH3-9 (e.g., UniProt ID:P01782).

[0155] The IL-10 agonist compounds of this disclosure (e.g., single-domain antibody polypeptides that are IL-10 agonist compounds) include a single-domain antibody ("IL-10Rα sdAb") that selectively binds to the extracellular domain of IL-10Rα and a single-domain antibody ("IL-10Rβ sdAb") that selectively binds to the extracellular domain of IL-10Rβ. As used herein, the terms "stablely associated" or "stable associated with ~" are used to refer to a variety of means by which one molecule (e.g., a polypeptide) can be thermodynamically and / or kinetically associated with another molecule. The stable association of one molecule to another can be achieved by a variety of means, including covalent and non-covalent interactions.

[0156] In some embodiments, the stable association of IL-10Rα sdAb and IL-10Rβ sdAb can be achieved by a covalent bond, such as a peptide bond. In some embodiments, the covalent linkage between the first binding domain and the second binding domain is a covalent bond between the C-terminus of the first binding domain and the N-terminus of the second binding domain.

[0157] In some embodiments, the covalent linkage between the IL-10 agonist proteins IL-10Rα sdAb and IL-10Rβ sdAb is brought about by a coordinate covalent linkage. This disclosure provides an example of a single-domain antibody containing a chelated peptide. The chelated peptide results in a coordinate covalent linkage to a transition metal ion. In some embodiments, the transition metal ion has the ability to form a coordinate covalent linkage with two or more chelated peptides. Consequently, each of the first and second binding domains contains a chelated peptide, and the stable association of the binding domains by each subunit can form a coordinate covalent complex with the transition metal ion. In some embodiments, the transition metal ion is selected from vanadium, manganese, iron, iridium, osmium, rhenium, platinum, palladium, cobalt, chromium, or ruthenium. The N-terminal domain of the single-domain antibody is presented to the environment to facilitate enhanced exposure of the sdAb's CDR to the target cytokine receptor ECD. The formation of coordinative covalent links between them is facilitated when the transition metal ion is in a kinetically unstable oxidation state, such as Co(II), Cr(II), or Ru(III). After complexation, the oxidation state of the transition metal can change to a kinetically inactive oxidation state (it can be oxidized or reduced). For example, Co(III), Cr(III), or Ru(II) give a kinetically inactive coordinative covalent complex. The formation of kinetically inactive and kinetically unstable coordinative covalent complexes between proteins containing chelated peptides via transition metals is described in detail in U.S. Patent No. 5,439,928 by Anderson et al.

[0158] In some embodiments, the covalent linkage between IL-10Rα sdAb and IL-10Rβ sdAb of an IL-10 agonist compound (e.g., a single-domain antibody polypeptide that is an IL-10 agonist compound) may further include a linker. The linker is a molecule selected from the group including (but not limited to) peptide linkers and chemical linkers. In some embodiments, the linker junctions the C-terminus of IL-10Rα sdAb to the N-terminus of IL-10Rβ sdAb. In some embodiments, the linker junctions the C-terminus of IL-10Rβ sdAb to the N-terminus of IL-10Rα sdAb.

[0159] In some embodiments, the linker is a peptide linker. The peptide linker may contain 1 to 50 amino acids (e.g., 2 to 50, 5 to 50, 10 to 50, 15 to 50, 20 to 50, 25 to 50, 30 to 50, 35 to 50, 40 to 50, 45 to 50, 2 to 45, 2 to 40, 2 to 35, 2 to 30, 2 to 25, 2 to 20, 2 to 15, 2 to 10, 2 to 5 amino acids). Glycine polymers and glycine-serine polymers are relatively structurally indeterminate and can therefore serve as neutral tethers between their constituent elements. As an example of a glycine polymer, (G) n , glycine-alanine polymer, alanine-serine polymer, glycine-serine polymer (for example, (G m S o ) n (SEQ ID NO:416, 417, 420, 421, 422, 423), (GSGGS) n (SEQ ID NO:434), (G m S o G m ) n (SEQ ID NO:418, 427, 428, 429, 430, 433, 445, 446, 447), (G m S o G m S o G m ) n (SEQ ID NO:444), (GSGGS) m ) n (SEQ ID NO:434), (GSGS)m G) n (SEQ ID NO:444) and (GGGS) m ) n Examples include (SEQ ID NO: 432, 448), as well as combinations thereof (where m, n, and o are integers from at least 1 to 20, e.g., integers from 1 to 18, 216, 3 to 14, 4 to 12, 5 to 10, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, each independently selected), and other flexible linkers. Exemplary flexible peptide linkers useful in the preparation of the IL-10 agonist compounds of this disclosure (e.g., single-domain antibody polypeptides that are IL-10 agonist compounds) include, but are not limited to, the linkers provided in Table 11.

[0160] In some embodiments, the covalent linkage between the first and second domains may be achieved by a chemical linker. Examples of chemical linkers include arylacetylenes, ethylene glycol oligomers containing 2 to 10 monomer units, diamines, diacitors, amino acids, or combinations thereof.

[0161] In some embodiments, the stable association of IL-10Rα sdAb and IL-10Rβ sdAb of the IL-10 agonist protein is brought about by non-covalent interactions. Examples of non-covalent interactions that give rise to a stable association between two molecules include electrostatic interactions (e.g., hydrogen bonds, ionic bonds, halogen bonds, dipole-dipole interactions, van der Waals forces, and p-effects including cation-p interactions, anion-p interactions, and pp interactions) and hydrophobic / hydrophilic interactions. In some embodiments, the stable association of the sdAb of the binding molecule of this disclosure may be brought about by non-covalent interactions.

[0162] In one embodiment, the non-covalent stable association of IL-10Rα sdAb and IL-10Rβ sdAb of an IL-10 agonist compound (e.g., a single-domain antibody polypeptide that is an IL-10 agonist compound) may be achieved by the conjugation of the sdAb monomers of each of the "knob-into-hole" operated Fc dimers. 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) that creates 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 (e.g., alanine or threonine) that thereby creates a cavity ("hole") at the interface in the second CH3 domain, within which the protruding side chain ("knob") of the first CH3 domain is accepted by the cavity in the second CH3 domain. The knob-into-hole modification is described in detail in Ridgway et al. 1996. Protein Engineering 9(7):617-621 and in U.S. Patent No. 5,731,168 issued on March 24, 1998, U.S. Patent No. 7,642,228 issued on January 5, 2010, U.S. Patent No. 7,695,936 issued on April 13, 2010, and U.S. Patent No. 8,216,805 issued on July 10, 2012. In one embodiment, the “knob-into-hole modification” includes the amino acid substitution 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 of the antibody heavy chains. Furthermore, the Fc domain may be modified by introducing cysteine ​​residues at positions S354 and Y349, which results in a stabilizing disulfide bridge between the two antibody heavy chains in the Fe region (Carter et al. 2001. Immunol Methods 248:7-15).The knob-into-hole format is used to facilitate the expression of heterodimer polypeptide conjugates by promoting the expression of a first polypeptide (e.g., IL-10Rβ-binding sdAb) on a first Fc monomer with a "knob" modification and a second polypeptide on a second Fc monomer with a "hole" modification. One embodiment of an IL-10 agonist compound in which IL-10Rα sdAb and IL-10Rβ sdAb are in a stable, non-covalent association state is, as described above, in which each sdAb of the IL-10 agonist compound (e.g., a single-domain antibody polypeptide that is an IL-10 agonist compound) is covalently bonded (optionally including a linker) to each subunit of the knob-into-hole Fc dimer.

[0163] Anti-IL-10R single-domain antibody In some embodiments, the IL-10 agonist compounds of this disclosure (e.g., single-domain antibody polypeptides that are IL-10 agonist compounds) are single-domain antibodies that have at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity with any one of the sequences of SEQ ID NO: 1-24 and SEQ ID NO: 500-523 in Table 1, or amino acid sequences having 0, 1, 2, or 3 amino acid changes, optionally conservative amino acid changes. In other embodiments, the IL-10 agonist compounds of this disclosure (e.g., single-domain antibody polypeptides that are IL-10 agonist compounds) are single-domain antibodies comprising an amino acid sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity with respect to any one of the sequences SEQ ID NO: 1-24 and SEQ ID NO: 500-523 in Table 1. In other embodiments, the IL-10 agonist compounds of this disclosure (e.g., single-domain antibody polypeptides that are IL-10 agonist compounds) are single-domain antibodies comprising an amino acid sequence having 0, 1, 2, or 3 amino acid changes compared to any one of the sequences SEQ ID NO: 1-24 and SEQ ID NO: 500-523 in Table 1. In other embodiments, the IL-10 agonist compounds of this disclosure (e.g., single-domain antibody polypeptides that are IL-10 agonist compounds) are single-domain antibodies comprising an amino acid sequence having one or more conserved amino acid changes compared to any one of the sequences with SEQ ID NO: 1-24 and SEQ ID NO: 500-523 in Table 1.

[0164] In some embodiments, the IL-10 agonist compounds of this disclosure (e.g., single-domain antibody polypeptides that are IL-10 agonist compounds) include CDR1, CDR2, and CDR3 with amino acid sequences of SEQ ID NO: 25-48 in Table 2. In some embodiments, the IL-10 agonist compounds of this disclosure (e.g., single-domain antibody polypeptides that are IL-10 agonist compounds) include CDR1, CDR2, and CDR3 with amino acid sequences of SEQ ID NO: 49-51 in Table 3.

[0165] In some embodiments, the disclosure provides IL-10 agonist compounds comprising polypeptides having at least 95% amino acid sequence identity with respect to amino acid sequences selected from the group consisting of SEQ ID NO: 1-24 and SEQ ID NO: 500-523.

[0166] In some embodiments, the Disclosure provides IL-10 agonist compounds comprising a first single-domain antibody polypeptide comprising a combination of CDR1, CDR2, and CDR3 amino acid sequences selected from one of the rows in Table A below, and a second single-domain antibody polypeptide comprising a combination of CDR1, CDR2, and CDR3 amino acid sequences selected from one of the rows in Table B below. (Table A) Anti-IL-10Rα VHH CDR TIFF2026521432000004.tif88166 (Table B) Anti-IL-10Rβ VHH CDR TIFF2026521432000005.tif14166

[0167] In some embodiments, the Disclosure provides an IL-10 agonist compound comprising a first single-domain antibody polypeptide containing CDR1 having the amino acid sequence of SEQ ID NO: 224, CDR2 having the amino acid sequence of SEQ ID NO: 229, and CDR3 having the amino acid sequence of SEQ ID NO: 236. In some embodiments, the Disclosure provides an IL-10 agonist compound comprising a second single-domain antibody polypeptide containing CDR1 having the amino acid sequence of SEQ ID NO: 296, CDR2 having the amino acid sequence of SEQ ID NO: 297, and CDR3 having the amino acid sequence of SEQ ID NO: 298.

[0168] In some embodiments, the Disclosure provides an IL-10 agonist compound comprising a first single-domain antibody polypeptide containing CDR1 having the amino acid sequence of SEQ ID NO: 224, CDR2 having the amino acid sequence of SEQ ID NO: 230, and CDR3 having the amino acid sequence of SEQ ID NO: 236. In some embodiments, the Disclosure provides an IL-10 agonist compound comprising a second single-domain antibody polypeptide containing CDR1 having the amino acid sequence of SEQ ID NO: 296, CDR2 having the amino acid sequence of SEQ ID NO: 297, and CDR3 having the amino acid sequence of SEQ ID NO: 298.

[0169] In some embodiments, the Disclosure provides an IL-10 agonist compound comprising a first single-domain antibody polypeptide containing CDR1 having the amino acid sequence of SEQ ID NO: 224, CDR2 having the amino acid sequence of SEQ ID NO: 231, and CDR3 having the amino acid sequence of SEQ ID NO: 236. In some embodiments, the Disclosure provides an IL-10 agonist compound comprising a second single-domain antibody polypeptide containing CDR1 having the amino acid sequence of SEQ ID NO: 296, CDR2 having the amino acid sequence of SEQ ID NO: 297, and CDR3 having the amino acid sequence of SEQ ID NO: 298.

[0170] In some embodiments, the Disclosure provides IL-10 agonist compounds comprising a polypeptide containing an amino acid sequence selected from the group consisting of SEQ ID NO: 1-24 or SEQ ID NO: 500-523. In some embodiments, the IL-10R-binding protein of the Disclosure contains one amino acid sequence from SEQ ID NO: 1-24 or SEQ ID NO: 500-523, wherein the amino acid sequence does not have a polyHis tag.

[0171] In some embodiments, the disclosure provides IL-10 agonist compounds comprising a polypeptide containing the amino acid sequence of SEQ ID NO:1.

[0172] In some embodiments, the disclosure provides IL-10 agonist compounds comprising a polypeptide containing the amino acid sequence of SEQ ID NO:2.

[0173] In some embodiments, the disclosure provides IL-10 agonist compounds comprising a polypeptide containing the amino acid sequence of SEQ ID NO:3.

[0174] In some embodiments, the disclosure provides IL-10 agonist compounds comprising a polypeptide containing the amino acid sequence of SEQ ID NO:4.

[0175] In some embodiments, the disclosure provides IL-10 agonist compounds comprising polypeptides having an amino acid sequence selected from the group consisting of SEQ ID NO: 500 to 523.

[0176] In some embodiments, the disclosure provides IL-10 agonist compounds comprising a polypeptide having the amino acid sequence of SEQ ID NO:500.

[0177] In some embodiments, the disclosure provides IL-10 agonist compounds comprising a polypeptide having the amino acid sequence of SEQ ID NO:501.

[0178] In some embodiments, the present disclosure provides IL-10 agonist compounds comprising a polypeptide having the amino acid sequence of SEQ ID NO:502.

[0179] In some embodiments, the disclosure provides IL-10 agonist compounds comprising a polypeptide having the amino acid sequence of SEQ ID NO:503.

[0180] In some embodiments, the amino acid sequences of the IL-10 agonist compounds of this disclosure do not include His tags or ASH6 ​​His tags (for example, if His tags or ASH6 ​​His tags are present, they are optionally removed).

[0181] Anti-IL-10Rα single-domain antibody In some embodiments, the IL-10Rα sdAb used in the IL-10 agonist compounds of this disclosure (e.g., single-domain antibody polypeptides that are IL-10 agonist compounds) includes a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity with any one of the amino acid sequences (SEQ ID NO: 25-48) of the hIL-10Rα sdAb disclosed in Table 2. In certain embodiments, the IL-10Rα sdAb includes a sequence that is substantially identical to any one of the amino acid sequences (SEQ ID NO: 25-48) of the hIL-10Rα sdAb provided in Table 2. In a particular embodiment, IL-10Rα sdAb contains a sequence that is identical to any one of the amino acid sequences (SEQ ID NO: 25-48) of hIL-10Rα sdAb provided in Table 2.

[0182] In some embodiments, the disclosure provides an IL-10Rα sdAb comprising a polypeptide having the amino acid sequence of SEQ ID NO: 25. In some embodiments, the disclosure provides an IL-10Rα sdAb comprising a polypeptide having the amino acid sequence of SEQ ID NO: 26. In some embodiments, the disclosure provides an IL-10Rα sdAb comprising a polypeptide having the amino acid sequence of SEQ ID NO: 27. In some embodiments, the disclosure provides an IL-10Rα sdAb comprising a polypeptide having the amino acid sequence of SEQ ID NO: 28.

[0183] Table 7 provides useful CDRs in the preparation of IL-10Rα sdAbs for incorporation into the IL-10 agonist compounds of this disclosure (e.g., single-domain antibody polypeptides that are IL-10 agonist compounds). In some embodiments, the IL-10Rα sdAb specifically binds to the ECD of hIL-10Rα. In some embodiments, IL-10Rα sdAb has at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity with any one sequence of SEQ ID NO: 224-228, or CDR1 having 0, 1, 2, or 3 amino acid changes, optionally conservative amino acid changes; CDR2 has at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity with any one sequence of SEQ ID NO: 229-235, or CDR2 having 0, 1, 2, or 3 amino acid changes, optionally conservative amino acid changes; and SEQ ID A single-domain antibody containing CDR3 having at least 90% sequence identity (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) compared to the sequence of NO:236, or having 0, 1, 2, or 3 amino acid changes, optionally conservative amino acid changes.

[0184] In some embodiments, IL-10Rα sdAb is a single-domain antibody containing a CDR1 having at least 95% (e.g., 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity with any one of the sequences SEQ ID NO:224-228 in Table 7, or having 0, 1, 2, or 3 amino acid changes, optionally conservative amino acid changes. In some embodiments, IL-10Rα sdAb is a single-domain antibody containing a CDR1 having any one of the sequences SEQ ID NO:224-228 in Table 7.

[0185] In some embodiments, IL-10Rα sdAb is a single-domain antibody containing a CDR2 having at least 95% (e.g., 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity with any one of the sequences SEQ ID NO: 229-235 in Table 7, or having 0, 1, 2, or 3 amino acid changes, optionally conservative amino acid changes. In some embodiments, IL-10Rα sdAb is a single-domain antibody containing a CDR2 having any one of the sequences SEQ ID NO: 229, 230, or 231 in Table 7.

