Methods and compositions for treating inflammatory and autoimmune disorders with ECM-affinity peptides linked to anti-inflammatory agents

Abstract

The present disclosure relates to collagen binding modification engineering of anti-inflammatory agents using collagen binding peptides (CBP) and vWF A3 to achieve targeted therapy of inflammatory diseases. Accordingly, embodiments of the present disclosure relate to compositions comprising an anti-inflammatory agent operably linked to an extracellular matrix (ECM) affinity peptide. Also disclosed are cytokines and anti-inflammatory agents, such as CD200, linked to serum proteins and / or ECM affinity peptides. Other aspects of the present disclosure relate to methods for treating an autoimmune or inflammatory disorder in a subject comprising administering to the subject a composition of the present disclosure.

Description

[0001] Cross-references to related applications

[0002] This application claims the benefit of priority to U.S. Provisional Patent Application No. 62 / 809,988, filed February 25, 2019, the entire contents of which are incorporated herein by reference. Background Technology

[0003] I. Field of Invention

[0004] This invention generally relates to the medical field. More particularly, it relates to compositions and methods involving nucleotide constructs and proteins, including engineered anti-inflammatory agents for targeting inflamed tissues.

[0005] II. Background

[0006] Treatments targeting cytokines and their receptors have significantly 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 can produce significant side effects by suppressing systemic immunity (6-10). To improve therapeutic efficacy and reduce systemic side effects, efficient drug delivery to inflamed areas is a promising therapeutic strategy for these diseases. Inflamed tissues release a range of mediators that induce enhanced permeability retention (EPR) effects (11-12). The EPR effect is caused by loose endothelial junctions allowing extravasation of macromolecules and nonfunctional lymphatic vessels, leading to prolonged retention times of macromolecules in solid tumors and inflamed tissues (11-15). Unlike tumor tissue, inflamed tissue has a functional lymphatic system from which drugs are excreted (14-16). Currently, there are no effective methods to target inflamed tissue in inflammatory and autoimmune diseases due to rapid clearance from inflamed tissue. Therefore, there is a need in the art for therapies that directly target inflamed tissue. Summary of the Invention

[0007] This disclosure relates to collagen-binding modification engineering using collagen-binding peptides (CBPs) and vWF A3 as anti-inflammatory agents to achieve targeted therapy for inflammatory diseases. Therefore, embodiments of this disclosure relate to compositions comprising anti-inflammatory agents operatively linked to extracellular matrix (ECM) affinity peptides. Aspects of this disclosure also relate to anti-inflammatory agents operatively linked to serum proteins and compositions comprising anti-inflammatory agents operatively linked to serum proteins. Other aspects of this disclosure relate to methods for treating an autoimmune or inflammatory condition in a subject, comprising administering a composition of this disclosure to the subject.

[0008] Other aspects of this disclosure relate to methods for alleviating inflammation in a subject, comprising administering to the subject a composition comprising an anti-inflammatory agent operatively linked to an extracellular matrix (ECM) affinity peptide. In some embodiments, the inflammation is due to an autoimmune or inflammatory condition, and wherein the autoimmune or inflammatory condition includes inflammatory bowel disease, idiopathic pulmonary fibrosis, multiple sclerosis, type 1 diabetes, or arthritis.

[0009] In some embodiments, the anti-inflammatory agent operably linked to the ECM affinity peptide comprises a collagen-binding domain conjugated to anti-TNFα. In some embodiments, the anti-inflammatory agent operably linked to the ECM affinity peptide comprises vWF-A3 operably linked to IL-4. In some embodiments, the anti-inflammatory agent operably linked to the ECM affinity peptide comprises a collagen-binding domain binding to anti-TGF-β. In some embodiments, the composition is administered systemically. In some embodiments, the composition is administered topically. In some embodiments, the administered dose of the anti-inflammatory agent operably linked to the ECM affinity peptide is at least 20% less than the minimum effective dose of an anti-inflammatory agent administered topically without the peptide.

[0010] 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, anti-IL-1, anti-IL-5, anti-IL-6, anti-IL-6R, anti-IL-12, anti-IL-17A, anti-IL-18, anti-IFN-γ, anti-GM-CSF, anti-CD3, anti-CD20, anti-VLA-4, anti-VLA-5, anti-VCAM-1, anti-TGF-β1, anti-α4-integrin, anti-α4β7-integrin, anti-connective tissue growth factor, anti-platelet-derived growth factor, anti-plasminogen activator inhibitor-1, or 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.

[0011] In some implementations, the anti-inflammatory agent comprises an antigen-binding fragment of the following: anti-TNF-α, anti-IL-1, anti-IL-5, anti-IL-6, anti-IL-6R, anti-IL-12, anti-IL-17A, anti-IL-18, anti-IFN-γ, anti-GM-CSF, anti-CD3, anti-CD20, anti-VLA-4, anti-VLA-5, anti-VCAM-1, anti-TGF-β1, anti-α4-integrin, anti-α4β7-integrin, anti-connective tissue growth factor, anti-platelet-derived growth factor, anti-plasminogen activator inhibitor-1, or anti-insulin-like growth factor binding protein antibody. The antigen-binding fragment may include a variable light chain region comprising CDR1, CDR2, and CD from antibodies against anti-TNF-α, anti-IL-1, anti-IL-5, anti-IL-6, anti-IL-6R, anti-IL-12, anti-IL-17A, anti-IL-18, anti-IFN-γ, anti-GM-CSF, anti-CD3, anti-CD20, anti-VLA-4, anti-VLA-5, anti-VCAM-1, anti-TGF-β1, anti-α4-integrin, anti-α4β7-integrin, anti-connective tissue growth factor, anti-platelet-derived growth factor, anti-plasminogen activator inhibitor-1, or anti-insulin-like growth factor binding protein. R3, and / or includes a variable heavy chain region comprising CDR1, CDR2, and CDR3 from antibodies against 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 comprises adalimumab, cetrus, infliximab, golimumab, tocilizumab, rituximab, ustekinumab, natezumab, vedolizumab, secukinumab, or ixazolizumab. In some implementations, the anti-inflammatory agent comprises an antigen-binding fragment derived from: adalimumab, sertozumab, infliximab, golimumab, tocilizumab, rituximab, ustekinumab, natezumab, vedolzumab, secukinumab, or isizumab.The antigen-binding fragment may include a variable light chain region comprising CDR1, CDR2, and CDR3 from adalimumab, sertozumab, infliximab, golimumab, tocilizumab, rituximab, ustekinumab, natezumab, vedolizumab, secukinumab, or isizumab, and / or a variable heavy chain region comprising CDR1, CDR2, and CDR3 from adalimumab, sertozumab, infliximab, golimumab, tocilizumab, rituximab, ustekinumab, natezumab, vedolizumab, secukinumab, or isizumab. Examples of antigen-binding fragments derived from intact antibodies include microantibodies, scFvs, chimeric antigen receptors, and biantibodies. It is also envisioned that the antibody be derived from one or more of the following bivalent or multispecific constructs: anti-TNF-α, anti-IL-1, anti-IL-5, anti-IL-6, anti-IL-6R, anti-IL-12, anti-IL-17A, anti-IL-18, anti-IFN-γ, anti-GM-CSF, anti-CD3, anti-CD20, anti-VLA-4, anti-VLA-5, anti-VCAM-1, anti-TGF-β1, anti-α4-integrin, anti-α4β7-integrin, anti-connective tissue growth factor, anti-platelet-derived growth factor, anti-plasminogen activator inhibitor-1, or anti-insulin-like growth factor binding protein antibodies. In some embodiments, the antibody is humanized. In some embodiments, the antibody is a chimeric antibody. One or more of these antibodies or antigen-binding fragments may be specifically excluded from the embodiments.

[0012] 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.

[0013] In some embodiments, the anti-inflammatory agent comprises an anti-inflammatory cytokine peptide. In some embodiments, the cytokine peptide comprises peptides derived 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 peptide is derived from human cytokine peptides. In some embodiments, the cytokine peptide is derived from non-human cytokine peptides. In some embodiments, the cytokine peptide is derived from mouse, dog, horse, pig, or goat cytokine peptides. In some embodiments, the cytokine peptide comprises an effector region derived 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 peptide comprises a peptide derived from IL-4. In some embodiments, the cytokine peptide comprises a peptide derived from IL-1ra. In some embodiments, the cytokine peptide comprises a peptide derived from IL-5. In some embodiments, the cytokine peptide comprises a peptide derived from IL-10. In some embodiments, the cytokine peptide comprises a peptide derived from IL-11. In some embodiments, the cytokine peptide comprises peptides derived from IL-23 and IL-35. In some embodiments, the cytokine peptide comprises a peptide derived from IL-36ra. In some embodiments, the cytokine peptide comprises a peptide derived from IL-37. In some embodiments, the cytokine peptide comprises a peptide derived from interferon-β. In some embodiments, the cytokine peptide comprises a peptide derived from TGF-β1. In some embodiments, the cytokine peptide comprises a peptide derived from TNF receptor I. In some embodiments, the cytokine peptide comprises a peptide derived from TNF receptor II. One or more of these anti-inflammatory peptides may be specifically excluded from the embodiments. In some embodiments, the cytokine peptide is a human cytokine peptide or a human cytokine peptide.

