Methods and compositions for treating inflammatory and autoimmune conditions using ECM-affinity peptides linked to anti-inflammatory agents.

Abstract

The present disclosure relates to engineering collagen-binding modifications of anti-inflammatory agents using collagen-binding peptides (CBPs) and vWF A3 to achieve targeted therapy of inflammatory diseases. Accordingly, embodiments of the present disclosure relate to compositions comprising anti-inflammatory agents operably linked to extracellular matrix (ECM) affinity peptides. Anti-inflammatory agents such as cytokines and CD200 linked to serum proteins and / or ECM affinity peptides are also disclosed. A further aspect of the present disclosure relates to a method for treating an autoimmune or inflammatory condition in a subject, comprising administering a composition of the present disclosure to the subject.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority to U.S. Provisional Patent Application No. 62 / 809,988, filed on 25 February 2019, which is incorporated herein by reference in its entirety.

[0002] I. Field of Invention The present invention broadly relates to the field of medicine. More specifically, the present invention relates to compositions and methods involving nucleotide constructs and proteins, including anti-inflammatory agents engineered to target inflamed tissue. [Background technology]

[0003] II. Background Therapies targeting cytokines and their receptors have dramatically altered outcomes in inflammatory and autoimmune diseases, particularly in anti-TNF therapy for rheumatoid arthritis (RA) and inflammatory bowel disease (IBD) (1-5). However, currently approved drugs do not completely cure most patients and may have significant side effects by suppressing systemic immunity (6-10). To enhance treatment outcomes and reduce systemic side effects, efficient drug delivery to the site of inflammation is a promising therapeutic strategy for such diseases. Inflammatory tissues release a set of mediators that induce enhanced permeability and retention (EPR) effects (11-12). The EPR effect is due to loose endothelial junctions that allow for extravasation of non-functional lymphatic vessels and macromolecules, resulting in the long-term retention of macromolecules within solid tumors and inflammatory tissues (11-15). Unlike tumor tissue, inflammatory tissues have a functional lymphatic system that drains substances from them (14-16). Currently, there is no effective method to target inflammatory tissue in inflammatory and autoimmune diseases for rapid clearance from the inflamed tissue. Therefore, there is a need in this field for therapies that directly target inflammatory tissue. [Overview of the Initiative]

[0004] This disclosure relates to the manipulation of collagen binding of anti-inflammatory agents using collagen-binding peptides (CBPs) and vWF A3 to achieve targeted therapy for inflammatory diseases. Accordingly, aspects of this disclosure relate to compositions comprising anti-inflammatory agents functionally linked to extracellular matrix (ECM) affinity peptides. Aspects of this disclosure also relate to compositions comprising anti-inflammatory agents functionally linked to serum proteins and anti-inflammatory agents functionally linked to serum proteins. Further aspects of this disclosure relate to methods for treating autoimmune or inflammatory conditions in a subject, including the step of administering the compositions of this disclosure to the subject.

[0005] A further aspect of this disclosure relates to a method for reducing inflammation in a subject, comprising the step of administering to the subject a composition comprising an anti-inflammatory agent functionally linked to an extracellular matrix (ECM) affinity peptide. In some embodiments, the inflammation is caused by an autoimmune condition or inflammatory state, where the autoimmune condition or inflammatory state includes inflammatory bowel disease, idiopathic pulmonary fibrosis, multiple sclerosis, type 1 diabetes, or arthritis.

[0006] In some embodiments, the anti-inflammatory agent functionally linked to an ECM-affinity peptide comprises a collagen-binding domain conjugated to anti-TNFα. In some embodiments, the anti-inflammatory agent functionally linked to an ECM-affinity peptide comprises vWF-A3 functionally linked to IL-4. In some embodiments, the anti-inflammatory agent functionally linked to an ECM-affinity peptide comprises a collagen-binding domain conjugated to anti-TGF-β. In some embodiments, the composition is administered systemically. In some embodiments, the composition is administered topically. In some embodiments, the dose of the anti-inflammatory agent functionally linked to an ECM-affinity peptide is at least 20% less than the minimum effective dose of the anti-inflammatory agent administered topically without the peptide.

[0007] In some embodiments, the anti-inflammatory agent comprises an anti-inflammatory antibody. In some embodiments, the anti-inflammatory antibody comprises an antibody specific to TNF-α, IL-1, IL-5, IL-6, IL-6R, IL-12, IL-17A, IL-18, IFN-γ, GM-CSF, CD3, CD20, VLA-4, VLA-5, VCAM-1, TGF-β1, α4-integrin, α4β7-integrin, connective tissue growth factor, platelet-derived growth factor, plasminogen activator inhibitor-1, or insulin-like growth factor-binding protein. In some embodiments, the antibody is an anti-TNF-α antibody, an anti-IL-1 antibody, an anti-IL-5 antibody, an anti-IL-6 antibody, an anti-IL-6R antibody, an anti-IL-12 antibody, an anti-IL-17A antibody, an anti-IL-18 antibody, an anti-IFN-γ antibody, an anti-GM-CSF antibody, an anti-CD3 antibody, an anti-CD20 antibody, an anti-VLA-4 antibody, an anti-VLA-5 antibody, an anti-VCAM-1 antibody, an anti-TGF-β1 antibody, an anti-α4-integrin antibody, an anti-α4β7-integrin antibody, an anti-connective tissue growth factor antibody, an anti-platelet-derived growth factor antibody, an anti-plasminogen activator inhibitor-1 antibody, or an anti-insulin-like growth factor-binding protein antibody. In some embodiments, the anti-inflammatory antibody is a blocking antibody. In some embodiments, the anti-inflammatory antibody is a neutralizing antibody. In some embodiments, the anti-inflammatory antibody is an antagonistic antibody. One or more of these antibodies may be specifically excluded from the embodiments.

[0008] In some embodiments, the anti-inflammatory agent comprises an antigen-binding fragment of an anti-TNF-α antibody, anti-IL-1 antibody, anti-IL-5 antibody, anti-IL-6 antibody, anti-IL-6R antibody, anti-IL-12 antibody, anti-IL-17A antibody, anti-IL-18 antibody, anti-IFN-γ antibody, anti-GM-CSF antibody, anti-CD3 antibody, anti-CD20 antibody, anti-VLA-4 antibody, anti-VLA-5 antibody, anti-VCAM-1 antibody, anti-TGF-β1 antibody, anti-α4-integrin antibody, anti-α4β7-integrin antibody, anti-connective tissue growth factor antibody, anti-platelet-derived growth factor antibody, anti-plasminogen activator inhibitor-1 antibody, or anti-insulin-like growth factor-binding protein antibody. The antigen-binding fragment is a variable light chain region containing CDR1, CDR2, and CDR3 from an anti-TNF-α antibody, anti-IL-1 antibody, anti-IL-5 antibody, anti-IL-6 antibody, anti-IL-6R antibody, anti-IL-12 antibody, anti-IL-17A antibody, anti-IL-18 antibody, anti-IFN-γ antibody, anti-GM-CSF antibody, anti-CD3 antibody, anti-CD20 antibody, anti-VLA-4 antibody, anti-VLA-5 antibody, anti-VCAM-1 antibody, anti-TGF-β1 antibody, anti-α4-integrin antibody, anti-α4β7-integrin antibody, anti-connective tissue growth factor antibody, anti-platelet-derived growth factor antibody, anti-plasminogen activator inhibitor-1 antibody, or anti-insulin-like growth factor-binding protein antibody. It may contain / or a variable heavy chain region including CDR1, CDR2, and CDR3 from an anti-TNF-α antibody, anti-IL-1 antibody, anti-IL-5 antibody, anti-IL-6 antibody, anti-IL-6R antibody, anti-IL-12 antibody, anti-IL-17A antibody, anti-IL-18 antibody, anti-IFN-γ antibody, anti-GM-CSF antibody, anti-CD3 antibody, anti-CD20 antibody, anti-VLA-4 antibody, anti-VLA-5 antibody, anti-VCAM-1 antibody, anti-TGF-β1 antibody, anti-α4-integrin antibody, anti-α4β7-integrin antibody, anti-connective tissue growth factor antibody, anti-platelet-derived growth factor antibody, anti-plasminogen activator inhibitor-1 antibody, or anti-insulin-like growth factor-binding protein antibody. In some embodiments, the antibody includes adalimumab, certolizumab, infliximab, golimumab, tocilizumab, rituximab, ustekinumab, natalizumab, vedolizumab, secukinumab, or ixekizumab.In some embodiments, the anti-inflammatory agent comprises antigen-binding fragments derived from adalimumab, certolizumab, infliximab, golimumab, tocilizumab, rituximab, ustekinumab, natalizumab, vedolizumab, secukinumab, or ixekizumab. Antigen-binding fragments may include variable light chain regions containing CDR1, CDR2, and CDR3 from adalimumab, certolizumab, infliximab, golimumab, tocilizumab, rituximab, ustekinumab, natalizumab, vedolizumab, secukinumab, or ixekizumab, and / or variable heavy chain regions containing CDR1, CDR2, and CDR3 from adalimumab, certolizumab, infliximab, golimumab, tocilizumab, rituximab, ustekinumab, natalizumab, vedolizumab, secukinumab, or ixekizumab. Examples of antigen-binding fragments derived from whole antibodies include minibodies, scFvs, chimeric antigen receptors, and diabodies. Divalent or multispecific constructs derived from one or more of the following antibodies are also intended: anti-TNF-α antibody, anti-IL-1 antibody, anti-IL-5 antibody, anti-IL-6 antibody, anti-IL-6R antibody, anti-IL-12 antibody, anti-IL-17A antibody, anti-IL-18 antibody, anti-IFN-γ antibody, anti-GM-CSF antibody, anti-CD3 antibody, anti-CD20 antibody, anti-VLA-4 antibody, anti-VLA-5 antibody, anti-VCAM-1 antibody, anti-TGF-β1 antibody, anti-α4-integrin antibody, anti-α4β7-integrin antibody, anti-connective tissue growth factor antibody, anti-platelet-derived growth factor antibody, anti-plasminogen activator inhibitor-1 antibody, or anti-insulin-like growth factor-binding protein antibody. In some embodiments, the antibodies are humanized. In some embodiments, the antibodies are chimeric antibodies. One or more of these antibodies or antigen-binding fragments may be specifically excluded from the embodiments.

[0009] In some embodiments, the antibody comprises an anti-TNF-α antibody. In some embodiments, the antibody comprises an anti-IL-1 antibody. In some embodiments, the antibody comprises an anti-IL-5 antibody. In some embodiments, the antibody comprises an anti-IL-6 antibody. In some embodiments, the antibody comprises an anti-IL-6R antibody. In some embodiments, the antibody comprises an anti-IL-12 antibody. In some embodiments, the antibody comprises an anti-IL-17A antibody. In some embodiments, the antibody comprises an anti-IL-18 antibody. In some embodiments, the antibody comprises an anti-IFN-γ antibody. In some embodiments, the antibody comprises an anti-GM-CSF antibody. In some embodiments, the antibody comprises an anti-CD3 antibody. In some embodiments, the antibody comprises an anti-CD20 antibody. In some embodiments, the antibody comprises an anti-VLA-4 antibody. In some embodiments, the antibody comprises an anti-VLA-5 antibody. In some embodiments, the antibody comprises an anti-VCAM-1 antibody. In some embodiments, the antibody comprises an anti-TGF-β1 antibody. In some embodiments, the antibody comprises an anti-α4-integrin antibody. In some embodiments, the antibody comprises an anti-α4β7-integrin antibody. In some embodiments, the antibody comprises an anti-connective tissue growth factor antibody. In some embodiments, the antibody comprises an anti-platelet-derived growth factor antibody. In some embodiments, the antibody comprises an anti-plasminogen activator inhibitor-1 antibody. In some embodiments, the antibody comprises an anti-insulin-like growth factor-binding protein antibody.

[0010] In some embodiments, the anti-inflammatory agent comprises an anti-inflammatory cytokine polypeptide. In some embodiments, the cytokine polypeptide comprises polypeptides from IL-4, IL-1ra, IL-5, IL-10, IL-11, IL-23, IL-35, IL-36ra, IL-37, interferon-β, TGF-β1, TNF receptor I, and TNF receptor II. In some embodiments, the cytokine polypeptide is derived from human cytokine polypeptides. In some embodiments, the cytokine polypeptide is derived from non-human cytokine polypeptides. In some embodiments, the cytokine polypeptide is derived from mouse, dog, horse, pig, or goat cytokine polypeptides. In some embodiments, the cytokine polypeptide comprises effector regions from one or more of IL-4, IL-1ra, IL-5, IL-10, IL-11, IL-23, IL-35, IL-36ra, IL-37, interferon-β, TGF-β1, TNF receptor I, and TNF receptor II. In some embodiments, the cytokine polypeptide comprises a polypeptide from IL-4. In some embodiments, the cytokine polypeptide includes a polypeptide from IL-1ra. In some embodiments, the cytokine polypeptide includes a polypeptide from IL-5. In some embodiments, the cytokine polypeptide includes a polypeptide from IL-10. In some embodiments, the cytokine polypeptide includes a polypeptide from IL-11. In some embodiments, the cytokine polypeptide includes polypeptides from IL-23 and IL-35. In some embodiments, the cytokine polypeptide includes a polypeptide from IL-36ra. In some embodiments, the cytokine polypeptide includes a polypeptide from IL-37. In some embodiments, the cytokine polypeptide includes a polypeptide from interferon-β. In some embodiments, the cytokine polypeptide includes a polypeptide from TGF-β1. In some embodiments, the cytokine polypeptide includes a polypeptide from TNF receptor I.In some embodiments, the cytokine polypeptide includes polypeptides from TNF receptor II. One or more of these anti-inflammatory polypeptides may be specifically excluded from the embodiments. In some embodiments, the cytokine polypeptide is a human cytokine polypeptide or is derived from a human cytokine polypeptide.

[0011] In some embodiments, the cytokine polypeptide comprises a polypeptide or fragment thereof from SEQ ID NO: 18-44, or a polypeptide having at least 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% (or any range within which this can be derived) sequence identity with a polypeptide or fragment thereof having one amino acid sequence from SEQ ID NO: 18-44. In some embodiments, the anti-inflammatory agent comprises a polypeptide or fragment thereof having SEQ ID NO: 58 or 59, or a polypeptide having sequence identity of at least 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% (or any range within which this can be derived) of a polypeptide or fragment thereof having a single amino acid sequence of SEQ ID NO: 58 or 59.

[0012] In some embodiments, the anti-inflammatory agent comprises a polypeptide derived from CD200. In some embodiments, the CD200 polypeptide comprises the extracellular domain of CD200. CD200 (UniProt identification number O54901) is a type I transmembrane protein capable of exerting immunosuppressive function through interaction with its receptor, CD200R1. Even when cleaved from the cell surface, the soluble extracellular domain of CD200 can bind to CD200R and be activated. Aspects of this disclosure relate to polypeptides comprising at least or more extracellular components of CD200 and serum proteins such as serum albumin. Polypeptides are useful in embodiments of the methods of this disclosure. Further embodiments relate to polypeptides comprising at least or more extracellular components of CD200, serum albumin, and ECM-affinity polypeptides.

[0013] In some embodiments, the ECM affinity peptide includes a collagen-binding domain. In some embodiments, the polypeptide includes a collagen-binding domain from decorin or von Willebrand factor (VWF). In some embodiments, the ECM affinity peptide includes a peptide from placental growth factor-2 (PlGF-2) or CXCL-12γ. In some embodiments, the ECM affinity peptide includes a peptide that is at least 85% identical to one of SEQ ID NO: 1-17, 47, or 52, or a peptide that is at least 85% identical to a fragment of one of SEQ ID NO: 1-17, 47, or 52. In some embodiments, the ECM affinity peptide is a peptide having sequence identity of at least 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% (or any range within which this can be derived) with a peptide having one amino acid sequence among SEQ ID NO: 1-17, 47, or 52, or SEQ ID NO: The peptide comprises a fragment having one amino acid sequence among 1 to 17, 47, or 52, and a peptide having at least 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% (or any range within which this can be derived) sequence identity.

[0014] In some embodiments, an anti-inflammatory agent functionally linked to an extracellular matrix (ECM) affinity peptide further comprises the peptide or a serum protein functionally linked to the agent. In some embodiments, the serum protein is functionally linked to the peptide. In some embodiments, the serum protein is functionally linked to the peptide via a peptide bond. In some embodiments, the serum protein comprises albumin. In some embodiments, the anti-inflammatory agent is located near the amino region of the serum protein. In some embodiments, the anti-inflammatory agent is located near the carboxyl region of the serum protein. In some embodiments, the ECM affinity peptide is located near the amino region of the anti-inflammatory agent. In some embodiments, the ECM affinity peptide is located near the carboxyl region of the anti-inflammatory agent. In some embodiments, the serum protein is located near the amino region of the ECM affinity peptide. In some embodiments, the serum protein is located near the carboxyl region of the ECM affinity peptide.

[0015] If the first region is bound to the carboxyl terminus of the second region, the first region is proximal to the carboxyl terminus of the second region. There may be further interposing amino acid residues between the first and second regions. Therefore, unless specifically stated otherwise, these regions do not need to be directly adjacent. The term "amino proximal" is similarly defined in that, if the first region is bound to the amino terminus of the second region, the first region is proximal to the amino terminus of the second region. Similarly, unless specifically stated otherwise, there may be further interposing amino acid residues between the first and second regions. In some embodiments, the composition comprises an amino-amino proximal collagen-binding domain of serum albumin protein and an IL-10 polypeptide proximal to the carboxyl terminus of serum albumin protein.

[0016] In some embodiments, peptides are covalently bonded to anti-inflammatory agents and / or other molecules such as serum proteins. In some embodiments, peptides are cross-linked to anti-inflammatory agents via bifunctional linkers. Linkers, such as amino acids or peptide-mimicking sequences, may be inserted between peptide and / or antibody sequences. In one embodiment, a finomer domain is linked to a heavy (H) or light (L) chain immediately after the last amino acid of the amino (NH2) or carboxy (C) terminus of the heavy (H) or light (L) chain. Linkers may have one or more properties, including mobility, inability to form regular secondary structures, or hydrophobicity or chargeability that can promote or interact with either domain. Examples of amino acids commonly found in mobile protein regions include Gly, Asn, and Ser. For example, a suitable peptide linker may be GGGSGGGS (SEQ ID NO: 48) or (GGGS)n (SEQ ID NO: 49), where n = 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 (or any derivable range within that range). Other nearly neutral amino acids, such as Thr and Ala, may also be used in the linker sequence. The length of the linker sequence can vary without significantly affecting the function or activity of the fusion protein (see, for example, U.S. Patent No. 6,087,329). In certain contexts, the heavy or light chains of peptides and antibodies are linked by a peptide sequence having approximately 1 to 25 amino acid residues. Examples of linkers include chemical moieties and conjugating agents such as sulfosuccinimidyl derivatives (sulfo-SMCC, sulfo-SMPB), disuccinimidyl suberate (DSS), disuccinimidyl glutarate (DSG), and disuccinimidyl tartrate (DST). NFurther examples include linear carbon chains such as N=1 to 100 carbon atoms, e.g., N=2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, the linker can be a dipeptide linker, such as valine-citrulline (val-cit), phenylalanine-lysine (phe-lys) linker, or maleimidocaproine-valine-citrulline-p-aminobenzyloxycarbonyl (vc) linker. In some embodiments, the linker is sulfosuccinimidyl-4-[N-maleimidomethyl]cyclohexane-1-carboxylate (smcc). The sulfo-smcc conjugation occurs via a maleimide group reacting with a sulfhydryl (thiol, --SH), but the sulfo-NHS ester is reactive with primary amines (lysine and those found at the N-terminus of proteins or peptides). Furthermore, the linker can be maleimidocaproyl (mc). In some embodiments, the peptide is linked to the anti-inflammatory agent via a peptide bond. The peptide may be linked to the amino or carboxyl terminus of the anti-inflammatory agent. In some embodiments, the peptide is linked to the heavy chain of the anti-inflammatory antibody. In some embodiments, the peptide is linked to the light chain of the anti-inflammatory antibody. In some embodiments, the ratio of the peptide to the anti-inflammatory agent is approximately 1:1 to 5:1. In some embodiments, the ratio of the peptide to the anti-inflammatory agent is approximately 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, or 20:1 (or any range within which it can be derived). One or more of these linkers may be specifically excluded from the embodiments.

[0017] In some embodiments, the composition further comprises a second anti-inflammatory agent functionally linked to an extracellular matrix (ECM) affinity peptide. In some embodiments, the composition further comprises a third, fourth, fifth, or sixth anti-inflammatory agent functionally linked to an extracellular matrix (ECM) affinity peptide.

[0018] In some embodiments, autoimmune or inflammatory conditions include inflammatory bowel disease, idiopathic pulmonary fibrosis, multiple sclerosis, type 1 diabetes, Crohn's disease, psoriasis, acute inflammation, chronic inflammation, neuritis, arthritis, rheumatoid arthritis, fibrosis, infections, allergies, adverse events associated with inflammatory treatment, and inflammatory diseases associated with inflammatory treatment. One or more of these conditions may be specifically excluded from the embodiments.

[0019] In some embodiments, the composition is administered systemically. In some embodiments, the composition is administered by intravenous injection. In some embodiments, the composition is administered topically. In some embodiments, the composition is administered to or adjacent to the site of inflammation.

[0020] In some embodiments, the dose of a composition containing an anti-inflammatory agent functionally linked to a peptide is less than the minimum effective dose of the anti-inflammatory agent administered without the peptide. In some embodiments, the dose of a composition containing an anti-inflammatory agent functionally linked to a peptide is less than the minimum effective dose of the anti-inflammatory agent administered without the peptide via the same route of administration. In some embodiments, the dose of an anti-inflammatory agent functionally linked to a peptide is at least 10% less than the minimum effective dose of the anti-inflammatory agent administered without the peptide. In some embodiments, the dose of an anti-inflammatory agent functionally linked to a peptide is at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, or 70% (or any range derivable therein) less than the minimum effective dose of the anti-inflammatory agent administered without the peptide.

[0021] In some embodiments, the subject has been previously treated with an anti-inflammatory agent, anti-inflammatory therapy, or autoimmune therapy. In some embodiments, the subject has been determined to be unresponsive to prior treatment. In some embodiments, the subject has not been previously treated for either an inflammatory disease or an autoimmune disease. In some embodiments, the method further comprises administering additional inflammatory therapy or autoimmune therapy. In some embodiments, the method further comprises administering a second anti-inflammatory agent functionally linked to an extracellular matrix (ECM) affinity peptide.

[0022] As used herein, the term "cytokine polypeptide" refers to a polypeptide that contains a cytokine or its receptor-binding domain, which retains a portion of the cytokine's activity.

[0023] The terms "protein," "polypeptide," and "peptide" are used interchangeably in this specification to refer to gene products containing polymers of amino acids.

[0024] The terms “subject,” “mammal,” and “patient” are used interchangeably. In some embodiments, the subject is a mammal. In some embodiments, the subject is a human. In some embodiments, the subject is a clinical trial animal such as a mouse, rat, rabbit, dog, donkey, or fruit fly, zebrafish, etc.

[0025] The methods and compositions described herein are intended to include any exclusion of any of the embodiments described herein.

[0026] As used herein, the terms “or” and “and / or” are used to describe multiple components in combination or mutually exclusive. For example, “x, y, and / or z” can mean “x” alone, “y” alone, “z” alone, “x, y, and z,” “(x and y) or z,” “x or (y and z),” or “x or y or z.” It is particularly intended that x, y, or z may be specifically excluded from the embodiments.

[0027] Throughout this application, the term “about” is used in accordance with its plain and common sense in the field of cell biology to indicate that the value includes the standard deviation of error with respect to the apparatus or method used to determine that value.

[0028] The term "comprising," which is synonymous with "including," "containing," or "characterized by," is comprehensive or free form and does not exclude further, uncited elements or stages of method. The phrase "consisting of" excludes any unspecified elements, stages, or components. The phrase "essentially consisting of" limits the scope of the described subject to those that do not substantially affect a particular material or stage and its fundamental and novel characteristics. A manner described in the context of the term "comprising" is also intended to be implemented in the context of the terms "consisting of" or "consisting essentially of."

[0029] It is particularly intended that any limitations discussed in relation to one aspect of the present invention may apply to any other aspect of the present invention. Furthermore, any composition of the present invention may be used in any method of the present invention, and any method of the present invention may be used to produce or utilize any composition of the present invention. Aspects of the aspects described in the examples may also be implemented elsewhere in different examples or elsewhere in this application, for example, in the context of aspects discussed in the summary of the invention, the detailed description of aspects, the claims, and the legend of the figures. [Invention 1001] A composition comprising an anti-inflammatory agent functionally linked to an extracellular matrix (ECM) affinity peptide. [Invention 1002] A composition comprising an anti-inflammatory agent functionally linked to a serum protein. [Invention 1003] The composition of the present invention 1001, wherein the anti-inflammatory agent contains an anti-inflammatory antibody. [Invention 1004] Anti-inflammatory antibodies include TNF-α, IL-1, IL-5, IL-6, IL-6R, IL-12, IL-17A, IL-18, IFN-γ, GM-CSF, CD3, CD20, VLA-4, VLA-5, VCAM-1, TGF-β1, α 4 -integrin, α 4 β 7 - A composition of the present invention 1003 comprising an antibody that specifically targets integrin, connective tissue growth factor, platelet-derived growth factor, plasminogen activator inhibitor-1, or insulin-like growth factor-binding protein. [Invention 1005] The composition of the present invention 1003, wherein the antibody is a humanized antibody or a chimeric antibody. [Invention 1006] A composition of the present invention 1004 or 1005, wherein the antibody comprises adalimumab, certolizumab, infliximab, golimumab, tocilizumab, rituximab, ustekinumab, natalizumab, vedolizumab, secukinumab, or ixekizumab. [Invention 1007] A composition according to Invention 1004 or 1006, wherein the anti-inflammatory agent comprises an anti-TNF-α antibody. [Invention 1008] A composition according to the present invention 1001 or 1002, wherein the anti-inflammatory agent comprises an anti-inflammatory cytokine polypeptide. [Invention 1009] The composition of the present invention 1002 or 1008, wherein the cytokine polypeptide comprises polypeptides from IL-4, IL-1ra, IL-5, IL-10, IL-11, IL-23, IL-35, IL-36ra, IL-37, interferon-β, TGF-β1, TNF receptor I, and TNF receptor II. [Invention 1010] The composition of the present invention 1009, wherein the cytokine polypeptide comprises a polypeptide derived from IL-4. [Invention 1011] The composition of the present invention 1009, wherein the cytokine polypeptide comprises a polypeptide derived from IL-10. [Invention 1012] A composition of the present invention 1001 or 1002, wherein the anti-inflammatory agent comprises a polypeptide derived from CD200. [Invention 1013] The composition of the present invention 1012, wherein the CD200 polypeptide contains the extracellular domain of CD200. [Invention 1014] A composition according to any one of the present invention 1001 or 1003-1011, wherein the ECM affinity peptide contains a collagen-binding domain. [Invention 1015] The composition of the present invention 1014, wherein the polypeptide comprises a collagen-binding domain from decorin or von Willebrand factor (VWF). [Invention 1016] A composition according to any one of the present invention 1001 or 1003-1010, wherein the ECM affinity peptide comprises a peptide from placental growth factor-2 (PlGF-2) or CXCL-12γ. [Invention 1017] A composition according to any one of the Invention 1001 or 1003-1016, comprising an ECM affinity peptide which is at least 85% identical to one of SEQ ID NO: 1-17, 47, or 52, or a peptide which is at least 85% identical to one of SEQ ID NO: 1-17, 47, or 52. [Invention 1018] A composition according to any one of the present invention 1001 or 1003-1017, further comprising an anti-inflammatory agent functionally linked to an extracellular matrix (ECM) affinity peptide, the peptide, or a serum protein functionally linked to the agent. [Invention 1019] The composition of the present invention 1018, wherein a serum protein is functionally linked to the peptide. [Invention 1020] A composition according to the present invention 1002 or 1019, wherein a serum protein is functionally linked to the peptide via a peptide bond. [Invention 1021] A composition according to any one of the present invention 1002 or 1018-1020, wherein the serum protein contains albumin. [Invention 1022] The composition of the present invention 1021, comprising a collagen-binding domain located in the amino-amino proximal position of serum albumin protein and an IL-10 polypeptide located in the carboxyl proximal position of serum albumin protein. [Invention 1023] A composition according to any one of the present invention 1001 or 1003-1021, wherein the peptide is covalently bonded to an anti-inflammatory agent. [Invention 1024] A composition according to any one of the present invention 1001 or 1003-1023, wherein the peptide is crosslinked to an anti-inflammatory agent via a bifunctional linker. [Invention 1025] A composition according to any one of the present invention 1001 or 1003-1023, wherein the peptide is linked to an anti-inflammatory agent via a peptide bond. [Invention 1026] A composition according to any of the present invention 1001 to 1025, wherein the ratio of peptide to anti-inflammatory agent or cytokine polypeptide is approximately 1:1 to 5:1. [Invention 1027] A composition according to any one of the present invention 1001 to 1026, further comprising a second anti-inflammatory agent functionally linked to an extracellular matrix (ECM) affinity peptide. [Invention 1028] A method for treating an autoimmune or inflammatory condition in a subject, comprising the step of administering any of the compositions of the present invention 1001 to 1027 to the subject. [Invention 1029] The method of the present invention 1028, wherein the autoimmune or inflammatory condition includes inflammatory bowel disease, idiopathic pulmonary fibrosis, multiple sclerosis, type 1 diabetes, Crohn's disease, psoriasis, acute inflammation, chronic inflammation, neuritis, arthritis, rheumatoid arthritis, fibrosis, infection, allergy, adverse events related to inflammatory treatment, and inflammatory diseases related to inflammatory treatment. [Invention 1030] The method of the present invention 1029, wherein the autoimmune or inflammatory condition includes multiple sclerosis. [Invention 1031] The method of the present invention 1029, wherein the autoimmune or inflammatory condition includes rheumatoid arthritis. [Invention 1032] A method according to any of the present invention 1028 to 1031, wherein the composition is administered systemically. [Invention 1033] The method of the present invention 1032, wherein the composition is administered by intravenous injection. [Invention 1034] The method according to invention 1028 or 1029, wherein the composition is administered topically. [Invention 1035] The method of the present invention 1034, wherein the composition is administered to or adjacent to the site of inflammation. [Invention 1036] Any method of the present invention 1028 to 1035, wherein the dose administered of a composition containing an anti-inflammatory agent functionally linked to the peptide is less than the minimum effective dose of the anti-inflammatory agent administered without the peptide. [Invention 1037] Any method of the present invention 1028 to 1035, wherein the dose administered of a composition containing an anti-inflammatory agent functionally linked to the peptide is less than the minimum effective dose of the anti-inflammatory agent administered without the peptide via the same route of administration. [Invention 1038] The method of the present invention 1037, wherein the dose of the anti-inflammatory agent functionally linked to the peptide is at least 10% less than the minimum effective dose of the anti-inflammatory agent administered without the peptide. [Invention 1039] A method according to any of items 1028 to 1038 of the present invention, wherein the subject has been previously treated with an anti-inflammatory agent, anti-inflammatory therapy, or autoimmune therapy. [Invention 1040] The method of the present invention 1039, wherein the subject has been determined to be unresponsive to previous treatment. [Invention 1041] A method according to any of items 1028 to 1038 of the present invention, wherein the subject has not been previously treated for either an inflammatory disease or an autoimmune disease. [Invention 1042] Any method of the present invention 1028-1041, further comprising administering additional inflammatory therapy or autoimmune therapy. [Invention 1043] Any method of the present invention 1028 to 1042 further comprising the administration of a second anti-inflammatory agent functionally linked to an extracellular matrix (ECM) affinity peptide. [Invention 1044] A method for reducing inflammation in a subject, comprising the step of administering a composition containing an anti-inflammatory agent functionally linked to an extracellular matrix (ECM) affinity peptide to the subject. [Invention 1045] The method of the present invention 1044, wherein the inflammation is caused by an autoimmune condition or inflammatory condition, and the autoimmune condition or inflammatory condition includes inflammatory bowel disease, idiopathic pulmonary fibrosis, multiple sclerosis, type 1 diabetes, arthritis, or rheumatoid arthritis. [Invention 1046] The method of the present invention 1045, wherein the autoimmune or inflammatory condition includes multiple sclerosis. [Invention 1047] The method of the present invention 1045, wherein the autoimmune or inflammatory condition includes rheumatoid arthritis. [Invention 1048] A method according to any one of the present invention 1044 to 1047, wherein the anti-inflammatory agent functionally linked to an ECM affinity peptide includes a collagen-binding domain conjugated to anti-TNFα. [Invention 1049] The method according to any one of the invention 1044 to 1047, wherein the anti-inflammatory agent functionally linked to an ECM affinity peptide includes vWF-A3 functionally linked to IL-4. [Invention 1050] A method according to any one of the present invention 1044 to 1047, wherein the anti-inflammatory agent functionally linked to an ECM affinity peptide includes a collagen-binding domain conjugated to anti-TGF-β. [Invention 1051] A method according to any of the present invention 1044 to 1050, wherein the composition is administered systemically. [Invention 1052] A method according to any of the present invention 1044 to 1050, wherein the composition is administered topically. [Invention 1053] The method of the present invention 1052, wherein the dose of an anti-inflammatory agent functionally linked to an ECM affinity peptide is at least 20% less than the minimum effective dose of the anti-inflammatory agent administered topically without the peptide. [Brief explanation of the drawing]

[0030] The following drawings constitute part of this specification and are included to further demonstrate certain aspects of the invention. The invention may be better understood by referring to one or more of these drawings together with the detailed description of the specific embodiments presented herein.