[0186] In some embodiments, IL-10Rα sdAb is a single-domain antibody containing a CDR3 having at least 95% (e.g., 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity with the sequence of SEQ ID NO:236 in Table 7, or having 0, 1, 2, or 3 amino acid changes, optionally conservative amino acid changes. In some embodiments, IL-10Rα sdAb is a single-domain antibody containing a CDR3 having the sequence of SEQ ID NO:236 in Table 7.

[0187] In some embodiments, the amino acid sequence of IL-10Rα sdAb of this disclosure does not include a His tag or an ASH6 ​​His tag (for example, if a His tag or an ASH6 ​​His tag is present, it is optionally removed).

[0188] Anti-IL-10Rβ single-domain antibody In some embodiments, the IL-10Rβ sdAb used in the IL-10 agonist compounds of this disclosure (e.g., single-domain antibody polypeptides that are IL-10 agonist compounds) includes a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity with any one of the amino acid sequences (SEQ ID NO: 49-51) of the hIL-10Rβ sdAb disclosed in Table 3. In certain embodiments, the IL-10Rβ sdAb includes a sequence that is substantially identical to any one of the amino acid sequences (SEQ ID NO: 49-51) of the hIL-10Rα sdAb provided in Table 3. In a particular embodiment, IL-10Rβ sdAb contains a sequence identical to one of the amino acid sequences (SEQ ID NO: 49-51) of hIL-10Rβ sdAb provided in Table 3.

[0189] In some embodiments, the disclosure provides an IL-10Rβ sdAb comprising a polypeptide having the amino acid sequence of SEQ ID NO: 49. In some embodiments, the disclosure provides an IL-10Rβ sdAb comprising a polypeptide having the amino acid sequence of SEQ ID NO: 50. In some embodiments, the disclosure provides an IL-10Rβ sdAb comprising a polypeptide having the amino acid sequence of SEQ ID NO: 51. In some embodiments, the disclosure provides an IL-10Rβ sdAb comprising a polypeptide having the amino acid sequence of SEQ ID NO: 52.

[0190] Table 8 provides CDRs useful in the preparation of IL-10Rβ sdAbs for incorporation into IL-10 agonist compounds of the present disclosure (e.g., single domain antibody polypeptides that are IL-10 agonist compounds). In some embodiments, the IL-10Rβ sdAb specifically binds to the ECD of hIL-10Rβ. In some embodiments, the IL-10Rβ sdAb has at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity compared to the sequence of SEQ ID NO:296, or has 0, 1, 2, or 3 amino acid changes, optionally conservative amino acid changes, and CDR1 having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity compared to the sequence of SEQ ID NO:297, or has 0, 1, 2, or 3 amino acid changes, optionally conservative amino acid changes, and CDR2 having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity compared to the sequence of SEQ ID NO:298, or has 0, 1, 2, or 3 amino acid changes, optionally conservative amino acid changes, and is a single domain antibody comprising CDR3.

[0191] In some embodiments, the IL-10Rβ sdAb has at least 95% (e.g., 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity compared to the sequence of SEQ ID NO:296 in Table 8, or has 0, 1, 2, or 3 amino acid changes, optionally conservative amino acid changes, and is a single domain antibody comprising CDR1. In some embodiments, the IL-10Rβ sdAb is a single domain antibody comprising CDR1 having the sequence of SEQ ID NO:296 in Table 8.

[0192] In some embodiments, the IL-10Rβ sdAb has at least 95% (e.g., 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity compared to the sequence of SEQ ID NO:297 in Table 8, or contains a CDR2 with 0, 1, 2, or 3 amino acid changes, optionally conservative amino acid changes, and is a single-domain antibody. In some embodiments, the IL-10Rβ sdAb is a single-domain antibody containing a CDR2 having the sequence of SEQ ID NO:297 in Table 8.

[0193] In some embodiments, the IL-10Rβ sdAb has at least 95% (e.g., 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity compared to the sequence of SEQ ID NO:298 in Table 8, or contains a CDR3 with 0, 1, 2, or 3 amino acid changes, optionally conservative amino acid changes, and is a single-domain antibody. In some embodiments, the IL-10Rβ sdAb is a single-domain antibody containing a CDR3 having the sequence of SEQ ID NO:298 in Table 8.

[0194] In some embodiments, the IL-10R binding protein of the present disclosure contains one of the amino acid sequences of SEQ ID NOs:500 - 523 shown in Table 17, and the amino acid sequence does not have a polyHis tag.

[0195] Nucleic acids encoding anti-IL-10 agonist compounds In some embodiments, the present disclosure provides an isolated nucleic acid encoding an IL-10 agonist compound having an amino acid sequence selected from one of SEQ ID NOs:1 - 24 and SEQ ID NOs:500 - 523 shown in Table 1 and Table 17.

[0196] In another aspect, the Disclosure provides isolated nucleic acids encoding IL-10 agonist compounds described herein (e.g., single-domain antibody polypeptides that are IL-10 agonist compounds). Table 6 provides DNA sequences (SEQ ID NO: 200-223) encoding the IL-10 agonist compounds (e.g., single-domain antibody polypeptides that are IL-10 agonist compounds) (SEQ ID NO: 1-24) of Table 1. In certain embodiments, the Disclosure provides isolated nucleic acids containing sequences having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the DNA sequences (SEQ ID NO: 200-223) of Table 6. In certain embodiments, the Disclosure provides isolated nucleic acids containing DNA sequences that are substantially identical to the DNA sequences (SEQ ID NO: 200-223) of Table 6. In a particular embodiment, the present disclosure provides isolated nucleic acids containing DNA sequences identical to the DNA sequences (SEQ ID NO: 200-223) in Table 6.

[0197] In another aspect, the Disclosure provides isolated nucleic acids encoding IL-10Rα sdAb as described herein. In certain embodiments, the Disclosure provides isolated nucleic acids containing sequences having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the DNA sequences encoding IL-10Rα sdAb in Table 9 (SEQ ID NO: 368-391). In certain embodiments, the Disclosure provides isolated nucleic acids containing DNA sequences that are substantially identical to the DNA sequences in Table 9 (SEQ ID NO: 368-391). In certain embodiments, the Disclosure provides isolated nucleic acids containing DNA sequences that are identical to the DNA sequences in Table 9 (SEQ ID NO: 368-391).

[0198] In another aspect, the Disclosure provides isolated nucleic acids encoding IL-10Rβ sdAb as described herein. In certain embodiments, the Disclosure provides isolated nucleic acids containing sequences having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the DNA sequences (SEQ ID NO: 392-415) in Table 10. In certain embodiments, the Disclosure provides isolated nucleic acids containing DNA sequences that are substantially identical to the DNA sequences (SEQ ID NO: 392-415) in Table 10. In certain embodiments, the Disclosure provides isolated nucleic acids containing DNA sequences that are identical to the DNA sequences (SEQ ID NO: 392-415) in Table 10.

[0199] In some embodiments, the nucleic acid sequence encoding the IL-10R binding protein of the present disclosure comprises one nucleic acid sequence from SEQ ID NO: 524 to 547, the nucleotide sequence encoding an amino acid sequence having a C-terminal Ala-Ser ("AS") linker and a hexameric histidine ("H6") polyHis-tagged chelated peptide (hereinafter also referred to as "ASH6" (SEQ ID NO: 513) and also referred to as the "His tag").

[0200] In some embodiments, the nucleic acid sequence encoding the IL-10R binding protein of this disclosure comprises one nucleic acid sequence from SEQ ID NO: 500 to 547, wherein the nucleotide sequence encodes an amino acid sequence containing an ASH6 ​​His tag.

[0201] In some embodiments, the nucleic acid sequence encoding the IL-10R binding protein of this disclosure comprises one nucleic acid sequence from SEQ ID NO: 548 to 571, wherein the nucleotide sequence encodes an untagged amino acid sequence.

[0202] In some embodiments, the Disclosure provides isolated nucleic acids encoding IL-10 agonist compounds having amino acid sequences selected from one of SEQ ID NO: 1-24 and SEQ ID NO: 500-523.

[0203] Generation of IL10R polypeptide-binding molecules The amino acid sequences of the IL-10 agonist compounds disclosed herein (e.g., single-domain antibody polypeptides that are IL-10 agonist compounds) are listed in Table 1 (SEQ ID NO: 1-24). The DNA sequences encoding these amino acid sequences used to express the IL-10 agonist compounds (e.g., single-domain antibody polypeptides that are IL-10 agonist compounds) are provided in Table 6 (SEQ ID NO: 200-223).

[0204] The IL-10 agonist proteins in Table 1 were prepared and their IL-10 activity was evaluated. Details regarding the expression and purification of these IL-10 agonist proteins are provided in the Examples. Briefly, nucleic acid sequences encoding SEQ ID NO: 1-24 were synthesized using the DNA sequences (SEQ ID NO: 200-223) in Table 7, respectively, substantially according to Examples 1 and 2. These sequences were then inserted into recombinant expression vectors, expressed in HEK293 cells, and purified. The supernatant containing the IL-10 receptor agonist proteins of SEQ ID NO: 1-24 was evaluated for binding activity substantially as described in Example 3. The results of these binding experiments are provided in Tables 17 and 18 of Example 3. The biological activity of the IL-10 receptor agonist proteins of SEQ ID NO: 1-24 was also evaluated substantially as described in Examples 4, 5, and 6.

[0205] Modulation of receptor-binding molecule activity In some embodiments, the design of the IL-10 agonist compounds of this disclosure (e.g., single-domain antibody polypeptides that are IL-10 agonist compounds) can be modulated by structural variations in the design of the receptor-binding molecules, for example, to achieve partial agonism or selective activation of a particular cell type. These variations in activity can be used to modulate the binding and activity of IL-10R receptor-binding molecules, or to optimize the activity of IL-10 agonist compounds (e.g., single-domain antibody polypeptides that are IL-10 agonist compounds) to achieve partial agonism, selective activation of a cell type, or to provide molecules having increased or decreased binding to their respective receptor subunits compared to cognitive ligands for each of IL-10Rα sdAb and IL-10Rβ sdAb, respectively.

[0206] The ability to modulate the activity of the IL-10 agonist compounds of this disclosure (e.g., single-domain antibody polypeptides that are IL-10 agonist compounds) offers substantial benefits in multiple therapeutic applications. IL-10 is a pleiotropic cytokine that modulates multiple immune responses through its actions on T cells, B cells, macrophages, and antigen-presenting cells (APCs) and its seemingly contradictory activities, which have limited its clinical development. IL-10 suppresses immune responses and inhibits the expression of IL-1α, IL-1β, IL-6, IL-8, TNF-α, GM-CSF, and G-CSF in activated monocytes and macrophages. IL-10 is associated with the suppression of IFN-γ production by NK cells. IL-10 also exhibits immunostimulatory properties, including enhanced stimulation of thymocytes treated with IL-2 and IL-4, enhanced B cell viability, and stimulation of MHC class II antigen presentation. As a result, the use of IL-10 has been identified as useful in treating a wide range of diseases, disorders, and conditions, including inflammatory conditions, immune-related disorders, fibrotic disorders, metabolic disorders, and cancer.

[0207] The IL-10 agonist proteins of this disclosure enable modulation of activity and offer significant benefits in the treatment of human diseases. The IL-10 agonist proteins described herein are useful in the treatment of 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 subjects requiring such treatment. In some embodiments, the IL-10 agonist proteins described herein can provide longer therapeutic efficacy than IL-10 (e.g., lower effective doses, reduced toxicity). The IL-10 agonist compounds of this disclosure (e.g., single-domain antibody polypeptides that are IL-10 agonist compounds) can trigger different levels of downstream signaling in different cell types. For example, by altering the linker length between the IL-10Rα sdAb antibody and the IL-10Rβ sdAb antibody in the IL-10R-binding molecule, an IL-10 agonist compound (e.g., a single-domain antibody polypeptide that is an IL-10 agonist compound) provides a higher level of downstream signaling in the desired cell type compared to the undesirable cell type. In some embodiments, an IL-10 agonist compound (e.g., a single-domain antibody polypeptide that is an IL-10 agonist compound) provides a higher level of downstream signaling in the desired cell type compared to the undesirable cell type. + It is a partial IL-10 agonist that selectively activates T cells. In some embodiments, activated T cells have upregulation of IFN gamma. In some embodiments, the IL-10 agonist protein, which is a partial agonist, can suppress autoimmune inflammatory diseases, such as ulcerative colitis and Crohn's disease.

[0208] In one aspect, the present disclosure provides an IL-10Rα-binding molecule that preferentially activates T cells, such as CD8+ T cells, compared to monocytes. In one aspect, the present disclosure provides an IL-10Rα-binding molecule having an affinity for the extracellular domain of IL-10Rα that is higher than the affinity of an IL-10Rβ sdAb for the extracellular domain of IL-10Rβ. In some aspects, the present disclosure provides an IL-10Rα-binding molecule having an affinity for the extracellular domain of IL-10Rα of about 10 -8 to about 10 -10 M, alternatively about 10 -9 to about 10 -10 M, or alternatively about 10 -10 M, and an IL-10Rβ sdAb having an affinity for the extracellular domain of IL-10Rβ of about 10 -6 to about 10 -9 M, alternatively about 10 -7 to about 10 -9 M, alternatively about 10 -7 to about 10 -8 M, alternatively about 10 -9 M, alternatively about 1,0 -8 M. In some aspects, the present disclosure provides an IL-10Rα-binding molecule having an affinity for the extracellular domain of IL-10Rα of about 10 -8 to about 10 -10 M, alternatively about 10 -9 to about 10 -10 M, or alternatively about 10 -10 M, and an IL-10Rβ sdAb having an affinity for the extracellular domain of IL-10Rβ of about 10 -6 to about 10 -9 M, alternatively about 10 -7 to about 10 -9 M, alternatively about 10 -7 to about 10 -8 M, alternatively about 10 -9 M, alternatively about 10 -8The present invention provides an IL-10Rα molecule of formula #1 having affinity for M, wherein the affinity of IL-10Rα sdAb to IL-10Rα ECD is more than twice as high, alternatively more than five times higher, alternatively more than ten times higher, alternatively more than twenty times higher, alternatively more than forty times higher, alternatively more than fifty times higher, alternatively more than sixty times higher, alternatively more than seventy times higher, alternatively more than eighty times higher, alternatively more than ninety times higher, alternatively more than one hundred times higher, alternatively more than fifty times higher, alternatively more than two hundred times higher, or alternatively more than five hundred times higher than the affinity of IL-10Rβ sdAb to IL-10Rβ ECD.

[0209] In some embodiments, for example by altering the linker length, IL-10 agonist compounds express both IL-10Rα and IL-10Rβ receptors, but if activated too strongly, they can cause anemia in cell types such as macrophages, where the level of downstream signaling is higher compared to T cells (e.g., CD8). + It can trigger downstream signaling in T cells. When downstream signaling in macrophages is activated to a high level, these activated macrophages may then eliminate aging red blood cells, potentially causing anemia. The ability of IL-10 agonist compounds (e.g., single-domain antibody polypeptides that are IL-10 agonists) to modulate the activity of T cells (e.g., CD8) can trigger downstream signaling at a higher level compared to the level of downstream signaling in macrophages. + The present disclosure provides molecules that facilitate downstream signaling in T cells (e.g., CD8 cells), thereby preventing anemia. In some embodiments, the IL-10 agonist compounds of this disclosure (e.g., single-domain antibody polypeptides that are IL-10 agonist compounds) facilitate downstream signaling in macrophages at levels at least 1.1, 1.5, 2, 3, 5, or 10 times higher than that of T cells (e.g., CD8 cells). +This results in downstream signaling in T cells. In other embodiments, the activity of an IL-10 agonist compound can be tuned using different IL-10Rα sdAb antibodies with different binding affinities and different IL-10Rβ sdAb antibodies with different binding affinities. Furthermore, if the IL-10 agonist compound (e.g., a single-domain antibody polypeptide that is an IL-10 agonist compound) is provided as a single polypeptide, the orientation of the two antibodies in the polypeptide can also be modified to alter the molecular properties. In some embodiments, E is caused by the cognitive ligand wild-type IL-10 of the IL-10 receptor. max E reduced compared to max It is desirable to provide an IL-10 agonist protein (i.e., an IL-10 agonist compound that is an IL-10 partial agonist) having E. max This reflects the maximum response level that can be obtained in a given cell type by a ligand (e.g., a binding protein or cognitive ligand described herein (e.g., IL-10)). In some embodiments, the IL-10 agonist proteins described herein are used to evoke E triggered by hIL-10. max Having at least 1% (for example, 1%-100%, 10%-100%, 20%-100%, 30%-100%, 40%-100%, 50%-100%, 60%-100%, 70%-100%, 80%-100%, 90%-100%, 1%-90%, 1%-80%, 1%-70%, 1%-60%, 1%-50%, 1%-40%, 1%-30%, 1%-20%, or 1%-10%).

[0210] Examples of means for modulating the activity and / or specificity of the receptor-binding molecules of the present disclosure include changing the sequential orientation of the IL-10Rα sdAb and the IL-10Rβ sdAb in the polypeptide IL-10 agonist compound, independently changing the binding affinity of each IL-10Rα sdAb and IL-10Rβ sdAb to their respective IL-10Rα and / or each target, and modulating the distance between the IL-10Rα sdAb and the IL-10Rβ sdAb, for example, by using a linker or changing the length (which affects the proximity of the intracellular signaling domains of the IL-10 receptor subunits, thereby achieving modulation of intracellular signaling characteristic of binding of the cognate ligand to the receptor, such as modulation of the level of intracellularly induced phospho-STAT3), but are not limited thereto. As shown by the data provided below, each of these variations can be used to tune the properties of the IL-10 agonist compound.