[0014] In some embodiments, the cytokine polypeptide comprises a polypeptide or fragment thereof of SEQ ID NO:18 to SEQ ID NO:44, or a polypeptide or fragment thereof having an amino acid sequence 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% sequence identity (or any of the derivative ranges thereof) with respect to one of SEQ ID NO:18 to SEQ ID NO:44. In some embodiments, the anti-inflammatory agent comprises a polypeptide or fragment thereof of SEQ ID NO:58 or SEQ ID NO:59, or a polypeptide or fragment thereof 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% sequence identity (or any of the derivative ranges thereof) with an amino acid sequence of one of SEQ ID NO:58 or SEQ ID NO:59.

[0015] In some embodiments, the anti-inflammatory agent comprises a peptide derived from CD200. In some embodiments, the CD200 peptide comprises the extracellular domain of CD200. CD200 (UniProt identifier O54901) is a type I transmembrane protein that exerts immunosuppressive functions by interacting with its receptor CD200R1. When cleaved from the cell surface, the soluble extracellular domain of CD200 can still bind to CD200R and activate CD200R. Embodiments of this disclosure relate to peptides comprising at least or at most an extracellular portion of CD200 and a serum protein such as serum albumin. The peptide can be used in the method embodiments of this disclosure. Other embodiments relate to peptides comprising at least or at most an extracellular portion of CD200, serum albumin, and an ECM-affinity peptide.

[0016] In some embodiments, the ECM-affinity peptide comprises a collagen-binding domain. In some embodiments, the peptide comprises a collagen-binding domain derived from a core proteoglycan or von Willebrand factor (VWF). In some embodiments, the ECM-affinity peptide comprises a peptide derived from placental growth factor-2 (PlGF-2) or CXCL-12γ. In some embodiments, the ECM-affinity peptide comprises a peptide having at least 85% identity with one of SEQ ID NO:1 to SEQ ID NO:17, SEQ ID NO:47, or SEQ ID NO:52, or a peptide having at least 85% identity with a fragment of one of SEQ ID NO:1 to SEQ ID NO:17, SEQ ID NO:47, or SEQ ID NO:52. In some embodiments, the ECM-affinity peptide comprises a peptide having an amino acid sequence 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% sequence identity (or any range from which it can be derived), or a peptide having an amino acid sequence ... A peptide fragment of one of the amino acid sequences NO:52 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% sequence identity (or any range from which it may be derived).

[0017] In some embodiments, the anti-inflammatory agent operably linked to the extracellular matrix (ECM) affinity peptide further comprises a serum protein operably linked to the peptide or reagent. In some embodiments, the serum protein is operably linked to the peptide. In some embodiments, the serum protein is operably linked to the peptide via a peptide bond. In some embodiments, the serum protein includes albumin. In some embodiments, the anti-inflammatory agent is adjacent to the amino group of the serum protein. In some embodiments, the anti-inflammatory agent is adjacent to the carboxyl group of the serum protein. In some embodiments, the ECM affinity peptide is adjacent to the amino group of the anti-inflammatory agent. In some embodiments, the ECM affinity peptide is adjacent to the carboxyl group of the anti-inflammatory agent. In some embodiments, the serum protein is adjacent to the amino group of the ECM affinity peptide. In some embodiments, the serum protein is adjacent to the carboxyl group of the ECM affinity peptide.

[0018] When the first region is attached to the carboxyl terminus of the second region, the first region is adjacent to the carboxyl group of the second region. Additional intermediate amino acid residues may be present between the first and second regions. Therefore, these regions do not need to be adjacent unless specifically stated otherwise. A similar definition of the term "adjacent to an amino group" is that when the first region is attached to the amino terminus of the second region, the first region is adjacent to the amino group of the second region. Similarly, unless otherwise stated, intermediate amino acid residues may also be present between the first and second regions. In some embodiments, the composition comprises an amino-amino group adjacent to the collagen-binding domain of serum albumin and a carboxyl group adjacent to the IL-10 polypeptide of serum albumin.

[0019] In some embodiments, the peptide is covalently linked to an anti-inflammatory agent and / or other molecules (e.g., serum proteins). In some embodiments, the peptide is cross-linked to the anti-inflammatory agent via a bifunctional linker. A linker, such as an amino acid or peptide-like sequence, may be inserted between the peptide and / or antibody sequence. In one embodiment, the fynomer domain is immediately attached to the heavy (H) chain or light (L) chain after the last amino acid at the amino (NH2)-terminus or carboxyl (C)-terminus of the heavy (H) chain or light (L) chain. The linker may have one or more properties, including flexible conformation, inability to form ordered secondary structures, or hydrophobic or charged properties that facilitate or interact with either domain. Examples of amino acids typically found in flexible protein regions may 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 range derived therefrom). Other near-neutral amino acids, such as Thr and Ala, can 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 one particular aspect, the peptide and antibody heavy or light chain are linked by a peptide sequence having about 1 to 25 amino acid residues. Examples of linkers may also include chemical moieties and conjugators, such as sulfosuccinimide derivatives (sulfon-SMCC, sulfon-SMPB), disuccinimide succinate (DSS), disuccinimide glutarate (DSG), and disuccinimide tartrate (DST). Examples of linkers also include linear carbon chains, such as C N(Where N = 1 to 100 carbon atoms, e.g., N = 2, 3, 4, 5, 6, 7, 8, 9, or 10). In some embodiments, the linker may be a dipeptide linker, such as a valine-citrulline (val-cit) linker, a phenylalanine-lysine (phe-lys) linker, or a maleimide hexanoic acid-valine-citrulline-p-aminobenzyloxycarbonyl (vc) linker. In some embodiments, the linker is sulfosuccinimide-4-[N-maleimidemethyl]cyclohexane-1-carboxylic acid ester (smcc). The sulfonyl-smcc conjugation occurs via a maleimide group that reacts with a mercapto (thiol, -SH) group, and its sulfonyl-NHS ester is reactive to primary amines (such as those found at the N-terminus of lysine and proteins or peptides). Furthermore, the linker may be maleimide hexanoyl (mc). In some embodiments, the peptide is linked to an anti-inflammatory agent via a peptide bond. The peptide may be linked to the amino or carboxyl terminus of an anti-inflammatory agent. In some embodiments, the peptide is linked to the heavy chain of an anti-inflammatory antibody. In some embodiments, the peptide is linked to the light chain of an anti-inflammatory antibody. In some embodiments, the ratio of peptide to anti-inflammatory agent is about 1:1 to 5:1. In some embodiments, the ratio of peptide to anti-inflammatory agent is about 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 derived therefrom). One or more of these linkers may be specifically excluded from the embodiments.

[0020] In some embodiments, the composition further comprises a second anti-inflammatory agent operably linked to an extracellular matrix (ECM) affinity peptide. In some embodiments, the composition further comprises a third, fourth, fifth, or sixth anti-inflammatory agent operably linked to an extracellular matrix (ECM) affinity peptide.

[0021] In some implementation schemes, autoimmune or inflammatory conditions include inflammatory bowel disease, idiopathic pulmonary fibrosis, multiple sclerosis, type 1 diabetes, Crohn's disease, psoriasis, acute inflammation, chronic inflammation, neuroinflammation, arthritis, rheumatoid arthritis, fibrosis, infection, allergy, adverse events related to inflammation treatment, and inflammatory diseases related to inflammation treatment. One or more of these conditions may be specifically excluded from the implementation scheme.

[0022] In some embodiments, the composition is administered systemically. In some embodiments, the composition is administered intravenously. In some embodiments, the composition is administered topically. In some embodiments, the composition is applied to or adjacent to the site of inflammation.

[0023] In some embodiments, the dosage of the composition comprising an anti-inflammatory agent operably linked to a peptide is less than the minimum effective dosage of an anti-inflammatory agent administered without the peptide. In some embodiments, the dosage of the composition comprising an anti-inflammatory agent operably linked to a peptide is less than the minimum effective dosage of an anti-inflammatory agent administered via the same route of administration without the peptide. In some embodiments, the dosage of the anti-inflammatory agent operably linked to the peptide is at least 10% less than the minimum effective dosage of an anti-inflammatory agent administered without the peptide. In some embodiments, the dosage of the anti-inflammatory agent operably linked to the peptide is at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, or 70% (or any range thereof) less than the minimum effective dosage of an anti-inflammatory agent administered without the peptide.