[0031] [Figure 1]Figures 1A-B. CBP conjugation resulted in collagen affinity for αTNF. (A) WT-αTNF and CBP-αTNF analyzed by MALDI-TOF MS. The x-axis is the mass-to-charge ratio (m / z), and the y-axis is the intensity of the bicharged ion. (B) WT-αTNF and CBP-αTNF binding affinity to type I, type II, and type III collagen analyzed by ELISA (n=3, mean + SD). [Figure 2-1] Figures 2A-D. CBP-αTNF accumulation in inflamed paws. CAIA (caused arthritis) was selectively induced in the right hind paw by passive immunization with anti-collagen antibodies, followed by subcutaneous injection of LPS into the right hind paw and PBS into the left hind paw. The day after LPS injection, Cy7-labeled CBP-αTNF and Cy7-labeled WT-αTNF were intravenously injected into naive and CAIA mice. Representative images of accumulation in arthritis or non-arthritis paws of mice injected with CBP-αTNF (A) and WT-αTNF (B). (C) Changes in radioefficiency ratio between arthritis (right posterior) and non-arthritis (left posterior) paws in naive and CAIA mice (n = 3-4, mean ± SD). (D) Representative histological images of joints in CAIA mice injected with CBP-αTNF (left, H&E staining; right, immunohistochemical staining against anti-rat IgG). [Figure 2-2] See the explanation in Figure 2-1. [Figure 3]Figures 3A-B. CBP-αTNF more effectively suppressed the development of arthritis than WT-αTNF. Arthritis was induced by passive immunization with anti-collagen antibodies followed by intraperitoneal injection of LPS. On the day of LPS injection, control IgG, WT-αTNF, or CBP-αTNF were intravenously injected into arthritis mice. (A) Arthritis score represents the mean + SE of 6 mice. * P<0.05, compared to control (Dunnett's multiple comparison test). # P<0.05, compared to the score on day 8 of each treatment group (Tukey's multiple comparison test). (B) Representative H&E images of the joints on day 8 in each treatment group. The severity of bone resorption and synovial hyperplasia was scored from 0 to 4, as described in Materials and Methods. Statistical analysis was performed using Dunnett's multiple comparison test for differences between the control group and the αTNF treatment group. [Figure 4] Figures 4A-B. Subcutaneous injection of CBP-αTNF also accumulated in arthritis-affected feet and suppressed the development of arthritis. (A) Arthritis was selectively induced in the right hind foot by passive immunization with anti-collagen antibodies, followed by subcutaneous injection of LPS into the right hind foot and PBS into the left hind foot. The day after LPS injection, Cy7-labeled CBP-αTNF was subcutaneously injected into the back of the mice. Representative images of accumulation (indicated by arrows) in arthritis-affected or non-arthritis-affected feet of mice injected with CBP-αTNF. (B) Arthritis was induced in all feet by passive immunization with anti-collagen antibodies, followed by intraperitoneal injection of LPS. On the day of LPS injection, control IgG, WT-αTNF, or CBP-αTNF was subcutaneously injected. Arthritis score represents the mean + SE of 5 mice. #P<0.05, compared to the score on day 8 of each group (Tukey's multiple comparison test). [Figure 5]Figures 5A-B. Effects of local injection on the development of arthritis. Arthritis was induced by passive immunization with anti-collagen antibodies followed by intraperitoneal injection of LPS. On the day of LPS injection, (A) Cy7-labeled PlGF-2123~144-αTNF and Cy7-labeled WT-αTNF or (B) control IgG, WT-αTNF, and PlGF-2123~144-αTNF were subcutaneously injected into the left hind leg of arthritis mice. (A) Representative images of retention at the injection site of WT-αTNF and PlGF-2123~144-αTNF in mice. (B) Arthritis scores represent the mean ± SE of 8 mice. #P<0.05, compared to the score on day 6 of each group (Tukey's multiple comparison test). [Figure 6] Figures 6A-E. A3-IL4 accumulated in the spinal cord and reduced the EAE score. The affinity of A3-IL4 to collagen type III (A) and the affinity between IL-4 and A3-IL4 proteins to IL-4Rα (B) were measured by ELISA. Graphs of [concentration] vs [signal] are shown (n = 4). EAE was induced by subcutaneous injection of MOG35-55 / CFA emulsion, followed by intraperitoneal injection of PTX on the immunization day and the following day. On postimmunization day 14, DyLight 800-labeled A3-IL4, A3 protein (C), or Cy7-labeled CBP-αTNF (D) were intravenously injected into naive or EAE mice. Spinal cord was collected 4 hours after injection and fluorescence intensity was measured. (E) From postimmunization day 14, normal IL-4 or A3-IL4 was intravenously injected every other day. The disease score for EAE is expressed as mean ± SE (n = 3-5). [Figure 7]Figures 7A-C. Localization of A3 protein and CBP conjugates in inflammatory tissues of other inflammatory disease models. (A) DyLight 800-labeled A3 protein or Cy7-labeled CBP-αTNF was intravenously injected into IL-10- / - × TLR-4- / - (DKO) mice that spontaneously developed or did not develop IBD, or into normal (C57BL / 6) mice. Colons were collected 4 hours after injection and provided for fluorescence imaging (top) and histological analysis (bottom). Colons of IBD-developing mice injected with Cy7-labeled CBP-αTNF were stained with H&E and periodate Schiff (PAS). In addition, the injected antibodies were detected by immunohistochemistry (IHC) against anti-rat IgG. (B) Cy7-labeled CBP-αTGF or Cy7-labeled αTGF was intravenously injected into naive mice or a bleomycin-induced pulmonary fibrosis model 7 days after bleomycin injection. Lungs were collected 4 hours after fluorescent injection, and fluorescence intensity was then measured. (C) DyLight 800-labeled A3 protein was intravenously injected into spontaneously developing type 1 diabetes (T1D) mice, cyclophosphamide-induced T1D mice, and non-diabetic mice. Pancreases were collected 15 minutes after fluorescent injection, and fluorescence intensity was then measured. [Figure 8]Figures 8A-D. Albumin fusion to IL-10 provided FcRn binding, leading to LN accumulation. (A) SDS-PAGE analysis of wt IL-10 and SA-IL-10. (B) Binding analysis of SA-IL-10 to FcRn. (C) Single cells from splenic cells (i) or popliteal LN (ii) were incubated with SA, SA-IL-10, or CBD-SA-IL-10 on ice for 30 minutes. For each cell type (x axis), (i) shows bars from left to right representing the % binding (y axis) of SA, SA-IL-10, and CBD-SA-IL-10. For each cell type (x axis), (ii) shows bars from left to right representing the % binding (y axis) of SA and SA-IL-10, respectively. Binding of each protein to immune cells was detected by co-staining with anti-SA antibodies and antibodies against specific markers of each immune cell population. (D) Immunofluorescence image of popliteal LN after intravenous injection of DyLight594-labeled wt IL-10 or SA-IL-10. T cells and high endothelial venules (HEVs) were stained with anti-CD3 antibody or anti-PNAd antibody, respectively. [Figure 9]Figures 9A-B. Albumin fusion to IL-10 provided long-term blood circulation, while CBD fusion improved in vivo distribution to inflamed joints. (A) wt IL-10, SA-IL-10, or CBD-SA-IL-10 (each equivalent to 35 μg of IL-10) was administered to BALB / c mice by tail vein injection. Serum was collected at the indicated time points. Serum concentrations of IL-10 were measured by ELISA (mean ± SEM; n = 5). The plasma half-life of IL-10 was calculated using biphasic exponential decay: MFI(t) = Ae-αt + Be-βt, t1 / 2, α, fast clearance half-life; t1 / 2, β, slow clearance half-life. Area under the curve (AUC) was analyzed by Graphpad Prism. (B) CAIA was selectively induced in the right hind paw by passive immunization with an anti-collagen antibody, followed by subcutaneous injection of LPS into the right hind paw (defined as day 3). The day after LPS injection, CAIA mice were intravenously injected with DyLight800-labeled wt IL-10, SA-IL-10, or CBD-SA-IL-10. Four hours after injection, the indicated organs were collected and analyzed using the IVIS imaging system (mean ± SEM; n = 4). Statistical analysis was performed using analysis of variance (ANOVA) with Tukey's test. *P < 0.05; **P < 0.01. For each organ (x axis), (B) shows bars from left to right representing the percentage distribution (y axis) of wt IL-10, SA-IL-10, and CBD-SA-IL-10, respectively. [Figure 10]Figures 10A-D. Albumin-fused IL-10 more effectively suppressed the development of arthritis than wt IL-10. (A) Arthritis (CAIA) was induced by passive immunization with anti-collagen antibody, followed by intraperitoneal injection of LPS. On the day of LPS injection, PBS, wt IL-10, SA-IL-10, or CBD-SA-IL-10 (equivalent to 43.5 μg of IL-10) was intravenously injected into arthritis mice. Arthritis scores represent the mean + SEM of 7 mice. (B) Comparison of therapeutic effects of wt IL-10 and CBD-SA-IL-10 and αTNF-α antibody in CAIA mice immunized with high doses (1.5 mg / mouse) of anti-collagen antibody. Arthritis scores represent the mean + SEM of 6-7 mice. (C) Representative H&E histological images of joints at day 13 in each treatment group. Scale bar, 500 μm. As described in Materials and Methods, the severity of bone resorption and synovial hyperplasia was scored from 0 to 4. (D) Effect of administration route on the therapeutic effect of SA-IL-10 and CBD-SA-IL-10. Arthritis scores represent the mean + SEM of 6-7 mice. Statistical analysis was performed using analysis of variance (ANOVA) with Tukey's test. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. [Figure 11]Figures 11A-D. Albumin-fused IL-10 showed improved therapeutic effects against established arthritis. Bovine collagen / CFA emulsion was subcutaneously injected into the tail base of DBA / 1J male mice. Three weeks later, bovine collagen / IFA emulsion was injected again as an additional immunization. When the arthritis score reached 2-4 (defined as day 0), mice were intravenously injected with PBS, SA-IL-10 or CBD-SA-IL-10 (each equivalent to 43.5 μg of IL-10), or 200 μg of anti-TNF-α antibody. In (A), the same treatment was administered to mice again on day 3. (A and B) Arthritis scores represent the mean + SEM of 9 mice. (C and D) Representative H&E histological images of the joints on day 16. Scale bar, 500 μm. As described in Materials and Methods, the severity of bone resorption and synovial hyperplasia was scored from 0 to 4. Statistical analysis was performed using analysis of variance (ANOVA) with Tukey's test in cases (A and B) and a two-tailed Student's t-test in cases (C and D). *P < 0.05; **P < 0.01; ***P < 0.001. [Figure 12-1]Figures 12A-F. Albumin-fused IL-10 accumulated in the LN and suppressed Th17 activation in the LN. CAIA (Central Arthritis Induced) was induced by passive immunization with an anti-collagen antibody, followed by intraperitoneal injection of LPS (defined as day 3). On the day of LPS injection, wt IL-10, SA-IL-10, or CBD-SA-IL-10 were intravenously injected into arthritis mice. IL-10 levels and Th17-related cytokines in the LN were measured using ELISA. (A) Comparison of IL-10 levels 4 hours after injection of each protein. (B) Pharmacokinetics of wt IL-10 or SA-IL-10 in the LN after intravenous injection (mean ± SEM; n = 4). (C) AUC of wt IL-10 and SA-IL-10 in various LNs. Th17-related cytokine levels in the joint inflow area (popliteal fossa) LN (D) and non-inflow area (cervical) LN (E). (F) GM-CSF levels in the popliteal LN. (Mean ± SEM; n = 7) Statistical analysis was performed using Tukey's test for analysis of variance (ANOVA). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; ns; Not significant. [Figure 12-2] See the explanation in Figure 12-1. [Figure 13-1]Figures 13A-C. Albumin-fused IL-10 suppressed the inflammatory response in the paw. CAIA was induced by passive immunization with anti-collagen antibodies followed by intraperitoneal injection of LPS. On the day of LPS injection (defined as day 3), PBS, wt IL-10, SA-IL-10, or CBD-SA-IL-10 were intravenously injected into arthritis mice. (A) Single cells were extracted from the hind paw on day 11 and subsequently analyzed by flow cytometry. The graph shows the frequencies of CD45+ cells, B cells (B220+ cells within CD45+ lymphocytes), dendritic cells (CD11c+ cells within CD45+ lymphocytes), monocytes (CD11b+ cells within CD45+ lymphocytes), granulocyte MDSCs / neutrophils (Ly6G+ Ly6C+ CD11b+ CD45+), monocyte MDSCs (Ly6G- Ly6C+ CD11b+ CD45+), macrophages (F4 / 80+ CD11b+ CD45+), M2 macrophages (CD206+ F4 / 80+ CD11b+ CD45+), and M1 macrophages (MHC II+ F4 / 80+ CD11b+ CD45+). (Mean ± SEM; n = 7) (B) Cytokine levels in the hind leg on day 11. (n = 5~7) (C) Representative H&E images of the joints on day 14 in each treatment group. Scale bar, 500 μm. As described in Materials and Methods, the severity of bone resorption and synovial hyperplasia was scored from 0 to 4 (mean ± SEM; n = 6~7). Statistical analysis was performed using Tukey's test of variance (ANOVA), except for %CD11c+ in (A). For the analysis of %CD11c+ in (A), the Kruskal-Wallis test followed by Dunn's multiple comparison test was used. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. [Figure 13-2] See the explanation in Figure 13-1. [Figure 14A] Figures 14A-B. CBD conjugation resulted in collagen affinity for IL-10. (A) SDS-PAGE analysis of CBD-SA-IL-10. (B) Binding analysis of CBD-SA-IL-10 to type I or type III collagen and FcRn. [Figure 14B]See the explanation in Figure 14A. [Figure 15A] Figures 15A-B. Effects of albumin-fused IL-10 on immune cell populations in the spleen (A) and pulmonary nephrocyte (LN) (B). CAIA (caused arthritis in arthritis) was induced by passive immunization with anti-collagen antibodies, followed by intraperitoneal injection of LPS (defined as day 3). On days 3 and 6, mice were intravenously injected with PBS, wt IL-10, or SA-IL-10. Single cells were extracted from the spleen and popliteal LN the day after the last injection and subsequently analyzed by flow cytometry. The graphs show the frequencies of CD3+ T cells, CD45+ lymphocytes, CD11b+ cells, CD11c+ cells, CD86+ cells, granulocyte MDSCs / neutrophils (Ly6G+ Ly6C+ in CD11b+ cells), monocyte MDSCs (Ly6G- Ly6C+ in CD11b+ cells), macrophages (F4 / 80+ in CD11b+ cells), CD86+ cells within macrophages, and M2 macrophages (CD206+ F4 / 80+ in CD11b+ cells). (Mean ± SEM; n = 6~7) Statistical analysis was performed using Tukey's test of ANOVA, except for the following graphs: (A) %CD11c+ in CD11b+ cells and (B) %Ly6G+ in CD11b+ cells. For the analysis of Ly6C+, the Kruskal-Wallis test followed by Dunn's multiple comparison test was used. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. [Figure 15B] See the explanation in Figure 15A. [Figure 16A]Figures 16A-B. Effects of albumin-fused IL-10 on T cell populations in the paws and blood. CAIA (caused arthritis) was induced by passive immunization with anti-collagen antibodies, followed by intraperitoneal injection of LPS (defined as day 3). On the day of LPS injection, mice were intravenously injected with PBS, wt IL-10, SA-IL-10, or CBD-SA-IL-10. (A) Single cells were extracted from the hind paws on day 11 and subsequently analyzed by flow cytometry. The graph shows the frequency of NK1.1+ CD3- NK cells, CD3+ T cells, CD3+ CD4+ T cells, Treg (Foxp3+ CD25+) CD3+ CD4+ T cells within CD45+ lymphocytes, CD3+ CD8+ T cells, effector memory T cells (CD62L- CD44+) within CD3+ CD8+ T cells, central memory T cells (CD62L+ CD44+) within CD3+ CD8+ T cells, and PD-1+ cells within CD3+ CD8+ T cells. (B) Lymphocytes were extracted from blood on day 11, followed by flow cytometry analysis. The graphs plot the frequencies of CD3+ T cells, CD3+CD4+ T cells, Treg (Foxp3+CD25+) CD3+CD4+ T cells, and CD3+CD8+ T cells within CD45+ lymphocytes (mean ± SEM; n = 5~7). Statistical analysis was performed using Tukey's test for analysis of %NK1.1+ in CD45+ cells, %Foxp3+ in CD4+ cells, %CD44+ / CD62L- in CD8+ cells, %CD44+ / CD62L+ in CD8+ cells, and %PD-1+ in CD8+ cells, except for the following graphs: Analysis of variance (ANOVA) was performed using Tukey's test, with the Kruskal-Wallis test followed by Dunn's multiple comparison test. *P < 0.05; **P < 0.01; ***P < 0.001. [Figure 16B] See the explanation in Figure 16A. [Figure 17A]Figures 17A-B. Safety evaluation of albumin-fused IL-10. wt IL-10, SA-IL-10, or CBD-SA-IL-10 were intravenously injected into healthy BALB / c mice. (A) Two days after injection, white blood cell count, red blood cell count, platelet count, serum hemoglobin concentration, and spleen weight were evaluated. (B) Alanine transaminase (ALT), amylase, blood urea nitrogen (BUN), serum calcium, creatine kinase (CK), CO2, total bilirubin (TBli), and serum total protein concentrations were evaluated using a biochemical analyzer. (Mean ± SEM; n = 5) Statistical analysis was performed using analysis of variance (ANOVA) with Tukey's test. *P < 0.05; **P < 0.01. [Figure 17B] See the explanation in Figure 17A. [Figure 18] Figures 18A-D. IL-4 retains activity even after SA fusion. (a) wt IL-4 and SA-IL-4 were analyzed by SDS-PAGE under reducing and non-reducing conditions using Coomassie blue staining. (b) Binding of SA-IL-4 to newly isolated immune cells from LN and spleen, measured by flow cytometry. (c) wt IL-4 and SA-IL-4 activity assay. STAT6 phosphorylation in T cells was analyzed by flow cytometry after culturing T cells in vitro with the indicated concentrations of wt IL-4 or SA-IL-4. (d) IL-17 concentration secreted under Th17 differentiation conditions in the presence of wt IL-4 or SA-IL-4, measured by ELISA. Data are mean ± SEM. Two experimental replicas. Statistical analysis was performed using one-way ANOVA with Tukey's test. **P < 0.01. [Figure 19-1]Figure 19A-H. SA fusion to IL-4 increased the amount of IL-4 in secondary lymphoid organs after intravenous injection. (a) Binding affinity of SA-IL-4 to FcRn as measured by SPR. (b) Plasma concentrations of wt IL-4 or SA-IL-4 after intravenous injection. 10 μg of wt IL-4 (n = 4) or equimolar SA-IL-4 (n = 3) was intravenously injected into naive mice. Blood was collected from 1 minute to 24 hours later, and plasma concentrations of IL-4 were measured by ELISA. (c-d) (c) IL-4 levels over time in the upper arm LN and lumbar LN, and (d) spleen. 40 μg of wt IL-4 or equimolar SA-IL-4 was intravenously injected into naive mice. LN and spleen were collected, and the amount of IL-4 was tested by ELISA (n = 5). (e) SA(P573K)-IL-4 levels were measured in the lumbar nephrocytes (LN) and spleen 1 hour after injection. wt IL-4 and SA-IL-4 data from (c~d) are re-presented. (f~g) Immunofluorescence images of lumbar LN 1 hour after intravenous injection of DyLight594-labeled IL-4 or SA-IL-4. T cells and high endothelial venules (HEVs) were stained with anti-CD3 antibody or anti-PNAd antibody, respectively. Scale bars represent (g) 200 μm and (h) 100 μm. Data are mean ± SEM. Two experimental copies. Statistical analysis was performed using one-way ANOVA with Tukey's test. **P < 0.01. [Figure 19-2] See the explanation in Figure 19-1. [Figure 20-1]Figures 20A-D. SA-IL-4 prevents the progression and onset of EAE disease in the acute phase. (b) Disease incidence and (c) Disease progression with (a) Weight change in C57BL / 6 myelin oligodendrocyte glycoprotein (MOG) 35-55 experimental autoimmune encephalomyelitis (EAE) mice. (b) Disease incidence and (c) Disease progression with (a) Weight change. n = 7 / group. (d) Representative spinal cord tissue image. Myelin expression was detected by immunohistochemistry using anti-myelin basic protein antibody (brown). Arrows indicate demyelination. The graph shows the percentage of mice that showed demyelination in each treatment group by blinded pathological analysis. Two experimental replicas. Data are mean ± SEM. Statistical analysis was performed using one-way ANOVA with Tukey's test. **P < 0.01. [Figure 20-2] See the explanation in Figure 20-1. [Figure 21]Figure 21A-H. SA-IL-4 treatment inhibits leukocyte infiltration into the spinal cord and induces immunosuppressive cells in the influx region (LN). Mice were administered wt IL-4, SA-IL-4, or PBS via intravenous injection (ip) or sc-injection of SA-IL-4 to mice every other day for 10 days starting 8 days post-immunization, or FTY720 1 mg / kg body weight was administered orally daily starting 8 days post-immunization. Cells were isolated from the influx region (LN) and spinal cord 17 days after immunization and analyzed by flow cytometry. (a-b) Frequency of (a) CD45+ leukocytes and (b) RoR□t+ Th17 cells in living cells in the spinal cord. (c~g) In the lumbar inflow region LN (dLN), the frequencies of (c) Ly6G+Ly6C+ G-MDSCs in CD11b+CD45+ cells, (d) Ly6G-Ly6C+ M-MDSCs in CD11b+CD45+ cells, (e) RoR□t+ Th17 cells in CD4+CD3+ T cells, (f) CD86+ M1 macrophages in F4 / 80+CD11b+ macrophages, (g) CD206+ M2 macrophages in F4 / 80+CD11b+ macrophages, and (h) B220+ B cells in CD11b+CD45+ cells were analyzed. Data are mean ± SEM. The experiment was performed once. Statistical analysis was performed using Tukey's test with one-way ANOVA. *P < 0.05, **P < 0.01. [Figure 22-1]Figures 22A-P show that SA-IL-4 treatment activates the PD-1 / PD-L1 axis and reduces integrin and cytokine expression in T cells. MOG35-55 induced EAE mice were injected with PBS, wt IL-4, or SA-IL-4 on days 8, 10, and 12 postimmunization. Spinal cord and spleen were isolated on day 13, and (a-i) immune cells were analyzed. (a) Frequency of tetramer+ (recognizing MOG35-55) cells in CD4+ T cells in the spinal cord. In the spleen, (b) αLβ2 integrin+ cells in tetramer+ CD4+ T cells, (c) α4β1 integrin+ cells in tetramer+ CD4+ T cells, (d) αLβ2 integrin+ cells in CD8+ T cells, and (e) α4β1 integrin+ cells in CD8+ T cells are shown. (f) Mean fluorescence intensity (MFI) of PD-1 in central memory (CM) CD44+CD62L+CD4+ T cells, (g) MFI of PD-1 in CM CD44+CD62L+CD8+ T cells, (h) MFI of PD-L1 in Ly6C+Ly6G-CD11b+ M-MDSCs, (i) Frequency of PD-L1+ in Ly6C+Ly6G-CD11b+ M-MDSCs, (j) MFI of PD-L1 in Ly6C+Ly6G+CD11b+ G-MDSCs, (k) Frequency of PD-L1+ in Ly6C+Ly6G+CD11b+ G-MDSCs, (l) Frequency of IL-23R+ cells in tetramer+ CD4+ T cells. (m~n) Splenocytes were cultured in vitro for 3 days in the presence of MOG protein. The concentrations of (m) IL-17A, (n) IFNγ, and (o) GM-CSF in the culture medium were analyzed by ELISA. (p) Splenocytes were cultured in vitro for 6 hours in the presence of MOG35-55 peptides. Cytokine expression in CD4+ T cells was characterized by flow cytometry. Data are mean ± SEM. The experiment was performed once. Statistical analysis was performed using one-way ANOVA with Tukey's test. *P < 0.05, **P < 0.01. [Figure 22-2] See the explanation in Figure 22-1. [Figure 23-1]Figures 23A-K. SA-IL-4 treatment in the chronic phase of EAE reduces clinical scores and prevents immune cell infiltration into the spinal cord. EAE was induced in C57BL / 6 mice using MOG35-55. PBS, wt IL-4, or SA-IL-4 were administered via ip injection every other day for 10 days starting 21 postimmunization. (a) Disease progression and (b) changes in body weight are shown (n = 6). (c-d) PBS, wt IL-4, or SA-IL-4 were administered via sc injection every other day for 12 days starting 21 postimmunization. (c) Disease progression and (d) changes in body weight are shown (n=8 for PBS and SA-IL-4; n=7 for other treatment groups). (e-h) On day 34, spinal cord and spleen were collected and immune cells were analyzed by flow cytometry. The graphs show the frequency of (e) CD45+ cells in living cells in the spinal cord, (f) CD4+CD3+CD45+ T cells in living cells in the spinal cord, (g) tetramer+ (recognizing MOG35~55) RoRγt+CD4+ Th17 cells in living cells in the spinal cord, and (h) IL-23R+ cells in tetramer+CD4+ cells in the spleen. (i~j) Splenocytes were cultured in vitro for 3 days in the presence of MOG protein. (i) IL-17A and (j) GM-CSF concentrations in the culture medium were analyzed by ELISA. (k) Splenocytes were cultured in vitro for 6 hours in the presence of MOG35~55 peptides. Cytokine expression in CD4+ T cells was characterized by flow cytometry. The experiment was performed once. Data are mean ± SEM. Statistical analysis was performed using Tukey's test with one-way ANOVA. *P < 0.05, **P < 0.01. [Figure 23-2] See the explanation in Figure 23-1. [Figure 24]Injected SA-IL-4 was analyzed by IVIS and accumulated in the lumbar nexa (LN) more than wt IL-4. In vivo distribution analysis of DyLight 800-labeled wt IL-4 and SA-IL-4 in the LN. 10 μg of wt IL-4 or SA-IL-4 was injected IV four hours after injection with an equal amount of fluorescence. Lumbar LN was collected and imaged using the IVIS in vivo imaging system (n = 6). Data are mean ± SEM. The experiment was performed once. Statistical analysis was performed using Student's t-test. [Figure 25] Figures 25A-B. The SA(P573K) mutation in SA-IL-4 reduces blood concentration and inactivates FcRn binding. Mice were intravenously injected with 40 μg of wt IL-4, SA-IL-4, or SA(P573K)-IL-4. Blood was collected 1 hour later, and plasma IL-4 concentrations were determined by ELISA. Data are mean ± SEM. (b) Binding affinity of SA(P573K)-IL-4 and FcRn as measured by SPR. Binding affinity could not be determined. 1st and 2nd mean that the concentration of 62.5 nM was tested twice to examine variability. Two experimental replicas. [Figure 26] Long-term treatment with SA-IL-4 suppresses the onset and progression of EAE disease. Disease progression (n=8) in C57BL / 6 MOG35-55 EAE mice treated with PBS or SA-IL-4 (10 μg relative to IL-4) via ip injection every other day for 16 days starting 8 day post-immunization. The number of EAE-developing mice per total mouse in each treatment group is shown. Mice were monitored until day 24. Data are mean ± SEM. Two experimental replicas. Statistical analysis was performed using Student's t-test. **P < 0.01. [Figure 27]FcRn binding is crucial for SA-IL-4 to suppress the onset and progression of EAE disease. Disease progression (n=6) in C57BL / 6 MOG35-55 EAE mice immunized with PBS or SA-IL-4 (10 μg relative to IL-4) via ip injection every other day starting 8 days post-immunization. Data are mean ± SEM. Two experimental replicas. Statistical analysis was performed using Student's t-test. **P < 0.01. [Figure 28] Figures 28A-B. SA-IL-4 did not affect the number of macrophages and dendritic cells in the spinal cord and influx region LN. Mice were administered wt IL-4, SA-IL-4, or PBS via intravenous injection (ip) or sc-injection (sc) every other day for 10 days starting 8 days post-immunization. FTY720 1 mg / kg body weight was administered orally daily starting 8 days post-immunization. Cells were isolated from the influx region LN (dLN) and spinal cord 17 days after immunization and analyzed by flow cytometry. (a) The frequency of F4 / 80+ macrophages in CD11b+ cells and (b) the frequency of CD11b+ CD11c+ DCs in CD45+ cells were analyzed. Data are mean ± SEM. The experiment was performed once. Statistical analysis was performed using one-way ANOVA with Tukey's test. [Figure 29-1] Figures 29A-O. Blood and organ analysis of SA-IL-4 demonstrated that SA-IL-4 is safe. Toxicity analysis of SA-IL-4. 10 μg of wt IL-4 or equimolar SA-IL-4 was intravenously injected into naive mice. Two days later, (a-i) serum was tested using a biochemical analyzer, and (j-m) blood was tested using a hemochemical analyzer. Lung water content was determined by weighing the lungs before and after lyophilization. Data are mean ± SEM. The experiment was performed once. Statistical analysis was performed using Tukey's test with one-way ANOVA. [Figure 29-2] See the explanation in Figure 29-1. [Figure 30A] Figure 30A-D. Gating strategies for flow cytometry. (A) Restimulation (cytokine expression). (B) Integrin expression. (C) MDSCs. (D) CD45+ and T cells. [Figure 30B] See the explanation in Figure 30A. [Figure 30C] See the explanation in Figure 30A. [Figure 30D] See the explanation in Figure 30A. [Modes for carrying out the invention]

[0032] Detailed explanation There is a great need to enhance the therapeutic effects of drugs for inflammatory and autoimmune diseases. One possible approach is to target anti-inflammatory drugs to the site of inflammation. Collagen is inaccessible in most tissues due to the low permeability of vascular structures, but is exposed to blood flow at the site of inflammation due to the hyperpermeability of vascular structures. This disclosure describes ECM-binding anti-inflammatory agents conjugated to ECM-affinity peptides. One such peptide is collagen-binding peptide (CBP). CBP conjugation resulted in collagen affinity for anti-TNFα antibody (αTNF). CBP-αTNF accumulated at the site of inflammation in a collagen antibody-induced arthritis model (Example 1). The development of arthritis was significantly suppressed by CBP-αTNF compared with the unmodified antibody. Furthermore, the collagen-binding domain derived from the fusion of von Willebrand factor (vWF) A3 domain to interleukin (IL)-4 (A3-IL4) enabled detection in the spinal cord of experimental autoimmune encephalomyelitis (EAE), a model of multiple sclerosis, after intravenous administration (Example 1). A3-IL4 reduced the clinical symptoms of EAE, while conventional IL-4 did not. The collagen-binding protein was detectable in inflammatory tissues of spontaneous inflammatory bowel disease, bleomycin-induced pulmonary fibrosis, and a model of type 1 diabetes. In summary, collagen affinity enables the targeting of anti-inflammatory drugs to inflammatory sites, demonstrating a novel approach to clinical technology transfer for treating inflammatory and autoimmune diseases.

[0033] III. Anti-inflammatory agents A. Antibodies The aspects of this disclosure relate to anti-inflammatory antibodies or fragments thereof. The term “antibody” means any isotype of intact immunoglobulin or fragment thereof that can compete with an intact antibody for specific binding to a target antigen, and includes chimeric antibodies, humanized antibodies, fully human antibodies, and bispecific antibodies. Where used herein, the terms “antibody” and “immunoglobulin” are interchangeable and refer to any of several classes of structurally related proteins that function as part of the immune response of animals, including IgG, IgD, IgE, IgA, IgM and related proteins, as well as polypeptides containing an antibody CDR domain that retains antigen-binding activity.

[0034] The term "antigen" refers to a molecule or part of a molecule that can be bound by a selective binder, such as an antibody. An antigen may possess one or more epitopes that can interact with different antibodies.

[0035] The term "epitope" refers to any region or portion of a molecule that can trigger an immune response by binding to an immunoglobulin or T cell receptor. Epitope determinants include chemically active surface groups such as amino acids, sugar side chains, phosphoryl or sulfonyl groups, and may possess specific three-dimensional structural and / or specific charge properties. Generally, antibodies specific to a particular target antigen selectively recognize epitopes on the target antigen within a complex mixture.