[0211] Fc Conjugate In some embodiments, the IL10R-binding molecule of the invention has the following formula [#1]: H2N-(huIL10 VHH1)-(L1) a (L1) b -(huIL10 VHH2)-COOH [#1] is a polypeptide, wherein "-" represents a covalent bond, L1 is a linker, a and b are independently selected from the integers 0 or 1, "H2N" represents the amino terminus, and "COOH" represents the carboxy terminus of the polypeptide.

[0212] As described above, the orientation of the sdAbs of an IL-10 agonist compound (e.g., a single-domain antibody polypeptide that is an IL-10 agonist compound) can be used to optimize the desired properties of the molecule. The orientation of the sdAbs of the IL-10R is, for example, the following formula: H2N-[IL-10Rα sdAb]-[L1] x -[IL-10Rβ sdAb]-[L2]y -[CP] z -COOH, H2N-[IL-10Rβ sdAb]-[L1] x -[IL-10Rα sdAb]-[L2] y -[CP] z -COOH, H2N-[IL-10Rα sdAb]-[L1] x -[IL-10Rβ sdAb]-[L2] y -[Fc] z -COOH, H2N-[IL-10Rβ sdAb]-[L1] x -[IL-10Rα sdAb]-[L2] y -[Fc] z -COOH In various different structures, such as those represented by the formula (wherein "IL-10Rα sdAb" is a single-domain antibody against the IL-10Rα subunit, "IL-10Rβ sdAb" is a single-domain antibody against the IL-10Rβ subunit, L1 and L2 are independently selected polypeptide linkers of 1 to 50 amino acids, x=0 or 1, y=0 or 1, CP is a chelated peptide, "Fc" is a monomeric Fc domain, and y=0 or 1), the following structures: [H2N-[IL-10Rα sdAb]-[L1] x -Fc1-COOH:H2N-[IL-10Rβ sdAb]-[L2] y -Fc2-COOH] A non-covalent complex of (wherein L1 and L2 are independently selected polypeptide linkers of 1 to 50 amino acids, x=0 or 1, y=0 or 1, CP is a chelated peptide, "Fc1" is a monomeric Fc domain, "Fc2" is a monomeric Fc domain, Fc1 and Fc2 form a stable non-covalent association, and y=0 or 1), and the following structure: H2N-[IL-10Rα sdAb]-[L1] x -(CP1)-M-(CP2)-(L2)-[IL-10Rβ sdAb]-NH2 The formula is provided as a coordination-covalent complex of (wherein L1 and L2 are independently selected polypeptide linkers of 1 to 50 amino acids, x and y are independently selected from 0 or 1, CP1 is a first chelated peptide, CP2 is a second chelated peptide, and M is a transition metal ion).

[0213] As discussed in this disclosure, in some embodiments, an IL-10 agonist compound (e.g., a single-domain antibody polypeptide that is an IL-10 agonist compound) is conjugated to a subunit of the Fc domain, as shown in formula #2 below: H2N-(IL-10 VHH#1)-(L1) a -(IL-10 VHH#1)-(L2) b -(Fc monomer) c -COOH [#2] It can be represented by the formula, where "IL-10 VHH#1" is the first IL-10Rα or IL-10Rβ VHH, "IL-10 VHH#2" is the second IL-10Rα or IL-10Rβ VHH different from IL-10 VHH#1, "-" represents a covalent bond, L1 is a linker, a, b, and c are independently selected from 0 or 1, "H2N" represents the amino terminus, and "COOH" represents the carboxyl terminus of the polypeptide.

[0214] For example, in each case, a representative IL-10 agonist of formula [#2] can be synthesized, where a=1, L1=G3S (SEQ ID NO:439), b=1, and L2=Ala-Ser, c=1. The Fc monomer can be prepared such as DR2485 (SEQ ID NO:2), which maintains a constant IL-10VHH#1 sequence while including variations in IL-10 VHH#2.

[0215] In some embodiments, humanized IL-10 agonist compound variants can be prepared, represented by formula [#1], in each case, a=1, L1=G3S (SEQ ID NO: 439), b=1 and L2=Ala-Ser, CP=Hisx6 (SEQ ID NO: 451), IL-10 VHH#1 is IL-10Rα VHH of DR2485 (SEQ ID NO: 26), and IL-10 VHH#2 is IL-10Rβ VHH of DR2485 (SEQ ID NO: 50).

[0216] In some embodiments, humanized IL-10 agonist compound variants can be prepared, represented by formula [#1], in each case, a=1, L1=G3S (SEQ ID NO:439), b=1 and L2=Ala-Ser, CP=Hisx6 (SEQ ID NO:451), IL-10 VHH#1 is IL-10Rα VHH of DR2519 (SEQ ID NO:27), and IL-10 VHH#2 is IL-10Rβ VHH of DR2519 (SEQ ID NO:51).

[0217] In other embodiments, humanized IL-10 agonist compound variants can be prepared, represented by formula [#1], in each case, a=1, L1=G3S (SEQ ID NO:439), b=1 and L2=Ala-Ser, C=Hisx6 (SEQ ID NO:451), IL-10 VHH#1 is IL-10Rα VHH of DR2520 (SEQ ID NO:28), and IL-10 VHH#2 is IL-10Rβ VHH of DR2520 (SEQ ID NO:52).

[0218] Humanization of sdAb The IL-10Rα VHH sdAb and IL-10Rβ VHH sdAb, which are useful for preparing the IL-10 agonist compounds of this disclosure, may be humanized. To demonstrate the usefulness of humanized versions of the VHH components of IL-10 agonist compounds (e.g., single-domain antibody polypeptides that are IL-10 agonist compounds), humanized IL-10Rα-IL-10Rβ VHH dimers were prepared and the binding of IL-10Rα and IL-10Rβ to ECD was evaluated.

[0219] When humanizing VHHs with respect to a target, a consideration for designing the humanized VHH is the distribution of amino acids at each position in the non-human framework, suggesting amino acid residues that may be substantially altered in the secondary and tertiary structures of the protein by the modification. To identify the amino acids in the highly conserved camel VHH framework region, the VHH sdAb sequence was obtained, and the distribution of amino acids at each position was evaluated using "R Script". Rare residues at each position were identified using an amino acid distribution chart. It was determined that certain residues in the camel VHH framework region are highly conserved.

[0220] Table 1 provides examples of the humanized IL-10 agonist compounds of this disclosure (SEQ ID NO: 1~24).

[0221] Humanization of the IL-10Rα-IL-10Rβ VHH dimer was performed to obtain the IL-10Rα-IL-10Rβ VHH dimer constructs DR2463, DR2485, DR2519, and DR2520. The amino acid sequences, nucleic acid sequences, and CDR domains (defined by various CDR identification / numbering schemes) of the VHH are shown in Tables 1-10 and 15-19 below.

[0222] In some embodiments, the present invention provides an sdAb having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to any one of the IL-10Rα sdAb and IL-10Rβ sdAb sequences in Table 1, Table 2, Table 3, or Table 17. In some embodiments, the present invention provides an sdAb that is substantially identical to any one of the sdAbs in Table 1, Table 2, Table 3, or Table 17. In some embodiments, the present invention provides an sdAb that is identical to any one of the sdAbs in Table 1, Table 2, Table 3, or Table 17.

[0223] The above-mentioned polypeptides are expressed by transfecting host cells with a vector containing synthetic nucleic acid sequences encoding amino acid sequences of SEQ ID NO: 1-24 and SEQ ID NO: 500-523 (these are synthetic nucleic acid sequences of the aforementioned amino acid sequences incorporating a 5' nucleic acid sequence encoding an IgH signal peptide).

[0224] Humanized IL-10 agonist compounds This disclosure provides IL-10 agonist compounds comprising humanized anti-IL-10Rα sdAb and anti-IL-10Rβ sdAb. Where used herein, the terms IL-10Rα sdAb and IL-10Rβ sdAb are also used to refer to anti-IL-10Rα sdAb and anti-IL-10Rβ sdAb.

[0225] Tables C and D below show the percentage of identical amino acids in the humanized IL-10R VHH dimer compared to the V3-23 and VH3-66 germline immunoglobulin heavy chain sequences. Table 1 provides the amino acid sequences of the humanized IL-10 agonist compounds of this disclosure. These include IL-10Rα sdAb and IL-10Rβs sdAb, with the CDRs underlined. Table 14 provides the amino acid sequence of the non-humanized DR841 compound (SEQ ID NO: 465). These include non-humanized IL-10Rα sdAb (SEQ ID NO: 466) and non-humanized IL-10Rβ sdAb (SEQ ID NO: 470), with the CDRs listed separately in SEQ ID NO: 467-469 and SEQ ID NO: 471-473, respectively. Table 14 also provides the nucleic acid sequence (SEQ NO: 474) encoding DR841.

[0226] (Table C) Human IL-10Rα VHH TIFF2026521432000006.tif41160

[0227] (Table D) Human IL-10Rβ VHH TIFF2026521432000007.tif41160

[0228] (Table E) VHH DR241(hIL10Rα) TIFF2026521432000008.tif42160

[0229] (Table F) VHH DR246(hIL10Rβ) TIFF2026521432000009.tif43160

[0230] In some embodiments, the present invention provides an IL-10 agonist compound having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to any one of the IL-10 agonist compounds in Table 1 or Table 17 (e.g., single-domain antibody polypeptides that are IL-10 agonist compounds). In some embodiments, the present invention provides an IL-10 agonist compound that is substantially identical to any one of the IL-10 agonist compounds in Table 1 or Table 17 (e.g., single-domain antibody polypeptides that are IL-10 agonist compounds). In some embodiments, the present invention provides an IL-10 agonist compound that is identical to the sequence of any one of the IL-10 agonist compounds in Table 1 or Table 17 (e.g., single-domain antibody polypeptides that are IL-10 agonist compounds).

[0231] Biased activity The inflammatory response is a series of biological events in mammals initiated in response to infectious and / or damaging stimuli that reduce the likelihood of systemic infection. Typically, the mammalian inflammatory response is mediated by myeloid cells, particularly macrophages, which are activated by exogenous stimuli, such as bacterial cell wall components like lipopolysaccharide (LPS) from Gram-negative bacteria. Activated myeloid cells act as harbingers of infection and / or injury by secreting various pro-inflammatory signaling molecules, such as, but not limited to, interleukin-6 (IL-6), interleukin-1 (IL-1, e.g., IL-1β), and tumor necrosis factor α (TNFα), which initiate and / or mediate several biological processes associated with the inflammatory response.

[0232] While inflammatory responses are essential for protecting mammalian subjects from infection, excessive and / or chronic activation of immune cells, such as myeloid cells, is associated with tissue damage, organ dysfunction, and autoimmune diseases. A wide variety of human diseases are associated with excessive and / or chronic inflammation, including, but not limited to, inflammatory bowel disease (IBD), rheumatoid arthritis (RA), Alzheimer's disease, asthma, type 1 and type 2 diabetes, and cancer. Among the many immune modulator molecules, interleukin-10 (IL-10) suppresses inflammatory activity and prevents adverse effects associated with excessive inflammation. For example, genetic deficiencies in IL-10 are associated with severe inflammatory bowel disease (IBD) in both mice and humans. Therefore, IL-10 expression and secretion correlate with the suppression of inflammatory responses in immune cells, such as the expression and / or secretion of pro-inflammatory cytokines, and the inhibition of antigen presentation by activated myeloid cells.

[0233] In addition to its central role in suppressing inflammatory responses, IL-10 also promotes inflammatory activity in several cell types, including activated CD8+ T cells. Contact of IL-10 with activated CD8+ T cells results in enhanced secretion of pro-inflammatory cytokines, including interferon-gamma (IFNγ), granzyme A, and granzyme B. The pro-inflammatory and anti-inflammatory activities associated with IL-10 present significant challenges to its therapeutic use in treating inflammatory diseases in mammalian subjects.

[0234] In some aspects of this disclosure, an IL-10 agonist compound (e.g., a single-domain antibody polypeptide that is an IL-10 agonist compound) is a “biased” IL-10 agonist or “partial” IL-10 agonist that alters the relative pro-inflammatory and anti-inflammatory properties of wild-type IL-10. As used herein, the term “biased” means that, when used in relation to an IL-10 agonist compound (e.g., an sdAb dimer or a VHH dimer), the biased IL-10 agonist compound exhibits a level of wild-type IL-10 activity in a first cell type that is proportionally higher than the level of wild-type IL-10 activity in a second cell type compared to wild-type IL-10. In one aspect, the first cell type is a cell of myeloid origin, such as a myeloid cell. In some aspects, the myeloid cell is a myeloid cell, a granulocyte (e.g., a neutrophil, eosinophil, or basophil), a mast cell, or a monocyte. In some embodiments, monocytes are macrophages or dendritic cells. In some embodiments, macrophages are Kupffer cells. In one embodiment, the first cell type is activated myeloid cells. In one embodiment, the first cell type is LPS-activated human myeloid cells. In some embodiments, the second cell type is T cells.

[0235] The IL-10 agonist compounds of this disclosure (e.g., single-domain antibody polypeptides that are IL-10 agonist compounds) can inhibit pro-inflammatory responses and / or STAT3-mediated signaling in a cell-type-dependent manner, such that activation of inflammatory macrophages is inhibited without substantially promoting the production of inflammatory cytokines such as interferon-γ by T cells. In some embodiments, the IL-10 agonist compounds of this disclosure are CD8 +While reducing the immunostimulatory function of wild-type hIL-10, such as IFNγ production by T cells, it retains the immunosuppressive function of wild-type hIL-10, such as inhibition of inflammatory cytokine production. For example, in some embodiments, the IL-10 agonist compounds of this disclosure (e.g., single-domain antibody polypeptides that are IL-10 agonist compounds) retain activity comparable to wild-type hIL-10 and suppress the activation of myeloid cells (e.g., indicated by increased STAT3-mediated signaling in myeloid cells), but substantially reduce the activation of PBMCs, T cells, B cells, and NK cells (e.g., indicated by decreased IFNγ production). In some embodiments, the IL-10 agonist compounds of this disclosure (e.g., single-domain antibody polypeptides that are IL-10 agonist compounds) are hIL-10 partial agonists.

[0236] Pro-inflammatory activity and anti-inflammatory activity In some embodiments, an IL-10 agonist compound (e.g., a single-domain antibody polypeptide that is an IL-10 agonist compound) is a biased IL-10 partial agonist that (a) exhibits a significant level of at least one anti-inflammatory property of wild-type IL-10, and (b) exhibits a significantly reduced level of at least one pro-inflammatory property of wild-type IL-10. In some embodiments, "significant level of at least one anti-inflammatory property" means that the Emax of the IL-10 agonist compound (e.g., a single-domain antibody polypeptide that is an IL-10 agonist compound) with respect to such anti-inflammatory property is greater than 10%, alternatively greater than 20%, alternatively greater than 30%, alternatively greater than 40%, alternatively greater than 50%, alternatively greater than 60%, alternatively greater than 70%, alternatively greater than 80%, or alternatively greater than 90% of the Emax level of such anti-inflammatory property exhibited by wild-type IL-10, when determined in the test system. Examples of anti-inflammatory properties that can be measured in the test system include, but are not limited to, (a) suppression of hIL-1β expression and / or secretion by activated human myeloid cells, (b) suppression of hIL-6 expression and / or secretion by activated human myeloid cells, and (c) suppression of hTNFα expression and / or secretion by activated human myeloid cells. In some embodiments, activated human myeloid cells are obtained by isolating human monocytes from the buffy coat of a centrifuged anticoagulant-treated human blood sample according to a procedure well known in the art, and activating the isolated monocytes by contacting them with lipopolysaccharide (LPS). The levels of hIL-1β, hIL-6, and hTNFα expressed and / or secreted by the activated monocytes can be determined by immunoassay or flow cytometry according to a procedure well known in the art. One protocol for evaluating the suppression of hIL-1β, hIL-6, and hTNFα expression and / or secretion by LPS-activated human monocytes is provided in the examples described herein.

[0237] In some embodiments, “significantly reduced level of at least one pro-inflammatory property” means that the Emax of an IL-10 agonist compound (e.g., a single-domain antibody polypeptide that is an IL-10 agonist compound) for such pro-inflammatory property, when determined in the test system, is less than 90%, alternatively less than 80%, alternatively less than 70%, alternatively less than 60%, alternatively less than 50%, alternatively less than 40%, alternatively less than 30%, alternatively less than 20%, and alternatively less than 10% of the Emax of the pro-inflammatory property of wild-type IL-10. Examples of pro-inflammatory properties include, but are not limited to, (a) suppression of IFNγ expression and / or secretion by activated human CD8+ T cells, (b) suppression of granzyme A expression and / or secretion by activated human CD8+ T cells, and (c) suppression of granzyme A expression and / or secretion by activated human CD8+ T cells. In some embodiments, activated human T cells are obtained by isolating CD8+ T cells from human whole blood according to procedures well known in the art, and activating the isolated CD8+ cells by contacting them with anti-CD3 antibodies and anti-CD28 antibodies. The levels of IFNγ, granzyme A, and granzyme B expressed and / or secreted by the isolated CD8+ T cells can be determined by immunoassay or flow cytometry according to procedures well known in the art. One protocol for evaluating the expression and / or secretion of IFNγ, granzyme A, and granzyme B expressed and / or secreted by CD3 / CD28 activated CD8+ T cells is disclosed herein in the examples.