[0024] In some embodiments, the subject has previously been treated with an anti-inflammatory agent, anti-inflammatory therapy, or autoimmune therapy. In some embodiments, it is determined that the subject has not responded to previous treatment. In some embodiments, the subject has not previously received treatment for an inflammatory or autoimmune disease. In some embodiments, the method further includes administering additional inflammatory or autoimmune therapy. In some embodiments, the method further includes administering a second anti-inflammatory agent operatively linked to an extracellular matrix (ECM) affinity peptide.

[0025] As used in this article, the term "cytokine polypeptide" refers to a polypeptide that is a cytokine or its receptor-binding domain and retains some of the cytokine activity.

[0026] When referring to gene products that contain amino acid polymers, the terms “protein,” “polypeptide,” and “peptide” are used interchangeably in this document.

[0027] The terms "object," "mammal," and "patient" are used interchangeably. In some embodiments, the object is a mammal. In some embodiments, the object is a human. In some embodiments, the object is a mouse, rat, rabbit, dog, donkey, or laboratory animal, such as a fruit fly or zebrafish.

[0028] The intended methods and compositions include any embodiments excluded herein.

[0029] As used herein, the terms “or” and “and / or” are used to describe multiple components that are combined or mutually exclusive. For example, “x, y, and / or z” can refer to a single “x”, a single “y”, a single “z”, “x, y, and z”, “(x and y) or z”, “x or (y and z)”, or “x or y or z”. In particular, x, y, or z may be specifically excluded from the implementation plan.

[0030] Throughout this specification, the term “about” is used according to its simple and general meaning in the field of cell biology to indicate the standard deviation of the error of the apparatus or method used to determine the value.

[0031] The term "comprising," "including," or "characterized by," is inclusive or open-ended and does not exclude additional, unlisted elements or method steps. The phrase "consisting of," excludes any unspecified elements, steps, or components. The phrase "substantially consisting of," limits the scope of the subject matter to the specified materials or steps that do not materially affect its essential and novel features. Embodiments contemplated in the context of the term "comprising" may also be implemented in the context of the terms "consisting of" or "substantially consisting of."

[0032] It is particularly anticipated that any limitations discussed with respect to one embodiment of the invention may be applied to any other embodiment of the invention. Furthermore, any composition of the invention can be used in any method of the invention, and any method of the invention can be used to produce or utilize any composition of the invention. Aspects of the embodiments set forth in the examples are also embodiments that may be implemented elsewhere in different embodiments or elsewhere in the application, for example, in the context of the embodiments discussed in the summary, detailed description, claims, and description of the drawings. Attached Figure Description

[0033] The following figures form part of this specification and are included to further illustrate certain aspects of the invention. A better understanding of the invention can be achieved by referring to one or more of these figures in conjunction with the detailed description of specific embodiments given herein.

[0034] Figures 1A to 1B. CBP conjugation provides collagen affinity for αTNF. (A) WT-αTNF and CBP-αTNF as analyzed by MALDI-TOF MS. The x-axis represents the mass-to-charge ratio (m / z), and the y-axis represents the intensity of the doubly charged ions. (B) Binding affinity of WT-αTNF and CBP-αTNF for type I, II, and III collagen was analyzed by ELISA (n = 3, mean + SD).

[0035] Figures 2A to 2D. CBP-αTNF accumulation in inflamed paws. Arthritis (CAIA) in the right hind paw was selectively induced by passive immunization with anti-collagen antibodies followed by subcutaneous injection of LPS in the right hind paw pad and subcutaneous injection of PBS in the left hind paw pad. On the day after LPS injection, Cy7-labeled CBP-αTNF and Cy7-labeled WT-αTNF were intravenously injected into naïve and CAIA mice. Representative images of accumulation in arthritic or non-arthritic paws of mice injected with CBP-αTNF (A) and WT-αTNF (B). (C) Changes in the ratio of radioefficiency of arthritic paw (right hind paw) to non-arthritic paw (left hind paw) in naïve 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 with anti-rat IgG).

[0036] Figure 3 A to Figure 3 B. CBP-αTNF inhibited the development of arthritis more effectively 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 was intravenously injected into arthritic mice. (A) Arthritis scores represent the mean + SE from six mice. *P<0.05, compared with the control (Dunnett's multiple comparison test). #P<0.05, compared with the scores on day 8 of each treatment group (Tukey's multiple comparison test). (B) Representative H&E images of joints on day 8 of each treatment group. The severity of synovial hyperplasia and bone resorption was scored from 0 to 4 as described in Materials and Methods. Differences between the control and αTNF treatment groups were statistically analyzed using Dunnett's multiple comparison test.

[0037] Figures 4A to 4B. Subcutaneous injection of CBP-αTNF also accumulated in arthritic paws and inhibited the development of arthritis. (A) Arthritis in the right hind paw was selectively induced by passive immunization with anti-collagen antibodies followed by subcutaneous injection of LPS in the right hind paw pad and subcutaneous injection of PBS in the left hind paw pad. Cy7-labeled CBP-αTNF was subcutaneously injected into the back of the mice the day after LPS injection. Representative images of accumulation in arthritic or non-arthritic paws of mice injected with CBP-αTNF (indicated by arrows). (B) Arthritis in all paws 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 was subcutaneously injected. Arthritis scores represent the mean + SE from five mice. #P < 0.05, compared with scores on day 8 in each group (Tukey's multiple comparison test).

[0038] Figures 5A to 5B. Effects of local injection on arthritis development. 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-2 123-144 -αTNF and Cy7-labeled WT-αTNF or (B) control IgG, WT-αTNF and PlGF-2 123-144 αTNF was subcutaneously injected into the left hind paw of arthritic mice. (A) WT-αTNF and PlGF-2 123-144 Representative images of αTNF retained at the injection site in mice. (B) Arthritis scores represent the mean ± SE of eight mice. #P<0.05, compared with scores on day 6 in each group (Tukey's multiple comparison test).

[0039] Figures 6A to 6E. A3-IL4 accumulates in the spinal cord and reduces EAE scores. The affinity of A3-IL4 for type III collagen (A) and the affinity of IL-4 and A3-IL4 proteins for IL-4Rα were measured by ELISA (B). A graph showing the relationship between [concentration] and [signal] is displayed (n=4). EAE was induced by subcutaneous injection of MOG. 35-55 / CFA emulsion, followed by intraperitoneal injection of PTX on the day of immunization and the second day. On day 14 post-immunization, 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 harvested 4 hours post-injection and fluorescence intensity was measured. (E) Starting from day 14 post-EAE immunization, normal form of IL-4 or A3-IL4 was intravenously injected every other day. Disease scores for EAE represent mean ± SE (n = 3–5).

[0040] Figures 7A to 7C. Localization of A3 protein and CBP conjugates in inflamed tissues of other inflammatory disease models. (A) Intravenous injection of DyLight 800-labeled A3 protein or Cy7-labeled CBP-αTNF into IL-10 in spontaneously or non-spontaneously developing IBD. - / - ×TLR-4 - / -(DKO) mice or normal (C57BL / 6) mice. Colons were harvested 4 hours after injection and used for fluorescence imaging (upper part) and histological analysis (lower part). Colons of mice with IBD development injected with Cy7-labeled CBP-αTNF were stained with H&E and periodic acid-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 were intravenously injected into naïve mice or bleomycin-induced pulmonary fibrosis models 7 days after bleomycin infusion. Lungs were harvested 4 hours after fluorescence injection and fluorescence intensity was measured. (C) DyLight 800-labeled A3 protein was intravenously injected into mice with spontaneously developing type 1 diabetes (T1D), cyclophosphamide-induced T1D mice, and non-diabetic mice. Pancreas was harvested 15 minutes after fluorescence injection and fluorescence intensity was measured.

[0041] Figures 8A through 8D. Albumin fusion with IL-10 provides FcRn binding and leads to LN accumulation. (A) SDS-PAGE analysis of wt IL-10 and SA-IL-10. (B) Analysis of SA-IL-10 binding to FcRn. (C) Single cells from spleen cells (i) or from popliteal LNs (ii) incubated on ice for 30 min with SA, SA-IL-10, or CBD-SA-IL-10. In (i), bar graphs representing the binding percentage (y-axis) of SA, SA-IL-10, and CBD-SA-IL-10 are shown from left to right for each cell type (x-axis). In (ii), bar graphs representing the binding percentage (y-axis) of SA and SA-IL-10 are shown from left to right for each cell type (x-axis). The binding of each protein to immune cells was detected by co-staining with anti-SA antibody and antibodies against specific markers for each immune cell population. (D) Immunofluorescence images of popliteal venules (LNs) 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 or anti-PNAd antibodies, respectively.