[0036] The epitope region of a given polypeptide can be identified using many different epitope mapping techniques known in the art, including X-ray crystallography, nuclear magnetic resonance spectroscopy, site-directed mutagenesis mapping, and protein display arrays. See, for example, Epitope Mapping Protocols, (Johan Rockberg and Johan Nilvebrant, Ed., 2018) Humana Press, New York, NY. Such techniques are publicly known in the art and are described, for example, in U.S. Patent No. 4,708,871; Geysen et al. Proc. Natl. Acad. Sci. USA 81:3998-4002 (1984); Geysen et al. Proc. Natl. Acad. Sci. USA 82:178-182 (1985); Geysen et al. Molec. Immunol. 23:709-715 (1986). See, for example, Epitope Mapping Protocols. Furthermore, antigenic regions of proteins can also be predicted and identified using standard antigenicity and hydrophobicity plots.

[0037] Intact antibodies generally consist of two full-length heavy chains and two full-length light chains, but may in some cases contain fewer chains, such as antibodies naturally occurring in camelids that may contain only heavy chains. The antibodies disclosed herein may originate from a single source or may be “chimeras,” meaning that different parts of the antibody may originate from two different antibodies. For example, the variable region or CDR region may originate from a rat or mouse source, while the constant region originates from a different animal source, such as a human. Antibodies or conjugated fragments may be produced in hybridomas by recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibodies. Unless otherwise specified, the term “antibody” includes its derivatives, variants, fragments, and mutaines, examples of which are described below (Sela-Culang et al. Front Immunol. 2013; 4: 302; 2013).

[0038] The term "light chain" includes full-length light chains and their fragments that have a variable region sequence sufficient to confer binding specificity. Full-length light chains have a molecular weight of approximately 25,000 daltons and include a variable region domain (abbreviated herein as VL) and a constant region domain (abbreviated herein as CL). There are two classifications of light chains, identified as kappa (κ) and lambda (λ). The term "VL fragment" refers to a fragment of a monoclonal antibody light chain that includes all or part of the light chain variable region, including the CDR. VL fragments may further include the light chain constant region sequence. The variable region domain of the light chain is located at the amino terminus of the polypeptide.

[0039] The term “heavy chain” includes full-length heavy chains and their fragments that have a variable region sequence sufficient to confer binding specificity. Full-length heavy chains have a molecular weight of approximately 50,000 daltons and include a variable region domain (abbreviated herein as VH) and three constant region domains (abbreviated herein as CH1, CH2, and CH3). The term “VH fragment” means a fragment of the heavy chain of a monoclonal antibody that includes all or part of the heavy chain variable region, including the CDR. VH fragments may further include the heavy chain constant region sequence. The number of heavy chain constant region domains will depend on the isotype. The VH domain is located at the amino terminus of the polypeptide, the CH domain is at the carboxy terminus, and CH3 is closest to the -COOH terminus. The isotype of an antibody can be IgM, IgD, IgG, IgA, or IgE, and is defined by the heavy chain in which the mu (μ), delta (δ), gamma (γ), alpha (α), or epsilon (ε) chains are present, respectively. IgG has several subtypes, including but not limited to IgG1, IgG2, IgG3, and IgG4. The IgM subtype includes IgM1 and IgM2. The IgA subtype includes IgA1 and IgA2.

[0040] Antibodies can be whole immunoglobulins of any isotype or classification, chimeric antibodies, or hybrid antibodies with specificity for two or more antigens. They can also be fragments containing hybrid fragments (e.g., F(ab')2, Fab', Fab, Fv, etc.). Immunoglobulins also include native, synthetic, or genetically modified proteins that act like antibodies by binding to specific antigens and forming complexes. The term antibody includes genetically modified or otherwise altered forms of immunoglobulins, such as:

[0041] The term "monomer" refers to an antibody containing only one Ig unit. Monomers are the basic functional units of antibodies. The term "dimer" refers to an antibody containing two Ig units linked to each other via the constant domain (Fc region or fragment-crystallizable region) of the antibody heavy chain. The complex can be stabilized by linking (J) chain proteins. The term "multimer" refers to an antibody containing more than two Ig units linked to each other via the constant domain (Fc region) of the antibody heavy chain. The complex can be stabilized by linking (J) chain proteins.

[0042] The term "bivalent antibody" refers to an antibody that contains two antigen-binding sites. The two binding sites may have the same antigen specificity, or they may be bispecific, meaning that the two antigen-binding sites have different antigen specificities.

[0043] Bispecific antibodies are a class of antibodies having two paratopes, each having different binding sites for two or more different epitopes. In some embodiments, bispecific antibodies can be biparatopic, where the bispecific antibody can specifically recognize different epitopes from the same antigen. In some embodiments, bispecific antibodies can be constructed from a pair of different single-domain antibodies referred to as "nanobodies." Single-domain antibodies are supplied and modified from cartilaginous fish and camelids. Nanobodies can be linked together by linkers using techniques common to those skilled in the art; such methods for the selection and linking of nanobodies are described in PCT publication numbers WO2015044386A1, WO2010037838A2, and Bever et al., Anal Chem. 86:7875-7882 (2014), which are respectively incorporated herein in their entirety by reference.

[0044] Bispecific antibodies can be constructed as whole IgG, Fab'2, Fab'PEG, diabody, or alternatively as scFv. Diabody and scFv can be constructed using only the variable domain and without the Fc region, potentially reducing the impact of anti-idiotype reactions. Bispecific antibodies can be produced by various methods, including but not limited to hybridoma fusion or Fab' fragment ligation. See, for example, Songsivilai and Lachmann, Clin. Exp. Immunol. 79:315-321 (1990); Kostelny et al., J. Immunol. 148:1547-1553 (1992), where their entirety is specifically incorporated by reference.

[0045] In certain aspects, antigen-binding domains can be multispecific or heterospecific by multimerizing with VH and VL region pairs that bind to different antigens. For example, an antibody may bind to (a) a cell surface antigen, (b) an Fc receptor on the surface of an effector cell, or (c) at least one other component, or interact with them. Thus, aspects may include, but are not limited to, bispecific, triplicate, quadruplicate, and other multispecific antibodies or their antigen-binding fragments directed to epitopes and other targets such as Fc receptors on effector cells.

[0046] In some embodiments, multispecific antibodies can be used by conventional methods known in the art and directly linked via short, mobile polypeptide chains. One such example is the diabody, a bivalent, bispecific antibody in which the VH and VL domains are expressed on a single polypeptide chain that is too short to allow pairing between domains on the same chain, thereby pairing the domains with complementary domains on another chain to create two antigen-binding sites. Linker functionality is applicable to triabodies, tetrabodies, and even higher-order antibody multimers. (See, for example, Hollinger et al., Proc Natl. Acad. Sci. USA 90:6444-6448 (1993); Polijak et al., Structure 2:1121-1123 (1994); Todorovska et al., J. Immunol. Methods 248:47-66 (2001)).

[0047] In contrast to bispecific whole antibodies, bispecific diabodies can also be advantageous because they can be readily constructed and expressed in Escherichia coli (E. coli). Diabodies (and other polypeptides such as antibody fragments) with appropriate binding specificity can be readily selected from a library using phage display (WO94 / 13804). If one arm of the diabody is kept constant, for example, if specificity to a protein is maintained, a library can be created to select antibodies with appropriate specificity by varying the other arm. Bispecific whole antibodies may also be produced by alternative methods, as described by Ridgeway et al. (Protein Eng., 9:616-621, 1996) and Krah et al. (N Biotechnol. 39:167-173, 2017), the whole of which is incorporated herein by reference.

[0048] A heteroconjugate antibody consists of two covalently bound monoclonal antibodies with different specificities. See, for example, U.S. Patent No. 6,010,902, which is incorporated herein by reference in its entirety.

[0049] The portion of the antibody molecule's Fv fragment that binds to the antigen's epitope with high specificity is referred to herein as the "paratope." The paratope consists of amino acid residues that come into contact with the antigen's epitope to promote antigen recognition. Each of the two Fv fragments of the antibody has two variable domains V in a dimerized configuration. H and V LIt is composed of. The primary structure of each variable domain is separated by a framework region (FR) and contains three adjacent hypervariable loops. The hypervariable loops are regions with the greatest variability in primary sequence among antibody molecules from any mammal. The term hypervariable loop may sometimes be used interchangeably with the term "complementary determining region (CDR)". The length of the hypervariable loop (or CDR) varies depending on the antibody molecule. The framework regions of all antibody molecules from a given mammal have a high degree of primary sequence similarity / consensus. The consensus of the framework region can be used by those skilled in the art to identify both the framework region and the hypervariable loops (or CDRs) scattered between the framework regions. The hypervariable loops are given identifying names that distinguish their positions within the polypeptide and the domains in which they occur. V L The CDRs in the V L domain are identified as L1, L2, and L3, with L1 being at the most distal end and L3 being closest to the C L domain. The CDRs may also be named CDR-1, CDR-2, and CDR-3. L3 (CDR-3) is generally the most variable region among all antibody molecules produced by a given organism. The CDRs are regions of the polypeptide chain that are linearly arranged in the primary structure and separated from each other by the framework regions. V L The amino-terminal (N-terminal) end of the chain is named FR1. The region identified as FR2 is located between the L1 and L2 hypervariable loops. FR3 is located between the L2 and L3 hypervariable loops, and the FR4 region is closest to the C H domain. This structure and nomenclature are repeated for the three CDRs identified as H1, H2, and H3 in the V H chain. Most of the amino acid residues in the variable domain, or Fv fragment (V L ), are part of the framework region (approximately 85%). The three-dimensional or tertiary structure of the antibody molecule is such that the framework region is more internal to the molecule and the CDRs are on the outer surface of the molecule, providing most of the structure.

[0050] Several methods have been developed to identify the precise amino acids that make up each of these regions, and these can be used by those skilled in the art. This is done using one of several sequence alignment methods and algorithms that identify conserved amino acid residues that make up framework regions, and therefore, CDRs located between framework regions, although they may differ in length, can be identified. Three commonly used methods have been developed for identifying the CDR of antibodies: Kabat (as described in TT Wu and EA Kabat, "AN 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, vol. 132, no. 2, pp. 211-250, Aug. 1970); Chothia (as described in C. Chothia et al., "Conformations of immunoglobulin hypervariable regions", Nature, vol. 342, no. 6252, pp. 877-883, Dec. 1989); and IMGT (as described in M.-P. Lefranc et al., "IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily (As described in "V-like domains," Developmental & Comparative Immunology, vol. 27, no. 1, pp. 55-77, Jan. 2003). Each of these methods involves a unique numbering system for identifying the amino acid residues that constitute the variable region. In most antibody molecules, the amino acid residues that actually contact the antigen epitope are located in the CDR, but in some cases, residues within the framework region contribute to antigen binding.

[0051] Those skilled in the art can use one of several methods to determine the paratope of an antibody. These methods include: 1) Prediction of the tertiary structure of antibody / epitope binding interactions by calculation based on the chemical properties of the amino acid sequence of the antibody variable region and the composition of the epitope. 2) Hydrogen-deuterium exchange and mass spectrometry 3) A polypeptide fragmentation and peptide mapping approach that generates multiple overlapping peptide fragments from the full length of a polypeptide and evaluates the binding affinity of these peptides to epitopes. 4) Antibody phage display library analysis in which mammalian antibody Fab fragment coding genes are expressed by bacteriophages so as to be incorporated into the phage coat. This population of Fab-expressing phages is then made to interact with antigens that are either immobilized or can be expressed by different exogenous expression systems. Unbound Fab fragments are washed away, thereby leaving only specifically bound Fab fragments attached to the antigen. Binding Fab fragments can be easily isolated, and the encoding gene can be determined. This approach can, if necessary, be used to identify Fv fragments or specific V fragments. H and V L It can also be used for even smaller regions of Fab fragments that include domains.

[0052] In certain aspects, affinity-mature antibodies are enhanced by one or more modifications in one or more CDRs, resulting in improved affinity of the antibody to the target antigen compared to parental antibodies that do not possess those modifications. Certain affinity-mature antibodies have nanomolar or picomolar affinity for the target antigen. Affinity-mature antibodies are produced by procedures known in the art, for example, Marks et al., Bio / Technology 10:779 (1992) describes affinity maturation by VH and VL domain shuffling, and random mutagenesis of CDRs and / or framework residues used in phage display is described by Rajpal et al., PNAS. 24: 8466-8471 (2005) and Thie et al., Methods Mol Biol. 525:309-22 (2009), in combination with computational methods demonstrated in Tiller et al., Front. Immunol. 8:986 (2017).

[0053] Chimeric immunoglobulins are the product of fusion genes derived from different species; "humanized" chimeras typically have a framework region (FR) from human immunoglobulins, with one or more CDRs from non-human sources.

[0054] In certain aspects, portions of the heavy and / or light chain are identical or homologous to corresponding sequences from another specific species or belonging to a specific antibody class or subclass, while the remainder of the chain is identical or homologous to corresponding sequences in fragments of antibodies originating from another species or belonging to another antibody class or subclass, and exhibiting the desired biological activity. See U.S. Patent No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA 81:6851 (1984). For methods relating to chimeric antibodies, see, for example, U.S. Patent No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA 81:6851-6855 (1985), which are respectively incorporated herein by reference in their entirety. CDR grafting is described, for example, in U.S. Patents 6,180,370, 5,693,762, 5,693,761, 5,585,089, and 5,530,101, all of which are incorporated herein by reference for all purposes.

[0055] In some embodiments, minimizing antibody polypeptide sequences from non-human species optimizes chimeric antibody function and reduces immunogenicity. Specific amino acid residues from the non-antigen recognition region of a non-human antibody are modified to be homologous to corresponding residues in a human antibody or isotype. One example is a "CDR-grafted" antibody, which contains one or more CDRs from a specific species or belonging to a specific antibody class or subclass, but the remainder of the antibody chain is identical or homologous to corresponding sequences in an antibody from a different species or belonging to a different antibody class or subclass. For use in humans, the V region, consisting of both CDR1, CDR2, and partial CDR3 from the light and heavy chain variable regions of a non-human immunoglobulin, is grafted onto the human antibody framework region, replacing the innate antigen receptor of the human antibody with a non-human CDR. In some cases, the corresponding non-human residues are replaced with framework region residues from the human immunoglobulin. Furthermore, humanized antibodies may contain residues not found in the recipient or donor antibody to further refine their performance. Humanized antibodies may also contain at least a portion of the constant region (Fc) of an immunoglobulin, typically that of a human immunoglobulin. See, for example, Jones et al., Nature 321:522 (1986); Riechmann et al., Nature 332:323 (1988); Presta, Curr. Op. Struct. Biol. 2:593 (1992); Vaswani and Hamilton, Ann. Allergy, Asthma and Immunol. 1:105 (1998); Harris, Biochem. Soc. Transactions 23; 1035 (1995); Hurle and Gross, Curr. Op. Biotech. 5:428 (1994); Verhoeyen et al., Science 239:1534-36 (1988).

[0056] Intrabodies are immunoglobulins that are localized within cells and bind to intracellular antigens, in contrast to secretory antibodies that bind to antigens in the extracellular space.

[0057] Polyclonal antibody preparations typically contain different antibodies against different determinants (epitopes). To produce polyclonal antibodies, a host, such as a rabbit or goat, is immunized with an antigen or antigenic fragment, usually with an adjuvant and, if necessary, conjugated to a carrier. The antibodies against the antigen are then recovered from the host's serum. Polyclonal antibodies can be affinity-purified against the antigen to achieve monospecificity.

[0058] A monoclonal antibody, or "mAb," is an antibody obtained from a homogeneous population of antibodies from a single parent cell, which is, for example, identical except for small amounts of native variants. Each monoclonal antibody is effective against a single antigenic determinant.

[0059] 1. Functional antibody fragments and antigen-binding fragments a. Antigen-binding fragment Certain aspects relate to antibody fragments, such as antibody fragments that bind to and / or neutralize inflammatory mediators. The term functional antibody fragment includes antigen-binding fragments of antibodies that retain the ability to specifically bind to an antigen. These fragments consist of various arrangements of variable region heavy chain (VH) and / or light chain (VL); in some embodiments, they include constant region heavy chain 1 (CH1) and light chain (CL); in some embodiments, they lack an Fc region consisting of heavy chain 2 (CH2) and 3 (CH3) domains. The antigen-binding fragments and their modifications may include: (i) Fab fragment types consisting of VL, VH, CL, and CHl domains; (ii) Fd fragment types consisting of VH and CHl domains; (iii) Fv fragment types consisting of VH and VL domains; (iv) single-domain fragment types dAb consisting of a single VH or VL domain (Ward, 1989; McCafferty et al., 1990; Holt et al., 2003); (v) isolated complementarity-determining regions (CDRs). Such terminology is described, for example, in Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, NY (1989); Molec. Biology and Biotechnology: A Comprehensive Desk Reference (Myers, RA (ed.), New York: VCH Publisher, Inc.); Huston et al., Cell Biophysics, 22:189-224 (1993); Pluckthun and Skerra, Meth. Enzymol., 178:497-515 (1989) and Day, ED, Advanced Immunochemistry, 2d ed., Wiley-Liss, Inc. New York, NY (1990); Antibodies, 4:259-277 (2015). All citations in this paragraph are incorporated by reference.

[0060] Antigen-binding fragments also include antibody fragments that precisely contain at least one, two, or three complementarity-determining regions (CDRs) from the light chain variable region. Fusion of CDR-containing sequences to the Fc region (or its CH2 or CH3 region) is included within this definition, for example, including scFv fused directly or indirectly to the Fc region as defined herein.

[0061] The term Fab fragment refers to a monovalent antigen-binding fragment of an antibody containing the VL, VH, CL, and CH1 domains. The term Fab' fragment refers to a monovalent antigen-binding fragment of a monoclonal antibody that is larger than a Fab fragment. For example, a Fab' fragment contains all or part of the VL, VH, CL, and CH1 domains as well as the hinge region. The term F(ab')2 fragment refers to a bivalent antigen-binding fragment of a monoclonal antibody containing two Fab' fragments linked by a disulfide crosslink at the hinge region. An F(ab')2 fragment may, for example, contain all or part of two VH and VL domains and further contain all or part of two CL and CH1 domains.

[0062] The term Fd fragment refers to a fragment of the heavy chain of a monoclonal antibody containing all or part of the VH, including the CDR. The Fd fragment may further contain the CH1 region sequence.

[0063] The term Fv fragment refers to a monovalent antigen-binding fragment of a monoclonal antibody that includes all or part of the VL and VH regions and lacks the CL and CH1 domains. The VL and VH regions include, for example, the CDR. A single-chain antibody (sFv or scFv) is an Fv molecule that forms a single polypeptide chain, with the VL and VH regions linked by a mobile linker to form the antigen-binding fragment. Single-chain antibodies are discussed in detail in International Patent Application Publication No. WO 88 / 01649 and U.S. Patents 4,946,778 and 5,260,203, their disclosures of which are incorporated herein by reference. The term (scFv)2 refers to a divalent or bispecific sFv polypeptide chain containing an oligomeric domain at the C-terminus, separated from sFv by a hinge region (Pack et al. 1992). The oligomerized domain contains a self-associating α-helix, such as a leucine zipper, which can be further stabilized by additional disulfide bonds. The (scFv)2 fragment is also known as a "mini-antibody" or "mini-body."

[0064] A single-domain antibody is an antigen-binding fragment containing only a VH or VL domain. In some cases, two or more VH regions are covalently linked by a peptide linker to create a bivalent-domain antibody. The two VH regions of a bivalent-domain antibody can target the same or different antigens.

[0065] b. Fragment crystallizable region Fc The Fc region comprises two heavy chain fragments containing the CH2 and CH3 domains of the antibody. The two heavy chain fragments are held together by two or more disulfide bonds and by hydrophobic interactions of the CH3 domain. As used herein, the term “Fc polypeptide” includes the native and mutaine forms of polypeptides derived from the Fc region of the antibody. This also includes cleavage forms of such polypeptides that contain a hinge region that facilitates dimerization.

[0066] 2. Antibody CDRs and polypeptides having scaffold domains that present CDRs. Antigen-binding peptide scaffolds, such as complementarity-determining regions (CDRs), are used to create protein-binding molecules according to their properties. Generally, those skilled in the art can determine the type of protein scaffold to which at least one CDR is grafted. It is known that scaffolds must, optimally, meet several criteria, including good phylogenetic conservation; known three-dimensional structure; small size; little to no post-transcriptional modification; and ease of production, expression, and purification. Skerra, J Mol Recognit, 13:167-87 (2000).

[0067] Protein scaffolds can be supplied from, but are not limited to, proteins having repeating motifs such as the fibronectin type III FN3 domain (known as the "monobody"), fibronectin type III domain 10, lipocalin, anticalin, the Z domain of Staphylococcus aureus protein A, thioredoxin A, or "ankyrin repeat," "armadillo repeat," "leucine-rich repeat," and "tetratricopeptide repeat." Such proteins are described in U.S. Patent Publication Nos. 2010 / 0285564, 2006 / 0058510, 2006 / 0088908, 2005 / 0106660, and PCT Publication No. WO2006 / 056464, which are respectively incorporated herein by reference in their entirety. Scaffolds derived from toxins from scorpions, insects, plants, mollusks, and neuronal nitric oxide synthase protein inhibitors (PINs) can also be used.

[0068] B. Cytokines Cytokines are a group of proteins released by cells when excited (only a small amount of cytokines are expressed on the cell membrane). Cytokines produced by cells can affect nearby target cells or, at very low concentrations, through the bloodstream. They have a wide range of functions in promoting the growth, differentiation, and activation of target cells. Many cytokines target immune cells and can play a role in the immune response. Based on structural and functional differences, cytokines can be broadly classified into chemokines, interleukins, growth factors, transforming growth factors, colony-stimulating factors, tumor necrosis factors, and interferons.

[0069] The following cytokines may be used as anti-inflammatory agents in the methods and compositions of this disclosure. Exemplary sequences are provided below, but equivalent or homologous proteins known in the art may also be used.

[0070] Human IL-1ra: TIFF0007870524000001.tif19159

[0071] Mouse IL-1ra: TIFF0007870524000002.tif19160

[0072] Human IL-4: TIFF0007870524000003.tif19159

[0073] Mouse IL-4: TIFF0007870524000004.tif19158

[0074] Human IL-5: TIFF0007870524000005.tif20160

[0075] Mouse IL-5: TIFF0007870524000006.tif19158

[0076] Human IL-10: TIFF0007870524000007.tif19159

[0077] Mouse IL-10: TIFF0007870524000008.tif12159

[0078] Human IL-11: TIFF0007870524000009.tif26159

[0079] Mouse IL-11: TIFF0007870524000010.tif26159

[0080] Human IL-23; p19 subunit: TIFF0007870524000011.tif26160p40 subunit: TIFF0007870524000012.tif41159

[0081] Human IL-35; p35 subunit: TIFF0007870524000013.tif26159Ebi3 subunit: TIFF0007870524000014.tif26159

[0082] Mouse IL-35; p35 subunit: TIFF0007870524000015.tif26159Ebi3 subunit: TIFF0007870524000016.tif26159

[0083] Human IL-36ra: TIFF0007870524000017.tif19159

[0084] Mouse IL-36ra: TIFF0007870524000018.tif19160

[0085] Human IL-37: TIFF0007870524000019.tif26159

[0086] Mouse IL-37: TIFF0007870524000020.tif27159

[0087] Human interferon-beta: TIFF0007870524000021.tif26160

[0088] Mouse interferon-beta: TIFF0007870524000022.tif26160

[0089] Human TGF-β1: TIFF0007870524000023.tif19157

[0090] Mouse TGF-β1: TIFF0007870524000024.tif19157

[0091] Human TNF receptor I: TIFF0007870524000025.tif26160

[0092] Human TNF receptor II: TIFF0007870524000026.tif55158

[0093] Mouse TNF receptor II: TIFF0007870524000027.tif34159

[0094] C. CD200 Aspects of this disclosure relate to polypeptides and compositions comprising the anti-inflammatory agent CD200. An exemplary CD200 polypeptide amino acid sequence is shown below.

[0095] The extracellular domain of mouse CD200 is represented by the following sequence: TIFF0007870524000028.tif26159

[0096] Mouse CD200-MSA fusion protein (linker is underlined): TIFF0007870524000029.tif107159

[0097] The extracellular domain of human CD200 (UniProt identification number P41217) is represented by the following sequence: TIFF0007870524000030.tif26160

[0098] An example human CD200 (lowercase)-human serum albumin (uppercase) fusion protein (linker is uppercase and underlined) is represented as follows: TIFF0007870524000031.tif99160

[0099] The mouse CD200 (lowercase)-CBD fusion protein (uppercase) (linker is uppercase and underlined) is represented as follows: TIFF0007870524000032.tif41159

[0100] The human CD200 (lowercase)-CBD fusion protein (uppercase) (linker is uppercase and underlined) is represented as follows: TIFF0007870524000033.tif49160

[0101] The mouse CD200 (uppercase)-mouse serum albumin (lowercase)-CBD (italic underlined uppercase) fusion protein is represented as follows (linker is uppercase and underlined): TIFF0007870524000034.tif99160

[0102] The human CD200 (uppercase)-human serum albumin (lowercase)-CBD (italic underlined uppercase) fusion protein is represented as follows (linker is uppercase and underlined): TIFF0007870524000035.tif100160

[0103] IV. ECM affinity peptides Collagen is an extracellular matrix (ECM) protein that regulates various cell biological functions, such as proliferation, differentiation, and adhesion, in both normal and tumor tissues (Ricard-Blum, Cold Spring Harb Perspect Biol 3:a004978, 2011). Collagen is the most abundant protein in mammals and is present in almost all tissues as one or more of its 28 isoforms (Ricard-Blum, Cold Spring Harb Perspect Biol 3:a004978, 2011). The subendothelial space of blood vessels is rich in collagen. Due to its insolubility under physiological conditions, collagen is hardly present in the blood (Dubois et al., Blood 107:3902-06, 2006; Bergmeier and Hynes, Cold Spring Harb Perspect Biol 4:a005132, 2012). Tumor vascular structures have been reported to be permeable due to their abnormal structure (Nagy et al., British journal of cancer 100:865, 2009). Therefore, collagen is exposed within the tumor due to its leaky vascular structure (Liang et al., Journal of controlled release 209:101-109, 2015; Liang et al., Sci Rep 6:18205, 2016; Yasunaga et al., Bioconjugate Chemistry 22:1776-83, 2011; Xu et al. The Journal of cell biology 154:1069-80, 2001; Swartz and Lund, Nat Rev Cancer 12:210-19). Furthermore, tumor tissue contains increased amounts of collagen compared to normal tissue (Zhou et al. J Cancer 8:1466-76, 2017; Provenzano et al. BMC Med 6:11, 2008).

[0104] Von Willebrand factor (vWF) is a blood coagulation factor that binds to both type I and type III collagen, as well as the adhesion receptor GPIb on platelets (Lenting et al., Journal of thrombosis and haemostasis: JTH 10:2428-37, 2012; Shahidi Advances in experimental medicine and biology 906:285-306, 2017). Upon injury, collagen beneath endothelial cells is exposed to the plasma, and vWF-collagen binding initiates the thrombus formation cascade (Shahidi Advances in experimental medicine and biology 906:285-306, 2017; Wu et al. Blood 99:3623-28, 2002). Among reported non-bacterial proteins / peptides, the vWF A domain has the highest affinity for collagen (Addi et al., Tissue Engineering Part B: Reviews, 2016). In particular, it has been reported that the A3 domain of vWF is a collagen-binding domain (CBD) within the A domain (Ribba et al. Thrombosis and Haemostasis 86:848-54, 2001). As described above, the inventors of this invention hypothesized that a fusion protein of vWF A3 CBD could enable targeted cytokine immunotherapy even in the case of systemic injection, due to the exposure of collagen by the leaky vascular structure of tumors.

[0105] In some embodiments, the ECM affinity peptide includes a collagen-binding domain derived from decorin. In some embodiments, the ECM affinity peptide includes a decorin peptide such as LRELHLNNNC (SEQ ID NO:1) derived from bovine or LRELHLDNNC (SEQ ID NO:2) derived from human.

[0106] In some embodiments, the ECM peptide has the following amino acid sequence: It contains a peptide fragment derived from starfish choline, represented by TIFF0007870524000036.tif48160.

[0107] In some embodiments, the ECM peptide comprises a peptide fragment derived from vWF. In some embodiments, the ECM peptide comprises vWF A1 or a fragment derived from a human sequence, residue numbers 1237-1458 (residue numbers 474-695 of mature VWF), and it is an amino acid sequence Represented by TIFF0007870524000037.tif34160.

[0108] In some embodiments, the ECM peptide has the following amino acid sequence: Includes all or part of vWF A3 represented by TIFF0007870524000038.tif55160.

[0109] In some embodiments, the ECM peptide has the following amino acid sequence: Includes all or part of vWF A3 represented by TIFF0007870524000039.tif26157.

[0110] In some embodiments, ECM affinity peptides are peptides derived from von Willebrand factor (vWF). The sequence of human vWF is as follows: Includes TIFF0007870524000040.tif201160TIFF0007870524000041.tif158159.

[0111] In some embodiments, the peptide is derived from the vWF A3 domain. The vWF A3 domain is derived from the human sequence, residue numbers 1670-1874 (residue numbers 907-1111 in mature vWF), and the following sequence: It has TIFF0007870524000042.tif26160.

[0112] In some aspects, ECM affinity peptides include peptides derived from PlGF-2. PlGF-2 has the following sequence: It has TIFF0007870524000043.tif26159.

[0113] Examples of PlGF-2 ECM affinity peptides are as follows: Includes TIFF0007870524000044.tif26160.

[0114] In some embodiments, the ECM affinity peptide is a peptide derived from CXCL-12γ. The sequence of CXCL-12γ is as follows: CXCL-12γ: The filename is TIFF0007870524000045.tif12159. Exemplary peptides include all or part of SEQ ID NO:12 and the following peptides: Includes TIFF0007870524000046.tif4128.

[0115] ECM affinity peptides may be peptides having 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% (or any range within which this can be derived) identity with the above-mentioned ECM or CBD peptides or fragments thereof.

[0116] Linker sequences may be present in anti-inflammatory peptide constructs. For example, at least, at most, or exactly 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59 Linkers having 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or more (or any range within which can be derived) amino acids can separate the antibody and peptide.

[0117] The ECM-affinity peptides of this disclosure may have affinity for one or more components of the extracellular matrix, such as fibronectin, collagen (type I collagen, type III collagen, and / or type IV collagen), tenascin C, fibrinogen, and fibrin. In certain aspects, the ECM-affinity peptides have affinity for collagen, and in other aspects, the ECM-affinity peptides do not bind to fibronectin.

[0118] In some embodiments, the ECM affinity peptides and / or anti-inflammatory agents of this disclosure are further ligated to serum proteins. Serum proteins include, for example, albumin, globulin, and fibrinogen. Globulins include alpha-1 globulin, alpha-2 globulin, beta-globulin, and gamma-globulin. Albumin may be mouse, human, bovine, or any other homologous albumin protein. In some embodiments, albumin is encoded by the ALB gene and has the following amino acid sequence: Contains human serum albumin as exemplified by TIFF0007870524000047.tif56159. In some embodiments, serum albumin has the following sequence: Contains a polypeptide having TIFF0007870524000048.tif77159.

[0119] In some embodiments, albumin is in the following sequence: Contains mouse albumin with the code TIFF0007870524000049.tif48160.

[0120] A related aspect is vWF A3 (uppercase) linked via mouse serum albumin (MSA) (lowercase is MSA) and a glycine serine peptide linker (uppercase and underlined in italics): Includes TIFF0007870524000050.tif77160.

[0121] Further related embodiments include vWF A3 (uppercase) linked via mouse serum albumin (MSA) (lowercase is MSA) and a glycine serine peptide linker (uppercase and underlined in italics). vWF A3 (uppercase) linked via human serum albumin (HSA) (lowercase is HSA) and a glycine serine peptide linker (uppercase and underlined in italics): TIFF0007870524000051.tif77160

[0122] V. Protein Compositions The polypeptides or polynucleotides of this disclosure, such as ECM affinity peptides, serum proteins, or cytokine polypeptides, contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 or more variant amino acids or nucleic acid substitutions, or SEQ ID NO: 1-66, at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 1 18, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 16 5, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212,213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 300, 400, 500, 550, 1000 or more, or any range of sequences that can be derived from them. The amino acids or nucleic acids may be at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% similar, identical, or homologous.

[0123] Polypeptides or polynucleotides of this disclosure, such as ECM affinity peptides, serum proteins, or cytokine polypeptides, are SEQ IDNO:1~66, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 14 6, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 2 It may contain 06, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 300, 400, 500, 550, 1000 or more consecutive amino acids or any range within which they can be derived.