[0238] In some embodiments, an IL-10 agonist compound (e.g., a single-domain antibody polypeptide that is an IL-10 agonist compound) is a biased hIL-10 partial agonist that exhibits a significant level of anti-inflammatory properties of at least one wild-type hIL-10 and, at the same time, a significantly reduced level of pro-inflammatory properties of at least one wild-type IL-10, wherein the significant level of anti-inflammatory properties of at least one wild-type hIL-10 is greater than 30% of the Emax of the anti-inflammatory properties exhibited by wild-type hIL-10, and the at least one anti-inflammatory properties include (i) suppression of hIL-1β expression and / or secretion in LPS-activated human monocytes, and (ii) The group is selected from (iii) suppression of hIL-6 expression and / or secretion, or (iii) suppression of hTNFα expression and / or secretion in LPS-activated human monocytes, wherein the significantly reduced level of at least one pro-inflammatory property of wild-type hIL-10 is the Emax of at least one anti-inflammatory property which is less than 30% of the Emax of the anti-inflammatory property exhibited by wild-type hIL-10, and the at least one pro-inflammatory property is selected from the group consisting of (i) suppression of IFNγ expression and / or secretion by activated human CD8+ T cells, (ii) suppression of granzyme A expression and / or secretion by activated human CD8+ T cells, and (iii) suppression of granzyme A expression and / or secretion by activated human CD8+ T cells.

[0239] In some embodiments, the IL-10 agonist compounds of the present disclosure (e.g., a single-domain antibody polypeptide that is an IL-10 agonist compound) are biased hIL-10 partial agonists that exhibit a significant level of anti-inflammatory properties of at least one wild-type hIL-10 and, at the same time, a significantly reduced level of pro-inflammatory properties of at least one wild-type IL-10, wherein the significant level of anti-inflammatory properties of at least one wild-type hIL-10 is greater than 30% of the Emax of the anti-inflammatory properties exhibited by wild-type hIL-10, and the at least one anti-inflammatory properties are (i) suppression of hIL-1β expression and / or secretion in LPS-activated human monocytes, and (ii) LPS-activated human monocytes The group is selected from (iii) suppression of hIL-6 expression and / or secretion in LPS-activated human monocytes, wherein the significantly reduced level of at least one pro-inflammatory property of wild-type hIL-10 is the Emax of at least one anti-inflammatory property which is less than 30% of the Emax of the anti-inflammatory property exhibited by wild-type hIL-10, and the at least one pro-inflammatory property is selected from the group consisting of (i) suppression of IFNγ expression and / or secretion by activated human CD8+ T cells, (ii) suppression of granzyme A expression and / or secretion by activated human CD8+ T cells, and (iii) suppression of granzyme A expression and / or secretion by activated human CD8+ T cells.

[0240] In some embodiments, the IL-10 agonist compounds of this disclosure (e.g., single-domain antibody polypeptides that are IL-10 agonist compounds) exhibit the following characteristics in an assay of anti-inflammatory activity, where the Emax of the IL-10 agonist compound (e.g., single-domain antibody polypeptides that are IL-10 agonist compounds) is selected from the group consisting of (i) suppression of hIL-1β expression and / or secretion in LPS-activated human monocytes, (ii) suppression of hIL-6 expression and / or secretion in LPS-activated human monocytes, or (iii) suppression of hTNFα expression and / or secretion in LPS-activated human monocytes, in a wild-type setting. The present invention includes an IL-10 agonist compound whose Emax is greater than 30% of the Emax of type hIL-10, and whose Emax is less than 10% of the Emax of wild-type hIL-10 in an assay for pro-inflammatory activity selected from the group consisting of (i) suppression of IFNγ expression and / or secretion by activated human CD8+ T cells, (ii) suppression of granzyme A expression and / or secretion by activated human CD8+ T cells, and (iii) suppression of granzyme A expression and / or secretion by activated human CD8+ T cells.

[0241] In some embodiments, the IL-10 agonist compound of this disclosure (e.g., a single-domain antibody polypeptide that is an IL-10 agonist compound) exhibits an anti-inflammatory activity assay in which the Emax of the IL-10 agonist compound (e.g., a single-domain antibody polypeptide that is an IL-10 agonist compound) is selected from the group consisting of (i) suppression of hIL-1β expression and / or secretion in LPS-activated human monocytes, (ii) suppression of hIL-6 expression and / or secretion in LPS-activated human monocytes, or (iii) suppression of hTNFα expression and / or secretion in LPS-activated human monocytes, compared to wild-type hI A biased IL-10 agonist compound is one in which the Emax of the IL-10 compound is greater than 50% of the Emax of L-10, and the Emax of the IL-10 agonist compound (e.g., a single-domain antibody polypeptide that is an IL-10 agonist compound) is less than 20% of the Emax of wild-type hIL-10 in an assay for pro-inflammatory activity selected from the group consisting of (i) suppression of IFNγ expression and / or secretion by activated human CD8+ T cells, (ii) suppression of granzyme A expression and / or secretion by activated human CD8+ T cells, and (iii) suppression of granzyme A expression and / or secretion by activated human CD8+ T cells.

[0242] In some embodiments, the IL-10 agonist compound of this disclosure (e.g., a single-domain antibody polypeptide that is an IL-10 agonist compound) exhibits an anti-inflammatory activity assay in which the Emax of the IL-10 agonist compound (e.g., a single-domain antibody polypeptide that is an IL-10 agonist compound) is selected from the group consisting of (i) suppression of hIL-1β expression and / or secretion in LPS-activated human monocytes, (ii) suppression of hIL-6 expression and / or secretion in LPS-activated human monocytes, or (iii) suppression of hTNFα expression and / or secretion in LPS-activated human monocytes, compared to wild-type hI A biased IL-10 agonist compound is one in which the Emax of the IL-10 compound is greater than 50% of the Emax of L-10, and the Emax of the IL-10 agonist compound (e.g., a single-domain antibody polypeptide that is an IL-10 agonist compound) is less than 10% of the Emax of wild-type hIL-10 in an assay for pro-inflammatory activity selected from the group consisting of (i) suppression of IFNγ expression and / or secretion by activated human CD8+ T cells, (ii) suppression of granzyme A expression and / or secretion by activated human CD8+ T cells, and (iii) suppression of granzyme A expression and / or secretion by activated human CD8+ T cells.

[0243] STAT3 As mentioned above, the interaction between IL-10 and the IL-10 receptor results in intracellular signaling characterized by enhanced intracellular production of phosphorylated STAT3 (phospho-STAT3). Therefore, one measure of IL-10 activity that can be evaluated using cells expressing the IL-10 receptor (composed of IL-10Rα and IL-10Rβ) is intracellular production of phospho-STAT3.

[0244] In one embodiment, the IL-10 agonist compound of the present invention (e.g., a single-domain antibody polypeptide that is an IL-10 agonist compound) is a biased hIL-10 partial agonist, where the first cell type is activated human myeloid cells and the second cell type is activated human T cells, and the level of IL-10 activity is measured by intracellular production of phospho-STAT3. In one embodiment, the IL-10 agonist compound (e.g., a single-domain antibody polypeptide that is an IL-10 agonist compound) is a biased hIL-10 partial agonist that retains hIL-10 activity against activated human monocytes in a larger proportion than that against activated human CD8+ T cells, and the level of IL-10 activity is measured by intracellular production of phospho-STAT3. In some embodiments, the levels of IL-10 activity in a first cell type and a second cell type are measured by the production of phospho-STAT3 in the first cell type in response to contact between the first and second cell types and an IL-10 agonist compound.

[0245] In some embodiments, the relative activation of STAT3 signaling by the IL-10 agonist compounds described herein in a first cell type compared to a second cell type differs from the relative activation of STAT3 signaling by wild-type human or mouse IL-10 in a first cell type compared to a second cell type. In some embodiments, the level of intracellular phospho-STAT3 induced in human myeloid cells in response to contact with an effective amount of human IL-10 agonist compound is at least 10-fold, alternatively at least 100-fold, and alternatively at least 1000-fold greater than the level of intracellular phospho-STAT3 induced in human lymphocytes in response to contact with the same amount of human IL-10 agonist compound. In one embodiment, the ratio of STAT3 signaling levels induced in lymphocytes in response to contact with human IL-10 agonist compounds to the ratio of STAT3 signaling levels induced in lymphocytes in response to contact with lymphocytes and human IL-10 agonist compounds to the ratio of STAT3 signaling levels induced in lymphocytes in response to contact with lymphocytes and wild-type hIL-10 to the ratio of STAT3 signaling levels induced in lymphocytes in response to contact with lymphocytes and wild-type hIL-10 to the ratio of STAT3 signaling levels induced in lymphocytes in response to contact with lymphocytes and wild-type hIL-10 (either greater or less). In some embodiments, the ratio of the activity of human IL-10 agonist compounds in human myeloid cells compared to human lymphocytes (determined by the level of intracellular phospho-STAT3) is greater than the ratio of the activity of wild-type human IL-10 in human myeloid cells compared to human lymphocytes. In some embodiments, myeloid cells are neutrophils, eosinophils, mast cells, basophils, or monocytes. In some embodiments, monocytes are macrophages or dendritic cells. In some embodiments, macrophages are Kupffer cells. In some embodiments, lymphocytes are CD8+ T cells, CD4+ T cells, B cells, or NK cells.

[0246] In some embodiments, the IL-10 agonist compounds of this disclosure (e.g., single-domain antibody polypeptides that are IL-10 agonist compounds) affect the pSTAT3 E of wild-type hIL-10 in myeloid cells. max pENTER3 E max In some embodiments, the IL-10 agonist compounds of the present disclosure exhibit reduced STAT3-mediated signaling in lymphocytes such as T cells, B cells, or NK cells compared to wild-type hIL-10. In some embodiments, the IL-10 agonist compounds of the present disclosure exhibit reduced pSTAT3 E in lymphocytes compared to wild-type hIL-10 in lymphocytes. max pSTAT3 E less than 70%, less than 60%, less than 50%, less than 40%, or less than 30% max It has. In some embodiments, the IL-10 agonist compound (e.g., a single-domain antibody polypeptide that is an IL-10 agonist compound) has the pSTAT3 E of the wild-type or parental IL-10 polypeptide in lymphocytes. max Less than 70% (e.g., less than 70%, less than 60%, less than 50%, less than 40%, or less than 30%) but greater than 20% pSTAT3 E max This results in the following: In some embodiments, lymphocytes are selected from CD8+ T cells, CD4+ T cells, B cells, or NK cells.

[0247] Modification of binding molecules and sdAb components Chelated peptides In one embodiment, the present disclosure provides an IL-10 agonist compound comprising one or more transition metal chelated polypeptide sequences known as chelated peptides. The chelated peptide has the formula: (His) a -(AA) b -(His) c (SEQ ID NO:448) The polypeptides, as well as their random copolymers, block copolymers, and alternating copolymers, wherein "His" is the amino acid histidine, "AA" is an amino acid other than proline, and is a histidine residue, with a=an integer from 0 to 10, b=an integer from 0 to 4, and c=an integer from 0 to 10. In some embodiments, the chelated peptide has an amino acid sequence selected from the group consisting of SEQ ID NO: 416 to 439. The incorporation of such transition metal chelation domains facilitates purification by immobilized metal affinity chromatography (IMAC) as described in U.S. Patent No. 4,569,794 by Smith et al., issued on February 11, 1986. Examples of transition metal chelated polypeptides useful in the implementation of the IL-10Rβ-binding molecules of this disclosure are described in Smith, et al. and U.S. Patent No. 5,320,663 by Dobeli et al., published on May 10, 1995 (the entire teachings of these patents are incorporated herein by reference). Specific transition metal chelated polypeptides useful in the implementation of the IL-10 agonist compounds of this disclosure are polypeptides containing 3 to 6 consecutive histidine residues (SEQ ID NO: 451, 461), e.g., 6-histidine (His)6 peptide (SEQ ID NO: 451), which are often referred to in the art as "His tags". In addition to providing a purification "handle" for recombinant proteins or facilitating immobilization onto SPR sensor chips, the conjugation of IL-10 agonist compounds (e.g., single-domain antibody polypeptides that are IL-10 agonist compounds) to chelated peptides facilitates targeted delivery of transition metal ions to IL-10R-expressing cells as kinetically inactive or kinetically unstable conjugates, substantially in accordance with the teachings of Anderson et al., U.S. Patent No. 5,439,829, issued August 8, 1995, and Hale, JE1996. Analytical Biochemistry 231(1):46-49.Regarding "dynamically inert complexes," the term "inert" refers to the degree of instability of a particular complexing ion involved in a reaction, which results in the substitution of one or more ligands in its coordination sphere by other ligands. In an aqueous environment, unoccupied coordination positions of transition metals are occupied by water molecules. For a [transition metal:chelated peptide] complex to form, these water molecules must be substituted by a chelated peptide or organic chelating agent. If such a reaction occurs rapidly, the reaction is called "unstable" (labile). However, if such a reaction occurs slowly, the complex is said to be dynamically "inert." Dynamic instability or inactivity is related to the reaction rate and should not be confused with thermodynamic stability or instability. A simple example of this (unstable vs. stable) distinction is [Co(NH3)6]. 3+ Despite being provided by an ion, which is thermodynamically unstable as indicated by the equilibrium constant below, it persists in an acidic medium for several days due to its lack of dynamic inactivity or instability. [Co(NH3)6] 3+ +6H3O + =[Co(H2O)6] 3+ +6NH4 + K=10 25

[0248] In contrast, [Ni(CN)4] 2- Its stability is extremely high. [Ni(CN)4] 2- =Ni 2+ +4CN - K=10 -22 However, the CN with isotope-labeled cyanide ions added to this solution -The rate of ion exchange is so rapid that it is impossible to measure by conventional techniques. Advanced Inorganic Chemistry, Cotton, FA and Wilkinson, G. (1972) 3rd ed. Interscience Publishers, p. 652. In some embodiments, the transition metal ion is a reporter molecule, e.g., a fluorescent compound or a radioactive substance, e.g., a radiological imaging agent or a radiological therapeutic agent. In some embodiments, the chelated peptide is a chelated peptide.

[0249] Elimination of N-linked glycosylation sites In some embodiments, the amino acid sequence (particularly the CDR sequence) of IL-10Rα sdAb or IL-10Rβ sdAb may contain a glycosylation motif, particularly an N-linked glycosylation motif of the sequence Asn-X-Ser(NXS) or Asn-X-Thr(NXT) (where X is any amino acid other than proline). In such cases, it is desirable to prevent glycosylation by eliminating such N-linked glycosylation motif by modifying the sequence of the N-linked glycosylation motif. In some embodiments, the elimination of the Asn-X-Ser(NXS)N-linked glycosylation motif may be achieved by incorporating a conservation amino acid substitution of the Asn(N) residue and / or Ser(S) residue of the Asn-X-Ser(NXS)N-linked glycosylation motif. In some embodiments, the exclusion of the Asn-X-Thr(NXT)N-linked glycosylation motif may be achieved by incorporating conserved amino acid substitutions of the Asn(N) and / or Thr(T) residues of the Asn-X-Thr(NXT)N-linked glycosylation motif. In some embodiments, when expressing an IL-10 agonist compound containing IL-10Rα sdAb or IL-10Rβ sdAb (e.g., a single-domain antibody polypeptide that is an IL-10 agonist compound) in a prokaryotic host cell, exclusion of the glycosylation site is not necessary. Since prokaryotic cells do not provide a mechanism for glycosylation of recombinant proteins, sequence modifications to exclude the N-linked glycosylation site may be unnecessary when using a prokaryotic expression system to produce recombinant IL-10 agonist compounds containing IL-10Rα sdAb or IL-10Rβ sdAb.

[0250] Association with carrier molecules to increase the duration of action. The IL-10 agonist compounds described herein (e.g., single-domain antibody polypeptides that are IL-10 agonist compounds) can be modified to provide an extended lifetime in vivo and / or an extended duration of action in a target. In some embodiments, the conjugating molecule can be conjugated to a carrier molecule to provide a desired pharmacological property, such as an extended half-life. In some embodiments, the conjugating molecule can be covalently linked to the Fc domain of IgG, albumin, or other molecules, as is known in the art, for example, by PEGylation and glycosylation, in order to extend its half-life. In some embodiments, an IL-10 agonist compound modified to provide an extended duration of action in mammalian subjects (e.g., a single-domain antibody polypeptide that is an IL-10 agonist compound) has a half-life in mammals of longer than 4 hours, alternatively longer than 5 hours, alternatively longer than 6 hours, alternatively longer than 7 hours, alternatively longer than 8 hours, alternatively longer than 9 hours, alternatively longer than 10 hours, alternatively longer than 12 hours, alternatively longer than 18 hours, alternatively longer than 24 hours, alternatively longer than 2 days, alternatively longer than 3 days, alternatively longer than 4 days, alternatively longer than 5 days, alternatively longer than 6 days, alternatively longer than 7 days, alternatively longer than 10 days, alternatively longer than 14 days, alternatively longer than 21 days, or alternatively longer than 30 days.