[0042] Figures 9A to 9B. Albumin-IL-10 fusion provides prolonged blood circulation, and CBD fusion improves biodistribution in inflamed joints. (A) BALB / c mice were administered wt IL-10, SA-IL-10, or CBD-SA-IL-10 (each equivalent to 35 μg IL-10) via tail vein injection. Serum was collected at specified time points. Serum IL-10 concentrations 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 . t 1 / 2,α Rapidly eliminates half-life; t1 / 2,β Slow clearance half-life. Area under the curve (AUC) was analyzed by Graphpad Prism. (B) Arthritis (CAIA) 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 pad (defined as day 3). On the second 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, designated organs were collected and analyzed using an IVIS imaging system. (mean ± SEM; n = 4). Statistical analysis was performed using ANOVA with Tukey's test. *P < 0.05; **P < 0.01. (B) shows bar graphs from left to right for each organ (x-axis) representing the distribution % (y-axis) of wt IL-10, SA-IL-10, and CBD-SA-IL-10, respectively.

[0043] Figures 10A to 10D. IL-10 fused with albumin inhibited the development of arthritis more effectively than wt IL-10. (A) Arthritis (CAIA) was induced by passive immunization with anti-collagen antibodies 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 IL-10) were intravenously injected into CAIA mice. Arthritis scores represent the mean + SEM of 7 mice. (B) Comparison of the therapeutic effects of wt IL-10 and CBD-SA-IL-10 versus αTNF-α antibody in CAIA mice immunized with a high dose (1.5 mg / mouse) of anti-collagen antibodies. Arthritis scores represent the mean + SEM of 6 to 7 mice. (C) Representative H&E histological images of joints on day 13 of each treatment group. Scale bar, 500 μm. As described in Materials and Methods, the severity of synovial hyperplasia and bone resorption was scored from 0 to 4. (D) Effect of administration routes SA-IL-10 and CBD-SA-IL-10 on treatment efficacy. Arthritis scores represent the mean + SEM of 6 to 7 mice. Statistical analysis was performed using analysis of variance (ANOVA) and Tukey's test. *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001.

[0044] Figures 11A to 11D. IL-10 fused with albumin showed therapeutic efficacy in improving established arthritis. Male DBA / 1J mice were subcutaneously injected with bovine collagen / CFA emulsion at the base of the tail. Three weeks later, bovine collagen / IFA emulsion was injected again as a booster. When the arthritis score became 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 IL-10) or 200 μg anti-TNF-α antibody. In (A), the same treatment was additionally administered to mice on day 3. (A and B) Arthritis scores represent the mean + SEM of 9 mice. (C and D) Representative H&E histological images of joints on day 16. Scale bar, 500 μm. Severity of synovial hyperplasia and bone resorption was scored from 0 to 4 as described in Materials and Methods. Statistical analysis was performed using analysis of variance (ANOVA), Tukey's test (A and B), and two-tailed Student's t-test (C and D). *P<0.05; **P<0.01; ***P<0.001.

[0045] Figures 12A to 12F. Albumin-fused IL-10 accumulates in the LN and inhibits Th17 activation. Arthritis (CAIA) 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, wt IL-10, SA-IL-10, or CBD-SA-IL-10 was intravenously injected into arthritic mice. IL-10 levels and Th17-related cytokines in the LN were measured using ELISA. (A) Comparison of IL-10 levels 4 hours after each protein injection. (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 articular drainage (popliteal) LN (D) and non-drained (cervical) LN (E). (F) GM-CSF levels in the popliteal LN. (Mean ± SEM; n = 7). Statistical analysis was performed using ANOVA and Tukey's test. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; ns: not significant.

[0046] Figure 13ASee Figure 13C. Albumin-fused IL-10 inhibits the inflammatory response in the paw. Arthritis (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), arthritic mice were intravenously injected with PBS, wtIL-10, SA-IL-10, or CBD-SA-IL-10. (A) Single cells were extracted from the hind paw on day 11 and analyzed by flow cytometry. The figure depicts CD45. + Cells, B cells (CD45) + B220 in lymphocytes + cells), dendritic cells (CD45) + CD11c in lymphocytes + Cells), monocytes (CD45) + CD11b in lymphocytes + Cells), granulocytes MDSC / neutrophils (Ly6G) + Ly6C + CD11b + CD45 + ), monocyte MDSC (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 + (A) Cytokine levels in the hind paw on day 11. (n=5–7) (B) Representative H&E images of the joints on day 14 for each treatment group. Scale bar, 500 μm. Severity of synovial hyperplasia and bone resorption was scored from 0 to 4 as described in Materials and Methods. (mean ± SEM; n=6–7) Except for %CD11c in (A). + In addition, statistical analysis was performed using analysis of variance (ANOVA) and Tukey's test. To analyze %CD11c in (A) + The Kruskal-Wallis test was used, followed by the Dunn multiple comparison test. *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001.

[0047] Figures 14A to 14B CBD conjugation provides collagen affinity for IL-10. (A) SDS-PAGE analysis of CBD-SA-IL-10. (B) Binding analysis of CBD-SA-IL-10 with type I or type III collagen and FcRn.

[0048] Figures 15A to 15B Effects of albumin-fused IL-10 on immune cell populations in spleen (A) and lenticule (LN) (B). Arthritis (CAIA) was induced by passive immunization with anti-collagen antibodies followed by intraperitoneal injection of LPS (defined as day 3). Mice were intravenously injected with PBS, wt% IL-10, or SA-IL-10 on days 3 and 6. Single cells were extracted from the spleen and popliteal LN the day after the last injection and analyzed by flow cytometry. The graphs depict the frequencies of CD3+ in live cells as follows: + T cells, intracellular CD45 + Lymphocytes, intracellular CD11b + Cells, CD11b + Intracellular CD11c + Cells, CD11c + Intracellular CD86 + Cells, granulocytes MDSC / neutrophils (CD11b) + Ly6G in cells + Ly6C + ), monocyte MDSC (CD11b + Ly6G in cells - Ly6C + ), macrophages (CD11b + Intracellular F4 / 80 + CD86 in macrophages + Cells and M2 macrophages (CD11b) + CD206 in cells + F4 / 80 + (mean ± SEM; n = 6–7). Statistical analysis was performed using ANOVA and Tukey's test, except for the following tables. For CD11b in (A) + %CD11c in cells + And CD11b in (B) + %Ly6G in cells + Ly6C +The analysis was performed using the Kruskal-Wallis test, followed by the Dunn multiple comparison test. *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001.

[0049] Figures 16A to 16B Effects of albumin-fused IL-10 on T cell populations in the paw and blood. Arthritis (CAIA) 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 paw on day 11 and analyzed by flow cytometry. The graph depicts the frequencies of NK1.1 in CD45+ lymphocytes. + CD3 - NK cells, CD45 + CD3 in lymphocytes + T cells, CD45 + CD3 in lymphocytes + CD4 + T cells, CD3 + CD4 + T cells' Treg (Foxp3) + CD25 + CD45 + CD3 in lymphocytes + CD8 + T cells, CD3 + CD8 + Effector memory T cells (CD62L) - CD44 + CD3 + CD8 + T cells are central memory T cells (CD62L) + CD44 + CD3 + CD8 + T cells PD-1 + Cells. (B) Lymphocytes were extracted from the blood on day 11 and then analyzed by flow cytometry. The graph depicts the frequencies as follows: CD45 + CD3 in lymphocytes + T cells, CD45 + CD3 in lymphocytes + CD4 + T cells, CD3 + CD4 + Treg (Foxp3) in T cells + CD25+ CD45 + CD3 in lymphocytes + CD8 + T cells. (mean ± SEM; n = 5–7). Statistical analysis was performed using ANOVA and Tukey's test, except for the following charts: for the analysis in (A) below: CD45 + Intracellular %NK1.1 + CD4 + %Foxp3 in cells + CD8 + %CD44 in cells + / CD62L - CD8 + %CD44 in cells + / CD62L + and CD8 + %PD-1 in cells + The Kruskal-Wallis test was used, followed by the Dunn multiple comparison test. *P<0.05; **P<0.01; ***P<0.001.

[0050] Figures 17A to 17B Safety assessment of albumin-fused IL-10. Healthy BALB / c mice were intravenously injected with wt IL-10, SA-IL-10, or CBD-SA-IL-10. (A) Two days after injection, white blood cell count, red blood cell count, platelet count, blood hemoglobin concentration, and spleen weight were assessed. (B) Serum concentrations of alanine aminotransferase (ALT), amylase, blood urea nitrogen (BUN), serum calcium, creatine kinase (CK), CO2, total bilirubin (TBli), and total protein were assessed using a biochemical analyzer. (mean ± SEM; n = 5). Statistical analysis was performed using analysis of variance (ANOVA) and Tukey's test. *P < 0.05; **P < 0.01.