[0124] In some embodiments, the polypeptides of this disclosure are SEQ ID NO: Amino acids 1 to 66 (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 8) 0, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145 ,146,147,148,149,150,151,152,153,154,155,156,157,158,159,160,161,162,163,164,165,166,167,168,169,170,171,172,173,174,175,176,177,178,179,180,181,182,183,184,185,186,187,188,189,190,191,192,193,194,195,196,197,198,199,200,201,202,203,204,205,2 06, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266,267、268、269、270、271、272、273、274、275、276、277、278、279、280、281、282、283、284、285、286、287、288、289、290、291、292、293、294、295、296、297、298、299、300、301、302、303、304、305、306、307、308、309、310、311、312、313、314、315、316、317、318、319、320、321、322、323、324、325、326、327、328、329、330、331、332、333、334、335、336、337、338、339、340、341、342、343、344、345、346、347、348、349、350、351、352、353、354、355、356、357、358、359、360、361、362、363、364、365、366、367、368、369、370、371、372、373、374、375、376、377、378、379、380、381、382、383、384、385、386、387、388、389、390、391、392、393、394、395、396、397、398、399、400、401、402、403、404、405、406、407、408、409、410、411、412、413、414、415、416、417、418、419、420、421、422、423、424、425、426、427、428、429、430、431、432、433、434、435、436、437、438、439、440、441、442、443、444、445、446、447、448、449、450、451、452、453、454、455、456、457、458、459、460、461、462、463、464、465、466、467、468、469、470、471、472、473、474、475、476、477、478、479、480、481、482、483、484、485、486、487、488、489、490、491、492、493、494、495、496、497、498、499、500、501、502、503、504、505、506、507、508、509、510、511、512、513、514、515、516、517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 537, 538, 539, 540, 541, 542, 543, 544, 545, 546, 547, 548, 549, 550, 551, 552, 553, 554, 555, 556, 557, 558, 559, 560, 561, 562, 563, 564, 565, 566, This may include 567, 568, 569, 570, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 596, 597, 598, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 612, 613, 614, or up to 615 (or any range that can be derived within that range).

[0125] In some embodiments, the polypeptides of this disclosure, such as ECM affinity peptides, serum proteins, or cytokine polypeptides, are at least, many, or exactly 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73 ,74,75,76,77,78,79,80,81,82,83,84,85,86,87,88,89,90,91,92,93,94,95,96,97,98,99,100,101,102,103,104,105,106,107,108,109,110,111,112,113,114,115,116,117,118,119,120,121,122,123,124,125,126,127,128,129,130,131,132,133,134,135,136,137,13 8, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196 ,197,198,199,200,201,202,203,204,205,206,207,208,209,210,211,212,213,214,215,216,217,218,219,220,221,222,223,224,225,226,227,228,229,230,231,232,233,234,235,236,237,238,239,240,241,242,243,244,245,246,247,248,249,250,251,252,253,254,255、256、257、258、259、260、261、262、263、264、265、266、267、268、269、270、271、272、273、274、275、276、277、278、279、280、281、282、283、284、285、286、287、288、289、290、291、292、293、294、295、296、297、298、299、300、301、302、303、304、305、306、307、308、309、310、311、312、313、314、315、316、317、318、319、320、321、322、323、324、325、326、327、328、329、330、331、332、333、334、335、336、337、338、339、340、341、342、343、344、345、346、347、348、349、350、351、352、353、354、355、356、357、358、359、360、361、362、363、364、365、366、367、368、369、370、371、372、373、374、375、376、377、378、379、380、381、382、383、384、385、386、387、388、389、390、391、392、393、394、395、396、397、398、399、400、401、402、403、404、405、406、407、408、409、410、411、412、413、414、415、416、417、418、419、420、421、422、423、424、425、426、427、428、429、430、431、432、433、434、435、436、437、438、439、440、441、442、443、444、445、446、447、448、449、450、451、452、453、454、455、456、457、458、459、460、461、462、463、464、465、466、467、468、469、470、471、472、473、474、475、476、477、478、479、480、481、482、483、484、485、486、487、488、489、490、491、492、493、494、495、496、497、498、499、500、501、502、503、504、505, 506, 507, 508, 509, 510, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 537, 538, 539, 540, 541, 542, 543, 544, 545, 546, 547, 548, 549, 550, 551, 552, 553, 554, 555, 556, 557, 558, 559, 560, 561, 562, 563, 564, 5 It may contain 65, 566, 567, 568, 569, 570, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 596, 597, 598, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 612, 613, 614, or 615 consecutive amino acids (or any range within which can be derived).

[0126] In some embodiments, polypeptides such as ECM affinity peptides, serum proteins, or cytokine polypeptides are at least, at most, or exactly 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% similar, identical, or homologous to any one of SEQ ID NO: 1-66. NO:1-66, at least, many, or exactly 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110 ,111,112,113,114,115,116,117,118,119,120,121,122,123,124,125,126,127,128,129,130,131,132,133,134,135,136,137,138,139,140,141,142,143,144,145,146,147,148,149,150,151,152,153,154,1 55, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199,200、201、202、203、204、205、206、207、208、209、210、211、212、213、214、215、216、217、218、219、220、221、222、223、224、225、226、227、228、229、230、231、232、233、234、235、236、237、238、239、240、241、242、243、244、245、246、247、248、249、250、251、252、253、254、255、256、257、258、259、260、261、262、263、264、265、266、267、268、269、270、271、272、273、274、275、276、277、278、279、280、281、282、283、284、285、286、287、288、289、290、291、292、293、294、295、296、297、298、299、300、301、302、303、304、305、306、307、308、309、310、311、312、313、314、315、316、317、318、319、320、321、322、323、324、325、326、327、328、329、330、331、332、333、334、335、336、337、338、339、340、341、342、343、344、345、346、347、348、349、350、351、352、353、354、355、356、357、358、359、360、361、362、363、364、365、366、367、368、369、370、371、372、373、374、375、376、377、378、379、380、381、382、383、384、385、386、387、388、389、390、391、392、393、394、395、396、397、398、399、400、401、402、403、404、405、406、407、408、409、410、411、412、413、414、415、416、417、418、419、420、421、422、423、424、425、426、427、428、429、430、431、432、433、434、435、436、437、438、439、440、441、442、443、444、445、446、447、448、449、450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493 ,494,495,496,497,498,499,500,501,502,503,504,505,506,507,508,509,510,511,512,513,514,515,516,517,518,519,520,521,522,523,524,525,526,527,528,529,530,531,532,533,534,535,536,537 ,538,539,540,541,542,543,544,545,546,547,548,549,550,551,552,553,554,555,556,557,558,559,560,561,562,563,564,565,566,567,568,569,570,571,572,573,574,575,576,577,578,579,580,58 It may contain 1, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 596, 597, 598, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 612, 613, 614, or 615 consecutive amino acids (or any range within which can be derived).

[0127] Polypeptides of the present disclosure, such as ECM affinity peptides, serum proteins, or cytokine polypeptides, may be at least, in many ways, or exactly 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% (or any range derivable therein) similar, identical, or homologous to one of SEQ ID NO: 1-66.

[0128] In some situations, position numbers 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 11 5, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 1 46, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176 ,177,178,179,180,181,182,183,184,185,186,187,188,189,190,191,192,193,194,195,196,197,198,199,200,201,202,203,204,205,206,20 7, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 2 38, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268,269、270、271、272、273、274、275、276、277、278、279、280、281、282、283、284、285、286、287、288、289、290、291、292、293、294、295、296、297、298、299、300、301、302、303、304、305、306、307、308、309、310、311、312、313、314、315、316、317、318、319、320、321、322、323、324、325、326、327、328、329、330、331、332、333、334、335、336、337、338、339、340、341、342、343、344、345、346、347、348、349、350、351、352、353、354、355、356、357、358、359、360、361、362、363、364、365、366、367、368、369、370、371、372、373、374、375、376、377、378、379、380、381、382、383、384、385、386、387、388、389、390、391、392、393、394、395、396、397、398、399、400、401、402、403、404、405、406、407、408、409、410、411、412、413、414、415、416、417、418、419、420、421、422、423、424、425、426、427、428、429、430、431、432、433、434、435、436、437、438、439、440、441、442、443、444、445、446、447、448、449、450、451、452、453、454、455、456、457、458、459、460、461、462、463、464、465、466、467、468、469、470、471、472、473、474、475、476、477、478、479、480、481、482、483、484、485、486、487、488、489、490、491、492、493、494、495、496、497、498、499、500、501、502、503、504、505、506、507、508、509、510、511、512、513、514、515、516、517、518、519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 537, 538, 539, 540, 541, 542, 543, 544, 545, 546, 547, 548, 549, 550, 551, 552, 553, 554, 555, 556, 557, 558, 559, 560, 561, 562, 563, 564, 565, 566, 567, 568, SEQ IDs starting with 569, 570, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 596, 597, 598, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 612, 613, 614, or 615 NO: Any of 1-66, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 ,50,51,52,53,54,55,56,57,58,59,60,61,62,63,64,65,66,67,68,69,70,71,72,73,74,75,76,77,78,79,80,81,82,83,84,85,86,87,88,89,90,91,92,93,94,95,96,97,98,9 9, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136 ,137,138,139,140,141,142,143,144,145,146,147,148,149,150,151,152,153,154,155,156,157,158,159,160,161,162,163,164,165,166,167,168,169,170,171,172,173,174、175、176、177、178、179、180、181、182、183、184、185、186、187、188、189、190、191、192、193、194、195、196、197、198、199、200、201、202、203、204、205、206、207、208、209、210、211、212、213、214、215、216、217、218、219、220、221、222、223、224、225、226、227、228、229、230、231、232、233、234、235、236、237、238、239、240、241、242、243、244、245、246、247、248、249、250、251、252、253、254、255、256、257、258、259、260、261、262、263、264、265、266、267、268、269、270、271、272、273、274、275、276、277、278、279、280、281、282、283、284、285、286、287、288、289、290、291、292、293、294、295、296、297、298、299、300、301、302、303、304、305、306、307、308、309、310、311、312、313、314、315、316、317、318、319、320、321、322、323、324、325、326、327、328、329、330、331、332、333、334、335、336、337、338、339、340、341、342、343、344、345、346、347、348、349、350、351、352、353、354、355、356、357、358、359、360、361、362、363、364、365、366、367、368、369、370、371、372、373、374、375、376、377、378、379、380、381、382、383、384、385、386、387、388、389、390、391、392、393、394、395、396、397、398、399、400、401、402、403、404、405、406、407、408、409、410、411、412、413、414、415、416、417、418、419、420、421、422、423、424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 47 4, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, 500, 501, 502, 503, 504, 505, 506, 507, 508, 509, 510, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524, 5 25, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 537, 538, 539, 540, 541, 542, 543, 544, 545, 546, 547, 548, 549, 550, 551, 552, 553, 554, 555, 556, 557, 558, 559, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569, 570, 571, 572, 573, 574, 575, There exist nucleic acid molecules or polypeptides containing 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 596, 597, 598, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 612, 613, 614, or 615 consecutive nucleotides or amino acids.

[0129] The polypeptides and nucleic acids of this disclosure are at least, at most, or exactly 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75 ,76,77,78,79,80,81,82,83,84,85,86,87,88,89,90,91,92,93,94,95,96,97,98,99,100,101,102,103,104,105,106,107,108,109,110,111,112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 1 44, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 1 75, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 20 6, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237 ,238,239,240,241,242,243,244,245,246,247,248,249,250,251,252,253,254,255,256,257,258,259,260,261,262,263,264,265,266,267,268,269、270、271、272、273、274、275、276、277、278、279、280、281、282、283、284、285、286、287、288、289、290、291、292、293、294、295、296、297、298、299、300、301、302、303、304、305、306、307、308、309、310、311、312、313、314、315、316、317、318、319、320、321、322、323、324、325、326、327、328、329、330、331、332、333、334、335、336、337、338、339、340、341、342、343、344、345、346、347、348、349、350、351、352、353、354、355、356、357、358、359、360、361、362、363、364、365、366、367、368、369、370、371、372、373、374、375、376、377、378、379、380、381、382、383、384、385、386、387、388、389、390、391、392、393、394、395、396、397、398、399、400、401、402、403、404、405、406、407、408、409、410、411、412、413、414、415、416、417、418、419、420、421、422、423、424、425、426、427、428、429、430、431、432、433、434、435、436、437、438、439、440、441、442、443、444、445、446、447、448、449、450、451、452、453、454、455、456、457、458、459、460、461、462、463、464、465、466、467、468、469、470、471、472、473、474、475、476、477、478、479、480、481、482、483、484、485、486、487、488、489、490、491、492、493、494、495、496、497、498、499、500、501、502、503、504、505、506、507、508、509、510、511、512、513、514、515、516、517、518、519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 537, 538, 539, 540, 541, 542, 543, 544, 545, 546, 547, 548, 549, 550, 551, 552, 553, 554, 555, 556, 557, 558, 559, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569, 570, 57 This may include 1, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 596, 597, 598, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 612, 613, 614, or 615 (or any range that can be derived from them).

[0130] Substitutions are made using one amino acid position number or nucleic acid position number from SEQ ID NO: 1 to 66, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 11 3, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 14 4, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 1 75, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 2 06, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267,268、269、270、271、272、273、274、275、276、277、278、279、280、281、282、283、284、285、286、287、288、289、290、291、292、293、294、295、296、297、298、299、300、301、302、303、304、305、306、307、308、309、310、311、312、313、314、315、316、317、318、319、320、321、322、323、324、325、326、327、328、329、330、331、332、333、334、335、336、337、338、339、340、341、342、343、344、345、346、347、348、349、350、351、352、353、354、355、356、357、358、359、360、361、362、363、364、365、366、367、368、369、370、371、372、373、374、375、376、377、378、379、380、381、382、383、384、385、386、387、388、389、390、391、392、393、394、395、396、397、398、399、400、401、402、403、404、405、406、407、408、409、410、411、412、413、414、415、416、417、418、419、420、421、422、423、424、425、426、427、428、429、430、431、432、433、434、435、436、437、438、439、440、441、442、443、444、445、446、447、448、449、450、451、452、453、454、455、456、457、458、459、460、461、462、463、464、465、466、467、468、469、470、471、472、473、474、475、476、477、478、479、480、481、482、483、484、485、486、487、488、489、490、491、492、493、494、495、496、497、498、499、500、501、502、503、504、505、506、507、508、509、510、511、512、513、514、515、516、517、518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 537, 538, 539, 540, 541, 542, 543, 544, 545, 546, 547, 548, 549, 550, 551, 552, 553, 554, 555, 556, 557, 558, 559, 560, 561, 562, 563, 564, 565, 566, 567, Possible substitutions include 568, 569, 570, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 596, 597, 598, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 612, 613, 614, or 615. One or more of these substitutions may be specifically excluded from the embodiment.

[0131] Peptides of the Disclosure, such as ECM-affinity peptides, serum proteins, or cytokine polypeptides, that have at least 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity with any one of SEQ ID NO: 1-66, or have at least 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity with any one of SEQ ID NO: 1-66. Polypeptide and proteins are amino acid position numbers 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 7 5, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 14 1, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, or 200Starting from (or any range within which can be derived), and with amino acid position numbers 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 5 9, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 1 15, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, Includes fragments or segments ending in 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, or 205 (or any range within which can be derived).

[0132] A substitution variant typically involves the exchange of one amino acid for another at one or more sites within a protein and may be designed to modify one or more properties of a polypeptide without losing or even losing other functions or characteristics. The substitution may be conservative, i.e., one amino acid may be replaced by an amino acid of similar shape and charge. Conservative substitutions are well-known in the art and include, for example, the substitution of alanine for serine, arginine for lysine, asparagine for glutamine or histidine, aspartic acid for glutamic acid, cysteine for serine, glutamine for asparagine, glutamic acid for aspartic acid, glycine for proline, histidine for asparagine or glutamine, isoleucine for leucine or valine, leucine for valine or isoleucine, lysine for arginine, methionine for leucine or isoleucine, phenylalanine for tyrosine, leucine or methionine, serine for threonine, threonine for serine, tryptophan for tyrosine, tyrosine for tryptophan or phenylalanine, and valine for isoleucine or leucine. Alternatively, the substitution may be non-conservative such that the function or activity of the polypeptide is affected. Non-conservative changes typically involve substituting one residue with a chemically different residue, such as using a polar or charged amino acid instead of a non-polar or uncharged amino acid and vice versa. One or more of these substitutions may be specifically excluded from an embodiment.

[0133] The protein may be recombinant or synthesized in vitro. Alternatively, a non-recombinant or recombinant protein may be isolated from bacteria. It is also contemplated that bacteria containing such variants can be practiced in compositions and methods. As a result, the protein may not need to be isolated.

[0134] As used herein, the term "functionally equivalent codons" refers to codons that encode the same amino acid, such as the six codons for arginine or serine, and similarly refers to codons that encode biologically equivalent amino acids.

[0135] Amino acid and nucleic acid sequences may each contain additional residues such as additional N-terminal or C-terminal amino acids, or 5' or 3' sequences, as long as they meet the above criteria including maintaining the biological protein activity involved in protein expression, and yet are still considered to be essentially as described in one of the sequences disclosed herein. The addition of terminal sequences applies particularly to nucleic acid sequences and can include various non-coding sequences adjacent to either the 5' or 3' portion of the coding region.

[0136] The following are considerations based on changing the amino acids of a protein to create equivalent or in some cases improved, second-generation molecules. For example, a particular amino acid can be used in place of another amino acid in the protein structure so as not to significantly lose its interaction binding ability. For example, structures such as enzyme catalytic domains or interaction components can have amino acids substituted to maintain such functions. Since the functional activity of a protein is defined by its interaction ability and properties, specific amino acid substitutions can be made in the protein sequence and in the underlying DNA coding sequence, and yet a protein with similar properties can be produced. Thus, the inventors contemplate that various changes can be made to the DNA sequence of a gene without significantly losing its biological utility or activity.

[0137] In other embodiments, the function of a polypeptide is intended to be modified by introducing one or more substitutions. For example, a particular amino acid can be used in place of other amino acids in a protein structure with the intention of modifying the interaction-binding ability of the interacting components. Structures such as protein interaction domains, nucleic acid interaction domains, and catalytic sites may have amino acids substituted to modify such functions. Since the interaction ability and properties of a protein determine its functional activity, specific amino acid substitutions can be made in the protein sequence and in its underlying DNA coding sequence, yet proteins with different properties can still be produced. In this way, the inventors intend that various changes can be made in the DNA sequence of a gene to significantly alter its biological utility or activity.

[0138] When making such changes, the hydrophobicity and hydrophilicity index of amino acids may be considered. The importance of the hydrophobicity and hydrophilicity index of amino acids in conferring interactive biological functions to proteins is generally understood in this art (Kyte and Doolittle, 1982). It is accepted that the relative hydrophobicity and hydrophilicity characteristics of amino acids contribute to the secondary structure of the resulting protein and further determine the interactions between the protein and other molecules, such as enzymes, substrates, receptors, DNA, antibodies, and antigens.

[0139] Similarly, it is understood in the art that substitutions of similar amino acids can be effectively made based on hydrophilicity. U.S. Patent No. 4,554,101, incorporated herein by reference, states that the maximum local mean hydrophilicity of a protein, governed by the hydrophilicity of its adjacent amino acids, correlates with the bioproperties of the protein. It is understood that one amino acid can be substituted with another amino acid having a similar hydrophilicity value, and yet a biologically and immunologically equivalent protein can still be produced.

[0140] As outlined above, amino acid substitutions are generally based on the relative similarities of amino acid side-chain substituents, such as hydrophobicity, hydrophilicity, charge, and size. Exemplary substitutions that take into account the various characteristics mentioned above are well known and include arginine and lysine; glutamic acid and aspartic acid; serine and threonine; glutamine and asparagine; as well as valine, leucine, and isoleucine.

[0141] In certain embodiments, all or part of the proteins described herein may also be synthesized in solution or on a solid support according to the prior art. Various automated synthesizers are commercially available and can be used according to known protocols. See, for example, Stewart and Young, (1984); Tarn et al., (1983); Merrifield, (1986); and Barany and Merrifield (1979), each of which is incorporated herein by reference. Alternatively, recombinant DNA techniques can be used, in which a nucleotide sequence encoding a peptide or polypeptide is inserted into an expression vector, which is then transformed or translocated into a suitable host cell and cultured under conditions suitable for expression.

[0142] In one embodiment, the method includes the use of gene transfer into cells, including microorganisms, for protein production and / or presentation. A gene for a protein of interest can be transferred into a suitable host cell, followed by cell culture under appropriate conditions. Nucleic acids encoding substantially all polypeptides can be used. The construction of recombinant expression vectors and the elements contained therein are discussed herein. Alternatively, the protein produced may be an endogenous protein normally synthesized by the cells used for protein production.

[0143] VI. Nucleic acids In certain embodiments, the disclosure relates to recombinant polynucleotides encoding proteins, polypeptides, and peptides of the present invention, such as ECM affinity peptides functionally linked to anti-inflammatory agents and / or other molecules. Therefore, certain embodiments relate to nucleotides encoding ECM affinity polypeptides or fragments thereof fused to ECM affinity polypeptides and / or anti-inflammatory agents or fragments thereof.

[0144] As used in this application, the term “polynucleotide” means a nucleic acid molecule that is either recombinant or isolated without a whole genome nucleic acid. Within the scope of the term “polynucleotide” are oligonucleotides (nucleic acids with a length of 100 residues or less), recombinant vectors, such as plasmids, cosmids, phages, and viruses. A polynucleotide contains a regulatory sequence that, in some respects, is substantially isolated from a native gene or protein-coding sequence. A polynucleotide may be single-stranded (coding or antisense) or double-stranded, and may be RNA, DNA (genomic, cDNA, or synthetic), its analogues, or a combination thereof. Further coding or non-coding sequences may or may not be present within a polynucleotide.

[0145] In this regard, the terms “gene,” “polynucleotide,” or “nucleic acid” are used to mean nucleic acids (including any sequences required for appropriate transcription, post-translational modification, or localization) that encode proteins, polypeptides, or peptides. As will be understood by those skilled in the art, these terms encompass genome sequences, expression cassettes, cDNA sequences, and smaller genetically engineered nucleic acid segments that express or can be adapted to express proteins, polypeptides, domains, peptides, fusion proteins, and variants. Nucleic acids encoding all or part of a polypeptide include all values ​​and ranges between 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230 of the polynucleotides encoding one or more amino acid sequences described or referenced herein. ,240,250,260,270,280,290,300,310,320,330,340,350,360,370,380,390,400,410,420,430,440,441,450,460,470,480,490,500,510,520,530,540,550,560,570,580,590,600,610,620,630,640,65 0, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, 1000, 1010, 1020, 1030, 1040, 1050, 106 It may include a sequence of nucleic acid sequences of nucleotides, nucleosides, or base pairs numbering 0, 1070, 1080, 1090, 1095, 1100, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 9000, 10000, or more (or any range within which can be derived).Furthermore, it is conceivable that a particular polypeptide may be encoded by nucleic acids that include variants encoding the same or substantially similar proteins, although they have slightly different nucleic acid sequences.

[0146] In certain embodiments, the present invention relates to isolated nucleic acid segments and recombinant vectors incorporating nucleic acid sequences encoding polypeptides or peptides of the present disclosure. The term “recombinant” can be used in combination with polynucleotide or polypeptide and generally refers to polypeptides or polynucleotides that are produced and / or manipulated in vitro, or polypeptides or polynucleotides that are replication products of such molecules.

[0147] In other embodiments, the present invention relates to isolated nucleic acid segments and recombinant vectors incorporating nucleic acid sequences encoding polypeptides or peptides of the present disclosure.

[0148] The nucleic acid segments used in this disclosure may be combined with other nucleic acid sequences, such as promoters, polyadenylation signals, additional restriction enzyme sites, multicloning sites, and other coding segments, and therefore their total length may vary considerably. Thus, nucleic acid fragments of almost any length can be used, and their total length is preferably limited by the ease of purification and their intended use in the recombinant nucleic acid protocol. In some cases, the nucleic acid sequence may encode a polypeptide sequence having further heterologous coding sequences, for example, to enable the purification, transport, secretion, or post-translational modification of the polypeptide, or to enable therapeutic utility such as targeting or efficacy. As discussed previously, tags or other heterologous polypeptides may be added to the sequence encoding the modified polypeptide, and "heterologous" means a polypeptide that is not the same as the modified polypeptide.

[0149] In certain embodiments, the Disclosure provides polynucleotide variants having substantial identity with the sequences disclosed herein; the variants, when compared to the polynucleotide sequences disclosed herein using the methods described herein (e.g., BLAST analysis with standard parameters), exhibit at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or higher sequence identity, including all values ​​and ranges between:

[0150] This disclosure similarly intends to use polynucleotides that are complementary to all of the polynucleotides described above.

[0151] A. Vector The polypeptides of this disclosure may be encoded by nucleic acid molecules contained in a vector. The term “vector” is used to mean a carrier nucleic acid molecule into which a heterogeneous nucleic acid sequence can be inserted for introduction into a cell into which it can be replicated and expressed. The nucleic acid sequence may be “heterogeneous,” meaning in this context that the nucleic acid sequence is heterogeneous to the cell into which the vector is introduced, or to the nucleic acid into which the nucleic acid sequence is incorporated, including sequences at a location within a host cell or nucleic acid that are homogeneous with sequences in the cell or nucleic acid, but not normally found. Vectors include DNA, RNA, plasmids, cosmids, viruses (bacteriophages, animal viruses, and plant viruses), and artificial chromosomes (e.g., YACs). Those skilled in the art will be able to assume that they have sufficient means to construct vectors through standard recombination techniques (e.g., Sambrook et al., 2001; Ausubel et al., 1996, both incorporated herein by reference). In addition to encoding the polypeptides of this disclosure, vectors may encode other polypeptide sequences, such as one or more other bacterial peptides, tags, or immunogenicity-enhancing peptides. Useful vectors encoding such fusion proteins include the pIN vector (Inouye et al., 1985), a vector encoding a sequence of histidines, and the pGEX vector for use in the creation of glutathione S-transferase (GST) soluble fusion proteins for subsequent purification and isolation or cleavage.

[0152] The term “expression vector” refers to a vector containing a nucleic acid sequence that codes for at least a portion of a gene product that can be transcribed. In some cases, it then translates the RNA molecule into a protein, polypeptide, or peptide. Expression vectors may contain various “regulatory sequences,” which are nucleic acid sequences necessary for the transcription and possibly translation of the functionally linked coding sequence in a particular host organism. In addition to regulatory sequences that govern transcription and translation, vectors and expression vectors may also contain nucleic acid sequences that perform other functions and are described herein.

[0153] B. Promoters and Enhancers A "promoter" is a control sequence. A promoter is typically a region of a nucleic acid sequence where the initiation and rate of transcription are controlled. This may include genetic elements to which regulatory proteins and molecules, such as RNA polymerase and other transcription factors, can bind. The phrases "functionally disposed," "functionally linked," "under control," and "under transcriptional control" mean that the promoter is in the correct functional position and / or orientation with respect to a nucleic acid sequence to control the initiation and expression of that sequence. A promoter may or may not be used together with an "enhancer," which refers to a cis-acting regulatory sequence involved in the transcriptional activation of a nucleic acid sequence.

[0154] Of course, it may be important to use a promoter and / or enhancer that efficiently directs the expression of a DNA segment in the cell type or organism selected for expression. Those skilled in the art of molecular biology are generally aware of using combinations of promoters, enhancers, and cell types to express proteins (see Sambrook et al., 2001, which is incorporated herein by reference). The promoter used may be constitutive, tissue-specific, or inducible, and in certain embodiments, can direct high-level expression of an introduced DNA segment under specific conditions, such as the large-scale production of a recombinant protein or peptide.

[0155] The specific promoter used to control the expression of a polynucleotide encoding a peptide or protein of the present invention is not considered to be critically important as long as the polynucleotide can be expressed in the target cell, preferably a bacterial cell. When human cells are targeted, it is preferred to position the polynucleotide coding region in the vicinity of and under the control of a promoter that can be expressed in human cells. Generally speaking, such a promoter can include any of a bacterial promoter, a human promoter, or a viral promoter.

[0156] C. Initiation signal and internal ribosome binding site (IRES) Specific start signals may also be required for efficient translation of coding sequences. These signals include ATG start codons or adjacent sequences. It may be necessary to provide exogenous translational control signals, including ATG start codons. Those skilled in the art will readily be able to determine this and provide the required signals.

[0157] In certain embodiments of the present invention, internal ribosome entry site (IRES) elements are used to construct multi-gene or polycistronic signaling codes. IRES elements can bypass the ribosome scanning model of 5'-methylated cap-dependent translation and initiate translation at an internal site (Pelletier and Sonenberg, 1988; Macejak and Sarnow, 1991). IRES elements can be ligated to heterologous read frames. Multiple read frames, each separated by IRESs, can be transcribed together to construct a polycistronic signaling code. Multiple genes can be efficiently expressed and a single signaling code transcribed using a single promoter / enhancer (see U.S. Patents 5,925,565 and 5,935,819, incorporated herein by reference).

[0158] D. Selectable and screenable markers In certain embodiments of the present invention, cells containing the nucleic acid constructs of this disclosure can be identified in vitro or in vivo by encoding a screenable or selectable marker in the expression vector. When transcribed and translated, the marker confers a identifiable change to the cell, enabling the easy identification of the cell containing the expression vector. Generally, a selectable marker confers a property that enables selection. A positive selectable marker is one whose presence enables selection, while a negative selectable marker is one whose presence hinders selection. An example of a positive selectable marker is a drug resistance marker.

[0159] E. host cell As used herein, the terms “cell,” “cell line,” and “cell culture” are interchangeable. All of these terms include any and all of its offspring in subsequent generations. It is understood that not all offspring may be identical due to intentional or accidental mutations. In the context of expressing heterologous nucleic acid sequences, “host cell” means prokaryotic or eukaryotic cell, including any transformable organism capable of replicating a vector or expressing heterologous genes encoded by a vector. Host cells may and may be used as recipients of vectors or viruses. Host cells may be “transplanted” or “transformed,” which refers to the process by which exogenous nucleic acids, such as sequences encoding recombinant proteins, are transferred to or introduced into host cells. Transformed cells include primary test cells and their offspring.

[0160] Host cells can be derived from prokaryotes or eukaryotes, including bacteria, yeast cells, insect cells, and mammalian cells, for vector replication or expression of part or all of the nucleic acid sequence. Numerous cell lines and cultures are available as host cells and can be obtained from the American Type Culture Collection (ATCC) (www.atcc.org), an organization that serves as an archive of living cultures and genetic material.

[0161] F. Expression System Numerous expression systems exist that contain at least some or all of the compositions discussed above. Systems based on prokaryotes and / or eukaryotes can be used in the present invention to produce nucleic acid sequences, or their covalent polypeptides, proteins, and peptides. Many such systems are commercially available and widely usable.

[0162] Insect cell / baculovirus systems can result in high levels of protein expression of heterologous nucleic acid segments, as described in U.S. Patents No. 5,871,986 and No. 4,879,236, both incorporated herein by reference, and can be purchased, for example, from INVITROGEN® under the name MAXBAC® 2.0 and from CLONTECH® under the name BACPACK® BACULOVIRUS EXPRESSION SYSTEM.

[0163] In addition to the expression systems disclosed in the present invention, other examples of expression systems include the STRATAGENE® COMPLETE CONTROL® Inducible Mammalian Expression System, which includes a synthetic ecdysone-inducible receptor or its pET expression system, i.e., an E. coli expression system. Another example of an inducible expression system is available from INVITROGEN®, which has the T-REX® (tetracycline-regulated expression) system, i.e., an inducible mammalian expression system using a full-length CMV promoter. INVITROGEN® also offers a yeast expression system called the Pichia methanolica Expression System, designed for high-level production of recombinant proteins in the methylotrope yeast Pichia methanolica. Those skilled in the art will be familiar with methods for expressing vectors such as expression constructs, and for producing nucleic acid sequences or their covalent polypeptides, proteins, or peptides.

[0164] VII. Combination Therapy The compositions and related methods of this disclosure, in particular the administration of ECM affinity peptides functionally linked to anti-inflammatory agents and / or other molecules, may be used in combination with the administration of further therapies, such as the further therapies described herein, or in combination with other conventional therapies known in the art for the treatment of autoimmune or inflammatory conditions.

[0165] The therapeutic compositions and treatments disclosed herein may precede, occur concurrently with, and / or follow other treatments or drugs, with intervals ranging from several minutes to several weeks. In embodiments in which drugs are applied separately to cells, tissues, or organisms, it will generally be ensured that no significant time elapses between each delivery point is exceeded so that the therapeutic agents can still exert favorable synergistic effects on the cells, tissues, or organisms. For example, in such cases, it is intended that cells, tissues, or organisms may come into contact with two, three, four or more drugs or treatments substantially simultaneously (i.e., within less than about one minute). In other contexts, one or more therapeutic agents or procedures are administered and / or performed for 1 minute, 5 minutes, 10 minutes, 20 minutes, 30 minutes, 45 minutes, 60 minutes, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 22 hours, 23 hours, 24 hours, 25 hours, 26 hours, 27 hours, 28 hours, 29 hours, 30 hours, 31 hours, 32 hours, 33 hours, 34 hours before and / or after the administration of another therapeutic agent or procedure. It may be administered or provided within or for a period of 35 hours, 36 hours, 37 hours, 38 hours, 39 hours, 40 hours, 41 hours, 42 hours, 43 hours, 44 hours, 45 hours, 46 hours, 47 hours, 48 ​​hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks or 8 weeks or longer, and within any range within which it can be derived.