[0251] Modifications of IL-10 agonist compounds (e.g., single-domain antibody polypeptides that are IL-10 agonist compounds) to provide an extended duration of action in mammalian subjects include, but are not limited to, conjugation of IL-10 agonist compounds to one or more carrier molecules, conjugation of IL-10 agonist compounds to protein carrier molecules in the form of fusion proteins with optionally additional polypeptide sequences (e.g., IL-10 agonist compound-Fc fusions), and conjugation to polymers (e.g., water-soluble polymers for providing PEGylated IL-10 agonist compounds).

[0252] It should be noted that two or more types of modifications may be used with respect to a given IL-10 agonist compound to provide an extended duration of action in mammalian subjects. For example, the IL-10 agonist compounds of this disclosure (e.g., a single-domain antibody polypeptide that is an IL-10 agonist compound) may include both amino acid substitutions that provide an extended duration of action and conjugation to a carrier molecule such as a polyethylene glycol (PEG) molecule.

[0253] Protein carrier molecules Examples of protein carrier molecules that can be covalently attached to IL-10 agonist compounds (e.g., single-domain antibody polypeptides that are IL-10 agonist compounds) to provide an extended duration of action in vivo include, but are not limited to, albumin, antibodies, and antibody fragments, such as the Fc domain of an IgG molecule.

[0254] Fc fusion: In some embodiments, IL-10 agonist compounds (e.g., single-domain antibody polypeptides that are IL-10 agonist compounds) are 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, thus reducing the frequency of administration of the biopharmaceutical product. Fc binds to the neonatal Fc receptor (FcRn) in endothelial cells lining blood vessels. Once bound, the Fc-fusion molecule is protected from degradation and re-released into circulation, allowing the molecule to circulate longer. This Fc binding is thought to be the mechanism by which endogenous IgG retains its long plasma half-life. More recent Fc-fusion technologies involve ligating only one copy of the biopharmaceutical to the Fc region of the antibody to optimize the pharmacokinetic and pharmacodynamic properties of the biopharmaceutical compared to traditional Fc-fusion conjugates. A useful "Fc region" in the preparation of Fc fusions can be a naturally occurring or synthetic polypeptide homologous to the IgG C-terminal domain produced by the digestion of IgG with papain. IgG Fc has a molecular weight of approximately 50 kDa. The conjugate molecules described herein can conjugate to the entire Fc region or to a smaller portion of the Fc region that retains the ability to extend the cyclic half-life of the chimeric polypeptide into which the conjugate molecule is incorporated. Additionally, the full-length or fragmented Fc region can be a variant of the wild-type molecule. In a typical form, each monomer of the dimeric Fc can carry heterologous polypeptides, which may be the same or different.

[0255] Linking of binding molecules to Fc monomers As described above, in linking an IL-10 agonist compound (e.g., a single-domain antibody polypeptide that is an IL-10 agonist compound) to the Fc subunit, a linker molecule, as described later, may be incorporated between the IL-10 agonist compound (e.g., a single-domain antibody polypeptide that is an IL-10 agonist compound) and the Fc subunit. In some embodiments, the IL-10 agonist compound (e.g., a single-domain antibody polypeptide that is an IL-10 agonist compound) is expressed as a fusion protein with an Fc domain incorporating the amino acid sequence of the hinge region of an IgG antibody. The Fc domain manipulated according to the above may be derived from the mammalian IgG species IgG1, IgG2, IgG3, and IgG4. In some embodiments, the Fc domain may be derived from the human IgG1, IgG2, IgG3, and IgG4 IgG species. In some embodiments, the hinge region is the hinge region of IgG1. In one particular embodiment, an IL-10 agonist compound (e.g., a single-domain antibody polypeptide that is an IL-10 agonist compound) is linked to an Fc domain using a human IgG1 hinge domain.

[0256] In some embodiments, the dimeric Fc molecule may be modified to have a "knob-into-hole modification" as detailed above.

[0257] Albumin carrier molecule In some embodiments, an IL-10 agonist compound (e.g., a single-domain antibody polypeptide that is an IL-10 agonist compound) is conjugated to an albumin molecule (e.g., human serum albumin) known in the art to promote prolonged in vivo exposure. In one embodiment of the present invention, an IL-10 agonist compound (e.g., a single-domain antibody polypeptide that is an IL-10 agonist compound) is conjugated to albumin via chemical linkage or expressed as a fusion protein with an albumin molecule referred to herein as an IL-10 agonist-albumin fusion. The term “albumin” when used in relation to the hzIL-10Rα / IL-10Rβ mutein albumin fusion includes albumins such as human serum albumin (HSA), cynomolgus monkey serum albumin, and bovine serum albumin (BSA). In some embodiments, the HSA contains the amino acid substitution C34S or K573P compared to the wild-type HSA sequence. According to this disclosure, albumin can be conjugated to IL-10 agonist compounds at the carboxyl terminus, amino terminus, both carboxyl and amino terminus, and internally (see, e.g., US 5,876,969 and US 7,056,701). In the HAS IL-10 agonist compounds envisioned by this disclosure, various forms of albumin can be used, such as albumin secretory presequences and their variants, fragments and their variants, and HSA variants. Such forms generally possess one or more desired albumin activities. In a further embodiment, this disclosure relates to a fusion protein comprising an IL-10 agonist compound directly or indirectly fused to albumin, albumin fragments, albumin variants, etc., wherein the fusion protein has higher plasma stability than the unfused drug molecule and / or the fusion protein retains the therapeutic activity of the unfused drug molecule.As an alternative to chemical linkage between an IL-10 agonist compound (e.g., a single-domain antibody polypeptide that is an IL-10 agonist compound) and an albumin molecule, the IL-10 agonist compound-albumin complex may be provided as a fusion protein comprising an albumin polypeptide sequence and an IL-10 agonist compound, optionally including a linker molecule between albumin and the IL-10 agonist compound, recombinantly expressed as a single polypeptide chain in host cells. Those skilled in the art can readily prepare such fusion proteins by recombinant techniques. Nucleic acid sequences encoding such fusion proteins may be ordered from any of the various commercial sources. The nucleic acid sequences encoding the fusion protein are incorporated into an expression vector and functionally linked to one or more expression regulatory elements, the vector is introduced into a suitable host cell, and the fusion protein is isolated from the host cell culture by techniques well known in the art.

[0258] Polymer carriers In some embodiments, an extended duration of action of an IL-10 agonist compound (e.g., a single-domain antibody polypeptide that is an IL-10 agonist compound) in vivo can be achieved by conjugation to one or more polymer carrier molecules, such as an XTEN polymer or a water-soluble polymer.

[0259] XTEN Conjugate The IL-10 agonist compound (e.g., a single-domain antibody polypeptide that is an IL-10 agonist compound) may further contain an XTEN polymer. Similar to PEGylation, to provide an extended duration in vivo, the XTEN polymer may be conjugated (chemically or as a fusion protein) to the IL-10 agonist compound (e.g., a single-domain antibody polypeptide that is an IL-10 agonist compound), which may be produced as a recombinant fusion protein in Escherichia coli (E. coli). XTEN polymers suitable for use with the IL-10 agonist compounds of this disclosure (e.g., single-domain antibody polypeptides that are IL-10 agonist compounds) are disclosed in Podust et al. 2016. Extension of in vivo half-life of biologically active molecules by XTEN protein polymers., J Controlled Release 240:52-66, and Haeckel et al. 2016. XTEN as Biological Alternative to PEGylation Allows Complete Expression of a Protease-Activatable Killin-Based Cytostatic. PLOS ONE|DOI:10.1371 / journal.pone.0157193 June 13, 2016. The XTEN polymer fusion protein may incorporate a protease-sensitive cleavage site, such as an MMP-2 cleavage site, between the XTEN polypeptide and the IL-10 agonist compound.

[0260] Water-soluble polymers In some embodiments, an IL-10 agonist compound (e.g., a single-domain antibody polypeptide of an IL-10 agonist compound) may be conjugated to one or more water-soluble polymers. Examples of water-soluble polymers useful in the practice of this disclosure include polyethylene glycol (PEG), polypropylene glycol (PPG), polysaccharides (polyvinylpyrrolidone, copolymers of ethylene glycol and propylene glycol, poly(oxyethylene polyol), polyolefin alcohols), polysaccharides, poly-alpha-hydroxy acids), polyvinyl alcohol (PVA), polyphosphazene, polyoxazoline (POZ), poly(N-acryloylmorpholine), or combinations thereof.

[0261] In some embodiments, an IL-10 agonist compound (e.g., a single-domain antibody polypeptide of an IL-10 agonist compound) can be conjugated to one or more polyethylene glycol molecules, or "PEGylated." The method or site of PEG attachment to the binding molecule can vary, but in certain embodiments, PEGylation does not alter, or only minimally alters, the activity of the binding molecule.

[0262] Suitable PEGs for conjugation into polypeptide sequences are generally soluble in water at room temperature and have a general formula. R(O-CH2-CH2) n Ure The formula has a configuration in which R is a hydrogen atom 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 carbon atoms. PEG can be linear or branched. Branched PEG derivatives, "star-PEG" and multi-armed PEG are envisioned in this disclosure.

[0263] In some examples, the sequences of the IL-10 agonist compounds of this disclosure provided in Tables 1, 2, and 3 harbor an N-terminal glutamine ("1Q") residue. The N-terminal glutamine residue has been observed to spontaneously cyclize, either under physiological conditions or in its vicinity, to form pyroglutamic acid (pE). (See, for example, Liu et al 2011. J Biol Chem. 286(13):11211-11217). In some embodiments, pyroglutamic acid formation complicates the N-terminal PEG conjugation, particularly when aldehyde chemistry is used for N-terminal PEGylation. As a result, when PEGylating the IL-10 agonist compounds of this disclosure (e.g., single-domain antibody polypeptides of IL-10 agonist compounds), particularly when aldehyde chemistry is used, IL-10 agonist compounds having an amino acid at position 1 (e.g., 1Q) are either substituted with another amino acid at position 1 or deleted at position 1 (e.g., des-1Q). In some embodiments, the IL-10 agonist compounds of this disclosure (e.g., single-domain antibody polypeptides of IL-10 agonist compounds) include amino acid substitutions selected from groups Q1E and Q1D.

[0264] In some embodiments, selective PEGylation of IL-10 agonist compounds (e.g., single-domain antibody polypeptides of IL-10 agonist compounds) may be utilized, for example, by incorporating non-natural amino acids having side chains that facilitate selective PEG conjugation. Specific PEGylation sites can be selected so that PEGylation of the binding molecule does not affect its binding to the target receptor.

[0265] In certain embodiments, an increase in half-life is greater than any decrease in biological activity. PEGs suitable for conjugation into polypeptide sequences are generally water-soluble at room temperature, with the general formula R(O-CH2-CH2) where R is a protecting group such as hydrogen or an alkyl or alkanol group, and n is an integer between 1 and 1000. nIt has OR. If R is a protecting group, it generally has 1 to 8 carbon atoms. The PEG conjugated with the polypeptide sequence can be linear or branched. Branched PEG derivatives, "star PEGs," and multi-armed PEGs are considered in this disclosure.

[0266] The molecular weight of PEG used in this disclosure is not limited to any particular range. The PEG component of the binding molecule may have a molecular mass greater than about 5 kDa, greater than about 10 kDa, greater than about 15 kDa, greater than about 20 kDa, greater than about 30 kDa, greater than about 40 kDa, or greater than about 50 kDa. In some embodiments, the molecular mass is about 5 kDa to about 10 kDa, about 5 kDa to about 15 kDa, about 5 kDa to about 20 kDa, about 10 kDa to about 15 kDa, about 10 kDa to about 20 kDa, about 10 kDa to about 25 kDa, or about 10 kDa to about 30 kDa. Linear or branched PEG molecules have molecular weights of approximately 2,000 to approximately 80,000 daltons, alternatively approximately 2,000 to approximately 70,000 daltons, alternatively approximately 5,000 to approximately 50,000 daltons, alternatively approximately 10,000 to approximately 50,000 daltons, alternatively approximately 20,000 to approximately 50,000 daltons, alternatively approximately 30,000 to approximately 50,000 daltons, alternatively approximately 20,000 to approximately 40,000 daltons, or alternatively approximately 30,000 to approximately 40,000 daltons. In one embodiment of this disclosure, PEG is a 40 kD branched PEG containing two 20 kD arms.

[0267] This disclosure also considers compositions of conjugates in which PEG has different n values ​​and therefore various different PEGs are present in specific ratios. For example, some compositions contain mixtures of conjugates where n = 1, 2, 3, and 4. In some compositions, the proportion of conjugates with n = 1 is 18–25%, the proportion of conjugates with n = 2 is 50–66%, the proportion of conjugates with n = 3 is 12–16%, and the proportion of conjugates with n = 4 is up to 5%. Such compositions can be produced by reaction conditions and purification methods known in the art. Chromatography may be used to separate the conjugate fractions, which are then identified, for example, containing conjugates with a desired number of PEGs and purified to be free of unmodified protein sequences and conjugates with other numbers of PEGs.

[0268] PEGs suitable for conjugation into polypeptide sequences are generally water-soluble at room temperature, with the general formula R(O-CH2-CH2) where R is a protecting group such as hydrogen, an alkyl group, or an alkanol group, and n is an integer between 1 and 1000. n It has OR. If R is a protecting group, it generally has 1 to 8 carbon atoms.

[0269] Two widely used first-generation activated monomethoxyPEGs (mPEGs) are succinimidylcarbonate PEG (SC-PEG; see, e.g., Zalipsky et al. 1992. Biotechnol Appl Biochem. 15:100-114) and benzotriazole carbonate PEG (BTC-PEG; see, e.g., Dolence et al., U.S. Patent No. 5,650,234), which preferentially react with lysine residues to form carbamate bonds, but are also known to react with histidine and tyrosine residues. The use of PEG aldehyde linkers targets a single site at the N-terminus of a polypeptide through reductive amination.

[0270] PEGylation most commonly occurs at the α-amino group at the N-terminus of a polypeptide, the epsilon-amino group at the side chain of a lysine residue, and the imidazole group at the side chain of a histidine residue. Since most recombinant polypeptides have a single α-amino group as well as several ε-amino and imidazole groups, numerous positional isomers can be generated depending on the chemical properties of the linker. General PEGylation strategies known in the art can be applied herein.

[0271] PEG can be attached to the binding molecules of this disclosure by terminal reactive groups ("spacers") that mediate the binding between a free amino group or carboxyl group of one or more polypeptide sequences and polyethylene glycol. PEG having spacers that can be attached to a free amino group includes N-hydroxysuccinilimide polyethylene glycol, which can be prepared by activating a succinate ester of polyethylene glycol with N-hydroxysuccinilimide.

[0272] In some embodiments, site-directed PEGylation of binding molecules is promoted by incorporating non-natural amino acids that support unique side chains. The incorporation of non-natural amino acids into polypeptides to provide functional moieties for achieving such site-directed PEGylation is known in the art. See, for example, Ptacin et al., PCT International Application No. PCT / US2018 / 045257 (WO 2019 / 028419Al).

[0273] PEG conjugated with a polypeptide sequence can be linear or branched. Branched PEG derivatives, "star PEGs," and multi-armed PEGs are considered in this disclosure. In certain embodiments, a PEG useful in the practice of this disclosure is a 10 kDa linear PEG-aldehyde (e.g., Sunbright® ME-100AL, NOF America Corporation, One North Broadway, White Plains,NY 10601 USA)), 10 kDa linear PEG-NHS esters (e.g., Sunbright (registered trademark) ME-100CS, Sunbright (registered trademark) ME-100AS, Sunbright (registered trademark) ME-100GS, Sunbright (registered trademark) ME-100HS, NOF), 20 kDa linear PEG-aldehydes (e.g., Sunbright (registered trademark) ME-200AL, NOF), 20 kDa linear PEG-NHS esters (e.g., Sunbright (registered trademark) ME-200CS, Sunbright (registered trademark) ME-200AS, Sunbright (registered trademark) ME-200GS, Sunbright (registered trademark) ME-200HS, NOF), 20 kDa 2-arm branched PEG-aldehydes containing two 10 kDa linear PEG molecules (e.g., Sunbright (registered trademark) GL2-200AL3, NOF), a 20 kDa two-arm branched PEG-NHS ester containing two 10 kDa linear PEG molecules (e.g., Sunbright® GL2-200TS, Sunbright® GL200GS2, NOF), a 40 kDa two-arm branched PEG-aldehyde containing two 20 kDa linear PEG molecules (e.g., Sunbright® GL2-400AL3), a 40 kDa two-arm branched PEG-NHS ester containing two 20 kDa linear PEG molecules (e.g., Sunbright® GL2-400AL3, Sunbright® GL2-400GS2, NOF), a linear 30 kDa It contains PEG-aldehydes (e.g., Sunbright (registered trademark) ME-300AL) and linear 30 kDa PEG-NHS esters.