[0051] Figures 18A to 18D. IL-4 remains active after SA fusion. (a) Analysis of Wt IL-4 and SA-IL-4 by SDS-PAGE with Coomassie blue staining under reducing and non-reducing conditions. (b) Binding of SA-IL-4 to freshly isolated immune cells from LN and spleen, measured by flow cytometry. (c) Assay of Wt IL-4 and SA-IL-4 activity. Phosphorylation of STAT6 in T cells was analyzed by flow cytometry after culturing T cells in vitro with specified concentrations of wt IL-4 or SA-IL-4. (d) Concentration of IL-17 secreted under Th17 differentiation conditions in the presence of wt IL-4 or SA-IL-4, measured by ELISA. Data are mean ± SEM. Two experiments were repeated. Statistical analysis was performed using one-way ANOVA and Tukey's test. **P < 0.01.

[0052] Figures 19A to 19H. The fusion of SA with IL-4 increases 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 intravenously injected wt IL-4 or SA-IL-4. 10 μg wt IL-4 (n=4) or equimolar SA-IL-4 (n=3) was intravenously injected into juvenile mice. Blood was collected from 1 minute to 24 hours later, and plasma IL-4 concentrations were measured by ELISA. (c) Changes in IL-4 levels over time in humeral and lumbar LNs and (d) spleen. 40 μg wt IL-4 or equimolar SA-IL-4 was intravenously injected into juvenile mice. Lymph nodes and spleen were harvested, and IL-4 levels were detected by ELISA (n=5). (e) Measured SA(P573K)-IL-4 levels in LNs and spleen 1 hour after injection. Data on Wt IL-4 and SA-IL-4 from (cd) are reproduced. Immunofluorescence images of lumbar LNs 1 hour after intravenous injection of DyLight594-labeled IL-4 or SA-IL-4 from (fg). T cells and high endothelial venules (HEVs) were stained with anti-CD3 or anti-PNAd antibodies, respectively. Scale bars represent 200 μm (g) and 100 μm (h). Data are mean ± SEM. Two experiments were repeated. Statistical analysis was performed using one-way ANOVA with Tukey's test. **P < 0.01.

[0053] Figure 20A to Figure 20D SA-IL-4 prevents the progression and development of acute acute exacerbation encephalopathy (EAE). C57BL / 6 myelin oligodendrocyte glycoprotein (MOG) 35-55(a) Disease progression, (b) disease incidence, and (c) weight change in experimental autoimmune encephalomyelitis (EAE) mice, which were administered the following every other day for 10 days starting from day 8 post-immunization: intraperitoneal injection (ip) of phosphate-buffered saline (PBS), intraperitoneal injection of wt IL-4 10 μg, or intraperitoneal or subcutaneous injection (sc) of SA-IL-4 10 μg molar equivalent, or oral administration of FTY720 1 mg / kg. n = 7 per group. (d) Representative histology of the spinal cord. Myelin expression was detected by immunohistochemistry using anti-myelin basic protein antibody (brown). Arrows indicate demyelination. The figure shows the percentage of mice showing demyelination in each treatment group by blinded pathological analysis. Two experiments were replicated. Data are mean ± SEM. Statistical analysis was performed using one-way ANOVA and Tukey's test. **P < 0.01.

[0054] Figures 21A to 21H. SA-IL-4 treatment inhibited leukocyte infiltration into the spinal cord and induced immunosuppressive cells in the draining LN. Mice were administered wt% IL-4, SA-IL-4, or PBS intraperitoneally, or SA-IL-4 subcutaneously, every other day for 10 days starting from day 8 post-immunization, or orally with FTY720 1 mg / kg body weight daily starting from day 8 post-immunization. On day 17 post-immunization, cells were isolated from the drained LN and spinal cord and analyzed by flow cytometry. (ab) Frequency of (a) CD45+ leukocytes and (b) RoRγt+ Th17 cells in live spinal cord cells. (cg) In lumbar drainage lymph nodes (dLN), the following frequencies were analyzed: (c) Ly6G+Ly6C+G-MDSCs in CD11b+CD45+ cells, (d) Ly6G-Ly6C+M-MDSCs in CD11b+CD45+ cells, (e) RoRγt+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. Data are mean ± SEM. The experiment was performed once. Statistical analysis was performed using one-way ANOVA and Tukey's test. *P<0.05, **P<0.01.

[0055] Figures 22A to 22P. SA-IL-4 treatment activates the PD-1 / PD-L1 axis and reduces integrin and cytokine expression in T cells. MOG 35-55EAE-induced mice were subcutaneously injected with PBS, wt IL-4, or SA-IL-4 on days 8, 10, and 12 post-immunization. On day 13, the spinal cord and spleen were isolated, and immune cells were analyzed. (a) CD4+ in the spinal cord. + T cell intratetramer + (Identify MOG) 35-55 (b) Cell frequency. In the spleen, the following frequencies were observed: (b) Tetramer + αLβ2 integrin in CD4+ T cells + Cell, (c) tetramer + CD4 + α4β1 integrin in T cells + Cells, (d)CD8 + αLβ2 integrin in T cells + Cells, and (e)CD8 + α4β1 integrin in T cells + Cells. (f) Central Memory (CM) CD44 + CD62L + CD4 + Mean fluorescence intensity (MFI) of PD-1 in T cells, (g)CM CD44 + CD62L + CD8 + MFI of PD-1 in T cells, (h)Ly6C + Ly6G - CD11b + MFI of PD-L1 in M-MDS, (i)Ly6C + Ly6G - CD11b + PD-L1 of M-MDSC + The frequency, (j)Ly6C + Ly6G + CD11b + MFI of PD-L1 in G-MDSC, (k)Ly6C + Ly6G + CD11b + G-MDSC's PD-L1 + The frequency of (l) tetramer + CD4 + T cell intracellular IL-23R + Cell frequency. (mn) Spleen cells 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) In MOG 35-55Spleen cells were cultured in vitro for 6 hours in the presence of peptides. CD4 + Cytokine expression in T cells was characterized by flow cytometry. Data are mean ± SEM. The experiment was performed once. Statistical analysis was performed using one-way ANOVA and Tukey's test. *P<0.05, **P<0.01.

[0056] Figures 23A to 23K. SA-IL-4 treatment in the chronic phase of EAE reduces clinical scores and prevents immune cell infiltration into the spinal cord. Using MOG... 35-55 EAE was induced in C57BL / 6 mice. Intraperitoneal injections of PBS, wt IL-4, or SA-IL-4 were administered every other day for 10 days, starting on day 21 post-immunization. (a) Disease progression and (b) weight changes are shown (n=6). (cd) Subcutaneous injections of PBS, wt IL-4, or SA-IL-4 were administered every other day for 12 days, starting on day 21 post-immunization. (c) Disease progression and (d) weight changes are shown (n=8 for PBS and SA-IL-4; n=7 for other treatment groups). (eh) On day 34, spinal cord and spleen were collected, and immune cells were analyzed by flow cytometry. Graphs show the frequencies of: (e) CD45 in spinal cord live cells. + Cells, (f) CD4 in living spinal cord cells + CD3 + CD45 + T cells, (g) spinal cord live cell intratetramer + (Identify MOG) 35-55 )RoRγt + CD4 + Th17 cells, (h) spleen tetramer + CD4 + Intracellular IL-23R + Cells. (ij) Spleen cells were cultured in vitro for 3 days in the presence of MOG protein. The concentrations of (i) IL-17A and (j) GM-CSF in the culture medium were analyzed by ELISA. (k) In MOG 35-55 Spleen cells were cultured in vitro for 6 hours in the presence of peptides. CD4 + Cytokine expression in T cells was characterized by flow cytometry. The experiment was performed once. Data are mean ± SEM. Statistical analysis was performed using one-way ANOVA and Tukey's test. *P<0.05, **P<0.01.

[0057] Figure 24In vitro imaging (IVIS) analysis showed that injected SA-IL-4 accumulated more in the lumbar spine (LN) than wt IL-4. Biodistribution analysis of DyLight800-labeled wt IL-4 and SA-IL-4 in the LN was performed. Equal amounts of fluorescent 10 μg wt IL-4 or SA-IL-4 were injected intravenously. Four hours after injection, lumbar LNs were collected and imaged using an IVIS in vivo imaging system (n=6). Data are presented as error ± SEM. Experiments were performed once. Statistical analysis was performed using Student's t-test.