[0166] Various combination regimens of therapeutic agents and treatments may be available. A non-limiting example of such combinations is given below, where the therapeutic agent, for example, the composition disclosed herein, is "A," and a second agent, for example, a further agent or treatment described herein or known in the art, is "B." TIFF0007870524000052.tif13128

[0167] In some embodiments, two or more treatment processes may be used. It is intended that multiple processes may be implemented.

[0168] VIII. Treatment method The compositions of this disclosure may be used for in vivo, in vitro, or ex vivo administration. The routes of administration of the compositions may be, for example, intradermal, subcutaneous, intravenous, topical, local, and intraperitoneal.

[0169] Autoimmune or inflammatory conditions suitable for treatment include diabetes (e.g., type 1 diabetes), graft rejection, arthritis (rheumatoid arthritis such as acute arthritis, chronic rheumatoid arthritis, gout or gouty arthritis, acute gouty arthritis, acute immune arthritis, chronic inflammatory arthritis, osteoarthritis, type II collagen-induced arthritis, infectious arthritis, Lyme arthritis, proliferative arthritis, psoriatic arthritis, Still's disease, spondyloarthritis, and systemic juvenile-onset rheumatoid arthritis, osteoarthritis, chronic progressive arthritis, osteoarthritis, polyarthritis, reactive arthritis, and ankylosing spondylitis), and inflammatory hyperproliferative skin Diseases, including psoriasis such as psoriasis vulgaris, guttate psoriasis, pustular psoriasis, and nail psoriasis, atopic dermatitis including hay fever and Job's syndrome, dermatitis including contact dermatitis, chronic contact dermatitis, exfoliative dermatitis, allergic dermatitis, allergic contact dermatitis, herpetiform dermatitis, nummular eczema, seborrheic dermatitis, nonspecific dermatitis, primary irritant contact dermatitis, and atopic dermatitis, X-linked hyper-IgM syndrome, allergic intraocular inflammatory disease, urticaria including chronic allergic urticaria and chronic idiopathic urticaria, myositis, polymyositis / dermatomyositis, and Sclerosis such as dermatomyositis, toxic epidermal necrolysis, scleroderma (including systemic sclerosis), systemic sclerosis, multiple sclerosis (MS) such as spinal optical MS, primary progressive MS (PPMS), relapsing-remitting MS (RRMS), progressive systemic sclerosis, atherosclerosis, arteriosclerosis, disseminated sclerosis, ataxic sclerosis, neuromyelitis optica (NMO), inflammatory bowel disease (IBD) (e.g., Crohn's disease, autoimmune-mediated gastrointestinal disease, ulcerative colitis, ulcerative colitis, microscopic colitis, collagenous colitis, polycolitis, necrotising colitis, and autoimmune inflammatory bowel disease), intestinal Inflammation of the uveum, pyoderma gangrenosum, erythema nodosum, primary sclerosing cholangitis, respiratory distress syndromes including adult or acute respiratory distress syndrome (ARDS), meningitis, inflammation of all or part of the uveum, iritis, choroiditis, autoimmune blood disorders, rheumatoid arthritis, hereditary angioedema, cranial nerve injuries such as meningitis, herpes zoster of pregnancy, bullous pemphigoid of pregnancy, pruritus scrotalis, autoimmune premature ovarian failure, sudden hearing loss due to autoimmune conditions, IgE-mediated diseases such as anaphylaxis and allergic and atopic rhinitis, encephalitis such as Rasmussen's encephalitis and limbic and / or brainstem encephalitis,Uveitis such as anterior uveitis, acute anterior uveitis, granulomatous uveitis, non-granulomatous uveitis, lens antigenic uveitis, posterior uveitis, or autoimmune uveitis; glomerulonephritis (GN) with or without nephrotic syndrome, such as chronic or acute glomerulonephritis including primary GN; immune-mediated GN; membranous GN (membranous nephropathy); idiopathic membranous GN or idiopathic membranous nephropathy; membranous or membranous proliferative disorders including types I and II. Sexual GN (MPGN), and rapidly progressive GN, proliferative glomerulonephritis, autoimmune polyglandular endocrine deficiency, balanoposthitis, plasmacytic balanoposthitis, balanoposthitis including balanoposthitis, erythema annulare centrifugal, erythema pigmentosum fixed, erythema multiforme, granuloma annulare, lichen sclerosing, lichen simplex chronicus, lichen spinalus, lichen planus, ichthyosis laminae, exfoliative hyperkeratosis, precancerous keratosis, pyoderma gangrenosum, allergic conditions and reactions, allergic reactions, allergies This may include, but is not limited to, conditions such as eczema including atopic or dyshidrotic eczema, dyshidrotic eczema, dyshidrotic eczema, and bullous palmoplantar eczema; asthma such as bronchial asthma, bronchial asthma and autoimmune asthma; conditions with T cell infiltration and chronic inflammatory response; immune responses to exogenous antigens such as fetal ABO blood group during pregnancy; chronic inflammatory pneumonia; autoimmune myocarditis; leukocytosis; lupus nephritis; lupus encephalitis; lupus in childhood; nonrenal lupus; extrarenal lupus; discoid lupus and discoid lupus erythematosus; alopecia lupus; systemic lupus erythematosus (SLE) including cutaneous SLE or subacute cutaneous SLE; lupus including neonatal lupus erythematosus syndrome (NLE) and disseminated lupus erythematosus; juvenile-onset (Type 1) diabetes mellitus including pediatric insulin-dependent diabetes mellitus (IDDM); adult-onset diabetes mellitus (Type 2) and autoimmune diabetes mellitus. Cytokine and T lymphocyte-mediated acute and delayed hypersensitivity, sarcoidosis, granulomatous vasculitis including lymphomatous granulomatosis and Wegener's granulomatosis, agranulocytic vasculitis, vasculitis including large vessel vasculitis (including polymyalgia rheumatica and giant cell (Takayasu) arteritis), medium vessel vasculitis (including Kawasaki disease and polyarteritis nodosa), microscopic polyarteritis, immunovasculitis, central nervous system vasculitis, cutaneous vasculitis, hypersensitivity vasculitis, necrotizing vasculitis such as necrotizing vasculitis,and ANCA-associated vasculitis and ANCA-associated small vessel vasculitis such as Churg-Strauss vasculitis or syndrome (CSS), temporal arteritis, aplastic anemia, autoimmune aplastic anemia, Coombs-positive anemia, Diamond-Blackfan anemia, hemolytic anemia or immune hemolytic anemia including autoimmune hemolytic anemia (AIHA), Addison's disease, autoimmune neutropenia, pancytopenia, leukopenia, diseases with extravascular migration of leukocytes, CNS inflammatory diseases, Alzheimer's disease, Parkinson's disease, sepsis, multi-organ injury syndromes such as those secondary to trauma or bleeding, antigenic Body complex-mediated diseases, anti-glomerular basement membrane diseases, antiphospholipid antibody syndromes, allergic neuritis, Behçet's disease / syndrome, Castleman syndrome, Goodpasture syndrome, Raynaud's syndrome, Sjögren's syndrome, Stevens-Johnson syndrome, bullous pemphigoid and cutaneous pemphigoid, pemphigoids (including pemphigus vulgaris, pemphigus phyllodes, mucous membrane pemphigus, and erythematous pemphigus), autoimmune polyglandular autoimmune syndromes, Reiter's disease or syndrome, burns, pre-eclampsia, immune complex disorders such as immune complex nephritis, antibody-mediated nephritis, polyneuropathy, IgM polyneuropathy or chronic neurological disorders such as IgM-mediated neuropathy, autoimmune or immune thrombocytopenia such as idiopathic thrombocytopenic purpura (ITP) including chronic or acute ITP, scleritis such as idiopathic scleritis and episcleritis, autoimmune diseases of the testes and ovaries including autoimmune orchitis and oophoritis, primary hypothyroidism, hypoparathyroidism, autoimmune thyroiditis, thyroiditis such as Hashimoto's disease and chronic thyroiditis (Hashimoto's thyroiditis), or autoimmune endocrine disorders including subacute thyroiditis and autoimmune thyroiditis, idiopathic hypothyroidism, Graves' disease, autoimmune polyglandular syndrome (or Polyglandular syndromes such as polyglandular endocrine disorders, paraneoplastic syndromes including Lambert-Eaton myasthenic syndrome or Eaton-Lambert syndrome, stiff person syndrome or stiff person syndrome, encephalomyelitis such as allergic encephalomyelitis or allergic encephalomyelitis and experimental allergic encephalomyelitis (EAE), experimental autoimmune encephalomyelitis, myasthenia gravis such as thymoma-associated myasthenia gravis, cerebellar degeneration, neuromuscular asthenia, opsoclonus or opsoclonus myoclonus syndrome (OMS), and sensory neuropathy.Multifocal motor neuropathy, Sheehan's syndrome, autoimmune hepatitis, chronic hepatitis, lupoid hepatitis, giant cell hepatitis, chronic active hepatitis or autoimmune chronic active hepatitis, lymphocytic interstitial pneumonia (LIP), bronchiolitis obliterans (non-transplant) vs. NSIP, Guillain-Barré syndrome, Berger's disease (IgA nephropathy), idiopathic IgA nephropathy, linear IgA dermatosis, acute febrile neutrophilic dermatosis, subcorneal pustular dermatosis, transient acanthospermic dermatosis, cirrhosis such as primary biliary cirrhosis or pulmonary cirrhosis, autoimmune enteropathy syndrome, celiac disease or celiac disease, celiac sprue (gluten enteropathy), refractory sprue, idiopathic sprue, cryoglobulinemia, amyotrophic lateral sclerosis (ALS; Lou Gehrig's disease), coronary artery disease, autoimmune inner ear disease (AIED), and other autoimmune conditions. Immunotherapy-related ear diseases, autoimmune hearing loss, polychondritis such as refractory or relapsing or relapsing polychondritis, alveolar proteinosis, Cogan syndrome / non-syphilitic interstitial keratitis, Bell's palsy, Sweet's disease / syndrome, rosacea autoimmune, pain associated with herpes zoster, amyloidosis, noncancerous lymphocytosis, primary lymphocytosis including monoclonal B-cell lymphocytosis (e.g., benign monoclonal immunoglobulinemia and monoclonal immunoglobulinemia of unknown importance, MGUS), peripheral neuropathy, paraneoplastic syndromes , channelopathy such as epilepsy, migraine, arrhythmia, muscle disorders, hearing loss, blindness, periodic paralysis, and central nervous system channelopathy, autism, inflammatory myopathy, focal or segmental or focal segmental glomerulosclerosis (FSGS), endocrine eye disease, uveoretinitis, chorioretinitis, autoimmune liver disease, fibromyalgia, multiple endocrine insufficiency, Schmidt syndrome, adrenal nephritis, gastric atrophy, presenile dementia, demyelinating diseases such as autoimmune demyelinating diseases and chronic inflammatory demyelinating polyneuropathy, Dressler syndrome, alopecia areata, alopecia totalis CREST syndrome (calcification, Raynaud's phenomenon, esophageal motility disorder, finger sclerosis, and telangiectasia), autoimmune infertility in men and women, for example, due to anti-sperm antibodies, mixed connective tissue disease, Chagas disease, rheumatic fever, recurrent miscarriage, farmer's lung, erythema multiforme, postcardiotomy syndrome, Cushing's syndrome, avian lung, allergic granulomatous vasculitis, benign lymphocytic vasculitis, Alport syndrome, alveolitis such as allergic pneumonia and fibrous pneumonia, interstitial lung disease, transfusion reactions, leprosy, malaria, leishmaniasis,Parasitic diseases such as cypanosomiasis, schistosomiasis, and aspergillosis, aspergillosis, Sumpter syndrome, Kaplan syndrome, dengue fever, endocarditis, intramyocardial fibrosis, diffuse interstitial pulmonary fibrosis, interstitial pulmonary fibrosis, pulmonary fibrosis, idiopathic pulmonary fibrosis, cystic fibrosis, endophthalmitis, persistent erythema elevata, fetal erythroblastosis, eosinophilic fasciitis, Schulman syndrome, Felty syndrome, filariasis, chronic circulatory inflammation, metachronous circulatory inflammation, iridocyclitis (acute or chronic), or circulatory inflammation such as Hooke's circulatory inflammation, Henoch-Schönlein purpura, human immunodeficiency virus (HIV) infection, SCID, acquired immunodeficiency syndrome (AIDS), etc. Covirus infection, sepsis, endotoxemia, pancreatitis, thyrotoxicosis, parvovirus infection, rubella virus infection, post-vaccination syndrome, congenital rubella infection, Epstein-Barr virus infection, mumps, Evans syndrome, autoimmune gonadal insufficiency, Sydenham's chorea, post-streptococcal nephritis, thromboangiitis obliterans, thyrotoxicosis, tabes dorsalis, choroiditis, giant cell polymyalgia, chronic hypersensitivity pneumonitis, keratoconjunctivitis sicca, epidemic keratoconjunctivitis, idiopathic nephritis syndrome, minimal change nephropathy, benign familial and ischemia-reperfusion injury, transplant organ reperfusion, retinal autoimmunity, arthritis, bronchitis, chronic obstructive airway / lung disease, silicosis, stomatitis, Aphthous stomatitis, arteriosclerotic disease, spermatogenesis deficiency, autoimmune hemolysis, Beck's disease, cryoglobulinemia, Dupuytren's contracture, lens hypersensitive endophthalmitis, allergic enteritis, erythema nodosum, idiopathic facial paralysis, chronic fatigue syndrome, rheumatic fever, Hamann-Ricci disease, sensorineural hearing loss, hemoglobinuria attacks, hypogonadism, focal enteritis, leukopenia, infectious mononucleosis, transverse myelitis, primary idiopathic myxedema, nephrotic syndrome, sympathetic ophthalmitis, granulomatous orchitis, pancreatitis, polyradiculitis, pyoderma gangrenosum, Quervain's thyroiditis, acquired splenic atrophy, non-malignant thymoma, vitiligo, toxic shock syndrome Food poisoning, conditions involving T cell infiltration, leukocyte adhesion disorders, immune responses associated with cytokine and T lymphocyte-mediated acute and delayed hypersensitivity, diseases involving extravasation of leukocytes, multiple organ injury syndrome, antigen-antibody complex-mediated diseases, anti-glomerular basement membrane diseases, allergic neuritis, autoimmune polyendocrine syndrome, folliculitis, primary myxedema, autoimmune atrophic gastritis, sympathetic ophthalmitis, rheumatic diseases, mixed connective tissue disease, nephrotic syndrome, insular pancreatitis, polyglandular autoimmune syndrome, polyglandular autoimmune syndrome type 1, adult-onset idiopathic hypoparathyroidism (AOIH), dilated Cardiomyopathy such as type 1 cardiomyopathy, epidermolysis bullosa (EBA), hemochromatosis, myocarditis, nephrotic syndrome, primary sclerosing cholangitis, suppurative or non-suppurative sinusitis, acute or chronic sinusitis, ethmoid, frontal, maxillary, or sphenoid sinusitis, eosinophil-related disorders such as eosinophilia, pulmonary infiltrative eosinophilia, eosinophilia-myalgia syndrome, Loeffler's syndrome, chronic eosinophilic pneumonia, tropical pulmonary eosinophilia, bronchopneumonitis aspergillosis, aspergillosis, or granulomas containing eosinophils, anaphylaxis, seronegative spondyloarthritis, autoimmune polyendocrine syndrome, sclerosing bile duct Inflammation, sclera, episclera, chronic mucocutaneous candidiasis, Bruton syndrome, transient hypogammaglobulinemia in infancy, Wiscott-Aldrich syndrome, ataxia with telangiectasia, collagen disease, rheumatism, autoimmune diseases associated with neurological disorders, lymphadenitis, decreased blood pressure response, vascular dysfunction, tissue damage, cardiovascular ischemia, hyperalgesia, renal ischemia, cerebral ischemia, and diseases with angiogenesis, allergic hypersensitivity, glomerulonephritis, reperfusion injury, ischemic reperfusion injury, reperfusion injury of myocardium or other tissues, lymphomatous tracheobronchitis, inflammatory skin diseases, skin diseases with acute inflammatory components, multiple organ failure,Immune responses associated with bullous diseases, renal cortical necrosis, acute purulent meningitis or other central nervous system inflammatory diseases, inflammatory diseases of the eye and orbit, granulocyte transfusion-associated syndromes, cytokine-induced toxicity, narcolepsy, acute severe inflammation, chronic refractory inflammation, pyelonephritis, intra-arterial hyperplasia, peptic ulcers, valvular heart disease, graft-versus-host disease, contact hypersensitivity, asthmatic airway hyperresponsiveness, and endometriosis are also targeted.

[0170] IX. Pharmaceutical compositions and methods In some embodiments, pharmaceutical compositions are administered to a subject. Different aspects involve administering an effective amount of the composition to the subject. In some embodiments, compositions comprising anti-inflammatory agents may be administered to a subject or patient to treat inflammation and / or autoimmunity. Furthermore, such compounds may be administered in combination with further treatments.

[0171] The composition can be formulated for parenteral administration, for example, for injection via intravenous, transcatheter, intra-arterial, intramuscular, subcutaneous, or intraperitoneal routes. Typically, such compositions can be prepared as either liquid solutions or suspensions for injection; solid forms suitable for use in preparing solutions or suspensions by adding liquid before injection can also be prepared; and the preparations can be emulsified. The preparation of such formulations will be known to those skilled in the art in light of this disclosure. Other routes of administration include injection into tumors, peritumortic, lymphoid, inflammatory tissue, or lymph nodes. In some embodiments, the administration is systemic.

[0172] Other routes of administration are also considered. For example, the construct and the drug may be administered in association with a carrier. In some embodiments, the carrier is a nanoparticle or microparticle.

[0173] Particles can have a variable-size structure and are variously known as microspheres, microparticles, nanoparticles, nanospheres, or liposomes. Such particulate formulations can be formed by covalent or non-covalent bonding of a construct to the particle. In this specification, “particle,” “microparticle,” “bead,” “microsphere,” and their grammatical equivalents mean small, individual particles that can be administered to a target. In certain embodiments, particles are substantially spherical in shape. As used herein, the term “substantially spherical” means that the shape of the particle does not deviate from a sphere by more than about 10%. Particles typically consist of a substantially spherical core and optionally one or more layers. The core may vary in size and composition. In addition to the core, particles may have one or more layers to provide appropriate functionality for the application of interest. The thickness of the layers, if present, may vary depending on the needs of the particular application. For example, layers may provide useful optical properties.

[0174] Suitable pharmaceutical forms for injection include sterile aqueous solutions or dispersions; formulations containing sesame oil, peanut oil, or aqueous propylene glycol; and sterile powders for the immediate preparation of sterile injectable solutions or dispersants. In all cases, the form must be sterile and fluid enough to be easily injected. It should also be stable under manufacturing and storage conditions and protected from contamination by microorganisms, such as bacteria and fungi.

[0175] Furthermore, the carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), suitable mixtures thereof, and vegetable oils. Proper fluidity can be maintained, for example, by coating, e.g., the use of lecithin, by maintaining the required particle size in the case of dispersants, and by the use of surfactants. Inhibition of microbial activity can be achieved by various antimicrobial and antifungal agents, e.g., parabens, chlorobutanol, phenol, sorbic acid, thimerosal, etc. In many cases, it is preferable to include isotonic agents, e.g., sugars, sodium chloride. Long-term absorption of the injectable composition can be achieved by using absorption retarders in the composition, e.g., aluminum monostearate and gelatin.

[0176] Sterile injectable solutions are prepared by incorporating the required amount of active compound into a suitable solvent containing the various other components listed above as needed, and then sterilizing by filtration. Generally, dispersants are prepared by incorporating various sterilizing active ingredients into a sterilizing medium containing a base dispersion medium and other necessary components listed above. In the case of sterilizing powders for the preparation of sterilizing injectable solutions, preferred preparation methods are vacuum drying and freeze-drying, which yield powders of the active ingredient plus additional desired components from the previously sterilized and filtered solution.

[0177] As used herein, the term “pharmaceutically acceptable” means that a compound, substance, composition and / or dosage form is suitable for contact with human and animal tissues, within the bounds of sound medical judgment, without excessive toxicity, irritation, allergic response or other problematic complications, and balanced by a reasonable benefit-to-risk ratio. The term “pharmaceutically acceptable carrier” means a pharmaceutically acceptable substance, composition or medium, e.g., liquid or solid fillers, diluents, excipients, solvents or encapsulants, involved in the loading or transport of a chemical agent.

[0178] As used herein, “pharmaceutically acceptable salt” means a derivative of the compound of this disclosure obtained by modifying the parent compound by converting the present acidic or basic moiety to its salt form. Examples of pharmaceutically acceptable salts include, but are not limited to, salts of mineral or organic acids of basic residues such as amines; and alkali or organic salts of acidic residues such as carboxylic acids. Examples of pharmaceutically acceptable salts include conventional non-toxic salts or quaternary ammonium salts of the parent compound formed with non-toxic inorganic or organic acids. A pharmaceutically acceptable salt may be synthesized from a parent compound containing a basic or acidic moiety by conventional chemical methods.

[0179] Some variation in dosage is inevitable depending on the condition of the subject. The person administering the medication determines the appropriate dose for each individual subject in any given event. The effective amount of a therapeutic or prophylactic composition is determined based on the intended purpose. The term "unit dose" or "dosage" refers to a physically independent unit suitable for use in the subject, each unit containing a predetermined amount of the composition calculated to produce the desired response discussed above, i.e., with its administration, i.e., the appropriate route and regimen. The amount administered depends on the desired effect according to both the number of treatments and the unit dose. The exact amount of the composition also depends on the practitioner's judgment and is unique to each individual. Factors influencing the dosage include the subject's physical and clinical condition, the route of administration, the intended treatment purpose (symptom relief or cure), and the potency, stability, and toxicity of the specific composition.

[0180] Once formulated, the liquid formulation is administered in a manner suitable for the drug formulation, in a dose that is therapeutically or prophylactically effective. The formulation is readily administered in various dosage forms, for example, as an injectable liquid formulation of the type described above.

[0181] Typically, a human adult (weighing approximately 70 kilograms) is administered a compound in doses of approximately 0.1 mg to 3000 mg (including any value and range within that range), or approximately 5 mg to 1000 mg (including any value and range within that range), or approximately 10 mg to 100 mg (including any value and range within that range). It should be understood that such dosage ranges are merely examples, and that administration may be adjusted according to factors known to those skilled in the art.

[0182] In certain aspects, subjects are administered the following amounts, at least, or at most, the following amounts of the drugs discussed herein: 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3 , 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9 0.6, 9.7, 9.8, 9.9, 10.0, 10.5, 11.0, 11.5, 12.0, 12.5, 13.0, 13.5, 14.0, 14.5, 15.0, 15.5, 16.0, 16.5, 17.0, 17.5, 18.0, 18.5, 19.0, 19.5, 20.0, 1, 2, 3 ,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,4 7, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210,215, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265, 270, 275, 280, 285, 290, 295, 300, 305, 310, 315, 320, 325, 330, 335, 340, 345, 350, 355, 360, 365, 370, 375, 380, 385, 390, 395, 400, 410, 420, 425, 430, 440, 441, 450, 460, 470, 475, 480, 490, 500, 510, 520, 525, 530, 540, 550, 560, 570, 575, 580, 590, 600, 610, 620, 625, 630, 640, 650, 660, 670, 675, 680, 690, 700, 710, 720, 725, 730, 740, 750, 760, 770, 775, 780, 790, 800, 810, 820, 825, 830, 840, 850, 860, 870, 875, 8 80, 890, 900, 910, 920, 925, 930, 940, 950, 960, 970, 975, 980, 990, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100, 3200, 3300, 3400, 3500, 3600, 370 0, 3800, 3900, 4000, 4100, 4200, 4300, 4400, 4500, 4600, 4700, 4800, 4900, 5000, 6000, 7000, 8000, 9000, 10000 milligrams (mg) or micrograms (mcg) or μg / kg or micrograms / kg / min or mg / kg / min or micrograms / kg / hour or mg / kg / hour, or μM or mM. Any derivable range within these is intended.

[0183] Medication may be administered as needed, or every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 18 or 24 hours (or any range within which it can be derived) or 1, 2, 3, 4, 5, 6, 7, 8, 9 or more times per day (or any range within which it can be derived). Medication may be administered first before or after the onset of symptoms. In some embodiments, the first dose of the regimen is administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 hours after the patient develops or shows signs or symptoms of the illness (or 1, 2, 3, 4, or 5 days after the patient develops or shows signs or symptoms of the illness (or 1, 2, 3, 4, or 5 days after the patient develops or shows signs or symptoms of the illness). Patients may be treated for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 days or longer (or any range within that range) until the symptoms of the illness disappear or decrease, or 6, 12, 18, or 24 hours after the symptoms of infection disappear or decrease, or 1, 2, 3, 4, or 5 days after. [Examples]

[0184] X. Examples The following embodiments are included to demonstrate preferred embodiments of the Disclosure. Those skilled in the art will recognize that the methods disclosed in the following embodiments are methods that the inventors have found to function well in the implementation of the Disclosure, and therefore may be considered to constitute preferred embodiments for implementation. However, those skilled in the art will recognize that, in view of the Disclosure, many modifications can be made in the specific embodiments disclosed without departing from the spirit and scope of the Disclosure, but similar or comparable results can still be obtained.

[0185] Example 1: The efficacy of the anti-inflammatory agent was enhanced by modifying the collagen binding. A. Results 1. CBP conjugation resulted in increased collagen affinity for anti-TNFα antibodies (αTNF). Anti-TNFα antibody (αTNF) was mixed with sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-SMCC), and then CBP was covalently crosslinked to the antibody. Up to five CBP molecules were found to be bound to the antibody, as calculated by matrix-assisted laser desorption / ionization-time-of-flight (MALDI-TOF) mass spectrometry (Figure 1A). To investigate the ability of CBP-conjugated αTNF (CBP-αTNF) to bind to collagen, the binding activity of CBP-αTNF and unmodified αTNF (WT-αTNF) to type I, II, and III collagen was determined by ELISA. CBP-αTNF bound to all types of collagen tested, while the binding signal of WT-αTNF to collagen was undetectable (Figure 1B).

[0186] 2. CBP conjugation allowed us to localize αTNF to the inflammatory foot in an arthritis model. The localization of CBP-αTNF in the inflammatory paw of a collagen antibody-induced arthritis (CAIA) model via binding to endogenous collagen was determined by in vivo in vivo distribution analysis. Arthritis was selectively induced in the right hind paw by passive immunization with an anti-collagen antibody, followed by subcutaneous injection of LPS into the right hind paw plantar. Local LPS injection induced severe arthritis in the right hind paw compared to the other paw. The day after LPS injection, fluorescently labeled CBP-αTNF and WT-αTNF were intravenously injected into CAIA and naive mice. Whole-body fluorescence levels were measured before antibody injection and at 0.5, 1, 2, 4, 6, 24, and 48 hours post-injection. Fluorescence levels in the arthritis-affected right hind paw of CAIA mice injected with CBP-αTNF and WT-αTNF increased immediately after injection, while fluorescence levels in naive mice were nearly identical in both arthritis-affected and non-arthritis-affected paws (Figures 2A and 2B). The ratio of CBD-αTNF levels in arthritis-affected feet to non-arthritis-affected feet was higher in mice injected with CBD-αTNF than in mice injected with WT-αTNF (Figure 2C). Injected CBD-αTNF was detected by immunohistochemistry in the synovial membrane and pannus, the major inflammatory sites of arthritis (Figure 2D). These data indicate that CBD-αTNF selectively localizes to inflamed tissue (i.e., arthritis-affected feet) after systemic injection, more so than its unmodified form.

[0187] 3. CBP conjugation enhanced the efficacy of αTNF in an arthritis model. Next, the inventors investigated the anti-inflammatory efficacy of CBP-αTNF in a CAIA model. Arthritis was induced in all paws by passive immunization with an anti-collagen antibody followed by intraperitoneal injection of LPS. On the day of LPS injection, control IgG, WT-αTNF, or CBP-αTNF was administered intravenously. Arthritis scores increased in control mice, and were reduced by WT-αTNF and CBP-αTNF (Figure 3A). The score reduction in CBP-αTNF-treated mice was significantly greater than that in WT-αTNF-treated mice. Histological observations revealed that joint destruction was significantly suppressed by CBP-αTNF (Figure 3B). The inventors further investigated whether CBP-αTNF could exert efficacy via subcutaneous injection. Similar accumulation tendencies and inhibitory efficacy were observed even with subcutaneous injection of CBP-αTNF (Figure 4). These data indicate that CBP-modified αTNF provides superior anti-inflammatory efficacy compared to its unmodified form.

[0188] 4. Local injection of ECM-bound α-TNF had a strong effect on the onset of arthritis. To evaluate the therapeutic efficacy of local therapy, the inventors of this invention used the same method as described above for CBP conjugation to administer placental growth factor 2 (specifically, PlGF-2) 123~144 A promiscuous ECM-binding peptide derived from ) was conjugated with αTNF (32, 33). PlGF-2 123~144 The peptide binds to multiple ECM proteins with high affinity and is therefore retained at the injection site. PlGF-2 injected into the left hind paw of a CAIA mouse. 123~144 Conjugation αTNF (PlGF-2) 123~144 PlGF-2 was retained at the injection site, but the signal from WT-αTNF decreased rapidly after injection (Figure 5A). To compare efficacy, control IgG, WT-αTNF, or PlGF-2 123~144 -αTNF was subcutaneously injected into the left hind paw of CAIA mice. Arthritis scores increased in both the right and left hind limbs of control IgG-treated mice. WT-αTNF did not suppress the score in this treatment regimen. However, PlGF-2123~144 -αTNF, even at a dose 100 times lower than WT-αTNF, almost completely suppressed the development of arthritis in the treated foot (left). Interestingly, PlGF-2 123~144 -αTNF did not suppress the development of arthritis in untreated feet (right), demonstrating its local efficacy (Figure 5B). These data suggest that drug accumulation at the site of inflammation is important for suppressing inflammation and maximizing the efficacy of anti-inflammatory drugs.

[0189] 5. CBD proteins derived from the vWF A3 domain can target inflamed spinal cord in experimental autoimmune encephalomyelitis (EAE) models. IL-4 is a cytokine that induces the differentiation of naive helper T cells (Th0) into Th2 cells. Application of IL-4 to the central nervous system via intrathecal or intranasal administration has been reported to improve clinical signs and axonal morphology in experimental autoimmune encephalomyelitis (EAE) models, a mouse model of multiple sclerosis. Therefore, to achieve EAE targeting of IL-4 from intravenous injection, a clinically relevant injection route, we synthesized a vWF A3 domain-fused IL-4 (A3-IL4) protein. A3-IL4 bound to collagen III with a dissociation constant (Kd) of 28.6 nM (Figure 6A). The vWF A3 domain fusion to IL-4 did not invalidate its binding affinity to its receptor, IL-4Rα (Figure 6B). Next, the localization of the vWF A3 domain protein in the inflamed spinal cord of the EAE model via binding to endogenous collagen was determined by fluorescence imaging. Fluorescently labeled A3 or A3-IL4 was intravenously injected into naive and EAE mice 14 days post-immunization, when the target tissue became inflamed. A3 and A3-IL4 were detected in the spinal cord of EAE mice but not in the spinal cord of naive mice (Figure 6C). Next, the inventors investigated the therapeutic effect of A3-IL4 on EAE symptoms. Starting 14 days post-immunization, when the first EAE symptoms appeared, PBS, normal IL-4, and A3-IL4 were intravenously injected every other day. A3-IL4 reduced the mean disease score, while normal IL-4 showed no therapeutic effect (Figure 6E).

[0190] 6. A3 protein and CBP conjugates can also target inflammatory tissues in other inflammatory disease models. To investigate the potential applications of collagen binding in inflammatory diseases, the localization of A3 protein and CBP conjugation antibodies was determined by fluorescence imaging in inflammatory tissues of spontaneous inflammatory bowel disease (IBD), bleomycin-induced idiopathic pulmonary fibrosis (IPF), and type 1 diabetes mellitus (T1D) models. At the point of inflammation in the target tissue, fluorescently labeled A3, CBP-αTNF, or CBP conjugation anti-TGF-β antibody (CBP-αTGF) was intravenously injected into EAE, IBD, IPF, and T1D models, and fluorescence levels in the target tissue were determined. A3 was detected in the colon of mice with IBD, as well as in the pancreas of spontaneous T1D and cyclophosphamide-induced T1D mice, but not in those of healthy mice (Figures 7A and 7C). Similarly, CBP-αTNF was detected in the spinal cord of EAE models and in the colon of IBD-developing mice, but not in those of healthy mice (Figures 6D and 7A). Histopathological analysis revealed that CBP-αTNF is localized in the lamina propria of the colon in IBD models where infiltrating cells are present (Figure 7A). In addition, CBP-αTGF was detected in the lungs of IPF models, but the unmodified antibody was not detected (Figure 7B). These results suggest that introducing collagen affinity to antibodies and cytokines may allow them to be targeted to inflammatory tissues.