[0274] In some embodiments, linkers can be used to link IL-10 agonist compounds (e.g., single-domain antibody polypeptides of IL-10 agonist compounds) and PEG molecules. A suitable linker generally comprises a modified polypeptide sequence and a “mobile linker” of sufficient length to allow some movement between the linked components and molecules. Linker molecules are generally about 6–50 atomic lengths. Linker molecules may be, for example, arylacetylenes, ethylene glycol oligomers containing 2–10 monomer units, diamines, diacitors, amino acids, or combinations thereof. A suitable linker can be readily selected and can be any suitable length, e.g., 1 amino acid length (e.g., Gly), 2, 3, 4, 5, 6, 7, 8, 9, 10, 10–20, 20–30, 30–50, or greater than 50 amino acid lengths. Examples of mobile linkers are described in Section IV. Furthermore, multimers of these linker sequences (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 10-20, 20-30, or 30-50) may be linked together to provide a mobile linker that can be used to conjugate two molecules. Instead of polypeptide linkers, the linker may be a chemical linker, such as a PEG-aldehyde linker. In some embodiments, the binding molecule is acetylated at its N-terminus by an enzymatic reaction with an N-terminal acetyltransferase and, for example, acetyl-CoA. Alternatively, or in addition to N-terminal acetylation, the binding molecule may be acetylated at one or more lysine residues by an enzymatic reaction with, for example, a lysine acetyltransferase. See, for example, Choudhary et al. 2009. Science 325 (5942):834840.

[0275] In some embodiments, the present invention provides a PEGylated IL-10 agonist compound of formula #1, wherein PEG is conjugated to an IL-10 agonist compound (e.g., a single-domain antibody polypeptide of an IL-10 agonist compound), and the PEG is a linear or branched PEG molecule having a molecular weight of about 2,000 to about 80,000 daltons, alternatively about 2,000 to about 70,000 daltons, alternatively about 5,000 to about 50,000 daltons, alternatively about 10,000 to about 50,000 daltons, alternatively about 20,000 to about 50,000 daltons, alternatively about 30,000 to about 40,000 daltons, or alternatively about 30,000 to about 40,000 daltons. In one embodiment of the present disclosure, the PEG is a 40kD branched chain PEG comprising two 20kD arms.

[0276] Fatty acid carriers In some embodiments, IL-10 agonist compounds that have an extended duration of action in mammalian subjects and are useful for carrying out the disclosure are achieved by covalently attaching an IL-10 agonist compound (e.g., a single-domain antibody polypeptide that is an IL-10 agonist compound) to a fatty acid molecule, as described in Resh. 2016. Progress in Lipid Research 63:120-131. Examples of fatty acids that can be conjugated include myristic acid, palmitic acid, and palmitoleic acid. Myristic acid is typically conjugated to the N-terminal glycine, but lysine can also be myristoylated. Palmitoylation is typically achieved by enzymatic modification of the -SH group of free cysteine, for example, the DHHC protein catalyzes S-palmitoylation. Palmitoleylation of serine and threonine residues is typically achieved enzymatically using the PORCN enzyme. In some embodiments, IL-10 agonist compounds (e.g., single-domain antibody polypeptides that are IL-10 agonist compounds) are acetylated at the N-terminus by an enzymatic reaction using an N-terminal acetyltransferase and, for example, acetyl-CoA. Alternatively, or in addition to N-terminal acetylation, IL-10 agonist compounds (e.g., single-domain antibody polypeptides that are IL-10 agonist compounds) are acetylated at one or more lysine residues by an enzymatic reaction, for example, using a lysine acetyltransferase. See, for example, Choudhary et al. 2009. Science 325 5942:834.

[0277] Modifications to provide additional functionality In some embodiments, the IL-10 agonist compound of this disclosure (e.g., a single-domain antibody polypeptide that is an IL-10 agonist compound) may include a functional domain of a chimeric polypeptide. The IL-10 agonist compound fusion protein of this disclosure can be readily produced by recombinant DNA methods using techniques known in the art, by constructing a recombinant vector containing a nucleic acid sequence encoding the IL-10 agonist compound such that it is in-frame with the nucleic acid sequence encoding the fusion partner at either the N-terminus or C-terminus of the IL-10 agonist compound (e.g., a single-domain antibody polypeptide that is an IL-10 agonist compound). The sequence optionally further comprises an in-frame nucleic acid sequence encoding a linker or spacer polypeptide.

[0278] FLAG tag In other embodiments, the IL-10 agonist compound (e.g., a single-domain antibody polypeptide that is an IL-10 agonist compound) may be modified to include an additional polypeptide sequence, such as a FLAG sequence, that functions as an antigenic tag. The FLAG sequence is recognized by a biotinylated, highly specific, anti-FLAG antibody as described herein (see, e.g., Blanar et al. 1992. Science. 256:1014 and LeClair et al. 1992. PNAS-USA. 89:8145). In some embodiments, the binding molecule further includes a C-terminal c-myc epitope tag.

[0279] Targeting section In some embodiments, an IL-10 agonist compound (e.g., a single-domain antibody polypeptide that is an IL-10 agonist compound) is conjugated to a molecule that provides a targeting domain to facilitate selective binding to a specific cell type or tissue expressing a cell surface molecule that specifically binds to the “targeting domain,” and optionally incorporates a linker molecule of 1 to 40 (alternatively 2 to 20, alternatively 5 to 20, or alternatively 10 to 20) amino acids between the IL-10 agonist compound sequence and the targeting domain sequence of the fusion protein.

[0280] In other embodiments, chimeric polypeptides comprising an IL-10 agonist compound and an antibody or its antigen-binding moiety can be generated. The antibody or antigen-binding component of the chimeric protein can serve as a targeting moiety. For example, it can be used to localize the chimeric protein to a specific subset of cells or target molecule. Methods for generating cytokine-antibody chimeric polypeptides are described, for example, in U.S. Patent No. 6,617,135.

[0281] In some embodiments, the targeting portion is an antibody that specifically binds to at least one cell surface molecule (i.e., at least one tumor antigen) associated with tumor cells, and the cell surface molecule associated with tumor cells is selected from the group consisting of GD2, BCMA, CD19, CD33, CD38, CD70, GD2, IL3Rα2, CD19, mesothelin, Her2, EpCam, Muc1, ROR1, CD133, CEA, EGRFR VIII, PSCA, GPC3, Pan-ErbB, and FAP.

[0282] C-terminal modification to reduce immunogenicity In some embodiments, this disclosure provides modifications to a newly exposed C-terminal VTVSS amino acid sequence (e.g., SEQ ID NO: 474-499) to eliminate or reduce recognition by existing antibodies. The exposed C-terminal VTVSS amino acid sequence (SEQ ID NO: 474) of camel-derived single-domain antibody fragments, e.g., VHH fragments and scFv fragments, is recognized by existing antibodies in the circulating immune system, resulting in an immunogenic response that limits the efficacy of therapeutic VHH and scFv drug therapies. This existing antibody immune response can be reduced by modifying the C-terminal amino acid sequence of single-domain antibodies having the exposed C-terminal VTVSS amino acid sequence (SEQ ID NO: 474). For example, Nieba et al. disclose mutating positions 11, 14, 41, 84, 87, and / or 89 (amino acid position numbers by Kabat) in the VH region. WO 11 / 07586 discloses mutations at positions 99, 101, and / or 148 in the VL domain, or at positions 12, 97, 98, 99, 103, and / or 144 in the VH domain (corresponding to amino acid positions 11, 83, 84, 85, 89, and 103 according to Kabat).

[0283] In some embodiments, such amino acid sequence modifications to the C-terminal VTVSS amino acid sequence (SEQ ID NO: 474) alter the neoepitope resulting from the newly exposed C-terminal VTVSS amino acid sequence (SEQ ID NO: 474) on bispecific antibodies containing single-chain, scFv, sdAb, Fab, diabody, scFab, or any other antigen-binding domain or Fc fusion protein, for example, exposing the normally unexposed N-terminal or C-terminal sequence, on single-chain antibodies such as sdAb, VHH, and on multi-domain antibodies including fusions of IgG or HSA with other single-chain antibodies.

[0284] In one aspect, the disclosure relates to an isolated single-domain antibody comprising a C-terminal modification, wherein the C-terminal modification comprises the substitution, addition, or deletion of at least one amino acid residue such that the substitution, addition, or deletion of at least one amino acid residue to the single-domain antibody eliminates interaction between at least one existing antibody and the single-domain antibody without interfering with the binding of the single-domain antibody to its target.

[0285] In one embodiment, the C-terminal amino acid sequence of a single-domain antibody is exposed in a way that allows the exposed C-terminus to be used for interaction with existing antibodies, and C-terminal modification reduces the exposure of the C-terminus to existing antibodies.

[0286] In one aspect, this disclosure relates to endogenous sdAb amino acid residues (T / L 108 )V 109 T 110 V 111 S 112 S 113 The present disclosure provides a polypeptide comprising a single-domain antibody (sdAb) containing a modified C-terminal amino acid sequence corresponding to (SEQ ID NO:474) (numbering according to the Kabat numbering scheme for human VH carboxy-terminal amino acid residues). According to this disclosure, the modified amino acid sequence is formula X 108 X 109 X 110 V 111 X 112 X 113 Including Y, During the ceremony, X 108 It is selected from the group consisting of L, T, and Q, X 109 It is selected from the group consisting of V, G, N, and L. X 110 It is selected from the group consisting of T and Q, X 111 is V, X 112 is selected from the group consisting of S, C, T, A, and G, or is arbitrary, does not exist, and X 113It is selected from the group consisting of S, C, A, G, and T, or it may not exist at all. however, X 109 If X is V, 112 and X 113 It is not possible for both to be S, and X 112 and X 113 It is not the case that both and are C, and Y is either absent or contains a polypeptide comprising 1 to 5 amino acids, the amino acids being independently selected from the group consisting of A, G, S, T, L, and V.

[0287] In some embodiments, sdAb is further modified to include amino acid substitutions selected from the group consisting of L11S, L11Q, L11G, and P14A, in numbering according to the Kabat numbering scheme.

[0288] In some aspects of this disclosure, X 108 L is X 109 X is selected from the group consisting of V, G, and L. 110 is T, and X 112 X is selected from the group consisting of S, T, and C. 113 X is selected from the group consisting of S, T, C, and A. 114 The polypeptide comprises 1 to 5 amino acids independently selected from the group consisting of A, G, S, T, L, and V, where Y is absent or is a dipeptide of amino acid sequence AA. In some embodiments, Y is AA. In other embodiments, amino acid sequence X 109 X 110 V 111 X 112 X 113 V 109 T 110 V 111 S 112 A 113Y is AA (disclosed as SEQ ID NO: 479 and SEQ ID NO: 480, respectively, "VTVSA" and "VTVSAAA"). In some embodiments, sdAb is an amino acid sequence X selected from the group consisting of SEQ ID NO: 121-135, 138-141, 144-155, 158-175, 179, 182-195, and 199 in Table 5, such as GTVSS (SEQ ID NO: 476), LTVSS (SEQ ID NO: 477), VTVCS (SEQ ID NO: 481), and VTVSC (SEQ ID NO: 483). 109 X 110 V 111 X 112 X 113 Includes.

[0289] In some embodiments, the polypeptides of this disclosure exhibit reduced binding to existing antibodies.

[0290] In another context, this disclosure relates to the formula: VHH1-L n -VHH2 Regarding the polypeptide, in the formula, VHH1 is the first VHH, L is a polypeptide linker containing 1 to 50 amino acids, n is 0 or 1, and VHH2 is the second VHH, which may be the same as or different from VHH1. The above formula VHH1-L n In -VHH2, the linker molecule L can be a GS linker. Various suitable GS linkers are listed and illustrated in Table 11 of this invention.

[0291] In some embodiments, VHH1 and VHH2 independently bind to the extracellular domain of the cytokine receptor. In some embodiments, the cytokine receptor to which VHH1 and VHH2 bind is selected from the group consisting of IL-2Rα, IL-2Rβ, IL-2Rγ, IL-10Rα, IL-10Rβ, IL-12Rβ1, IL-12Rβ2, IL-18Rα, IL-18Rβ, IL-22R1, IL-27Rα, gp130, IL-23R, IL-28Rα, IFNRγ1, and IFNRγ2. In some embodiments, VHH1 and VHH2 selectively bind to cytokine receptor pairs selected from the following pairs: IL-10Rα / IL-10Rβ, IL-27Rα / gp130, IFNγR1 / IFNγR2, IL-10Rβ / IL-28Rα, IL-2Rβ / IL2Rγ, IL-18Rα / IL-18Rβ, IL-22R1 / IL-10Rβ, IL-10Rα / IL-2Rγ, IL-2Rβ / IL-2Rγ, IL-10R1 / IFNRγ, IFNRγ / IL-28Rα, IL-12Rβ1 / IL-12Rβ2, IL-12Rβ1 / IL-23R, and IL-10Rα / IL-2Rγ.

[0292] In some embodiments, a single-domain antibody and its antigen-binding fragment can be conjugated to various other chemical entities, such as antibody-drug conjugates (ADCs).

[0293] Examples of modifications to the C-terminal VTVSS motif (SEQ ID NO: 474) of the IL10R agonist compounds of this disclosure are provided in SEQ ID NO: 121-135, 138-141, 144-155, 158-175, 179, 182-195, and 199 in Table 5.

[0294] Recombinant manufacturing Alternatively, the humanized IL-10 agonist compounds of this disclosure are produced by recombinant DNA technology. In a typical implementation of recombinant polypeptide production, a nucleic acid sequence encoding the desired polypeptide is incorporated into an expression vector suitable for the host cell in which expression will be achieved. The nucleic acid sequence is functionally ligated by the vector to one or more expression regulatory sequences that are encoded and functional in the target host cell. The recombinant protein may be recovered by host cell disruption or, if a secretory leader sequence (signal peptide) is incorporated into the polypeptide, from cell culture.

[0295] Nucleic acid sequence encoding an IL-10 agonist compound In some embodiments, the Disclosure provides nucleic acid sequences encoding IL-10 agonist compounds with SEQ ID NO: 1-24, 121-135, 138-141, 144-155, 158-175, 179, 182-195, 199, and 500-523.

[0296] In some embodiments, humanized IL-10 agonist compounds are produced by recombinant methods using nucleic acid sequences encoding an IL-10 agonist compound (e.g., a single-domain antibody polypeptide that is an IL-10 agonist compound) (or a fusion protein containing an IL-10 agonist compound (e.g., a single-domain antibody polypeptide that is an IL-10 agonist compound)). The nucleic acid sequence encoding the desired hzIL-10 agonist compound can be synthesized by chemical means using an oligonucleotide synthesizer.

[0297] Nucleic acid molecules are not limited to sequences that encode polypeptides, but can include some or all of the non-coding sequences upstream or downstream of the coding sequence (e.g., the coding sequence for IL-2). Those skilled in the art of molecular biology are familiar with common procedures for isolating nucleic acid molecules. They can be produced, for example, by processing genomic DNA with restriction endonucleases or by performing polymerase chain reactions (PCR). If the nucleic acid molecule is ribonucleic acid (RNA), the molecule can be produced, for example, by in vitro transcription.

[0298] Nucleic acid molecules encoding IL-10 agonist compounds (e.g., single-domain antibody polypeptides that are IL-10 agonist compounds) (and their fusions) may contain naturally occurring sequences or sequences that are different from naturally occurring ones but encode the same polypeptide due to genetic degeneracy. These nucleic acid molecules may consist of RNA or DNA (e.g., genomic DNA, cDNA, or synthetic DNA, e.g., those produced by phosphoramidite-based synthesis), or combinations or modifications of nucleotides within these types of nucleic acids. Additionally, nucleic acid molecules may be double-stranded or single-stranded (i.e., either sense strands or antisense strands).

[0299] Nucleic acid sequences encoding IL-10 agonist compounds (e.g., single-domain antibody polypeptides that are IL-10 agonist compounds) can be obtained from various commercial sources that provide custom nucleic acid sequences. Amino acid sequence variants of the IL-10 agonist compounds of this disclosure (e.g., single-domain antibody polypeptides that are IL-10 agonist compounds) are prepared by introducing appropriate nucleotide changes into the coding sequence based on the genetic code known in the art. Such variants represent insertions, substitutions, and / or specified deletions of residues, as described above. Any combination of insertions, substitutions, and / or specified deletions is made to arrive at the final construct, insofar as the final construct has the desired biological activity as defined herein.

[0300] Methods for constructing DNA sequences encoding IL-10 agonist compounds and expressing them in appropriately transformed hosts include, but are not limited to, the use of PCR-based mutagenesis techniques. Mutations consisting of deletions or additions of amino acid residues to IL-10 agonist compounds can also be performed using standard recombination techniques. In the case of nucleotide deletions or additions, the nucleic acid molecule encoding the IL-10 agonist compound is optionally digested with an appropriate restriction endonuclease. The resulting fragment can be expressed as is or further manipulated, for example, by ligating it into a second fragment. Ligation may be prompted by the presence of complementary nucleotides where two ends of the nucleic acid molecule overlap, but blunt-end fragments can also be ligated. Nucleic acids generated by PCR can also be used to generate various mutant sequences.