[0058] Figures 25A to 25B. SA(P573K) mutation to SA-IL-4 reduces blood concentration and eliminates FcRn binding. Mice were intravenously injected with 40 μg wt IL-4, SA-IL-4, or SA(P573K)-IL-4. Blood was collected 1 hour later and plasma IL-4 concentration was 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. First (1 st ) and the second (2) nd The 62.5 nM concentration was tested twice to verify variability. The two experiments were repeated.

[0059] Figure 26 Long-term treatment with SA-IL-4 inhibits the development and progression of EAE. Intraperitoneal injection of C57BL / 6MOG. 35-55 Disease progression in EAE mice was assessed by injection every other day for 16 days, starting on day 8 after immunization with PBS or SA-IL-4 (10 μg, IL-4-based) (n=8). The number of mice developing EAE per mouse in each treatment group is indicated. Mice were monitored until day 24. Data are presented as mean ± SEM. Two experiments were replicated. Statistical analysis was performed using Student's t-test. **P < 0.01.

[0060] Figure 27 FcRn binding is crucial for SA-IL-4 inhibition in the development and progression of EAE. Intraperitoneal injection of C57BL / 6MOG... 35-55 Disease progression in EAE mice was measured by injection every other day starting from day 8 after immunization with PBS or SA-IL-4 (10 μg, based on IL-4) (n=6). Data are mean ± SEM.

[0061] The two experiments were replicated. Statistical analysis was performed using Student's t-test. **P < 0.01.

[0062] Figures 28A to 28B. SA-IL-4 did not affect the number of macrophages and dendritic cells in the spinal cord and draining LN. Mice were intraperitoneally injected with wt IL-4, SA-IL-4, or PBS, or subcutaneously injected with SA-IL-4, every other day for 10 days starting from day 8 post-immunization. FTY720 1 mg / kg body weight was orally administered daily starting from day 8 post-immunization. Cells were isolated from the draining LN (dLN) and spinal cord 17 days post-immunization and analyzed by flow cytometry. The following frequencies were analyzed: (a) CD11b + Intracellular F4 / 80 + Macrophages and (b)CD45 + CD11b in cells + CD11c + DC. Data are mean ± SEM. The experiment was conducted once. Statistical analysis was performed using one-way ANOVA and Tukey's test.

[0063] Figures 29A to 29O. Blood and organ analyses of SA-IL-4 showed that SA-IL-4 is safe. Toxicity analysis of SA-IL-4. Naïve mice were intravenously injected with 10 μg wt IL-4 or equimolar SA-IL-4. Two days later, (ai) serum was tested using a biochemical analyzer, and (jm) blood was tested using a hematology analyzer. Lung water volume was determined by weighing the lungs before and after lyophilization. Data are mean ± SEM. Experiments were performed once. Statistical analysis was performed using one-way ANOVA with Tukey's test.

[0064] Figures 30A to 30D Gating strategies in flow cytometry. (A) Restimulation (cytokine expression). (B) Integrin expression. (C) MDSC. (D) CD45. + And T cells. Detailed Implementation

[0065] There is a great need to improve the therapeutic efficacy of drugs for inflammatory and autoimmune diseases. One possible approach is to target anti-inflammatory drugs to inflamed areas. Collagen is inaccessible in most tissues due to the low permeability of the vascular system, but is exposed to the blood flow in inflamed areas due to the high permeability of the vascular system. This disclosure describes ECM-binding anti-inflammatory agents conjugated with ECM affinity peptides. One such peptide is collagen-binding peptide (CBP). The CBP conjugation provides collagen affinity against TNFα antibody (αTNF). CBP-αTNF accumulates in inflamed areas in a collagen antibody-induced arthritis model (Example 1). Compared with the unmodified antibody, CBP-αTNF significantly inhibits the development of arthritis. Furthermore, the fusion of the collagen-binding domain derived from the A3 domain of von Willebrand factor (vWF) with interleukin (IL)-4 (A3-IL4) enables its detection in the spinal cord of experimental autoimmune encephalomyelitis (EAE), a multiple sclerosis model following intravenous administration (Example 1). A3-IL4 reduces the clinical symptoms of EAE, while normal IL-4 does not. Collagen-binding proteins were detected in inflamed tissues of spontaneous inflammatory bowel disease, bleomycin-induced pulmonary fibrosis, and type 1 diabetes models. In conclusion, collagen affinity enables anti-inflammatory drugs to target inflamed areas, suggesting a novel clinical translational approach for treating inflammatory and autoimmune diseases.

[0066] III. Anti-inflammatory agents

[0067] A. Antibodies

[0068] This disclosure relates to anti-inflammatory antibodies or fragments thereof. The term "antibody" refers to any isotype of intact immunoglobulin or fragment thereof that can competitively bind specifically to a target antigen with an intact antibody, including chimeric, humanized, fully humanized, and bispecific antibodies. As used herein, the terms "antibody" and "immunoglobulin" are used interchangeably to refer to any of several classes of structure-related proteins that function as part of an animal's immune response, including IgG, IgD, IgE, IgA, IgM, and related proteins, as well as polypeptides containing an antibody CDR domain that retains antigen-binding activity.

[0069] 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 have one or more epitopes that can interact with different antibodies.

[0070] The term "epitope" includes any region or portion of a molecule capable of triggering an immune response by binding to immunoglobulins or T-cell receptors. Epitope determinants may include chemically active surface groups, such as amino acids, sugar side chains, phosphoryl groups, or sulfonyl groups, and may have specific three-dimensional structural features and / or specific charge features. Typically, antibodies specific to a particular target antigen will preferentially recognize epitopes on the target antigen in a complex mixture.

[0071] Many different epitope mapping techniques well known in the art can be used to identify epitope regions of a given polypeptide. These techniques include 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 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, ibid. In addition, standard antigenicity and hydrophilicity maps can be used to predict and identify antigenic regions of proteins.

[0072] Intact antibodies typically consist of two full-length heavy chains and two full-length light chains, but in some cases may include fewer chains; for example, antibodies naturally occurring in camels may contain only heavy chains. The antibodies disclosed herein may be derived from a single source or may be “chimeric,” meaning that different portions of the antibody may be derived from two different antibodies. For example, the variable region or CDR region may be derived from rat or mouse sources, while the constant region may be derived from different animal sources, such as humans. Antibodies or binding fragments may be produced in hybridomas via recombinant DNA technology or by enzymatic or chemical cleavage of intact antibodies. Unless otherwise stated, the term “antibody” includes its derivatives, variants, fragments, and mutant proteins, as exemplified below (Sela-Culang et al. Front Immunol. 2013; 4:302; 2013).

[0073] The term "light chain" includes full-length light chains and fragments thereof, possessing sufficient variable region sequence to confer binding specificity. Full-length light chains have a molecular weight of approximately 25,000 Daltons and include a variable region domain (VL) and a constant region domain (CL). Light chains are classified into two classes, identified as kappa (κ) and lambda (λ). The term "VL fragment" refers to a light chain fragment of a monoclonal antibody that includes all or part of the light chain variable region, including the CDR. VL fragments may also include the light chain constant region sequence. The variable region domain of the light chain is located at the amino terminus of the polypeptide.

[0074] The term "heavy chain" includes the full-length heavy chain and its fragments, possessing sufficient variable region sequence to confer binding specificity. The full-length heavy chain has a molecular weight of approximately 50,000 Daltons and includes a variable region domain (abbreviated as VH) and three constant region domains (abbreviated as CH1, CH2, and CH3). The term "VH fragment" refers to a heavy chain fragment of a monoclonal antibody, including all or part of the heavy chain variable region, including the CDR. The VH fragment may also 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 at the carboxyl terminus, and CH3 is closest to the -COOH terminus. Antibody isotypes can be IgM, IgD, IgG, IgA, or IgE, and are defined by the presence of the heavy chain, which is classified into five classes: μ-chain, δ-chain, γ-chain, α-chain, or ε-chain. IgG has multiple isotypes, including but not limited to IgG1, IgG2, IgG3, and IgG4. IgM isotypes include IgM1 and IgM2. IgA subtypes include IgA1 and IgA2.

[0075] Antibodies can be any isotype or classification of intact immunoglobulin, chimeric antibodies, or hybrid antibodies specific to two or more antigens. They can also be fragments (e.g., F(ab′)2, Fab′, Fab, Fv, etc.), including hybrid fragments. Immunoglobulins also include natural, synthetic, or genetically engineered proteins that act like antibodies by binding to specific antigens to form complexes. The term antibody includes genetically engineered or otherwise modified forms of immunoglobulins, such as the following:

[0076] The term "monomer" refers to an antibody containing only one Ig unit. A monomer is the basic functional unit of an antibody. The term "dimer" refers to an antibody containing two Ig units interconnected by a heavy chain constant domain (Fc, or crystallizable fragment region). The complex can be stabilized by a linker (J) chain protein. The term "multimer" refers to an antibody containing more than two Ig units interconnected by a heavy chain constant domain (Fc region). The complex can be stabilized by a linker (J) chain protein.