[0191] B. Discussion This study demonstrated that introducing collagen-binding affinity to anti-inflammatory antibodies enhances their retention in inflamed tissues and their therapeutic efficacy. Intravenous and subcutaneously administered CBP-αTNF accumulated in inflamed feet in a CAIA model. Furthermore, CBP conjugation to the antibody enabled detection at multiple inflammatory sites in EAE, IBD, and IPF models. This suggests that collagen affinity can target inflammatory sites and is broadly applicable to various inflammatory diseases. This is because collagen is universally and abundantly present around vascular structures, but in inflamed tissues, it is only exposed to blood flow when hyperpermeability of vascular structures occurs. Therefore, collagen affinity as a drug delivery method is a general inflammation-specific approach, rather than a tissue, molecular expression, or disease-specific approach. More importantly, CBP-αTNF more effectively reduced arthritis scores in the CAIA model than unmodified αTNF. αTNF for inflammatory diseases such as RA and IBD does not result in complete remission in most patients and can cause serious side effects (6-10). Therefore, CBP-αTNF has the potential for technological transfer to achieve advanced treatments for inflammatory and autoimmune diseases.

[0192] To evaluate whether other collagen-binding proteins can also target inflammatory sites after systemic injection, the inventors used a vWF A3 domain recombinant protein. A3-IL4 accumulated in the spinal cord in the EAE model and reduced disease scores, whereas normal IL-4 did not. Targeting neuronal IL-4 signaling is expected to be a novel therapeutic strategy to halt the progression of damage in multiple sclerosis (34). While intrathecal or intranasal pathways have been proposed for IL-4 treatment to cross the blood-brain barrier, this embodiment demonstrates that providing IL-4 with collagen-binding ability makes it possible to reduce clinical symptoms in the EAE model even via an intravenous pathway. The inventors also show accumulation of A3 protein in inflammatory tissue in IBD and T1D models. The inventors used the CAIA model for autoantibody-induced acute inflammation, the EAE model for autoimmune-mediated chronic inflammation, the IBD model for spontaneous inflammation, the IPF model for inflammatory fibrosis, and the T1D model for spontaneous T-cell-mediated autoimmune disease as their primary inflammation models. These data suggest that collagen-binding antibodies and cytokines can accumulate at the site of inflammation, enabling effective antibody and cytokine therapy in multiple inflammatory diseases.

[0193] This example uses PlGF-2 123~144 -αTNF local injection solution was retained at the injection site and showed potent therapeutic efficacy at low doses. However, PlGF-2 123~144-α-TNF therapy is only effective when administered by local injection due to its chaotic ECM affinity. The main technology transfer advantage of collagen-binding approaches for inflammation-targeted therapy is that it allows targeting of inflammatory sites from systemic delivery pathways. In the case of clinical technology transfer of collagen-binding anti-inflammatory drugs, the advantage is that using the A3 domain of vWF or CBP of decorin limits the possibility of recognition by the immune system because both are naturally present in the human body. In addition, CBP can be conjugated to antibodies by a simple chemical reaction. The advantage of this feature is that it is easy to produce, as it can function with antibodies whose production has already been optimized. In this embodiment, we have shown that CBP can conjugate both anti-TNFα antibodies and anti-TGFβ antibodies. The CBP conjugation synthesis reaction of antibodies can be carried out in just 90 minutes using a chemical reaction similar to protein PEGylation. The same reaction is used in antibody-drug conjugation, such as in the production of trastuzumab emtansine (35, 36). Regarding A3-IL4, considering that cytokines are small molecules and generally easy to produce, the inventors chose to recombinantly fuse A3 with IL-4 rather than conjugate it. These features may facilitate the development of collagen-binding drug therapies to overcome barriers to clinical technology transfer.

[0194] In conclusion, it was found that introducing collagen affinity into antibodies and cytokines enables them to target inflammatory sites. Furthermore, collagen-binding anti-inflammatory drugs showed higher therapeutic efficacy compared to their unmodified counterparts. This simple approach of engineered collagen-binding drugs may hold potential for clinical transfer as an anti-inflammatory therapy.

[0195] C. Materials and Methods 1. Synthesis of CBP conjugation antibodies A rat anti-mouse TNF-α antibody (clone XT3.11, BioXcell) was incubated with 20 equivalents of sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-SMCC) at room temperature for 30 minutes. Excess sulfo-SMCC was removed using a Zeba spin desalting column (Thermo Fisher Scientific). Next, 30 equivalents of a collagen-binding sequence peptide (CBP, LRELHLNNNC) derived from decorion were added and reacted at room temperature for 1 hour for conjugation to the thiol moiety of the C residue. The peptide had been synthesized by Genscript with a purity of over 95%.

[0196] 2. Production and purification of recombinant vWF A3 domain and A3-fusion IL-4 protein Human vWF A3 domain residues Cys1670-Gly1874 (907-1111 in mature vWF), as well as sequences encoding the human vWF A3 domain and mouse IL-4 fusion protein, were synthesized and subcloned into the mammalian expression vector pcDNA3.1(+) by Genscript. For further purification of the recombinant protein, a sequence encoding 6His was added to the N-terminus. HEK-293F cells adapted to suspension were routinely maintained in serum-free FreeStyle 293 expression medium (Gibco). On the day of transfection, cells were 1 × 10⁶ 6Fresh medium was inoculated at a density of 10 cells / ml. Plasmid DNA at 2 μg / ml, linear 25 kDa polyethyleneimine at 2 μg / ml (Polysciences), and OptiPRO SFM medium (final concentration 4%, Thermo Fisher) were added sequentially. The culture flask was agitated by orbital shaking at 135 rpm at 37°C in the presence of 5% CO2. Six days after transfection, the cell medium was collected by centrifugation and filtered through a 0.22 μm filter. The medium was loaded onto a HisTrap HP 5 ml column (GE Healthcare) using AKTA pure 25 (GE Healthcare). After washing the column with wash buffer (20 mM imidazole, 20 mM NaH2PO4, 0.5 M NaCl, pH 7.4), proteins were eluted using a gradient of 500 mM imidazole (in 20 mM NaH2PO4, 0.5 M NaCl, pH 7.4). The eluted solution was further purified by size exclusion chromatography using a HiLoad Superdex 200PG column (GE Healthcare). All purification steps were performed at 4°C. Expression of A3 and A3-IL-4 was determined by Western blotting using an anti-His tag antibody (BioLegend), and the proteins were confirmed to be over 90% pure by SDS-PAGE.

[0197] 3. Detection of CBP-αTNF or A3-IL4 binding to collagen proteins The measurements were carried out as described above (32). A 96-well ELISA plate (Greiner Bio One) was coated overnight at 37°C with type I, type II, and type III human collagen (10 μg / mL each in PBS, Millipore Sigma), followed by blocking with 1% BSA in PBS containing 0.05% Tween 20 (PBS-T) at room temperature for 1 hour. Next, the wells were washed with PBS-T and further incubated with 1 μM CBP-αTNF, 1 μM unmodified αTNF, or 0-740 nM A3-IL4 at room temperature for 1 hour. After three washes with PBS-T, antibodies were detected by incubating with HRP conjugate antibody against rat IgG (Jackson ImmunoResearch) at room temperature for 1 hour. A3-IL4 was detected by specific antibody against mouse IL-4 (R&D Systems). After washing, the bound proteins were detected by measuring the absorbance at 450 nm after subtracting the absorbance at 570 nm using a tetramethylbenzidine substrate.

[0198] 4. Detection of A3-IL4 binding to its receptor The measurements were carried out as described above (32). A 96-well ELISA plate (Greiner Bio One) was coated overnight at 37°C with recombinant mouse IL-4Rα protein (10 μg / mL each in PBS, R&D Systems), followed by blocking with 1% BSA in PBS (PBS-T) at room temperature for 1 hour. Next, the wells were washed with PBS-T and incubated with 0–740 nM A3-IL4 or IL-4 at room temperature for 1 hour. After three washes with PBS-T, IL-4 was detected using a specific antibody against mouse IL-4 (R&D Systems). After washing, the bound protein was detected using a tetramethylbenzidine substrate by subtracting the absorbance at 570 nm and measuring the absorbance at 450 nm.

[0199] 5. MALDI-TOF MS Antibodies were analyzed by MALDI-TOF MS (Bruker Ultraflextreme MALDI TOF / TOF). All spectra were collected using the acquisition software Bruker flexControl® and processed with the analysis software Bruker flexAnalysis®. First, a saturated solution of the matrix, α-cyano-4-hydroxycinnamic acid (Sigma-Aldrich), was prepared in 50:50 acetonitrile:1% TFA in water as the solvent. Next, the analyte (5 μL, 0.1 mg / mL) and matrix solution (25 μL) in PBS were mixed, and 1 μL of this mixture was deposited onto an MTP 384 ground steel target plate. The droplets were dried in a stream of nitrogen gas, resulting in the formation of a homogeneous sample / matrix coprecipitate. All samples were analyzed using a high-mass linear positive mode assay with 2500 laser shots at 75% laser intensity. The measurement results were externally calibrated at three points using a mixture of carbonic anhydrase, phosphorylase B, and bovine serum albumin.

[0200] 6. In vivo biodistribution studies Anti-TNFα antibody (clone XT3.11, BioXcell) and anti-TGF-β antibody (clone 1D11.16.8, BioXcell) were administered in 8 equivalents of SM(PEG) 24 The cells were incubated with (Thermo Fisher Scientific) for 30 minutes at room temperature. Excess SM(PEG) was removed using a Zeba spin desalting column (Thermo Fisher Scientific). 24The ions were removed. Next, 30 equivalents of Cy7-labeled CBP ([Cy7]LRELHLNNNC[COOH]) were added and reacted at room temperature for 30 minutes for conjugation to the thiol portion of the C residue. The peptide was synthesized with over 95% purity using Genscript. Unreacted dyes were removed by dialysis against PBS. For CBP non-conjugated antibodies, αTNF and αTGF were labeled using sulfo-Cy7 NHS ester (Lumiprobe) according to the manufacturer's instructions. A3 and A3-IL4 were labeled using DyLight 800 NHS ester (Thermo Fisher Scientific) according to the manufacturer's instructions. When the target tissue of the model mouse became inflamed, 10–100 μg of Cy7-labeled WT-αTNF, WT-αTGF, CBP-αTNF, and CBP-αTGF, or DyLight 800-labeled A3 and A3-IL4 were intravenously injected. Mouse organs were collected and imaged using the Xenogen IVIS Imaging System 100 (Xenogen) under the following conditions: f / stop: 2; optical filter excitation 745 nm; excitation 800 nm; exposure time: 5 seconds; small binning.

[0201] 7. Mouse collagen antibody-induced arthritis (CAIA) model Arthritis was induced in female Balb / c mice (7 weeks old) by intraperitoneal injection of an anti-collagen antibody cocktail (1.5 mg / mouse, Chondrex) on day 3, followed by intraperitoneal injection of LPS (50 μg / mouse, Chondrex) on day 0. On the day of LPS injection, control IgG (200 μg / mouse), WT-αTNF (200 μg / mouse), or CBP-αTNF (200 μg / mouse) was administered intravenously or subcutaneously to the back of the mice; or control IgG (100 μg / mouse), WT-αTNF (100 μg / mouse), or PlGF-2 was administered to the left hind paw. 123~144-αTNF (1 μg / mouse) was administered subcutaneously. Joint swelling was scored daily as described elsewhere (31). On day 8, the hind legs were fixed in 10% neutral formalin (Sigma-Aldrich), decalcified in Decalcifer II (Leica), and then subjected to histological analysis.

[0202] 8. Experimental Autoimmune Encephalomyelitis (EAE) Model EAE is MOG in full Freund adjuvant on day 0. 35~55 EAE was induced in 13-week-old female C57BL / 6 mice by immunization with an emulsion (200 μg / mouse, Hooke Laboratories), followed by administration of pertussis toxin in PBS (100 ng / mouse) on the day of immunization and the following day. On postimmunization day 14, when EAE symptoms appeared, treatment with 1 μg / mouse recombinant mouse IL-4 (Peprotech) or 1 μg / mouse A3-IL4 (equivalent to 0.4 μg / mouse on a molar basis) was initiated and repeated every other day. Individual mice were scored daily for disease severity based on the following scale: 0, no clinical disease; 0.5, tail weakness; 1, tail paralysis; 2, hindlimb weakness; 3, hindlimb paralysis; 3.5, forelimb weakness; 4, forelimb paralysis; or 5, mortal or dead.

[0203] 9. A spontaneously occurring colitis model of inflammatory bowel disease (IBD) IL-10 - / - × TLR-4 - / - The (DKO) mice were kindly provided by Cathyn Nagler (University of Chicago). DKO mice spontaneously develop colitis and have a high incidence of rectal prolapse. At 29 weeks of age, when the first signs of rectal prolapse appeared, the mice were used as IBD-affected mice for imaging analysis. As non-IBD controls, 16-week-old DKO mice and mice from the genetic background strain (C57BL / 6) were used.

[0204] 10. Bleomycin-induced idiopathic pulmonary fibrosis (IPF) model Inflammatory pulmonary fibrosis was induced in female C57BL / 6 mice (9 weeks old) by intranasal injection of bleomycin (Sigma-Aldrich) at a dose of 100 μg / mouse in physiological saline. Once the lungs became inflamed 7 days after bleomycin administration, the mice were used for imaging analysis.

[0205] 11. NOD mouse model of Type 1 diabetes (T1D) Non-obese diabetic (NOD) mice are known as a spontaneous model of T-cell-mediated autoimmune insulin-dependent diabetes (37, 38). Cyclophosphamide can promote the development of diabetes in NOD mice (39). For image analysis, we used non-diabetic controls, spontaneously diabetic NOD mice, and Balb / c mice as diabetic mice in which intraperitoneal injection of 300 mg / kg of cyclophosphamide (Sigma-Aldrich) accelerates the development of diabetes. Blood glucose levels were used as an indicator of diabetes development.

[0206] 12. Histological analysis and immunohistochemistry Paraffin-embedded joint tissue from CAIA mice and the colon from IBD-affected mice were sliced ​​to a thickness of 5 μm and stained with H&E and / or PAS for pathological analysis. The severity of bone resorption and synovial hyperplasia in the arthritis models was scored using a three-point scale (0-2) following previously reported criteria with slight modifications: 0, normal to minimal infiltration of the pannus in the subchondral bone of the cartilage and marginal zone; 1, mild to moderate infiltration of the marginal zone with mild cortical and medullary bone destruction; 2, severe infiltration associated with complete or near-complete destruction of the joint structure. The scores for both hind paws were totaled for each mouse (total score per mouse, 0-4).

[0207] Immunohistochemical staining was performed according to standard procedures. Briefly, sections were incubated in 0.3% H2O2 for 20 minutes, blocked in PBS-buffered 1% BSA for 1 hour, incubated with HRP-labeled anti-rat IgG (Jackson ImmunoResearch) for 1 hour at room temperature and overnight at 4°C, and visualized with diaminobenzidine.

[0208] 13. Statistical analysis Statistical analysis was performed using GraphPad Prism software, and P < 0.05 was considered statistically significant. Changes in arthritis scores over time were evaluated using repeated measures two-way ANOVA, and if the interaction was considered significant, data were evaluated using Dunnett's multiple comparison test at each time point. To compare the efficacy of CBP-αTNF with WT-αTNF, data on day 8 were re-analyzed using Tukey's multiple comparison test. For histological scores in the CAIA model, Dunnett's multiple comparison test was used to compare the differences between the control IgG injection group and the αTNF treatment group.

[0209] D. References The following references and publications referenced throughout this specification are incorporated herein by reference to the extent that they provide exemplary procedures or other details that supplement what is described herein. TIFF0007870524000053.tif202160TIFF0007870524000054.tif238160TIFF0007870524000055.tif238160TIFF0007870524000056.tif116160

[0210] Example 2: Enhanced lymph node transport of manipulated IL-10 suppresses a mouse model of rheumatoid arthritis. Rheumatoid arthritis (RA) is a major autoimmune disease. Clinical trials using interleukin-10 (IL-10) have been conducted as a potential treatment for RA, but its therapeutic effect has been limited due to the possibility of insufficient retention in lymphoid organs where antigen recognition primarily occurs. In this specification, we have engineered IL-10 as a fusion with serum albumin (SA) with and without collagen-binding domain (CBD) fusion. SA-IL-10 and CBD-SA-IL-10 showed longer circulation times after intravenous injection than unmodified IL-10; furthermore, SA fusion led to enhanced lymph node (LN) accumulation compared to unmodified IL-10. Intravenous SA-IL-10 and CBD-SA-IL-10 treatment restored the immune cell composition in the feet to a normal state, increased the frequency of suppressive M2 macrophages, and protected joint morphology. Intravenous SA-IL-10 and CBD-SA-IL-10 demonstrated similar efficacy to treatment with anti-TNF-α antibodies. SA fusion to IL-10 is a simple yet effective manipulation strategy for achieving LN accumulation and RA control.

[0211] Rheumatoid arthritis (RA) is an autoimmune disease that is currently controlled by treatment with inhibitors of inflammatory pathways. The pathological features of RA are synovitis and joint destruction, which cause severe pain and joint dysfunction (1, 2). The causative antigens of RA are not fully understood, but collagen recognition by immune cells plays a crucial role. During the progression of RA, autoantigen-specific T cells, particularly Th17 cells, are activated and produce inflammatory cytokines, including IL-17. In the joints, inflammatory cytokines such as TNF-α and IL-6 induce the activation of macrophages and neutrophils as mediators of the inflammatory response. These inflammatory cells infiltrate the joints and trigger various inflammatory responses, including the activation of osteoclasts that destroy bone within the joints (3). Current strategies for treating RA are symptomatic, and given that many inflammatory cytokines are involved in the progression of RA, various biological therapies, such as antibodies or soluble receptors for TNF-α, have been developed and approved for clinical use (4).

[0212] Another type of biological therapy involves the administration of anti-inflammatory cytokines to treat RA in order to induce tolerance or systemic suppression of inflammation. IL-10 is one such anti-inflammatory cytokine (5-7), and various attempts have been made to explore IL-10-based therapies for autoimmune diseases (6-8). However, the therapeutic effects of IL-10 in autoimmune diseases remain controversial, possibly due to its short circulating half-life and uncontrolled biodistribution after systemic administration (8).

[0213] In this study, the inventors manipulated IL-10 to provide prolonged blood circulation through fusion with serum albumin (SA), and enhanced vascular permeability and binding affinity to inflammatory sites where extracellular matrix proteins, including collagen, are exposed to proteins from the blood through fusion with a collagen-binding domain (CBD) (16, 17). Therefore, the inventors hypothesized that CBD fusion to SA adds inflammatory site targeting to the SA-fused cytokine. By doing so, the inventors sought to explore whether the enhanced blood circulation and vascular structure targeting at disease sites synergistically improve the therapeutic effect of IL-10 on RA. However, the inventors observed that SA fusion to IL-10 not only enhanced circulation time but also enhanced accumulation in lymph nodes (LNs). Hereinafter, the arthritis-suppressing effect of manipulated IL-10 was evaluated using passive collagen antibody-induced arthritis (CAIA) and active CIA models in mice. The inventors found that CBD fusion enhanced accumulation in inflamed feet, and further found that SA fusion to IL-10 enhanced the transport of IL-10 to the LN after intravenous injection. SA-fused IL-10 significantly improved the anti-inflammatory effects of IL-10 in two mouse RA models and functioned similarly to TNF-α blockade.

[0214] A. Albumin-fused IL-10 binds to FcRn and APC and accumulates in LN. Wild-type (wt) mouse IL-10, SA-fusion mouse IL-10, and CBD-SA-fusion IL-10 were expressed by recombination, and the molecular weight of the fusion proteins was correspondingly higher than that of wt IL-10 as determined by SDS-PAGE; furthermore, the majority of SA-IL-10 and CBD-SA-IL-10 existed as monomers under non-reducing conditions (Figures 8A and 14A). Surface plasmon resonance (SPR) analysis revealed that SA-IL-10 and CBD-SA-IL-10 had micromolar K levels. d It was revealed that it binds to the neonatal Fc receptor (FcRn) (Figures 8B and 14B). Furthermore, CBD-SA-IL-10 has a nanomolar order of K d It was shown to bind to type I and type III collagen (Figure 14B). The binding ability of these proteins to splenocytes and unicellular cells isolated from popliteal LN was further evaluated by flow cytometry (Figure 8C). SA-fused IL-10 showed high binding to macrophages and dendritic cells in both splenocytes and LN-derived cells. After intravenous administration of fluorescently labeled SA-IL-10, a significantly higher fluorescence signal was observed in popliteal LN compared to wt IL-10 (Figure 8D). Interestingly, the higher fluorescence signal was located around high endothelial venules (HEVs) where antigen-presenting cells (APCs) were present (18).

[0215] B. Albumin-fused IL-10 shows long-term blood circulation, while CBD-fused IL-10 leads to accumulation in inflamed feet. SA is known to exhibit long-lasting circulation in vascular endothelial cells via FcRn-mediated regeneration (19, 20). As expected, SA-IL-10 showed significantly longer-lasting circulation compared to wt IL-10; CBD-SA-IL-10 also had comparable circulation to SA-IL-10 (Figure 9A). Figure 9B shows the fluorescence signals from major organs of mice intravenously injected with DyLight800-labeled proteins. SA-IL-10 and CBD-SA-IL-10 showed higher signals in the heart, lungs, and spleen than wt IL-10, reflecting their long-lasting circulation characteristics. Furthermore, CBD-SA-IL-10 showed a significantly higher signal from inflamed paws of mice treated with CBD-SA-IL-10 than from wt IL-10, but no significant difference in fluorescence was observed between wt IL-10 and CBD-SA-IL-10 in non-inflamed paws, indicating the inflammation-targeting ability of CBD-SA-IL-10 through collagen affinity, as reported in previous studies in other contexts (16, 21).

[0216] C. Albumin-fused IL-10 suppresses the onset of arthritis. The therapeutic effects of manipulated IL-10 in a passive collagen antibody-induced arthritis (CAIA) model were evaluated (Figure 10). Intravenous injection of SA-IL-10 or CBD-SA-IL-10 significantly suppressed the development of arthritis, while mice injected with PBS or wt IL-10 showed severe inflammation in the paws (Figure 10A). The therapeutic effect of CBD-SA-IL-10 was compared with treatment with anti-TNF-α antibody (αTNF-α), a mouse model of an antibody drug clinically used for RA treatment, and here CBD-SA-IL-10 induced CAIA suppression comparable to αTNF-α (Figure 10B). Histological analysis revealed that both CBD-SA-IL-10 and αTNF-α suppressed joint destruction compared to PBS or wt IL-10 treatment (Figure 10C). Histological scores were also significantly reduced by treatment with CBD-SA-IL-10 and αTNF-α (Figure 10C). The effect of the administration route on therapeutic efficacy was also investigated by comparing intravenous, topical (sole) and subcutaneous (distal and mid-dorsal) administration (Figure 10D). Sole injection of CBD-SA-IL-10 induced a relatively high inhibitory effect on CAIA compared to its intravenous injection results shown in Figures 10A and 10B, suggesting retention of CBD-SA-IL-10 in the inflamed foot via collagen affinity (Figure 8B). Surprisingly, SA-IL-10 showed a very high inhibitory effect on CAIA via all administration routes tested (Figure 10D).

[0217] As a second arthritis model, an active collagen-induced arthritis (CIA) model was used to evaluate CBD-SA-IL-10 and SA-IL-10 against RA treatment. CBD-SA-IL-10 significantly suppressed the increase in clinical scores compared to CIA mice treated with PBS (Figure 11A), and its therapeutic effect was comparable to αTNF-α treatment. Joint histological features and scores also revealed the suppression of arthritis establishment by CBD-SA-IL-10 (Figure 11B). Importantly, 5 out of 10 mice treated with CBD-SA-IL-10 showed a score of 1 or less, suggesting a high therapeutic effect of CBD-SA-IL-10. In the absence of the CBD domain, a single injection of SA-IL-10 into CIA mice induced a significant suppression of arthritis establishment compared to PBS (Figure 11C). Most mice treated with PBS showed severe inflammation in their paws, as indicated by histological features and scores (Figure 11D). In contrast, mice treated with SA-IL-10 showed paw conditions nearly identical to naive mice, and most mice had a histological score of 1 or less. In summary, these results demonstrate very high anti-inflammatory effects from topical or intravenous administration of CBD-SA-IL-10, and even from subcutaneous administration of SA-IL-10.

[0218] D. Albumin-fused IL-10 reduces immune activity after accumulating in the linden. SA-fused IL-10 showed micromolar affinity for FcRn (Figure 8B) and accumulated in the LN after intravenous injection (Figure 8D). Next, the amount of IL-10 in the LN and its pharmacokinetics were quantitatively evaluated (Figures 12A-C). After intravenous injection of wt IL-10, SA-IL-10, or CBD-SA-IL-10 into CAIA mice, IL-10 concentrations in the LN at various time points were detected using ELISA. Four hours after injection, SA-IL-10 showed significantly higher IL-10 signals in the joint inflow area (popliteal fossa) LN and mesenteric LN, and relatively high signals in the non-inflow area (cervical) LN compared to wt IL-10 and CBD-SA-IL-10, while CBD-SA-IL-10 showed higher accumulation than wt IL-10 (Figure 12A). Mice injected with SA-IL-10 also showed a peak in IL-10 concentration approximately one hour after injection (Figure 12B) and exhibited a 5- to 10-fold higher AUC in the LN compared to wt IL-10 (Figure 12C). These data suggest that SA-IL-10 accumulates in the LN immediately after intravenous injection and exhibits a higher retention rate in the LN compared to wt IL-10.

[0219] High concentrations and AUC of SA-IL-10 in the LN may affect the phenotypes of various immune cells in the LN and other secondary lymphoid organs. Therefore, immune cell populations in the spleen and popliteal LN were analyzed by flow cytometry (Figure 15). Intravenous injection of SA-IL-10 affected CD3 in the spleen. + T cells and CD45 + This induced a significant decrease in lymphocyte frequency (Figure 15A). Furthermore, CD86 + Dendritic cells, granulocyte-derived bone marrow suppressor cells (G-MDSCs), and CD86 + The frequency of M1 macrophages decreased, and CD206 +The frequency of M2 macrophages increased after injection of SA-IL-10 compared to PBS or wt IL-10. A similar trend was observed in the popliteal LN (Figure 15B). These data suggest that SA-IL-10 suppressed APC activity while simultaneously activating immunosuppressive M2 macrophages. Inactivation of APCs and high accumulation of IL-10 in the LN may suppress the activity of Th17 cells, which play a crucial role in the development of RA (22, 23). The inventors measured Th17-related cytokines (IL-17, IL-6, and TGF-β) in the joint inflow area (popliteal fossa) and non-inflow area (cervical) lymph nodes (LNs) and compared them with treatment with wt IL-10. IL-17 levels were statistically reduced in popliteal LNs after treatment with SA-IL-10, but not statistically reduced by CBD-SA-IL-10, and levels in cervical LNs were not statistically reduced by any IL-10 variant (Figures 12D and 12E). Treatment with SA-IL-10 reduced the concentration of GM-CSF in popliteal LNs, but not wt IL-10 (Figure 12F).

[0220] E. Albumin-fused IL-10 suppresses inflammatory responses in the feet. Next, the immune cell population in the hind paw was analyzed using flow cytometry (Figure 13A). CD45 was analyzed after intravenous injection of SA-IL-10 or CBD-SA-IL-10. + The frequency of immune cells was significantly reduced compared to the group treated with PBS or wt IL-10. CD45 + Within the cell, the frequencies of B cells and dendritic cells became comparable to those in healthy mice, and CD11b + The number of cells also decreased significantly to the level of healthy mice. CD11b + Within the cells, the G-MDSC population decreased, and the frequency of macrophages recovered to the level of healthy mice. Furthermore, CD206 + The frequency of M2 macrophages was significantly increased by SA-IL-10 injection compared to PBS or wt IL-10 treatment, even exceeding the frequency in healthy mice. Analysis of T cell populations in the paw revealed that SA-IL-10 increased CD4 in CAIA mice.+ Cells and Foxp3 + It was revealed that changes in Treg were suppressed (Figure 16A). Furthermore, SA-IL-10 suppressed the decrease in Treg frequency in the blood (Figure 16B). Reflecting these changes in the immune cell population, various inflammatory cytokines in the paws were significantly reduced by intravenous injection of SA-IL-10 or CBD-SA-IL-10, and their levels were comparable to those in healthy mice (Figure 13B). Histological analysis showed that intravenous administration of SA-IL-10 significantly suppressed the inflammatory response in the paws and reduced joint lesions compared to PBS-treated mice (Figure 13C).

[0221] F. Albumin-fused IL-10 does not show toxicity after injection. Finally, a safety assessment was conducted to determine if the manipulated IL-10 had any adverse effects. Representative blood parameters and spleen weight, measured by a blood analyzer, did not show significant changes between treatment groups (Figure 17A). Various biochemical markers in serum were also examined using a biochemical analyzer (Figure 17B). In the manipulated IL-10 treatment group, most markers, with the exception of amylase (which did not increase and rather decreased slightly), were at similar levels compared to the PBS treatment group, indicating that manipulated IL-10 is highly safe after systemic administration.

[0222] G. Discussion Current treatment for rheumatoid arthritis (RA) is based on symptomatic therapy, focusing on pain relief, control of synovitis, and suppression of joint damage. Antibodies that neutralize inflammatory cytokines, particularly TNF-α, or competing soluble receptors, have shown high therapeutic efficacy in patients with RA (4). These biologics primarily act on inflamed joints to capture inflammatory cytokines. However, these inhibitors are known to increase the risk of infection because their targets are pleiotropic in immune function, and these drugs are repeatedly administered to provide their anti-inflammatory effects at the disease site (24-27). Furthermore, the administration of antibody drugs can also induce neutralizing anti-drug antibodies, which reduces therapeutic efficiency (28). Therefore, the development of alternative approaches with structurally different molecular classes and different immunosuppressive molecular mechanisms, such as tolerance, is desirable.

[0223] In this specification, the inventors are exploring a novel approach to treating RA through enhanced lymph node transport using engineered IL-10, a representative anti-inflammatory cytokine that modulates the phenotype of RA-associated immune cells toward an immunosuppressive state. Clinical trials using recombinant IL-10 have already been conducted to treat autoimmune diseases, including RA (6-8, 29). One drawback of IL-10 is its short half-life in the blood (8). In this specification, the inventors genetically fused SA to IL-10 to extend its retention in the blood and even in secondary lymphoid organs. Furthermore, to enhance binding at inflammatory sites where the microvascular system is hyperpermeable, the inventors genetically fused CBD, derived from the blood protein von Willebrand factor (16, 17). The inventors evaluated both SA-IL-10 and CBD-SA-IL-10 compared to wt IL-10 for improvement of arthritis in two models. CAIA is a macrophage and neutrophil-mediated acute RA model, while CIA is a T-cell-mediated RA model, particularly Th17. Given that RA is a heterogeneous disease in clinical settings and these models complement each other, it is encouraging to see that SA-fused IL-10 suppresses disease severity in both models. SA fusion to IL-10 is crucial for achieving a significant therapeutic effect comparable to αTNF-α antibody treatment, a common treatment in clinical settings. Furthermore, to the best of our knowledge, this study is the first to demonstrate the therapeutic effect of IL-10 in the CAIA model.

[0224] SA fusion to IL-10 resulted in enhanced accumulation in the LN after intravenous injection compared to wt IL-10, maintaining high IL-10 concentrations in the LN over extended periods (Figure 12A). To date, LN transport of SA or albumin-bound nanoparticles has been achieved primarily by intradermal or subcutaneous administration, where the LN is accessed via afferent lymphatic vessels downstream of the collecting lymphatic vessels at the injection site (30-33). In connection with studies of the in vivo distribution of inflammatory cytokines, one paper demonstrated high localization of human SA-fused IL-2 after intravenous injection into the spleen, liver, and LN, where IL-2 receptor-expressing T cells are present, but the precise mechanism of this high localization remains unclear (34). Hereinafter, we reveal enhanced transport of SA-fused IL-10 to the LN after intravenous injection, in which SA enters the LN via the blood vascular structure. CBD-SA-IL-10 showed less accumulation in LN than SA-IL-10, likely due to binding to collagen in other tissues. Both SA-IL-10 and CBD-SA-IL-10 showed high binding affinity to FcRn (as expected, micromolar K). d This is shown (Figures 8B and 14B), which provides long-term circulating properties to both proteins via regeneration mediated by FcRn expressed in vascular endothelial cells (Figure 9A). Regeneration via transcytoplasmic transport of IgG (from basal to luminal) mediated by FcRn is a well-established phenomenon, although the same phenomenon for SA has only recently been reported (19, 20). Here, in LN, molecular transport appears to be in the opposite direction, from luminal to basal. Interestingly, histological analysis revealed the accumulation of SA-IL-10 surrounding HEV in LN (Figure 8D). Further experiments are needed to elucidate the more detailed mechanisms of enhanced LN accumulation and its relationship with FcRn, such as LN accumulation analysis using mutant SA-fused IL-10 to inactivate FcRn binding.