[0301] The IL-10 agonist compounds of this disclosure can be synthesized recombinantly not only directly, but also as fusion polypeptides with heterologous polypeptides at the N-terminus or C-terminus of a mature IL-10 agonist compound, such as a signal sequence or another polypeptide having a specific cleavage site. Generally, the signal sequence may be a component of the vector, or it may be part of the coding sequence inserted into the vector. The selected heterologous signal sequence is preferably one that is recognized and processed (i.e., cleaved by a signal peptidase) by a host cell. The incorporation of the signal sequence depends on whether it is desired that the IL-10 agonist compound (e.g., a single-domain antibody polypeptide that is an IL-10 agonist compound) be secreted from the recombinant cell that produces it. If the selected cell is a prokaryotic cell, it is generally preferable that the DNA sequence does not encode a signal sequence. If the recombinant host cell is a yeast cell, such as Saccharomyces cerevisiae, extracellular secretion of an IL-10 agonist compound (e.g., a single-domain antibody polypeptide that is an IL-10 agonist compound) into the culture medium may be achieved using an alpha-conjugation factor secretion signal sequence, as described in Singh's U.S. Patent No. 7,198,919 (B1), issued on April 3, 2007.

[0302] When expressing an IL-10 agonist compound (e.g., a single-domain antibody polypeptide that is an IL-10 agonist compound) as a chimera (e.g., a fusion protein containing an IL-10 agonist compound and a heterologous polypeptide sequence), the chimeric protein can be encoded by a hybrid nucleic acid molecule containing a first sequence encoding all or part of the IL-10 agonist compound (e.g., a single-domain antibody polypeptide that is an IL-10 agonist compound) and a second sequence encoding all or part of the heterologous polypeptide. For example, the subject IL-10 agonist compounds described herein may be fused to hexa- / octa-histidine tags (H6, H7, H8 disclosed as SEQ ID NO: 451-453, respectively) to facilitate the purification of proteins expressed by bacteria, or to hemagglutinin tags to facilitate the purification of proteins expressed in eukaryotic cells. The terms "first" and "second" should not be understood as limitations on the orientation of the elements of the fusion protein, as heterologous polypeptides can be ligated to either the N-terminus and / or C-terminus of an IL-10 agonist compound (e.g., a single-domain antibody polypeptide that is an IL-10 agonist compound). For example, the N-terminus may be ligated to a targeting domain, and the C-terminus to a hexahistidine tag (SEQ ID NO: 451) purification handle.

[0303] The gene to be backtranslated can be constructed using the complete amino acid sequence of the polypeptide (or fusion / chimera) to be expressed. DNA oligomers containing the nucleotide sequence encoding the IL-10 agonist compound can be synthesized. For example, several small oligonucleotides encoding a portion of the desired polypeptide can be synthesized and then ligated. Individual oligonucleotides typically contain a 5' or 3' overhang for complementary assembly.

[0304] Codon optimization In some embodiments, nucleic acid sequences encoding IL-10 agonist compounds (e.g., single-domain antibody polypeptides of IL-10 agonist compounds) may be “codon-optimized” to promote expression in specific host cell types. Techniques for codon optimization in diverse expression systems, including mammalian, yeast, and bacterial host cells, are well known in the art, and online tools are available to provide codon-optimized sequences for expression in various host cell types. For example, see Al-Hawash et al. 2017. Gene Reports. 9:46-53 and Mauro and Chappell, Recombinant Protein Expression in Mammalian Cells:Methods and Protocols See David Hacker (Human Press New York). Additionally, there are various web-based online software packages freely available to assist in the preparation of codon-optimized nucleic acid sequences.

[0305] Expression vector Once assembled (by synthesis, site-directed mutagenesis, or other means), the nucleic acid sequence encoding the IL-10 agonist compound is inserted into the expression vector. Various expression vectors are available for use in different host cells and are typically selected based on the host cell for expression. An expression vector typically includes, but is not limited to, one or more of the following: an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence. Vectors include viral vectors, plasmid vectors, and embedded vectors. Plasmids are an example of non-viral vectors.

[0306] To promote the efficient expression of recombinant polypeptides, the nucleic acid sequence encoding the polypeptide sequence to be expressed is functionally ligated to a functional transcriptional and translational regulatory sequence in a selected expression host.

[0307] Selection Marker Expression vectors typically contain a selection gene, also known as a selection marker. This gene encodes a protein necessary for the survival or proliferation of transformed host cells grown in a selective culture medium. Host cells not transformed with a vector containing the selection gene will not survive in the culture medium. Typical selection genes encode proteins that (a) confer resistance to antibiotics or other toxins, such as ampicillin, neomycin, methotrexate, or tetracycline; (b) compensate for nutritional deficiencies; or (c) supply essential nutrients not available from the complex medium.

[0308] Regulatory control array The expression vectors for IL-10 agonist compounds of this disclosure contain a regulatory sequence that is recognized by a host organism and functionally linked to a nucleic acid sequence encoding an IL-10 agonist compound (e.g., a single-domain antibody polypeptide of an IL-10 agonist compound). The terms “regulatory sequence,” “regulatory sequence,” or “expression regulatory sequence” are used interchangeably herein and refer to promoters, enhancers, and other expression regulatory elements (e.g., polyadenylation signals). See, for example, Goeddel. 1990. Gene Expression Technology: Methods in Enzymology 185 (Academic Press, San Diego, CA USA). Regulatory sequences include those that direct the constitutive expression of a nucleotide sequence in many types of host cells and those that direct the expression of a nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences). It will be understood by those skilled in the art that the design of an expression vector may depend on factors such as the selection of host cells to be transformed and the level of expression of the desired protein. Various factors understood by those skilled in the art should be considered in the selection of the expression regulatory sequence. These include, for example, the relative strength of the sequence, its controllability, and its compatibility with the actual DNA sequence encoding the IL-10 agonist compound (e.g., a single-domain antibody polypeptide of an IL-10 agonist compound), particularly its potential secondary structure.

[0309] promoter In some embodiments, regulatory sequences are promoters, which are selected, for example, based on the cell type to which expression is desired. Promoters are untranslated sequences located upstream (5') (generally within about 100–1000 bp) of the start codon of structural genes that control the transcription and translation of specific nucleic acid sequences to which they are functionally linked. Such promoters typically fall into two classes: inductive and constitutive. Inductive promoters are those that initiate an increased level of transcription from their controlled DNA in response to some change in culture conditions, such as the presence or absence of nutrients or changes in temperature. A large number of promoters recognized by various potential host cells are well known.

[0310] The T7 promoter is available for use in bacteria, the polyhedrin promoter for use in insect cells, and the cytomegalovirus or metallothionein promoter for use in mammalian cells. Furthermore, in higher eukaryotes, tissue-specific and cell-type-specific promoters are widely available. These promoters are so named for their ability to direct the expression of nucleic acid molecules in a given tissue or cell type within the body. Those skilled in the art are well aware of the numerous promoters and other regulatory elements that can be used to direct the expression of nucleic acids.

[0311] Transcription from a vector in mammalian host cells may be controlled by promoters obtained from the genomes of viruses, e.g., polyomaviruses, fowlpox viruses, adenoviruses (e.g., human adenovirus serotype 5), bovine papillomavirus, aerosarcoma viruses, cytomegaloviruses, retroviruses (e.g., mouse stem cell viruses), hepatitis B viruses, most preferably Simian virus 40 (SV40), heterozoan promoters, e.g., actin promoters, PGK (phosphoglycerate kinase), or immunoglobulin promoters, heat shock promoters, insofar as such promoters are compatible with the host cell system. Early and late promoters of the SV40 virus can be conveniently obtained as SV40 restriction fragments that also contain the SV40 virus origin of replication.

[0312] Enhancer Transcription in higher eukaryotes is often increased by inserting enhancer sequences into vectors. Enhancers are cis-acting elements of DNA, typically about 10–300 bp in length, that act on the promoter to increase transcription. Enhancers are relatively orientation and position-independent and can be found within the coding sequence itself, as well as at the 5' and 3' positions of the transcription unit, within introns, and in the coding sequence itself. Many enhancer sequences from mammalian genes (globin, elastase, albumin, alpha-fetoprotein, and insulin) are now publicly known. Typically, however, enhancers from eukaryotic viruses are used. Examples include the SV40 enhancer on the late side of the origin of replication, the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the origin of replication, and the adenovirus enhancer. Enhancers may be spliced ​​into the expression vector at the 5' or 3' position of the coding sequence, but are preferably located at the 5' site of the promoter. Expression vectors used in eukaryotic host cells also contain sequences necessary for transcription termination and mRNA stabilization. Such sequences are commonly available from the 5' and occasionally 3' untranslated regions of eukaryotic or viral DNA or cDNA. Standard techniques are used to construct suitable vectors containing one or more of the components listed above.

[0313] In addition to sequences that promote the transcription of the inserted nucleic acid molecule, vectors may contain other genes encoding replication origins and selection markers. For example, the neomycin resistance (neoR) gene confers G418 resistance to cells in which it is expressed, thus enabling phenotypic selection of transfected cells. Examples of additional marker or reporter genes include beta-lactamase, chloramphenicol acetyltransferase (CAT), adenosine deaminase (ADA), dihydrofolate reductase (DHFR), hygromycin-B-phosphotransferase (HPH), thymidine kinase (TK), lacZ (encoding beta-galactosidase), and xanthine guanine phosphoribosyltransferase (XGPRT). Those skilled in the art can readily determine whether a given regulatory element or selection marker is suitable for use in a particular experimental context.

[0314] The proper assembly of the expression vector can be confirmed by nucleotide sequencing, restriction mapping, and expression of a biologically active polypeptide in a suitable host.

[0315] host cell This disclosure further provides prokaryotic or eukaryotic cells containing and expressing nucleic acid molecules encoding IL-10 agonist compounds. The cells of this disclosure are transfected cells, i.e., cells into which nucleic acid molecules, such as those encoding mutant IL-2 polypeptides, have been introduced by means of recombinant DNA technology. Progeny of such cells are also considered to be within the scope of this disclosure.

[0316] Host cells are typically selected according to their compatibility with the selected expression vector, the toxicity of the products encoded by the DNA sequence of the present invention, their secretory characteristics, their ability to accurately fold polypeptides, their fermentation or culture requirements, and the ease of purifying the products encoded by the DNA sequence. Suitable host cells for DNA cloning or expression in the vectors herein are prokaryotes, yeasts, or higher eukaryotic cells.

[0317] In some embodiments, recombinant IL-10 agonist compounds can also be produced in eukaryotes, such as yeast or human cells. Suitable eukaryotic host cells include insect cells (examples of baculovirus vectors available for protein expression in cultured insect cells (e.g., Sf9 cells) include the pAc series (Smith et al. 1983. Mol Cell Biol. 3:2156-2165) and the pVL series (Lucklow and Summers. 1989. Virology. 170:31-39)); yeast cells (examples of vectors for expression in yeast S. cerevisiae include pYepSecl (Baldari et al. 1987. EMBO J.6:229-234), pMFa (Kurjan and Herskowitz. 1982. Cell. 30:933-943), pJRY88 (Schultz et al. 1987. Gene. 54:113-123), and pYES2 (Invitrogen). This includes pPicZ (Invitrogen Corporation, San Diego, Calif.) and pPicZ (Invitrogen Corporation, San Diego, Calif.); or mammalian cells (mammalian expression vectors include pCDM8 (Seed. 1987. Nature. 329:840) and pMT2PC (Kaufman et al. 1987. EMBO J. 6:187:195)).

[0318] Examples of useful mammalian host cell lines include: mouse L cells (LM[TK-], ATCC#CRL-2648); monkey kidney CV1 cell line transformed with SV40 (COS-7, ATCC CRL 1651); human embryonic kidney cell line (HEK293 or HEK293 cells subcloned for growth in suspension culture); baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary cells-DHFR(CHO); mouse Sertoli cells (TM4); monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1 587); human cervical cancer cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat hepatocytes (BRL 3A, ATCC CRL 1442); and human lung cells (W138, ATCC CCL 75). These include human hepatocytes (Hep G2, HB 8065); mouse mammary tumor cells (MMT 060562, ATCC CCL51); TRI cells; MRC 5 cells; FS4 cells; and human hepatoma strain (Hep G2). In mammalian cells, the regulatory function of expression vectors is often provided by viral regulatory elements. For example, commonly used promoters are derived from polyoma, adenovirus 2, cytomegalovirus, and Simianvirus 40.

[0319] IL-10 agonist compounds (e.g., single-domain antibody polypeptides of IL-10 agonist compounds) may be produced in prokaryotic hosts, e.g., *Escherichia coli*, or eukaryotic hosts, e.g., insect cells (e.g., Sf21 cells), or mammalian cells (e.g., COS cells, NIH 3T3 cells, or HeLa cells). These cells are available from many suppliers, including the American Type Culture Collection (Manassas, Va.). In selecting an expression system, the only concern is that the components are compatible with each other. Those skilled in the art can make such a decision. Furthermore, if guidance is needed in selecting an expression system, those skilled in the art can refer to Ausubel et al. 1993. Current Protocols in Molecular Biology. John Wiley and Sons, New York, NY) and Pouwels et al. 1987. Cloning Vectors: A Laboratory Manual. Suppl.).

[0320] In some embodiments, the resulting IL-10 agonist compound is glycosylated or deglycosylated depending on the host organism used to produce the mutaine. When bacteria are selected as the host, the IL-10 agonist compound produced (e.g., a single-domain antibody polypeptide of the IL-10 agonist compound) is deglycosylated. Eukaryotic cells, on the other hand, typically result in glycosylation of the IL-10 agonist compound (e.g., a single-domain antibody polypeptide of the IL-10 agonist compound).

[0321] In some embodiments, the amino acid sequence of sdAb to be incorporated into the IL-10 agonist compound (particularly the CDR sequence) may contain a glycosylation motif, particularly an N-linked glycosylation motif of the sequence Asn-X-Ser(NXS) or Asn-X-Thr(NXT) (where X is any amino acid other than proline). In such cases, it is desirable to eliminate such an N-linked glycosylation motif by modifying its sequence to prevent glycosylation. In some embodiments, the N-linked glycosylation motif is disrupted by incorporating a conserved amino acid substitution of the Asn(N) residue of the N-linked glycosylation motif.

[0322] For other additional expression systems for both prokaryotic and eukaryotic cells, see Chapters 16 and 17 of Sambrook et al. 1989. Molecular Cloning: A Laboratory Manual (2nd ed., Cold Spring Harbor Laboratory Press, Plainview, NY) and Goddel. 1990. Gene Expression Technology: Methods in Enzymology 185 (Academic Press, San Diego, Calif.).

[0323] Transfection Expression constructs of the disclosed herein can be introduced into host cells to produce the IL-10 agonist compounds disclosed herein. Expression vectors containing nucleic acid sequences encoding IL-10 agonist compounds (e.g., single-domain antibody polypeptides of IL-10 agonist compounds) are introduced into prokaryotic or eukaryotic host cells via conventional transformation or transfection techniques. Preferred methods for transforming or transfecting host cells can be found in Sambrook et al. 1989. Molecular Cloning: A Laboratory Manual (2nd ed., Cold Spring Harbor Laboratory Press, Plainview, NY) and other standard molecular biology laboratory manuals. To facilitate transfection of target cells, target cells may be directly exposed to nonviral vectors under conditions that promote uptake of nonviral vectors. Examples of conditions that promote uptake of foreign nucleic acids by mammalian cells are well known in the art and include, but are not limited to, chemical means (e.g., Lipofectamine®, Thermo-Fisher Scientific), high levels of salt, and magnetic fields (electroporation).

[0324] cell culture Cells may be cultured in conventional nutrient media, modified as appropriate for inducing promoters, selecting transformants, or amplifying genes encoding desired sequences. Mammalian host cells may be cultured in a variety of media. Commercially available media, e.g., Ham F10 (Sigma), Minimum Essential Medium ((MEM), Sigma), RPMI 1640 (Sigma), and Dulbecco's Modified Eagle Medium ((DMEM), Sigma), are suitable for culturing host cells. Any of these media may be supplemented as needed with hormones and / or other growth factors (e.g., insulin, transferrin, or epidermal growth factor), salts (e.g., sodium chloride, calcium, magnesium, and phosphates), buffers (e.g., HEPES), nucleosides (e.g., adenosine and thymidine), antibiotics, trace elements, and glucose or equivalent energy sources. Any other necessary supplements may also be included in appropriate concentrations known to those skilled in the art. Culture conditions, e.g., temperature and pH, are those previously used with host cells selected for expression and are apparent to those skilled in the art.

[0325] Recombinant protein recovery Recombinantly produced IL-10 agonist compound polypeptides can be recovered from the culture medium as secreted polypeptides when a secretion leader sequence is used. Alternatively, IL-10 agonist compound polypeptides (e.g., single-domain antibody polypeptides of IL-10 agonist compounds) can also be recovered from host cell lysates. Protease inhibitors, such as phenylmethylsulfonyl fluoride (PMSF), may be used during the recovery phase from cell lysates to inhibit proteolysis during purification, and antibiotics may be included to prevent the growth of exogenous contaminants.

[0326] Various purification processes, such as affinity chromatography, are known and have applications in the art. Affinity chromatography separates molecules based on their ability to bind to specific ligands, using highly specific binding sites that are normally present in biological macromolecules. Covalent bonding involves attaching ligands to an insoluble, porous support medium in a manner that explicitly presents the ligand to the protein sample, thereby separating and purifying a second species from a mixture using the innate specific binding of one molecular species. Antibodies are commonly used in affinity chromatography. Size selection processes are also available, such as gel filtration chromatography (also known as size exclusion chromatography or molecular sieve chromatography), which is used to separate proteins according to their size. In gel filtration, a protein solution is passed through a column packed with a semipermeable, porous resin. The semipermeable resin has a range of pore sizes that determine the size of the proteins that can be separated by the column.