[0077] 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.

[0078] Bispecific antibodies are a class of antibodies with two complementary sites that have different binding sites targeting two or more different epitopes. In some embodiments, bispecific antibodies may be bicomponent, wherein the bispecific antibody can specifically recognize different epitopes from the same antigen. In some embodiments, bispecific antibodies may be constructed from a pair of different single-domain antibodies called “nanobodies.” Single-domain antibodies are derived from and modified from cartilaginous fish and camelids. Nanobodies can be linked together using techniques commonly known to those skilled in the art; such methods of selecting and linking antibodies are described in PCT publications WO2015044386A1, WO2010037838A2 and Bever et al., Anal Chem. 86:7875–7882 (2014), each of which is incorporated herein by reference in its entirety.

[0079] Bispecific antibodies can be constructed as: complete IgG, Fab'2, Fab'PEG, double-chain antibodies, or scFv. Double-chain antibodies and scFv can be constructed using only the variable domain without the Fc region, which may reduce the effect on anti-idiotype reactions. Bispecific antibodies can be generated by a variety of methods, including but not limited to hybridoma fusion or Fab' fragment linkage. See, for example, Songsivilai and Lachmann, Clin. Exp. Immunol. 79:315-321 (1990); Kostelny et al., J. Immunol. 148:1547-1553 (1992), each of which is incorporated herein by reference in its entirety.

[0080] In some respects, antigen-binding domains can be multispecific or heterospecific by polymerizing with VH and VL regions that bind different antigens. For example, antibodies can bind to or interact with (a) cell surface antigens, (b) Fc receptors on the surface of effector cells, or (c) at least one other component. Thus, aspects may include, but are not limited to, bispecific, trispecific, tetraspecific, and other multispecific antibodies or antigen-binding fragments thereof targeting epitopes and other targets, such as Fc receptors on effector cells.

[0081] In some implementations, multispecific antibodies can be used and directly linked via short, flexible polypeptide chains using conventional methods known in the art. One such example is a bispecific antibody, a divalent bispecific antibody in which the VH and VL domains are expressed on a single polypeptide chain, and a linker, too short to pair between domains on the same chain, forces the domains to pair with complementary domains on the other chain, creating two antigen-binding sites. This linker function is applicable to implementations of triple-chain, quadruple-chain, and higher-order antibody polymers. (See, for example, Hollinger et al., ProcNatl.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)).

[0082] In contrast to bispecific whole antibodies, bispecific biantibodies can also be advantageous because they can be readily constructed and expressed in *E. coli*. Double-chain antibodies (and other peptides, such as antibody fragments) with appropriate binding specificity can be readily selected from a library using phage display (WO94 / 13804). A library can be prepared where the other arm is altered and an antibody with appropriate specificity is selected, provided one arm of the double-chain antibody remains unchanged, for example, having specificity against a protein. Bispecific whole antibodies can be prepared using alternative engineering methods as described in Ridgeway et al. (Protein Eng., 9:616-621, 1996) and Krah et al. (N Biotechnol. 39:167-173, 2017), each of which is incorporated herein by reference in its entirety.

[0083] Heteroconjugated antibodies consist of two monoclonal antibodies covalently linked together, each having different specificities. See, for example, U.S. Patent No. 6,010,902, which is incorporated herein by reference in its entirety.

[0084] The Fv fragment portion of an antibody molecule that binds to an antigenic epitope with high specificity is referred to herein as a “complementary site.” A complementary site consists of amino acid residues that contact the antigenic epitope to facilitate antigen recognition. Each of the two Fv fragments of an antibody consists of two variable domains, VH and VL, in a dimerized configuration. The primary structure of each variable domain includes three hypervariable loops, separated by and flanked by a frame region (FR). The hypervariable loop is the region of highest primary sequence variability in antibody molecules from any mammal. The term hypervariable loop is sometimes used interchangeably with the term “complementarity-determining region (CDR).” The length of the hypervariable loop (or CDR) varies from antibody molecule to antibody. Frame regions from all antibody molecules from a given mammal exhibit high primary sequence similarity / identity. Those skilled in the art can use the commonalities of the frame regions to identify the frame regions and the hypervariable loops (or CDRs) scattered between them. Hypervariable loops are given identification names that distinguish their position in the polypeptide and which domain they appear in. CDRs in the VL domain are identified as L1, L2, and L3, with L1 appearing at the furthest end and L3 appearing closest to the CL domain. CDRs can also be named CDR-1, CDR-2, and CDR-3. L3 (CDR-3) is typically the most variable region among all antibody molecules produced by a given organism. CDRs are linearly aligned regions of polypeptide chains in the primary structure, separated from each other by framework regions. The amino terminus (N-terminus) of the VL chain is called FR1. The region identified as FR2 appears between the L1 and L2 hypervariable loops. FR3 appears between the L2 and L3 hypervariable loops, and the FR4 region is closest to the CL domain. The VH chain repeats this structure and nomenclature, comprising three CDRs identified as H1, H2, and H3. The majority of amino acid residues in the variable domains or Fv fragments (VH and VL) are part of the framework region (approximately 85%). The three-dimensional or tertiary structure of antibody molecules places the framework region more internally within the molecule and provides most of the structure, with CDRs located on the outer surface of the molecule.

[0085] Several methods have been developed and can be used by those skilled in the art to identify the exact amino acids that make up each of these regions. This can be accomplished using any of a variety of multiple sequence alignment methods and algorithms that identify conserved amino acid residues that make up the frame regions, and thus identify CDRs that may vary in length but are located between the frame regions. Three commonly used methods have been developed to identify the CDR of antibodies: Kabat (see below: TTWu and EAKabat, “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 (see below: C. Chothia et al., “Conformations of immunoglobulin hypervariable regions,” Nature, vol. 342, no. 6252, pp. 877–883, Dec. 1989); and IMGT (see below: M.-P. Lefranc et al., “IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like). Domains, “Developmental & Comparative Immunology, vol. 27, no. 1, pp. 55–77, Jan. 2003”. These methods each include 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 appear in the CDR, although in some cases, residues within the frame region contribute to antigen binding.

[0086] Those skilled in the art can use any of a variety of methods to determine the complementary site of an antibody. These methods include:

[0087] 1) Calculation and prediction of the tertiary structure of antibody / epitope binding interactions based on the chemical properties of the amino acid sequence of the antibody variable region and the composition of the epitope.

[0088] 2) Hydrogen-deuterium exchange and mass spectrometry.

[0089] 3) Peptide fragmentation and peptide mapping analysis methods, in which multiple overlapping peptide fragments are generated from the full length of the peptide and the binding affinity of these peptides to epitopes is evaluated.

[0090] 4) Antibody phage display library analysis, in which antibody Fab fragments encoding mammalian genes are expressed by phages incorporating into the phage coat. The phage population expressing the Fab is then allowed to interact with the immobilized antigen, or expressed therein via different exogenous expression systems. Unbound Fab fragments are washed away, leaving only the specifically bound Fab fragments attached to the antigen. The bound Fab fragments can be easily isolated and the genes encoding them identified. This method can also be used for smaller regions of Fab fragments, including Fv fragments or specific VH and VL domains, depending on the case.

[0091] In some respects, affinity-matured antibodies are enhanced by one or more modifications to one or more of their CDRs, resulting in increased affinity for the target antigen compared to parental antibodies without those modifications. Some affinity-matured antibodies will have nanomolar or picomolar affinity for the target antigen. Affinity-matured antibodies are produced by procedures known in the art, such as those described in Marks et al., Bio / Technology 10:779 (1992), which describe affinity maturation via VH and VL domain shuffling. Random mutagenesis of CDRs and / or framework residues used in phage display is described as follows: Rajpal et al., PNAS.24:8466-8471 (2005) and Thie et al., Methods Mol Biol.525:309-22 (2009), combined with the computational methods described in Tiller et al., Front. Immunol.8:986 (2017).

[0092] Chimeric immunoglobulins are products of fusion genes from different species; “humanized” chimeras typically have a framework region (FR) derived from human immunoglobulins, and one or more CDRs derived from non-human sources.

[0093] In some respects, portions of the heavy and / or light chains are identical or homologous to corresponding sequences from another particular species or belonging to a particular antibody class or subclass, while the remainder of the chains is identical or homologous to corresponding sequences in antibodies from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, provided they exhibit the desired biological activity. See, for example, 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), each specifically incorporated herein by reference in its entirety. CDR porting is described in, for example, U.S. Patent Nos. 6,180,370, 5,693,762, 5,693,761, 5,585,089 and 5,530,101, which are incorporated herein by reference for all purposes.