[0225] SA-IL-10 exhibits high binding to APCs (Figure 8B). After accumulation in the LN, SA-IL-10 molecules are taken up by APCs present in the LN, leading to suppression of dendritic cell and M1 macrophage activity and induction of M2 macrophages (Figure 15). M2 macrophages can alter the differentiation fate of Th0 cells into Treg cells in the LN (35). Furthermore, the immunosuppressive environment created by high concentrations of IL-10 in the LN can lead to further polarization of macrophages into the M2 phenotype and suppression of Th17 differentiation (36, 37), resulting in the decrease of IL-17, GM-CSF, or other cytokines in the LN observed by the inventors (Figures 12D and 12F). GM-CSF is a cytokine that is a marker of pathogenic Th17, and its inhibitory antibodies are currently being tested in clinical trials (38). Therefore, the decrease in GM-CSF due to SA-IL-10 treatment indicates a decrease in immune activation in the joint inflow region of the LN. Th17 cells have been reported to express the IL-10 receptor, and IL-10 binding suppresses IL-17 expression and secretion (14, 36). Since antigen recognition by Th17 cells is mainly carried out in lymphoid tissue, SA-IL-10 may directly bind to Th17 cells and suppress the IL-17 pathway. These changes in the LN also suppressed the infiltration of immune cells, particularly G-MDSCs and macrophages, into the feet (Figure 13A), induced an increase in M2 macrophages (Figure 13A), and resulted in a decrease in inflammatory cytokines (Figure 13B) and suppression of joint inflammation (Figures 10, 11, and 13C).

[0226] SA-IL-10 induced a high anti-inflammatory response after administration via any of the tested routes, namely intravenous, subcutaneous (distal site), or plantar (local) injection, suggesting that SA-IL-10 can enter the LN systemically after being taken up by the lymphatic vessels in the area of ​​local injection site, pass through the lymphatic vessels, and return to the systemic circulation via the thoracic duct. The high therapeutic effect of subcutaneous injection suggests a specific clinical benefit of SA-IL-10. Intravenous CBD-SA-IL-10 also showed an inhibitory effect on the development of CAIA and CIA, comparable to anti-TNF-α antibodies, which are the current standard biological treatment for RA. However, the therapeutic effect of CBD-SA-IL-10 was lower than that observed with SA-IL-10, which corresponds to lower LN transport of CBD-SA-IL-10 (Figure 12A). CBD-SA-IL-10 may bind to collagen in inflamed tissue after penetrating it, leading to accumulation in the inflamed foot (Figure 9B). However, such collagen affinity may interfere with LN transport of CBD-SA-IL-10 (16, 39). Therefore, SA fusion is a simple but effective method for preparing cytokines engineered to achieve enhanced LN transport.

[0227] In this study, SA fusion to IL-10 resulted in increased persistence of autoimmune-related immune recognition within the lymphocyte (LN), leading to its expression and sustained function. As a result, SA-IL-10 suppressed the main inflammatory pathways of RA progression but did not inhibit pleomorphic inflammatory cytokines such as TNF-α. Furthermore, SA-IL-10 showed no significant toxicity in preliminary safety assessments (Figure 17). SA-IL-10 demonstrated significant therapeutic effects in both CIA and CAIA models. Therefore, the data suggest the potential of SA-IL-10 for clinical application in suppressing RA, and our findings more broadly suggest the ability of LNs to modulate immunity through systemic tolerogenic manipulation in other autoimmune and inflammatory diseases.

[0228] H. Amino acid sequences of mouse wt IL-10, SA-IL-10, and CBD-SA-IL-10 TIFF0007870524000057.tif232160

[0229] I. Materials and Methods 1. Research Design This study was designed to test a strategy for targeting anti-inflammatory cytokines to the lymphocyte (LN) through manipulated affinity for FcRn. Specifically, the inventors tested whether LN targeting of the anti-inflammatory cytokine IL-10 via serum albumin fusion was superior to untargeted wt IL-10 and currently available anti-inflammatory antibody therapeutics (α-TNF-α) in a mouse model of rheumatoid arthritis (RA). To evaluate the efficacy of wt IL-10, SA-IL-10, and anti-TNF-α antibodies, the inventors scored arthritis symptoms and joint tissue features in both passive and active CAIA models. The inventors also measured various aspects of in vivo distribution and LN transport, immune responses in the LN and foot, and post-treatment toxicity. Statistical methods were not used to predetermine the required sample size, but the sample size was selected based on estimates from pilot experiments so that statistically significant results could be obtained by appropriate statistical tests. The production of wt IL-10, SA-IL-10, and CBD-SA-IL-10 was performed by multiple individuals to ensure reproducibility. All experiments were repeated at least twice. In animal experiments, mice were randomly divided into treatment groups in cages immediately before the first drug injection and treated in the same manner. The n values ​​used to calculate statistics are shown in the legend of the figures. Drug administration and pathological analysis were performed using a blinding method. Statistical methods are described in the "Statistical Analysis" section.

[0230] 2. Production and purification of recombinant proteins Sequences encoding mouse serum albumin without propeptides (amino acid numbers 25-608 of whole serum albumin), mouse IL-10, human VWF A3 domain residues Cys1670-Gly1874 (referred to herein as CBD, numbers 907-1111 of mature VWF), and the (GGGS)2 linker were synthesized and subcloned into the mammalian expression vector pcDNA3.1(+) using Genscript. For further purification of the recombinant protein, a sequence encoding 6His was added to the C-terminus. HEK-293F cells adapted to the suspension were routinely maintained in serum-free FreeStyle 293 expression medium (Gibco). On the day of transfection, cells were 1 × 10⁶ 6Fresh medium was inoculated at a density of 100 cells / mL. Plasmid DNA at 2 μg / mL, linear 25 kDa polyethyleneimine at 2 μg / mL (Polysciences), and OptiPRO SFM medium (final concentration 4%, Thermo Fisher) were added sequentially. The culture flask was agitated by orbital shaking at 135 rpm at 37°C in the presence of 5% CO2. Seven days after transfection, the cell medium was collected by centrifugation and filtered through a 0.22 μm filter. The medium was loaded onto a HisTrap HP 5 ml column (GE Healthcare) using AKTA pure 25 (GE Healthcare). After washing the column with wash buffer (20 mM NaH2PO4, 0.5 M NaCl, pH 8.0), the protein was eluted with a gradient of 500 mM imidazole (in 20 mM NaH2PO4, 0.5 M NaCl, pH 8.0). The protein was further purified by size exclusion chromatography using a HiLoad Superdex 200PG column (GE Healthcare) with PBS as the eluent. All purification steps were performed at 4°C. Protein expression was confirmed to be over 90% pure by SDS-PAGE. The purified protein was tested for endotoxin via the HEK-Blue TLR4 receptor cell line, and the endotoxin level was confirmed to be less than 0.01 EU / mL. Protein concentration was determined from absorbance at 280 nm using NanoDrop (Thermo Scientific).

[0231] 3. Detection of binding to collagen and FcRn SPR measurements were performed using a Biacore X100 instrument. In the collagen binding experiment, recombinant human type I or type III collagen (Millipore Sigma) was immobilized on a CM5 sensor tip by a standard amine coupling method (approximately 1500 resonance units (RU)) and blocked with ethanolamine. The reference cell was also blocked with ethanolamine. The binding assay was performed at room temperature, and the K of CBD-SA-IL-10 was measured. dThe values ​​were determined by fitting a 1:1 Langmuir binding model to the data using BIAevaluation software (GE Healthcare). In the FcRn binding experiment, recombinant mouse FcRn (Acro Biosystems) was immobilized to a C1 chip (GE Healthcare) via amine coupling for approximately 200 RU according to the manufacturer's instructions. SA-IL-10 or CBD-SA-IL-10 was flowed at room temperature at 30 μL / min in running buffer (0.01 M monobasic anhydrous sodium phosphate, pH 5.8, 0.15 M NaCl) while decreasing the concentration. The sensor chip was regenerated with PBS, pH 7.4 after each cycle. Specific binding of the SA fusion protein to FcRn was calculated by comparing it with a defunctionalized channel used as a reference. The K values ​​of SA-IL-10 and CBD-SA-IL-10 were determined by fitting a 1:1 Langmuir binding model to the data using BIAevaluation software (GE Healthcare). d The value was determined.

[0232] 4. Mouse Seven-week-old BALB / c female mice and eight-week-old DBA / 1J male mice were obtained from the Jackson Laboratory. The experiment was conducted with the approval of the University of Chicago's Institutional Animal Care and Use Committee.

[0233] 5. Binding of proteins to spleen cells or LN-derived cells Single-cell suspensions were obtained by gently disrupting the spleen and popliteal LN through a 70 μm cell strainer. Red blood cells were lysed with ACK lysis buffer for splenocytes (Quality Biological). Cells were counted and resuspended in RPMI-1640 supplemented with 10% FBS and 1% penicillin / streptomycin (all from Life Technologies). 1 × 10⁶ 5Cells were seeded at 1 / well in a 96-well microplate and incubated on ice for 30 minutes with 2 μg / 100 μL of SA, SA-IL-10, or CBD-SA-IL-10. After four washes with PBS, the cells were further incubated on ice for 20 minutes with anti-mouse albumin antibody (abcam). After three washes with PBS, cells were incubated on ice for 20 minutes with 1 μg / mL AlexaFluor 647-labeled anti-rabbit IgG (Jackson ImmunoResearch), anti-B220 (RA3-6B2, BioLegend), anti-CD3 (145-2C11, BD Biosciences), anti-CD4 (RM4-5, BD Biosciences), anti-CD8 (53-6.7, BD Biosciences), anti-CD11c (HL3, BD Biosciences), anti-CD45 (30-F11, BD Biosciences), and anti-F4 / 80 (T45-2342, BD Biosciences) antibodies. Cells were analyzed by flow cytometry as described below.

[0234] 6. Plasma pharmacokinetics of proteins IL-10, SA-IL-10, or CBD-SA-IL-10 (equivalent to 35 μg of IL-10) was intravenously injected into female BALB / c mice. Blood samples were collected in low-protein-binding tubes at 1, 5, 10, and 30 minutes post-injection, as well as at 1, 4, 8, and 24 hours, and then incubated overnight at 4°C. Serum IL-10 concentrations were measured using the IL-10 Mouse Uncoated ELISA kit (Invitrogen) according to the manufacturer's protocol. Exponential biphase decay (Y = Ae -αt + Be -βt The half-life was calculated using fitting. Fast clearance half-life, t 1 / 2,α ; Slow clearance half-life, t 1 / 2,β The data was analyzed using Prism software (v8, GraphPad).

[0235] 7. CAIA Model Arthritis was induced in female BALB / c mice by intraperitoneal injection of an anti-collagen antibody cocktail (1.0 mg / mouse, Chondrex) on day 0, followed by intraperitoneal injection of LPS (25 μg / mouse, Chondrex) on day 3. In the case of Figures 3B-C only, 1.5 mg / mouse of the anti-collagen antibody cocktail was injected. On day 3, prior to LPS injection, mice were injected intravenously, subcutaneously (mid-dorsal), or via the sole of the foot with PBS, wt IL-10, SA-IL-10, CBD-SA-IL-10 (each equivalent to 43.5 μg of IL-10), or 200 μg of rat anti-mouse TNF-α antibody (clone XT3.11, Bio X Cell). Joint swelling was scored daily according to the manufacturer's protocol (Chondrex). On the final day of scoring, the hind paws were fixed in 10% neutral formalin (Sigma-Aldrich), decalcified in Decalcifer II (Leica), and then subjected to histological analysis. The paraffin-embedded paws were sliced ​​to a thickness of 5 μm and stained with H&E. Images were scanned with a Pannoramic digital slide scanner and analyzed using Pannoramic Viewer software. The severity of bone resorption and synovial hyperplasia in the arthritis model was scored using a three-point scale (0-2) following previously reported criteria with slight modifications: 0, normal to minimal infiltration of the pannus in the subchondral bone of the cartilage and marginal zone; 1, mild to moderate infiltration of the marginal zone with mild cortical and medullary bone destruction; 2, severe infiltration associated with complete or near-complete destruction of the joint structure. The scores for both hind paws were totaled for each mouse (total score per mouse, 0-4). Histopathological analysis was performed blinded.

[0236] 8. CIA Model Male DBA / 1J mice (8 weeks old) were immunized by subcutaneous injection of bovine collagen / complete Freund's adjuvant (CFA) emulsion (Hooke Kit, Hooke Laboratories) at the base of the tail. Three weeks later, a replenishment antigen injection of bovine collagen / incomplete Freund's adjuvant (IFA) emulsion (Hooke Kit, Hooke Laboratories) was administered. After the replenishment antigen injection, the mice were examined daily, and joint swelling was scored according to the manufacturer's protocol (Hooke Laboratories). When the total score was between 2 and 4 (defined as day 0), the mice were intravenously injected with PBS, SA-IL-10, CBD-SA-IL-10 (each equivalent to 43.5 μg of IL-10), or 200 μg of rat anti-mouse TNF-α antibody (clone XT3.11, Bio X Cell). On the final day of scoring, the hind legs were collected and histological analysis was performed as described above.

[0237] 9. In vivo biodistribution studies To produce fluorescently labeled proteins, wt IL-10, SA-IL-10, and CBD-SA-IL-10 were incubated in an 8-fold molar excess of DyLight 800 NHS ester (Thermo Fisher) at room temperature for 1 hour. Unreacted dyes were removed using a Zebaspin spin column (Thermo Fisher) according to the manufacturer's instructions. BALB / c mice were intraperitoneally injected with an anti-collagen antibody cocktail (1.0 mg / mouse) on day 0, followed by an injection of 10 μg of LPS into the right hind leg on day 3. The following day, 20 μg of DyLight 800-labeled protein was intravenously injected. Four hours later, organs taken from the disease model were imaged using the Xenogen IVIS Imaging System 100 (Xenogen) under the following conditions: f / stop: 2; optical filter excitation 745 nm; excitation 800 nm; exposure time: 5 seconds; small binning. To normalize the fluorescence signals from each organ, the weight of each organ was measured.

[0238] 10. LN microscopy BALB / c mice were intravenously injected with equimolar amounts of DyLight594-labeled wt IL-10 (43.5 μg) or SA-IL-10 labeled with dye. 24 hours after injection, popliteal lymph nodes (LNs) were harvested and frozen in dry ice with an optimal cutting temperature (OCT) compound. Tissue slices (10 μm) were obtained from the frozen sections. The tissue was fixed with 2% paraformaldehyde in PBS at room temperature for 15 minutes. After washing with PBS-T, the tissue was blocked with 2% BSA in PBS-T at room temperature for 1 hour. The tissue was stained with anti-mouse CD3 antibody (1:100, 145-2C11, BioLegend) or anti-mouse peripheral lymph node addressin (PNAd) antibody (1:200, MECA79, BioLegend) and Alexa Fluor 488 donkey anti-rat antibody (1:400, Jackson ImmunoResearch). After washing the tissue three times, it was covered with Prolong gold antifade mountant containing 4',6-diamidino-2-phenylindole (DAPI; Thermo Fisher Scientific). For CD3 staining, an IX83 microscope (Olympus) was used for imaging at 10x magnification, and for PNAd staining, a Leica SP8 3D laser scanning confocal microscope was used at 20x magnification. Images were processed using ImageJ software (NIH).

[0239] 11. LN Pharmacokinetics CAIA mice were intravenously injected with wt IL-10, SA-IL-10, or CBD-SA-IL-10 (each equivalent to 35 μg of IL-10). LNs from the popliteal fossa, mesentery, and neck were collected 30 minutes after injection, and at 1, 4, 8, and 24 hours. These were then homogenized using Lysing Matrix D and FastPrep-24 5G (MP Biomedical) at 5000 beats / min for 40 seconds in T-PER tissue protein extraction reagent (Thermo Scientific) containing cOmplete® proteinase inhibitor cocktail (Roche). After homogenization, samples were incubated overnight at 4°C. Samples were centrifuged (5000 g, 5 min), and total protein and IL-10 concentrations were analyzed using a BCA assay kit (Thermo Fisher) and an IL-10 Mouse Uncoated ELISA kit (Invitrogen), respectively. Simultaneously, cytokine levels in LN extracts were measured using either the Mouse Uncoated ELISA kit (Invitrogen) or the Ready-SET-Go! ELISA kit (eBioscience) according to the manufacturer's protocol. To detect GM-CSF, CAIA mice were intravenously injected twice at 3-day intervals with either wt IL-10 or SA-IL-10 (equivalent to 35 μg of IL-10 each). Popliteal LN was collected the day after the last injection for GM-CSF detection.

[0240] 12. Flow cytometry CAIA mice were intravenously injected with PBS, wt IL-10, SA-IL-10, or CBD-SA-IL-10 (each equivalent to 43.5 μg of IL-10). After 8 days, blood and hind feet were collected. Red blood cells in the blood were lysed with ACK lysis buffer (Quality Biological) and then stained with antibodies for flow cytometry. The feet were digested at 37°C for 60 minutes in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 2% FBS, 2 mg / mL collagenase D, and 40 μg / mL DNase I (Roche). Single-cell suspensions were obtained by gently disrupting the cells through a 70 μm cell strainer. Antibodies against the following molecules were used: anti-mouse CD3 (145-2C11, BD Biosciences), CD4 (RM4-5, BD Biosciences), anti-mouse CD8α (53-6.7, BD Biosciences), anti-mouse CD25 (PC61, BD Biosciences), anti-mouse CD45 (30-F11, BD Biosciences), CD44 (IM7, BD Biosciences), CD62L (MEL-14, BD Biosciences), PD-1 (29F.1A12, BD Biosciences), NK1.1 (PK136, BD Biosciences), Foxp3 (MF23, BD Biosciences), F4 / 80 (T45-2342, BD Biosciences), MHC II (M5 / 114.15.2, BioLegend), CD206 (C068C2, The cells used were BioLegend, Ly6G (1A8, BioLegend), Ly6C (HK1.4, BioLegend), CD11b (M1 / 70, BioLegend), CD11c (HL3, BD Biosciences), and B220 (RA3-6B2, BioLegend). Identification of fixable live / dead cells was performed using Fixable Viability Dye eFluor 455 (eBioscience) according to the manufacturer's instructions.Unless otherwise instructed, staining was performed on ice for 20 minutes, and intracellular staining was carried out using the Foxp3 staining kit according to the manufacturer's instructions (BioLegend). After the washing step, cells were stained with specific antibodies on ice for 20 minutes before fixation. All flow cytometry analyses were performed using a Fortessa (BD Biosciences) flow cytometer and analyzed using FlowJo software (Tree Star).

[0241] 13. Safety Evaluation BALB / c mice were intravenously injected with PBS, wt IL-10, SA-IL-10, or CBD-SA-IL-10 (each equivalent to 43.5 μg of IL-10). Two days after injection, blood samples collected from the mice were analyzed using a COULTER Ac·T 5diff CP blood analyzer (Beckman Coulter) according to the manufacturer's instructions. Spleen weight was also measured. Serum samples collected from mice injected with protein were analyzed using a Biochemistry Analyzer (Alfa Wassermann Diagnostic Technologies) according to the manufacturer's instructions.

[0242] 14. Statistical analysis Statistical significance between experimental groups was determined using Prism software (v8, GraphPad). Using one-way ANOVA followed by Tukey's HSD post-hoc test, the variances between groups were found to be similar by the Brown-Forsyth test. For nonparametric data, the Kruskal-Wallis test followed by Dunn's multiple comparison test was used. For single comparisons, the two-tailed Student's t-test was used. (Symbol) * , ** , *** and **** These values ​​show p-values ​​less than 0.05, 0.01, 0.001, and 0.0001, respectively; ns, not significant.

[0243] J. References The following references and publications referenced throughout this specification are incorporated herein by reference to the extent that they provide exemplary procedures or other details that supplement what is described herein. TIFF0007870524000058.tif121160TIFF0007870524000059.tif245160TIFF0007870524000060.tif245160TIFF0007870524000061.tif165160

[0244] Example 3: Long-term retention of albumin-fused IL-4 in secondary lymphoid organs via FcRn improves experimental autoimmune encephalomyelitis. Multiple sclerosis (MS) is a common and severe demyelinating autoimmune disease of the central nervous system. Interleukin (IL)-4 suppresses the development of pathogenesis in experimental autoimmune encephalomyelitis (EAE), a mouse model of MS, but IL-4 has not been clinically transferred. Herein, we manipulate a serum albumin (SA)-IL-4 fusion protein (SA-IL-4) to target secondary lymphoid organs (SLOs), where antigen-specific T cell priming primarily occurs. SA-IL-4 showed higher accumulation and residence time in lymph nodes (LNs) and spleens compared to wild-type (wt) IL-4 via neonatal Fc receptor (FcRn) binding. Subcutaneous administration of SA-IL-4 prevented the development of EAE disease in all mice and demonstrated higher therapeutic efficacy compared to FTY720 and wt IL-4. SA-IL-4 prevents the infiltration of immune cells into the spinal cord, maintaining spinal cord structure and consequently promoting neurological function. SA-IL-4 is antigen-reactive CD4 +SA-IL-4 reduced integrin expression in T cells and impaired cell migration ability. In the spinal cord inflow area (dLN), SA-IL-4 increased the number of granulocyte-like myeloid suppressor cells, the major suppressors of EAE disease, and the expression of programmed death ligand-1 on these cells. SA-IL-4 reduced the number of Th17 cells, the pathogenic cell population of EAE disease. In the chronic phase of EAE, SA-IL-4 also showed remarkable therapeutic effects, including inhibition of immune cell infiltration into the spinal cord and complete suppression of the immune response to myelin antigens in the spleen. Manipulated SA-IL-4 demonstrates the potential for technology transfer of MS as both a prophylactic and therapeutic measure via accumulation in the spinal cord.

[0245] Multiple sclerosis (MS) is a potentially disabling autoimmune disease affecting millions of people worldwide. Autoreactive immune cells migrate to the central nervous system (CNS), causing demyelination and resulting in localized damage to the white matter (1). Lymphocytes and macrophages infiltrating the CNS cause axonal damage. Recent studies have shown that Th17 cells activated in secondary lymphoid organs (SLOs) migrate to the spinal cord and brain, playing a crucial role in the onset and severity of MS (20). Therefore, inhibiting lymphocyte migration to the CNS and inducing an immunosuppressive microenvironment in SLOs would offer an effective treatment for MS. FTY720 and anti-integrin α4 antibodies are used in clinics to inhibit lymphocyte migration (3,4). Experimental autoimmune encephalomyelitis (EAE) is a widely accepted mouse model of MS that reflects many features of disease progression and pathogenesis, including lymphocyte migration to the CNS and demyelination.

[0246] A. SA-IL-4 binds to immune cells and inhibits Th17 differentiation. The inventors recombinantly expressed wild-type (wt) mouse IL-4 and mouse SA-fused mouse IL-4 (Figure 18A). SDS-PAGE revealed that SA fusion to IL-4 increased its molecular size. When added to newly isolated immune cells from the lung nematode (LN) and spleen, SA-IL4 selectively bound to antigen-presenting cells (APCs), such as macrophages and dendritic cells (DCs), in vitro compared to other immune cells (Figure 18B).

[0247] The IL-4 receptor is expressed in T cells when stimulated (12). SA-IL-4 induced downstream phosphorylation of STAT6 in T cells at an EC50 32 times higher than wt IL-4. This suggests that wt IL-4 is more active than SA-IL-4 in vitro (Figure 18C). We have shown that both wt IL-4 and SA-IL-4 induced downstream phosphorylation of naive CD4 cells cultured in Th17 cell differentiation medium. + We found that it inhibits Th17 differentiation of T cells (Figure 18D). In summary, these results demonstrate that the inventors have successfully created a functionally active SA-IL-4 fusion protein.

[0248] SA-IL-4 increases its blood half-life and persistence in the spleen in both the pulmonary nephrocyte (LN) and the spleen.

[0249] Surface plasmon resonance (SPR) analysis revealed that SA-IL-4 has a dissociation constant (K) of 385 nM. D) demonstrated binding to FcRn (Figure 19A). The plasma half-life of SA-IL-4 after intravenous (iv) injection was remarkably prolonged, in contrast to wt IL-4 which was removed from the plasma within minutes (Figure 19B). Next, we tested whether intravenously injected SA-IL-4 accumulated in the spleen and LN using naive mice. SA-IL-4 significantly increased the amount of IL-4 after intravenous injection in both the lumbar and brachial LNs and in the spleen (Figure 2cd). In vivo distribution analysis based on fluorescence also showed enhanced SA-IL-4 accumulation in the lumbar LN compared to wt IL-4 (Figure 24). To test the involvement of FcRn in SA-IL-4 accumulation in the LN, we created a P573K point mutation in SA in fusion to IL-4 that inhibits FcRn binding (Figure 25A) (13). The SA(P573K) mutation reduced the amount of IL-4 in the LN compared to SA-IL-4, bringing it down to a level similar to wt IL-4 (Figure 19E). Although SA(P573K)-IL-4 has a longer half-life in the blood compared to wt IL-4 due to its increased molecular size, it has a shorter half-life than SA-IL-4 due to impaired FcRn binding (Figure 25B). In summary, these data suggest that FcRn binding is required for SA transport to the LN.

[0250] Following intravenous injection of SA-IL-4, histological examination of the lumbago showed increased signaling for SA-IL-4 in the lumbar lumbago compared to wt IL-4. SA-IL-4 is associated with CD3 + It did not co-localize with T cells, and was particularly localized in the subcapsular space (Figure 19G). From higher magnification, SA-IL-4 was found to be localized in peripheral lymph node addressingsin. + (PNAd + It was revealed that SA(P573K)-IL-4 partially co-localizes with the high endothelial venules shown by the cells (Figure 19H). The amount of SA(P573K)-IL-4 in the spleen was lower than that of SA-IL-4 (Figure 19F). In summary, we have shown that SA fusion to IL-4 increases the persistence of IL-4 in the LN and spleen through FcRn binding.

[0251] B. SA-IL-4 treatment is a powerful preventive measure against the development of EAE disease. Next, the inventors treated myelin oligodendrocyte glycoprotein (MOG) antigen-induced EAE with SA-IL-4 during the acute phase of EAE (Figure 20). SA-IL-4 was injected subcutaneously (sc) or intraperitoneally (ip). The inventors chose subcutaneous injection because it is clinically advantageous and the drug is slowly absorbed from the injection site. Intraperitoneal injection was performed instead of intravenous injection because the tail becomes flaccid in mice that develop EAE, making tail vein injection difficult. The inventors further compared the therapeutic effect of SA-IL-4 with that of FTY720. FTY720 is a clinically approved drug (fingolimod) for treating MS, which sequesters lymphocytes in the LN and prevents them from reacting with autoantigens in target tissues (3). Subcutaneous injection of SA-IL-4 completely suppressed the development of the disease in all mice (Figure 20A, B). Intraperitoneal injection of SA-IL-4 and FTY720 prevented the development of EAE in 4 out of 7 mice and suppressed disease severity in the remaining mice. wt IL-4 treatment did not suppress EAE clinical scores compared to the PBS-treated group, and all mice in that group developed the disease. Using body weight change as a clinical indicator of health, mice treated with PBS and wt IL-4 were observed to lose significantly more body weight (Figure 20C). Mice subcutaneously injected with SA-IL-4 gained more body weight than all other groups and indicated better health. The intraperitoneal SA-IL-4 group gained weight on average, while FTY720-treated mice maintained their body weight. Next, we analyzed spinal demyelination, the main morphological sign of EAE disease (Figure 20D). Importantly, mice subcutaneously injected with SA-IL-4 showed no detectable demyelination, demonstrating prevention of spinal cord injury. All wt IL-4 treated mice showed demyelination. The inventors then monitored the mice for an extended period up to day 24 and found that SA-IL-4 intraperitoneal injection suppressed disease onset and progression (Figure 26). Importantly, SA(P573K)-IL-4 did not suppress disease scores (Figure 27). These data suggest that SA fusion to IL-4 strongly improves the therapeutic effect of IL-4 on suppressing EAE disease.

[0252] C. SA-IL-4 treatment suppresses immune cell infiltration into the spinal cord and induces an immunosuppressive environment in the dLN. The inventors then analyzed immune cells in the spinal cord and dLN after treatment. Surprisingly, subcutaneous injection of SA-IL-4 significantly suppressed the infiltration of immune cells into the CNS; CD45 detected in the spinal cord. + Immune cells were present in very small numbers (Figure 21A). Consequently, Th17 cells in the spinal cord were hardly detectable in the subcutaneous SA-IL-4 treated group (Figure 21B). Intraperitoneal injection of SA-IL-4 suppressed immune cell infiltration in 4 out of 7 mice, which corresponds to the incidence of EAE disease in that group. As expected, FTY720 administration also suppressed immune cell infiltration into the spinal cord. wt IL-4 showed no effect on immune cell infiltration, including Th17 cells, into the spinal cord compared to PBS treatment.

[0253] The inventors then analyzed immune cells in lumbar dLN. SA-IL-4 increased granulocyte-like bone marrow-derived suppressor cells (G-MDSCs) but decreased monocyte-like MDSCs (M-MDSCs) (Figure 21C, D). CD4 in dLN + The frequency of Th17 cells within T cells was also reduced by SA-IL-4 treatment (both intraperitoneal and subcutaneous) compared to FTY720 treatment (Figure 21E). FTY720 treatment tended to increase the frequency of Th17 cells in dLNs compared to the PBS group, presumably because FTY720 inhibits lymphocyte outflow from LNs. SA-IL-4 treatment reduced the frequency of M1 macrophages and increased M2 macrophages in dLNs (Figure 21F). wt IL-4 did not reduce the frequency of M1 macrophages but increased M2 macrophages. CD11b + Frequency of macrophages within cells (Figure 28A), and CD45 +The intracellular dendritic cell (DC) frequency (Supplementary Figure 4b) was maintained. B cells have been reported to promote EAE induction by promoting T cell reactivation (14). SA-IL-4 (subcutaneous) reduced the frequency of B cells compared to both the PBS and FTY720 treatment groups (Figure 21H). In summary, these data demonstrate that SA-IL-4 treatment creates an immunosuppressive environment in the dLN, preventing the infiltration of immune cells into the spinal cord.

[0254] D. SA-IL-4 treatment is for antigen-reactive CD4 + Reduces IL-17-related cytokine and integrin expression in T cells. The inventors then analyzed the molecular mechanisms of reduced immune cell infiltration in the spinal cord and complete prevention of EAE disease by subcutaneous injection of SA-IL-4. 35~55 We found that the number of reactive T cells was maintained in all treatment groups, suggesting that SA-IL-4 does not alter antigen recognition (Figure 22A). Therefore, we hypothesized that SA-IL-4 alters T cell functionality. We first tested the T cell migration capacity. Expression levels of αLβ2 and α4β1 integrins, adhesion molecules important for lymphocyte migration (15), have been reported to be reduced by IL-4 (16). We hypothesized that SA-IL-4 treatment alters the function of T cells. 35~55 Reactive CD4 + It significantly reduced αLβ2 integrin expression on T cells, but total CD8 + We found that it was not reduced in T cells (Figure 22B-E). α4β1 integrin expression was found to be related to MOG 35~55 Reactive CD4 + In T cells, total CD8 + No significant changes were observed in T cells. Since αLβ2 integrin is essential for Th17 cell infiltration into the spinal cord (15), this suggests that downregulation of integrin expression is one of the mechanisms by which SA-IL-4 reduces lymphocyte migration into the spinal cord.

[0255] The inventors then tested PD-1 expression in T cells and PD-L1 expression in MDSCs, since the association of PD-1 and PD-L1 suppresses T cell activation (Figure 22F-K) (17). Surprisingly, SA-IL-4, not wt IL-4, was found to be in the central memory CD4 + T cells and central memory CD8 + PD-1 expression was increased in both T cells (Figure 22F,G). Furthermore, SA-IL-4, rather than wt IL-4, increased PD-L1 expression levels and the frequency of PD-L1-expressing cells in both M-MDSCs and G-MDSCs (Figure 22H-K). These data suggest that T cell suppression can be induced through MDSCs and the PD-1 / PD-L1 axis.

[0256] The inventors then analyzed the expression of Th17-related proteins. IL-23 is an important cytokine for Th17 functionality. IL-4 has been reported to bind to APC and silence IL-23 and corresponding Th17 differentiation (18). The inventors found that SA-IL-4 treatment is effective in MOG 35~55 IL-23R in the reactive T cell repertoire + We found that it reduced the frequency of cells (Figure 22L).

[0257] Next, the inventors restimulated splenocytes with MOG protein (Figure 22M,N). ELISA of the culture supernatant revealed a decrease in IL-17A expression with SA-IL-4 treatment compared to wt IL-4 treatment, compared to PBS (Figure 22M). The reduction in IL-17 expression was due to MOG in the SA-IL-4 treatment group. 35~55 This indicates a decrease in the number and / or activity level of reactive Th17 cells. Since IFNγ concentration was maintained, it was suggested that SA-IL-4 had little effect on Th1 cells (Figure 22N). SA-IL-4 tended to decrease the level of GM-CSF, which has been reported to be a pathogenic cytokine of EAE (19) (Figure 22O). Next, the inventors investigated MOG in splenic cells. 35~55Cytokine expression in T cells was examined by flow cytometry after peptide restimulation (Figure 22P). The inventors analyzed the total pathogenic cytokines of EAE: GM-CSF, IL-17, IFNγ, and TNFα. SA-IL-4 showed CD4 expression compared to other treatments. + The frequency of cytokine-expressing cells within the T cell compartment was reduced. These results strongly suggest that Th17 cells in SA-IL-4 treated mice were less pathogenic and less virulent compared to other treatment groups. In summary, these data suggest that SA-IL-4 modulates multiple immune cell responses in SLO and suppresses the development of EAE disease by preventing immune cell infiltration into the spinal cord, particularly T cell infiltration.