[0327] Recombinant IL-10 agonist compounds from transformed hosts can be purified according to any preferred method. Recombinant IL-10 agonist compounds can be isolated from inclusion bodies produced in Escherichia coli or from conditional media from a given mutain-producing mammalian or yeast culture using cation exchange, gel filtration, and / or reverse-phase liquid chromatography. Substantially purified forms of recombinant IL-10 agonist compounds can be purified from expression systems using common biochemical procedures and may be used as therapeutic agents, for example, as described herein.

[0328] In some embodiments, when an IL-10 agonist compound (e.g., a single-domain antibody polypeptide of an IL-10 agonist compound) is expressed with the purification tag discussed above, this purification handle can be used for the isolation of the IL-10 agonist compound (e.g., a single-domain antibody polypeptide of an IL-10 agonist compound) from cell lysates or cell culture media. If the purification tag is a chelated peptide, methods for isolating such molecules using immobilized metal affinity chromatography are well known in the art. See, for example, Smith, et al., U.S. Patent No. 4,569,794.

[0329] The biological activity of the recovered IL-10 agonist compound (e.g., a single-domain antibody polypeptide of an IL-10 agonist compound) can be assayed for activation by any suitable method known in the art and may be evaluated in a substantially purified form or as a portion of the cell lysate or cell culture medium when a secretion leader sequence is used for expression.

[0330] Pharmaceutical preparations In some embodiments, the subject IL-10 agonist compound (and / or nucleic acid encoding the IL-10 agonist compound (e.g., a single-domain antibody polypeptide of the IL-10 agonist compound), or recombinant cells incorporating a nucleic acid sequence and modified to express the IL-10 agonist compound (e.g., a single-domain antibody polypeptide of the IL-10 agonist compound)) may be incorporated into a composition comprising a pharmaceutical composition. Such a composition typically comprises a polypeptide or nucleic acid molecule and a pharmaceutically acceptable carrier. The pharmaceutical composition is formulated to be compatible with its intended route of administration and is suitable for therapeutic use in which the IL-10 agonist compound (e.g., a single-domain antibody polypeptide of the IL-10 agonist compound) is administered to a subject requiring treatment or prophylaxis.

[0331] Carrier The carrier comprises a sterile diluent, such as water for injection, saline solution, fixative oil, polyethylene glycol, glycerin, propylene glycol, or other synthetic solvent. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof. Appropriate fluidity can be maintained, for example, by the use of a coating such as lecithin, by maintaining the required particle size in the case of a dispersion, and by the use of a surfactant, such as sodium dodecyl sulfate. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, NJ), or phosphate-buffered saline (PBS).

[0332] cushioning agent The term buffering agent includes buffering agents such as acetic acid (salt), citric acid (salt), or phosphoric acid (salt), and agents for adjusting tonicity, such as sodium chloride or dextrose. pH can be adjusted using an acid or base, such as monobasic and / or dibasic sodium phosphate, hydrochloric acid, or sodium hydroxide (for example, to a pH of about 7.2–7.8, e.g., 7.5).

[0333] dispersion Generally, dispersions are prepared by incorporating the active compound into a sterile medium containing a basic dispersion medium and other required components from those listed above. For sterile powders for the preparation of sterile injectable solutions, preferred methods of preparation are vacuum drying and freeze-drying, which yield a powder of the active ingredient and any additional desired components from its previously sterile filtered solution.

[0334] Preservatives Pharmaceutical formulations for parenteral administration to subjects should be sterile and fluid to facilitate easy passage through injection needles. They should be stable under production and storage conditions and protected from contaminants. Prevention of microbial action can be achieved by various antimicrobial and antifungal agents, e.g., benzyl alcohol or methylparaben; antioxidants, e.g., ascorbic acid or sodium bisulfite; chelating agents, e.g., ethylenediaminetetraacetic acid, parabens, chlorobutanol, phenol, ascorbic acid, and thimerosal. Sterile solutions can be prepared, as required, by incorporating the required amount of the active compound in a suitable solvent along with one or a combination of the components listed above, followed by sterile filtration.

[0335] Tensioning agent In many cases, it is preferable to include isotonic agents, such as sugars, polyalcohols, such as mannitol, sorbitol, and sodium chloride, in the composition.

[0336] Route of administration Some aspects of the therapeutic methods of the present disclosure involve the administration of a pharmaceutical formulation comprising an IL-10 agonist compound (and / or a nucleic acid encoding an IL-10 agonist compound (e.g., a single-domain antibody polypeptide of an IL-10 agonist compound) or a recombinantly modified host cell expressing an IL-10 agonist compound (e.g., a single-domain antibody polypeptide of an IL-10 agonist compound)) to a subject in need of treatment. The pharmaceutical formulation comprising an IL-10 agonist compound of the present disclosure may be administered to a subject in need of treatment or prophylaxis by various routes of administration, including parenteral administration, oral administration, topical application, or inhalation.

[0337] Parenteral administration In some embodiments, the methods of the present disclosure involve parenteral administration of a pharmaceutical formulation comprising an IL-10 agonist compound (and / or a nucleic acid encoding an IL-10 agonist compound (e.g., a single-domain antibody polypeptide of an IL-10 agonist compound) or a recombinantly modified host cell expressing an IL-10 agonist compound (e.g., a single-domain antibody polypeptide of an IL-10 agonist compound)) to a subject requiring treatment. Examples of parenteral administration routes include, for example, intravenous, intradermal, subcutaneous, transdermal (topical), transmucosal, and rectal administration. Parenteral formulations comprise solutions or suspensions used for parenteral application and may include media, carriers, and buffers. Pharmaceutical formulations for parenteral administration comprise sterile aqueous solutions (if water-soluble) or dispersions and sterile powders for the immediate preparation of sterile injection solutions or dispersions. Parenteral preparations may be enclosed in ampoules, disposable syringes, or multi-dose vials made of glass or plastic. In one embodiment, the formulation is provided in a pre-filled syringe for parenteral administration.

[0338] Oral administration In some embodiments, the methods of the present disclosure involve the oral administration of a pharmaceutical formulation comprising an IL-10 agonist compound (and / or a nucleic acid encoding an IL-10 agonist compound (e.g., a single-domain antibody polypeptide of an IL-10 agonist compound) or a recombinantly modified host cell expressing an IL-10 agonist compound (e.g., a single-domain antibody polypeptide of an IL-10 agonist compound)) to a subject requiring treatment. When an oral composition is used, it generally includes an inert diluent or food carrier. For the purpose of oral therapeutic administration, the active compound can be incorporated together with excipients and may be used in the form of tablets, lozenges, or capsules, e.g., gelatin capsules. The oral composition may also be prepared using a fluid carrier for use as a mouthwash. A pharmaceutically compatible binder and / or adjuvant material may be included as part of the composition. Tablets, pills, capsules, and lozenges may contain any of the following ingredients or compounds of similar properties: binders, e.g., microcrystalline cellulose, gum tragacanth, or gelatin; excipients, e.g., starch, or lactose; disintegrants, e.g., alginic acid, Primogel®, or corn starch; lubricants, e.g., magnesium stearate or Sterotes®; lubricants, e.g., colloidal silicon dioxide; sweeteners, e.g., sucrose, or saccharin; or flavoring agents, e.g., peppermint, methyl salicylate, or orange flavoring agents.

[0339] Inhalation preparations In some embodiments, the methods of the present disclosure involve inhalation administration of a pharmaceutical formulation comprising an IL-10 agonist compound (and / or a nucleic acid encoding an IL-10 agonist compound (e.g., a single-domain antibody polypeptide of an IL-10 agonist compound) or recombinantly modified host cells expressing an IL-10 agonist compound (e.g., a single-domain antibody polypeptide of an IL-10 agonist compound)) to a subject requiring treatment. In the case of inhalation administration, the subject IL-10 agonist compound, or the nucleic acid encoding it, is delivered in the form of an aerosol spray from a pressurized container or dispenser containing a suitable propellant, e.g., a gas, e.g., carbon dioxide, or from a nebulizer. Such methods include those described in U.S. Patent No. 6,468,798.

[0340] Mucosal and transdermal preparations In some embodiments, the methods of the present disclosure involve mucosal or transdermal administration of a pharmaceutical formulation comprising an IL-10 agonist compound (and / or a nucleic acid encoding an IL-10 agonist compound (e.g., a single-domain antibody polypeptide of an IL-10 agonist compound) or a recombinantly modified host cell expressing an IL-10 agonist compound (e.g., a single-domain antibody polypeptide of an IL-10 agonist compound)) to a subject requiring treatment. For mucosal or transdermal administration, a permeabilizing agent suitable for the barrier to be permeated is used in the formulation. Such permeabilizing agents are generally known in the art and include, for example, surfactants, bile salts, and fusidic acid derivatives for mucosal administration. Mucosal administration can be achieved through the use of a nasal spray or suppository (e.g., using a conventional suppository base, e.g., cocoa butter and other glycerides) or a retaining enema for rectal delivery. For transdermal administration, the active compound may be formulated into an ointment, paste, gel, or cream, as is generally known in the art, and may also incorporate a permeability enhancer, such as ethanol or lanolin.

[0341] Sustained-release and depot formulations In some embodiments of the methods of this disclosure, an IL-10 agonist compound (e.g., a single-domain antibody polypeptide of an IL-10 agonist compound) is administered to a subject ...

Claims

1. An IL-10 agonist compound comprising a first single-domain antibody polypeptide conjugated to a second single-domain antibody polypeptide, The first single-domain antibody polypeptide specifically binds to the α subunit (IL-10Rα) of the IL-10 receptor, and CDR1 containing an amino acid sequence selected from the group consisting of SEQ ID NO: 224-228. CDR2 containing an amino acid sequence selected from the group consisting of SEQ ID NO: 229-235, and CDR3 containing an amino acid sequence selected from the group consisting of SEQ ID NO: 236 Includes, The second single-domain antibody polypeptide specifically binds to the β subunit (IL-10Rβ) of the IL-10 receptor, and CDR1 containing an amino acid sequence selected from the group consisting of SEQ ID NO:296, CDR2 containing an amino acid sequence selected from the group consisting of SEQ ID NO:297, and CDR3 containing an amino acid sequence selected from the group consisting of SEQ ID NO: 298 including, The aforementioned IL-10 agonist compound.

2. The first single-domain antibody polypeptide is as follows: Includes a combination of CDR1, CDR2, and CDR3 amino acid sequences selected from one of the rows, The second single-domain antibody polypeptide is as follows: Includes a combination of CDR1, CDR2, and CDR3 amino acid sequences selected from one of the rows, The IL-10 agonist compound according to claim 1.

3. The first single-domain antibody polypeptide described above, CDR1 containing the amino acid sequence of SEQ ID NO:224, CDR2 containing the amino acid sequence of SEQ ID NO:229, and CDR3 containing the amino acid sequence of SEQ ID NO:236 Includes, The aforementioned second single-domain antibody polypeptide CDR1 containing the amino acid sequence of SEQ ID NO:296, CDR2 containing the amino acid sequence of SEQ ID NO:297, and CDR3 containing the amino acid sequence of SEQ ID NO:298 including, The IL-10 agonist compound according to claim 2.

4. The first single-domain antibody polypeptide described above, CDR1 containing the amino acid sequence of SEQ ID NO:224, CDR2 containing the amino acid sequence of SEQ ID NO:230, and CDR3 containing the amino acid sequence of SEQ ID NO:236 Includes, The aforementioned second single-domain antibody polypeptide CDR1 containing the amino acid sequence of SEQ ID NO:296, CDR2 containing the amino acid sequence of SEQ ID NO:297, and CDR3 containing the amino acid sequence of SEQ ID NO:298 including, The IL-10 agonist compound according to claim 2.

5. The first single-domain antibody polypeptide CDR1 containing the amino acid sequence of SEQ ID NO:224, CDR2 containing the amino acid sequence of SEQ ID NO:231, and CDR3 containing the amino acid sequence of SEQ ID NO:236 Includes, The aforementioned second single-domain antibody polypeptide CDR1 containing the amino acid sequence of SEQ ID NO:296, CDR2 containing the amino acid sequence of SEQ ID NO:297, and CDR3 containing the amino acid sequence of SEQ ID NO:298 including, The IL-10 agonist compound according to claim 2.

6. A first single-domain antibody polypeptide containing an amino acid sequence selected from the group consisting of SEQ ID NO: 25 to 48, A second single-domain antibody polypeptide containing an amino acid sequence selected from the group consisting of SEQ ID NO:49 and SEQ ID NO:50, and An IL-10 agonist compound according to any one of claims 1 to 5, comprising:

7. The IL-10 agonist compound according to any one of claims 1 to 6, wherein the first single-domain antibody polypeptide and the second single-domain antibody polypeptide are linked by a linker.

8. The IL-10 agonist compound according to any one of claims 1 to 7, wherein the C-terminus of the first single-domain antibody polypeptide is conjugated to the N-terminus of the second single-domain antibody polypeptide, and optionally, the C-terminus of the first single-domain antibody polypeptide is conjugated to the N-terminus of the second single-domain antibody polypeptide via a linker.

9. The IL-10 agonist compound according to any one of claims 1 to 7, wherein the N-terminus of the first single-domain antibody polypeptide is conjugated to the C-terminus of the second single-domain antibody polypeptide, and optionally, the N-terminus of the first single-domain antibody polypeptide is conjugated to the C-terminus of the second single-domain antibody polypeptide via a linker.

10. An IL-10 agonist compound according to any one of claims 1 to 9, comprising a polypeptide having at least 95% amino acid sequence identity with respect to an amino acid sequence selected from the group consisting of SEQ ID NO: 1 to 24 or SEQ ID NO: 500 to 523.

11. The IL-10 agonist compound according to claim 10, comprising a polypeptide containing an amino acid sequence selected from the group consisting of SEQ ID NO: 1 to 24 or SEQ ID NO: 500 to 523.

12. The IL-10 agonist compound according to claim 11, comprising a polypeptide containing the amino acid sequence SEQ ID NO:

500.

13. The IL-10 agonist compound according to claim 11, comprising a polypeptide containing the amino acid sequence of SEQ ID NO:

501.

14. The IL-10 agonist compound according to claim 11, comprising a polypeptide containing the amino acid sequence of SEQ ID NO:

502.

15. The IL-10 agonist compound according to claim 11, comprising a polypeptide containing the amino acid sequence of SEQ ID NO:

503.

16. The IL-10 agonist compound according to claim 11, comprising a polypeptide containing the amino acid sequence of SEQ ID NO:

504.

17. An IL-10 agonist compound according to any one of claims 1 to 16, comprising a C-terminal amino acid modification that reduces immunogenicity caused by existing antibodies.

18. An IL-10 agonist compound according to any one of claims 1 to 17, comprising a C-terminal amino acid modification and a C-terminal amino acid sequence selected from the group consisting of SEQ ID NO: 474 to 499.

19. A compound according to any one of claims 1 to 18, selected from the group consisting of SEQ ID NO: 121-135, 138-141, 144-155, 158-175, 179, 182-195, and 199.

20. The following formula #1: H2N-(huIL10 VHH1)-(L1) a (L1) b -(huIL10 VHH2)-COOH [#1] An IL-10 agonist compound comprising an IL-10R-binding molecular polypeptide, During the ceremony, The "-" indicates a covalent bond. L1 is the linker, a and b are chosen independently from the integers 0 or 1. "H2N" represents the amino terminus, and "COOH" represents the carboxyl terminus of the polypeptide. The aforementioned IL-10 agonist compound.

21. A pharmaceutically acceptable formulation of an IL-10 agonist compound according to any one of claims 1 to 20.

22. A nucleic acid sequence encoding an IL-10 agonist compound according to any one of claims 1 to 20.

23. A recombinant vector comprising the nucleic acid described in claim 22.

24. A method for treating a mammalian subject suffering from an autoimmune disease, an infectious disease, or an inflammatory disease by administering a therapeutically effective amount of an IL-10 agonist compound according to any one of claims 1 to 20.

25. A method for treating a mammalian subject suffering from a neoplastic disease by administering a therapeutically effective amount of an IL-10 agonist compound according to any one of claims 1 to 20.

26. A means for inducing intracellular signaling in cells expressing IL-10Rα and IL-10Rβ, comprising: (ii) a single-domain antibody (IL-10Rβ sdAb) conjugated to a single-domain antibody (IL-10Rα sdAb) that binds to IL-10Rβ and is humanized compared to UniProt VH3-66 (UniProt A0A0C4DH42); and (i) a single-domain antibody (IL-10Rα sdAb) that binds to IL-10Rα and is humanized compared to UniProt V3-23 (UniProt number P01764); Pharmacologically acceptable carriers and A composition containing the following:

27. The composition according to claim 26, wherein the cells expressing IL-10Rα and IL-10Rβ are monocytes.

28. The composition according to claim 26, wherein pSTAT3 signaling is induced by dimerizing the extracellular domain of the IL-10Rα subunit and the extracellular domain of the IL-10Rβ subunit of the IL-10 receptor (IL-10R) on a cell.

29. The composition according to claim 26, wherein intracellular pSTAT-3 signaling in monocyte cells expressing IL10Rα and IL10R is superior to pSTAT-3 signaling in T cells expressing IL10Rα and IL10Rβ.

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