[0258] E. SA-IL-4 restores paralysis caused by chronic EAE. To determine whether SA-IL-4 has a therapeutic effect in the chronic phase of EAE, the inventors designed an experiment involving the treatment of mice that had already reached the stage of severe paralysis. The inventors initiated intraperitoneal injection of IL-4 from day 21 after induction (Figure 23A,B). Surprisingly, SA-IL-4 demonstrated therapeutic efficacy even when administered at this late point, while wt IL-4 did not. SA-IL-4 treated mice gained weight, while wt IL-4 treated mice did not, indicating recovery from the disease. The inventors then tested the effect of SA-IL-4 in the chronic phase via subcutaneous injection and compared it with oral FTY720 treatment (Figure 23C,D). As a result, mice treated with SA-IL-4 showed a tendency toward lower clinical scores compared to the other treatment groups. Mice administered with SA-IL-4 gained weight compared to the other groups. FTY720-treated mice and wt IL-4-treated mice did not gain body weight compared to PBS-treated mice.

[0259] The inventors then tested immune cell infiltration into the spinal cord 34 days after induction by flow cytometry (Figure 23E-G). SA-IL-4 and FTY720 treatments compared with PBS and wt IL-4 treatments in terms of CD4 +T cells and MOG 35~55 SA-IL-4 reduced the number of spinal cord infiltrating immune cells, including reactive Th17 cells, compared to other treatment groups. 35~55 Reactive CD4 + The number of IL-23R-expressing cells in T cells was reduced (Figure 23H). Finally, splenocytes were restimulated with MOG protein. ELISA of the culture supernatant revealed a decrease in IL-17A and GM-CSF concentrations in the SA-IL-4 treated group compared to PBS, but this was not the case after wt IL-4 and FTY720 treatment (Figure 23I,J). MOG 35~55 Flow cytometry analysis after peptide restimulation showed that SA-IL-4 was more effective in reducing CD4 compared to other treatments. + It was shown to reduce the frequency of cytokine-expressing cells within the T cell compartment (Figure 23K). In summary, these results suggest that SA-IL-4 treatment has a potent therapeutic effect in the chronic phase of EAE.

[0260] F. SA-IL-4 did not show significant toxicity after systemic injection. To test whether SA-IL-4 exhibits any adverse effects, the inventors analyzed serum using a biochemical analyzer and blood using a hematological analyzer (Figure 29). SA-IL-4 treatment did not increase organ damage markers or alter blood cell counts (Figures 29A-M). SA-IL-4 and wt IL-4 induced splenomegaly (Figure 29N). wt IL-4 induced pulmonary edema, indicated by increased pulmonary water content, but SA-IL-4 did not (Figure 29N). These findings suggest that SA-IL-4 is safe after systemic administration.

[0261] While several MS treatments are currently available in clinics, the disease remains underdeveloped. Several effective therapies, such as those with FTY720 (fingolimod) and anti-α4 integrin (natalizumab), inhibit the infiltration of effector lymphocytes into lesional tissue, which is associated with an increased risk of immune-related adverse events (20). IFNβ functions through multiple mechanisms, including reducing lymphocyte migration into the CNS (21). Although IFNβ is not as effective as fingolimod (22), it is used clinically to modulate the pathogenesis of MS (23). Herein, we seek therapies that can shape the immune response toward a more tolerative phenotype without adverse effects, using IL-4 to deviate from the Th17 pathway known to be involved in the pathogenesis of the disease.Herein, we utilize molecular manipulation techniques to target IL-4 to SLO and improve the underlying autoimmune response to myelin antigens.

[0262] In this study, subcutaneous injection of SA-IL-4 prevented the development of EAE in all mice tested. Surprisingly, SA-IL-4 potently reduced lymphocyte infiltration into the spinal cord when administered at both early and late time points. Analysis of dLN revealed that SA-IL-4 increased M2 macrophages and G-MDSCs, as well as decreasing Th17 cells. However, wt IL-4 also increased the number of M2 macrophages, suggesting that IL-4-mediated suppression of inflammatory macrophages is insufficient to control EAE under these experimental conditions.

[0263] IL-4 has been reported to maintain and increase the immunosuppressive properties of MDSC (24). G-MDSC is an important population that suppresses the onset of EAE (17). G-MDSC expresses PD-L1 and induces functional suppression of T cells. On the other hand, M-MDSC was decreased by SA-IL-4 treatment. The role of M-MDSC in EAE is debatable, and pathogenic effects have been reported (25). Interestingly, SA-IL-4 enhanced the expression of PD-L1 in both G-MDSC and M-MDSC in the SLO, while wt IL-4 did not. At the same time, SA-IL-4 enhanced the expression of PD-1 in both CD4 + and CD8 + central memory T cells in the SLO, while wt IL-4 did not. The inventors believe that this PD-1 / PD-L1 induction effect is the mechanism by which MDSC suppresses pathogenic T cells in SA-IL-4-treated mice, and the observation of this phenomenon in the SLO demonstrates the value of the SLO targeting ability conferred by SA fusion.

[0264] Th17 cells play an important role in the severity and onset of EAE disease (26). SA-IL-4 treatment reduced the frequency of Th17 cells in the dLN compared to FTY720 treatment. IL-4 directly inhibits the differentiation of naive T cells into Th17 cells, as shown by the inventors in Figure 18D. Furthermore, there is a possibility of an APC-mediated Th17 inhibition pathway. IL-23 is expressed by APCs (26), generates pathogenic Th17 cells, and induces the expression of IL-17 (27). IL-4 has been reported to reduce the expression of IL-17 through silencing of IL-23 in APCs (18). Therefore, it is possible that SA-IL-4 acts on APCs to reduce the expression of IL-23, thereby blocking the generation of pathogenic Th17 cells in the SLO. This model is consistent with the inventors' data that SA-IL-4 reduces the pathogenicity of T cells via GM-CSF + since GM-CSF + is a marker of pathogenic Th17 cells.

[0265] Furthermore, SA-IL-4 treatment can lead to MOG-reactive CD4 + A decrease in integrin expression in the T cell compartment was induced. Although immunized mice had already initiated an anti-MOG response before treatment, SA-IL-4 was able to prevent the migration of autoimmune cells to the CNS. Therefore, SA-IL-4 treatment inactivated T cell function rather than altering the number of antigen-reactive T cells. Overall, these data indicate that SA-IL-4 acts through multiple immune pathways to create an immunosuppressive environment in SLOs.

[0266] FTY720 is one of the FDA-approved drugs for MS, and it works by inducing lymphopenia, thus limiting the migration of effector lymphocytes to the CNS disease site. Subcutaneously administered SA-IL-4 showed higher efficacy than FTY720 in this study. This improvement with SA-IL-4 is strong evidence of its potential for clinical technology transfer. FTY720 has the disadvantage of not being able to be administered to infected patients. Also, among the approved MS treatments that modulate lymphocyte migration, such as FTY720 and integrin α4 blockade, induction of John Cunningham virus (JCV) activity can lead to progressive multifocal leukoencephalopathy (PML), which can ultimately cause serious adverse events, including death. SA-IL-4 is antigen-reactive CD4 + Integrin expression was reduced only in T cells, but CD8 + It was not reduced in T cells. This is because SA-IL-4 is antigen-reactive CD4 +It exerts a further inhibitory effect on T cell migration, suggesting that this may contribute to the suppression of inflammation in the spinal cord. Hematological studies showed that SA-IL-4 did not induce lymphopenia. This may be a further advantage over approved drugs, as SA-IL-4 is not expected to strongly suppress the immune response to infection, and therefore may be useful for patients who are not suitable for treatment with FTY720. SA-IL-4 is likely to suppress autoimmunity through various mechanisms that do not overlap with current therapies. In this study, FTY720 did not suppress Th17 development in dLN, but SA-IL-4 did. Therefore, SA-IL-4 may be useful for patients who do not achieve sufficient therapeutic effects with current treatments.

[0267] Intradermal or subcutaneous injection of SA has been well studied for its accumulation in the injection site-inflow region (LN) (28). In this specification, we have shown that SA-IL-4 accumulates in the LN after intravenous injection, i.e., from the blood rather than via the afferent lymphatic vessels. Previous in vivo distribution studies of SA-fused IL-2 have shown splenic, hepatic, and LN accumulation of IL-2 via intravenous injection (29), but the molecular mechanisms and its localization within the LN remained unclear. Histological analysis has shown that intravenously injected SA-IL-4 accumulates and co-localizes around high endothelial venules (HEVs) (30). While transcytoplasmic transport of IgG via FcRn has been well studied, transcytoplasmic transport of albumin via FcRn binding has only recently been reported by several research groups (31-33). Since FcRn is highly expressed in the LN (34), intravenously injected SA-IL-4 was likely transported to the LN via transcytoplasmic transport. In this study, SA(P573K)-IL-4 achieved even lower levels of IL-4 in the LN compared to SA-IL-4, as well as wt IL-4. This suggests that FcRn binding plays a crucial role in the transport of SA-IL-4 from the blood to the LN. From our observation of SA-IL-4 concentrating around the medullary space where APCs are present, we hypothesize that SA-IL-4 enters through the HEV and then binds to DCs and macrophages, as we show in Figure 18. Since SA(P573K)-IL-4 did not suppress the disease score in Figure 27, it is suggested that FcRn binding and subsequent increased persistence in the SLO are important for suppressing the symptoms of EAE disease. Therefore, the inventors believe that SA-cytokine fusion immunosuppressive molecules function not primarily by extending blood circulation time, but by extending recycling time in SLOs, such as the LN and spleen. Thus, SA-fusion cytokines can be collected by one immune cell, recycled by that cell, and used to stimulate other immune cells. Consequently, the lifespan of blood circulation is not directly related to the lifespan of SLO presence.The inventors believe that this biological finding can open up a new field of research on LN molecule transport, and further research will reveal the detailed mechanism.

[0268] SA-IL-4 demonstrated therapeutic efficacy in the chronic phase of EAE. Nasal or lumbar administration of IL-4 has previously been reported to result in direct binding of IL-4 to neurons in attempts to regenerate the nervous system in EAE (9). The therapeutic effect of intraperitoneal injection of SA-IL-4 was comparable to that of nasal-administered wt IL-4 in previous reports. This suggests that SA-IL-4 may also bind to neurons and induce regeneration. Because the blood-brain barrier is disrupted in the EAE model, SA-IL-4 may be able to access neurons within the spinal cord. Further research into the direct effects of SA-IL-4 on nerve regeneration would be interesting.

[0269] Subcutaneous administration of SA-IL-4 showed a higher preventive effect than intraperitoneal administration. This route is convenient for clinical technology transfer and generally exhibits sustained release from tissues. The inventors hypothesize that this sustained release was a further factor in the suppression of EAE development.

[0270] SA-IL-4 administered by systemic injection showed no apparent toxicity in blood biochemical and hematological analyses. The inventors observed splenomegaly, but this was typically transient and not considered a serious toxicity. While wt IL-4 induced pulmonary edema, this was more concerning. However, pulmonary edema was not observed with SA-IL-4; this difference may be due to the reduced activity of SA-IL-4 in activating STAT6. These results indicate a low risk of inducing adverse events with SA-IL-4 administration in a clinical setting.

[0271] In conclusion, engineered SA-IL-4 demonstrated persistence in SLOs through FcRn binding. SA-IL-4 showed significant therapeutic effects at multiple stages of EAE after systemic injection, modulating key immune pathways in EAE, such as Th17 cell reduction and increased G-MDSC and PD-L1 expression by MDSCs in SLOs. SA-IL-4 has potential for technology transfer in both prophylactic and therapeutic uses through a novel biological approach that utilizes a different mechanism than currently approved therapies.

[0272] Amino acid sequences of G. wt IL-4 and SA-IL-4 TIFF0007870524000062.tif106160

[0273] H. Materials and Methods 1. Production and purification of recombinant proteins Sequences encoding mouse SA (amino acid numbers 25-608 of whole serum albumin), mouse IL-4, and the (GGGS)2 linker, which lack propeptides, were synthesized and subcloned into the mammalian expression vector pcDNA3.1(+) using Genscript. For further purification of the recombinant protein, the sequence encoding 6Hi was added to the C-terminus. The amino acid sequence of the protein is shown in Supplementary Table 1. HEK-293F cells adapted to suspension were routinely maintained in serum-free FreeStyle 293 expression medium (Gibco). On the day of transfection, cells were reduced to 1 × 10⁶ 6Fresh medium was inoculated at a density of 10 cells / ml. Plasmid DNA at 2 μg / ml, linear 25 kDa polyethyleneimine at 2 μg / ml (Polysciences), and OptiPRO SFM medium (final concentration 4%, Thermo Fisher) were added sequentially. The culture flask was agitated by orbital shaking at 135 rpm at 37°C in the presence of 5% CO2. Seven days after transfection, the cell medium was collected by centrifugation and filtered through a 0.22 μm filter. The medium was loaded onto a HisTrap HP 5 ml column (GE Healthcare) using AKTA pure 25 (GE Healthcare). After washing the column with wash buffer (20 mM NaH2PO4, 0.5 M NaCl, pH 8.0), the protein was eluted with a gradient of 500 mM imidazole (in 20 mM NaH2PO4, 0.5 M NaCl, pH 8.0). The protein was further purified by size exclusion chromatography using a HiLoad Superdex 200PG column (GE Healthcare) with PBS as the eluent. All purification steps were performed at 4°C. The expressed protein was confirmed to be over 90% pure by SDS-PAGE. The purified protein was tested for endotoxin via the HEK-Blue TLR4 receptor cell line, and the endotoxin level was confirmed to be less than 0.01 EU / mL. The protein concentration was determined from the absorbance at 280 nm using NanoDrop (Thermo Scientific).

[0274] 2. Mouse Eight-week-old C57BL / 6 female mice were obtained from Charles River Laboratories. The mice were housed at the University of Chicago's animal facility for at least one week prior to immunization. All experiments were conducted with the approval of the University of Chicago's Animal Care and Use Committee.

[0275] 3. Binding of proteins to spleen cells or LN-derived cells A single cell suspension was obtained by gently disrupting the spleen or popliteal lymph nodes through a 70 μm cell strainer. Red blood cells were lysed with ACK lysis buffer (Quality Biological) for splenocytes. Cells were counted and resuspended in RPMI-1640 supplemented with 10% FBS and 1% penicillin / streptomycin (all from Life Technologies). 1×10 5 cells / well were seeded into 96-well microplates and incubated with 2 μg / 100 μl of SA, SA-IL4 on ice for 30 minutes. After four washes with PBS, cells were further incubated with rabbit monoclonal anti-mouse serum albumin antibody (clone EPR20195 abcam) on ice for 20 minutes. After three washes with PBS, cells were incubated with 1 μg / ml AlexaFluor 647-labeled anti-rabbit IgG antibody, anti-B220 antibody, anti-CD3 antibody, anti-CD4 antibody, anti-CD8 antibody, anti-CD11c antibody, anti-CD45 antibody and anti-F4 / 80 antibody on ice for 20 minutes. Cells were analyzed by flow cytometry as described below.

[0276] 4. Analysis of STAT6 phosphorylation by flow cytometry EasySep Mouse CD4 + T cell isolation kit (Stem Cell) was used to purify mouse CD4 + T cells from the spleens of C57BL / 6 mice. The purified CD4 + T cells (10 6 cells / ml) were pre-coated with 5 μg / ml anti-CD3 antibody (clone 17A2, Bioxcell) and activated in 6-well plates supplemented with soluble 2 μg / ml anti-CD28 antibody (clone 37.51, BioLegend) for 2 days. The medium was IMDM (Gibco) containing 10% heat-inactivated FBS, 1% penicillin / streptomycin and 50 μM 2-mercaptoethanol (Sigma Aldrich). After 2 days of culture, activated CD4 +T cells were stimulated with 50 ng / ml recombinant mouse IL-2 (Peprotech) for 3 hours to induce IL-4Rα expression. After IL-2 stimulation, the cells were washed and allowed to stand in fresh medium for 3 hours. The cells were then transferred to a 96-well plate (50,000 cells / well). The indicated amount of wt IL-4 or SA-IL-4 was added to CD4. + T cells were exposed to 37°C for 15 minutes to induce STAT6 phosphorylation. The cells were immediately fixed with BD Phosflow Lyse / Fix buffer at 37°C for 10 minutes, and then permeabilized on ice with BD Phosflow Perm Buffer III for 30 minutes. Cells were stained with AlexaFluor 647 anti-pSTAT6 antibody (clone J71-773.58.11, BD), which recognizes Tyr641 phosphorylation. Staining was performed in the dark at room temperature (RT) for 1 hour. Cells were acquired using BD LSR, and data were analyzed using FlowJo (Treestar). + The mean fluorescence intensity (MFI) of the population was plotted against cytokine concentration. Dose-response curves were fitted using Prism (v8, GraphPad).

[0277] 5. Surface Plasmon Resonance (SPR) SPR measurements were performed using a Biacore X100 SPR system (GE Healthcare). Mouse FcRn recombinant protein (Acro Biosystems) was immobilized on a C1 chip (GE Healthcare) via amine coupling at approximately 200 resonance units (RUs) according to the manufacturer's instructions. SA-IL4 was flowed at 30 μL / min in running buffer (0.01 M monobasic anhydrous sodium phosphate, pH 5.8, 0.15 M NaCl) while decreasing the concentration. The sensor chip was regenerated with PBS, pH 7.4 after each cycle. Specific binding of the SA fusion protein to FcRn was calculated by comparing it with a defunctionalized channel used as a reference. The experimental results were fitted to Langmuir binding kinetics using BIAevaluation software (GE Healthcare).

[0278] 6. Differentiation of Th17 cells in vitro under culture conditions Follow the manufacturer's instructions for the EasySep® Mouse Naive CD4. + Using the T Cell Isolation Kit (STEMCELL Technologies), naive CD4 cells were extracted from spleen cells. + T cells were isolated. 10 5 The cells were plated into a 96-well plate and cultured for 3 days. As Th17 induction medium,

[0279] The concentration of IL-17A in the culture medium was measured using the IL-17 Ready-Set-Go! Mouse Uncoated ELISA Kit (Invitrogen) according to the manufacturer's protocol. The data was analyzed using Prism software (v6, GraphPad).

[0280] 7. Plasma pharmacokinetics of proteins wt IL-4 or SA-IL-4 (equivalent to 10 μg of IL-4) was intravenously injected into female C57BL / 6 mice. Blood samples were collected in low-protein-binding tubes at 1 minute, 10 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, and 24 hours after injection. Plasma IL-4 concentrations were measured using the IL-4 Ready-Set-Go! Mouse Uncoated ELISA kit (Invitrogen) according to the manufacturer's protocol.

[0281] 8. Pharmacokinetics of proteins in lymph nodes and spleen wt IL-4, SA-IL-4, or SA(P573K)-IL-4 (equivalent to 40 μg of IL-4) were intravenously injected into healthy C57BL / 6 mice. LNs from the lumbar and upper arm, as well as the spleen, were collected at 1, 4, and 24 hours post-injection. These samples were then homogenized using Lysing Matrix D and FastPrep-24 5G (MP Biomedical) at 5000 beats / min for 40 seconds in T-PER tissue protein extraction reagent (Thermo Scientific) containing the cOmplete® proteinase inhibitor cocktail (Roche). After homogenization, samples were incubated overnight at 4°C. Samples were centrifuged (5000 g, 5 min), and total protein and IL-4 concentrations were analyzed using a BCA assay kit (Thermo Fisher) and an IL-4 Mouse Uncoated ELISA kit (Invitrogen), respectively. Simultaneously, cytokine levels in LN extracts were measured using either the Mouse Uncoated ELISA kit (Invitrogen) or the Ready-SET-Go! ELISA kit (eBioscience) according to the manufacturer's protocol.

[0282] 9. Fluorescence-based detection of IL-4 in LN To produce fluorescently labeled wt IL-4 and SA-IL-4, the proteins were incubated in an 8-fold molar excess of DyLight 800 NHS ester (Thermo Fisher) at RT for 1 hour, and unreacted dye was removed using a Zebaspin spin column (Thermo Fisher) according to the manufacturer's instructions. 10 μg of DyLight 800-labeled wt IL-4 and SA-IL-4 with equivalent fluorescence were intravenously injected into naive C57BL / 6 mice. Four hours later, the iliac lining (LN) was imaged using a Xenogen IVIS Imaging System 100 (Xenogen) under the following conditions: f / stop: 2; optical filter excitation 745 nm; excitation 800 nm; exposure time: 5 seconds; small binning.

[0283] 10. Immunofluorescence As described above, wt IL-4 and SA-IL-4 were fluorescently labeled with DyLight 594 NHS ester (Thermo Fisher). Mice were euthanized 1 hour after intravenous injection of fluorescently labeled IL-4 (40 μg for wt IL-4 and the same fluorescence dose for SA-IL-4). Mouse linolones (LNs) were collected, fixed overnight in 2% PFA in PBS, and washed with PBS. After overnight incubation in 30% sucrose solution, the LNs were embedded in an Optimum Cutting Temperature compound. Next, 5 μm frozen sections were cut using a cryostat. Next, sections were blocked in RT with 2% BSA in PBS and incubated in RT for 2 hours with the following primary antibodies: 10 μg / ml hamster anti-mouse CD3ε antibody (clone: ​​145-2C11, BioLegend) and 2.5 μg / ml rat anti-mouse PNAd (clone: ​​MECA-79, BioLegend). After washing with PBS-T, the tissue was stained in RT for 1 hour with the following fluorescently labeled secondary antibodies: Alexa Fluor 647 goat anti-hamster (1:400, Jackson ImmunoResearch) and Alexa Fluor 488 donkey anti-rat (1:400, Jackson ImmunoResearch). After washing the tissue three times, it was covered with Prolong gold antifade mountant containing 4',6-diamidino-2-phenylindole (DAPI; Thermo Fisher Scientific). For CD3 staining, an IX83 microscope (Olympus) was used for imaging at 10x magnification, and for PNAd staining, a Leica SP8 3D laser scanning confocal microscope was used at 20x magnification. Images were processed using ImageJ software (NIH).

[0284] 11. EAE Model MOG was injected into the dorsal ventral flank of 9-12 week old C57BL / 6 juvenile female mice. 35~55Subcutaneous immunization with a complete Freund's adjuvant (CFA) emulsion was followed by intraperitoneal administration of pertussis toxin (PTX) in PBS, first on the day of immunization and then again on the following day. MOG 35~55 CFA emulsion and PTX were purchased from Hooke Laboratories. Following the initial immunization, the severity of EAE was monitored, and clinical scores were measured daily from day 8 post-immunization. Clinical scores were determined by AI, MN, or AS based on Hooke Laboratories criteria in a blinded manner for treatment group division. IL-4, SA-IL-4, and PBS were administered intraperitoneally or subcutaneously (into the mouse's back) in 100 μl of PBS every other day. FTY720 (1 mg / kg body weight) was administered orally daily.

[0285] 12. Histological features of the spinal cord Thoracic and lumbar vertebrae were harvested from EAE mice and dissected at the thoracolumbar junction. The tissue was fixed overnight in 2% PFA. After washing with PBS, the tissue was decalcified overnight using Decalcifier II (Leica Biosystem). Next, the tissue was embedded in paraffin. After paraffin embedding, the block was cut into 5 mm sections. After deparaffinization and rehydration, the tissue sections were treated with Target Recovery Solution (S1699, DAKO) and heated in a steamer at a temperature of over 95°C for 20 minutes. The tissue sections were incubated with anti-mouse aMBP (abcam ab40390) in a humidity chamber at RT for 1 hour. After washing with TBS, the tissue sections were incubated with biotinylated anti-rat IgG (10 mg / mL, Vector Laboratories) at RT for 30 minutes. Antigen-antibody binding was detected by the Elite Kit (PK-6100, Vector Laboratories) and the DAB (DAKO, K3468) system. The slides were imaged using EVOS FL Auto (Life Technologies).

[0286] 13. Flow cytometry EAE mice were treated with PBS, wt IL-4, or SA-IL-4 (equivalent to 10 μg of IL-4) every other day starting 8 days after immunization. Spinal cord, spleen, and lumbar spinal cord tissue were collected 13, 17, or 34 days after immunization. Spinal cord tissue was digested at 37°C for 30 minutes in Dulbecco's modified Eagle medium (DMEM) supplemented with 2% FBS, 2 mg / mL collagenase D (Sigma-Aldrich), and 40 μg / mL DNase I (Roche). Single-cell suspensions were obtained by gently disrupting cells through a 70 μm cell strainer. For the spleen, erythrocytes in the blood were lysed with ACK lysis buffer (Quality Biological) and then stained with antibodies for flow cytometry.Antibodies were used against the following molecules: anti-mouse CD3ε (145-2C11, BD Biosciences), CD4 (RM4-5, BD Biosciences), anti-mouse CD8α (53-6.7, BD Biosciences), anti-mouse CD45 (30-F11, BD Biosciences), CD44 (IM7, BD Biosciences), CD62L (MEL-14, BD Biosciences), F4 / 80 (T45-2342, BD Biosciences), CD86 (GL1, BD Biosciences), CD206 (C068C2, BioLegend), Ly6G (1A8, BioLegend), Ly6C (HK1.4, BioLegend), CD11b (M1 / 70, BioLegend), CD11c (HL3, BD Biosciences), B220 (RA3-6B2, BioLegend, PD-1 (29F.1A12, BD Biosciences), PD-L1 (MIH7, BioLegend), IL-23R (O78-1208, BD Biosciences), Integrin αL (HI111, BD Biosciences), Integrin β2 (M18 / 2, BD Biosciences), Integrin β1 (HMb1-1, BD Biosciences), Integrin α4 (R1-2, BD Biosciences), GM-CSF (MP1-22E9, BD Biosciences), IL-17 (TC11-18H10.1, BD Biosciences), IFNγ (XMG1.2, BD Biosciences), TNFα (eBioscience, MP6-XT22), and RoRγt antibody (Q31-378, BD Biosciences). For the detection of MOG-recognizing T cells, use T-Select I-Ab MOG. 35~55 Tetramer-PE (MBL International Corporation) or MOG 38~49Tetramer-PE (NIH Tetramer Core Facility) was used. Identification of fixable live / dead cells was performed using Fixable Viability Dye eFluor 455 (eBioscience), Live / Dead Fixable Violet (eBioscience), or Live / Dead Fixable Aqua (eBioscience) according to the manufacturer's instructions. Staining was performed on ice for 20 minutes. For intracellular staining, cells were fixed at 4°C for 20 minutes using Cytofix / Cytoperm (BD Bioscience). For permeabilization, cells were stained in perm / wash buffer (BD Bioscience) at 4°C for 30 minutes. After the washing step, cells were stained with specific antibodies on ice for 20 minutes before fixation. All flow cytometry analyses were performed using a Fortessa (BD Biosciences) flow cytometer and analyzed using FlowJo software (Tree Star).

[0287] 14. Restimulation of splenic cells Single-cell suspensions were prepared from dLN and spleen. For cytokine production analysis, 5 × 10⁶ cells were collected. 5 Individual lymphocytes and 2 × 10⁶ 6 Individual splenocytes were plated into a 96-well round-bottom plate. Cells were treated with 10 μM MOG. 35~55 Cells were stimulated with a peptide (Genscript). After 2 hours, GolgiPlug (brefelzin A) and GolgiStop (monensin) were added according to the manufacturer's protocol to block intracellular cytokine secretion. Four hours after the addition of GolgiPlug and GolgiStop, cells were stained for flow cytometry. For fixation, Cytofix / Cytoperm (BD Bioscience) was used at 4°C for 20 minutes. For permeabilization, Perma / wash buffer (BD Bioscience) was used, and cells were stained in Perma / wash buffer at 4°C for 30 minutes. For 3 days of restimulation, 2.5 × 10⁶ cells were used. 5 individual lymphocytes or 1 × 10⁶ 6Individual splenocytes were plated into a 96-well round-bottom plate. Cells were treated with 10 μM MOG. 35~55 The mice were stimulated with either 100 μg / ml MOG protein (Anaspec) (for 6 hours of culture followed by flow cytometry) or 100 μg / ml MOG protein (for 72 hours of culture). After 72 hours, the supernatant was collected for ELISA analysis using the Ready-Set-Go! Kit (Invitrogen) or the LEGEND MAX mouse GM-CSF ELISA kit (BioLegend).

[0288] 15. Safety assessment of SA-IL-4 C57BL / 6 mice were intravenously injected with PBS, wt IL-4, or SA-IL-4 (equivalent to 10 μg of IL-4). Two days later, blood samples collected from the mice were analyzed using a COULTER Ac·T 5diff CP blood analyzer (Beckman Coulter) according to the manufacturer's instructions. Lungs and spleens were collected and weighed. Lung water content was determined by weighing before and after overnight freeze-drying using a FreeZone 6 Benchtop Freeze Dryer (Labconco). Serum samples collected from mice injected with PBS, wt IL-4, and SA-IL-4 were analyzed using a Biochemistry Analyzer (Alfa Wassermann Diagnostic Technologies) according to the manufacturer's instructions.

[0289] 16. Statistical analysis Statistical significance between experimental groups was determined using Prism software (v6 GraphPad). Using one-way ANOVA followed by Tukey's HSD post-hoc test, the variances between groups were found to be similar by the Brown-Forsyth test. For single comparisons, a two-tailed Student's t-test was used. (Symbol) * and ** These represent p-values ​​less than 0.05 and 0.01, respectively.

[0290] I. References TIFF0007870524000063.tif92160TIFF0007870524000064.tif245160TIFF0007870524000065.tif245160

[0291] While certain embodiments have been described above with some degree of specificity, or with reference to one or more individual embodiments, those skilled in the art will be able to make numerous modifications to the embodiments of this disclosure without departing from the scope of the invention. Furthermore, where appropriate, aspects of any of the embodiments described above may be combined with aspects of any other embodiments described to form further embodiments having equivalent or different characteristics and addressing the same or different problems. Similarly, it will be understood that the above benefits and advantages may relate to one embodiment or to several embodiments. Any reference to a published patent application or other publication is incorporated herein by reference to the disclosures of said published application / publication. The claims should not be construed as including means-plus-function or step-plus-function limitations unless such limitations are explicitly expressed in a given claim using the phrase(s) “means for” or “step for”.

Claims

1. A composition comprising serum albumin protein, IL-4 covalently bound to the carboxyl terminus of serum albumin protein, and a collagen-binding domain located near the amino region of serum albumin protein, wherein the collagen-binding domain is derived from a von Willebrand factor (VWF) A3 domain or decorin.

2. The composition according to claim 1, wherein the serum albumin protein comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 60 or SEQ ID NO:

46.

3. The composition according to claim 1, wherein the serum albumin protein comprises the amino acid sequence of SEQ ID NO: 60 or SEQ ID NO:

46.

4. The composition according to any one of claims 1 to 3, wherein IL-4 comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 20 or SEQ ID NO:

21.

5. The composition according to any one of claims 1 to 3, wherein IL-4 comprises the amino acid sequence of SEQ ID NO: 20 or SEQ ID NO:

21.

6. The composition according to claim 1, wherein the collagen-binding domain comprises a peptide having an amino acid sequence of SEQ ID NO: 1, 7, or 47, or a peptide that is at least 95% identical to one of the fragments of SEQ ID NO: 1, 7, or 47.

7. A pharmaceutical composition for treating an autoimmune or inflammatory condition in a subject, comprising the composition according to any one of claims 1 to 6.

8. The pharmaceutical composition according to claim 7, wherein the autoimmune or inflammatory state includes inflammatory bowel disease, idiopathic pulmonary fibrosis, multiple sclerosis, type 1 diabetes, Crohn's disease, psoriasis, acute inflammation, chronic inflammation, neuritis, arthritis, rheumatoid arthritis, fibrosis, infection, allergy, adverse events related to inflammatory treatment, and inflammatory diseases related to inflammatory treatment.

9. The pharmaceutical composition according to claim 8, wherein the autoimmune condition is multiple sclerosis.

10. A pharmaceutical composition according to any one of claims 7 to 9, which is administered systemically.

11. A pharmaceutical composition according to any one of claims 7 to 10, administered by intravenous injection.

12. A pharmaceutical composition according to any one of claims 7 to 9, which is administered topically.

13. The pharmaceutical composition according to claim 12, which is administered to the site of inflammation or adjacent to the site of inflammation.

14. A pharmaceutically acceptable composition according to any one of claims 7 to 13, used in combination with additional inflammatory therapy or autoimmune therapy, and / or in combination with a second anti-inflammatory agent functionally linked to an extracellular matrix (ECM) affinity peptide.

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