[0094] In some implementations, minimizing the antibody peptide sequence from a non-human species can optimize chimeric antibody function and reduce immunogenicity. Specific amino acid residues in 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 transplant” antibody, in which the antibody contains one or more CDRs from a specific species or belonging to a specific antibody class or subclass, while the remainder of the antibody chain is identical or homologous to the corresponding sequence in an antibody derived from another species or belonging to another antibody class or subclass. For human use, the V region, consisting of CDR1, CDR2, and a portion of CDR3 from the variable regions of the light and heavy chains of a non-human immunoglobulin, is grafted onto the framework region of the human antibody, replacing the antigen receptor of the naturally occurring human antibody with a non-human CDR. In some cases, corresponding non-human residues replace framework region residues of the human immunoglobulin. Furthermore, humanized antibodies may include residues not found in the recipient or donor antibody to further improve performance. Humanized antibodies may also contain at least a portion of the immunoglobulin constant region (Fc), typically the constant region 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).

[0095] Antibodies in the body are immunoglobulins that are localized within cells and bind to intracellular antigens, rather than being secreted by the body, which binds to antigens in the extracellular space.

[0096] Polyclonal antibody formulations typically consist of different antibodies targeting different determinants (epitopes). To produce a polyclonal antibody, a host, such as a rabbit or goat, is immunized with an antigen or antigen fragment, usually with an adjuvant, and conjugated to a vector if necessary. The antibody against the antigen is then collected from the host's serum. Polyclonal antibodies can be affinity-purified against the antigen to achieve single specificity.

[0097] Monoclonal antibodies, or "mAbs," are antibodies derived from a homogeneous population of antibodies derived from unique parental cells. For example, the population is identical, differing only in the presence of naturally occurring mutations that may exist in small amounts. Each monoclonal antibody targets a single antigenic determinant.

[0098] 1. Functional antibody fragments and antigen-binding fragments

[0099] a. Antigen-binding fragments

[0100] Some aspects involve 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 bind specifically to an antigen. These fragments consist of various arrangements of variable region heavy chains (VH) and / or light chains (VL); and in some embodiments, include constant region heavy chain 1 (CH1) and light chain (CL). In some embodiments, they lack the Fc region consisting of heavy chain 2 (CH2) and 3 (CH3) domains. Implementation schemes for 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); and (v) separated complementarity-determining regions (CDR). These terms are described, for example, in the following: 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 way of reference.

[0101] The antigen-binding fragment also includes antibody fragments that precisely retain at least one, two, or three complementarity-determining regions (CDRs) from the light chain variable region. Fusion of a CDR-containing sequence to the Fc region (or its CH2 or CH3 region) is included within this definition, including, for example, scFvs fused directly or indirectly to the Fc region.

[0102] The term Fab fragment refers to a monovalent antigen-binding fragment of an antibody containing 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 includes VL, VH, CL, and CH1 domains, as well as all or part of the hinge region. The term F(ab')2 fragment refers to a bivalent antigen-binding fragment of a monoclonal antibody that comprises two Fab' fragments linked by disulfide bonds in the hinge region. An F(ab')2 fragment includes, for example, all or part of two VH and VL domains, and may further include all or part of two CL and CH1 domains.

[0103] The term Fd fragment refers to the heavy chain fragment of a monoclonal antibody, which includes all or part of the VH (volume chain height) and CDR (corresponding radix dendritic). Fd fragments may also include the CH1 region sequence.

[0104] The term Fv fragment refers to a monovalent antigen-binding fragment of a monoclonal antibody, comprising all or part of the VL and VH domains, and lacking the CL and CH1 domains. The VL and VH domains include, for example, the CDR domain. A single-chain antibody (sFv or scFv) is an Fv molecule in which the VL and VH regions are linked by a flexible linker to form a single polypeptide chain, thereby forming an antigen-binding fragment. Single-chain antibodies are discussed in detail in International Patent Application Publication No. WO 88 / 01649 and U.S. Patent Nos. 4,946,778 and 5,260,203, the disclosures of which are incorporated herein by reference. The term (scFv)2 refers to a bivalent or bispecific sFv polypeptide chain that includes an oligomerizing domain at its C-terminus, separated from the sFv by a hinge region (Pack et al. 1992). The oligomerizing domain contains a self-associated α-helix, such as a leucine zipper, which can be further stabilized by additional disulfide bonds. The (scFv)2 fragment is also referred to as a “microantibody” or “micro-antibody.”

[0105] Single-domain antibodies are antigen-binding fragments containing only VH or VL domains. In some cases, two or more VH regions are covalently linked to peptide linkers to produce bivalent-domain antibodies. The two VH regions of a bivalent-domain antibody can target the same or different antigens.

[0106] b. Fragment crystallization region, Fc

[0107] The Fc region contains two heavy chain segments, including the CH2 and CH3 domains of the antibody. The two heavy chain segments are held together by two or more disulfide bonds and hydrophobic interactions with the CH3 domain. As used herein, the term "Fc polypeptide" includes polypeptide forms derived from both native and mutant protein forms of the antibody's Fc region. Truncated forms of such polypeptides containing hinge regions that promote dimerization are also included.

[0108] 2. Peptides possessing antibody CDRs and scaffold domains displaying CDRs

[0109] According to the implementation scheme, an antigen-binding peptide scaffold, such as a complementarity-determining region (CDR), is used to generate a protein-binding molecule. Typically, those skilled in the art can determine the type of protein scaffold to which at least one CDR is grafted. It is well known that optimal scaffolds must meet many criteria, such as: good phylogenetic conservation; known three-dimensional structure; small size; little or no post-transcriptional modification; and / or ease of production, expression, and purification. (Skerra, J Mol Recognit, 13:167-87 (2000)).

[0110] Protein scaffolds may be derived from, but are not limited to: fibronectin type III FN3 domains (referred to as “monomers”), fibronectin type III domain 10, lipid carrier proteins, anti-carrier proteins (anticalin), the Z domain of Staphylococcus aureus protein A, thioredoxin A, or proteins with repetitive motifs, such as “ankylin repeat sequences,” “armadillo repeat sequences,” “leucine-rich repeat sequences,” and “thirty-four peptide repeat sequences.” Such proteins are described in U.S. Patent Publications 2010 / 0285564, 2006 / 0058510, 2006 / 0088908, 2005 / 0106660, and PCT Publication WO2006 / 056464, each of which is incorporated herein by reference in its entirety. Scaffolds derived from toxins from scorpions, insects, plants, mollusks, etc., and protein inhibitors of neuronal nitric oxide synthase (PIN) may also be used.

[0111] B. Cytokines

[0112] Cytokines are a group of proteins released from cells upon excitation (only a few cytokines are expressed on the cell membrane). Cell-produced cytokines can affect nearby target cells at very low concentrations or circulate in the bloodstream. They have a wide range of functions in promoting the growth, differentiation, and activation of target cells. Many cytokines can target immune cells and play a role in the immune response. Based on differences in structure and function, cytokines can be broadly classified into chemokines, interleukins, growth factors, transforming growth factors, colony-stimulating factors, tumor necrosis factors, and interferons.

[0113] The following cytokines can be used as anti-inflammatory agents in the methods and compositions of this disclosure. While exemplary sequences are provided below, equivalent or homologous proteins known in the art may also be used.

[0114] Human IL-1ra:

[0115] Mouse IL-1ra:

[0116] Human IL-4:

[0117] Mouse IL-4:

[0118] Human IL-5:

[0119] Mouse IL-5:

[0120]

[0121] Human IL-10:

[0122]

[0123] Mouse IL-10:

[0124]

[0125] Human IL-11:

[0126]

[0127] Mouse IL-11:

[0128]

[0129] Human IL-23; p19 subunit:

[0130]

[0131] Human IL-35; p35 subunit:

[0132]

[0133] Mouse IL-35; p35 subunit:

[0134]

[0135] Human IL-36ra:

[0136]

[0137] Mouse IL-36ra:

[0138]

[0139] Human IL-37:

[0140]

[0141] Mouse IL-37:

[0142]

[0143] Human interferon-β:

[0144]

[0145] Mouse interferon-β:

[0146]

[0147] Human TGF-β1:

[0148]

[0149] Mouse TGF-β1:

[0150]

[0151] Human TNF receptor I:

[0152]

[0153] Human TNF receptor II:

[0154]

[0155] Mouse TNF receptor II:

[0156]

[0157] C.CD200

[0158] Embodiments of this disclosure relate to peptides and compositions comprising the anti-inflammatory agent CD200. An exemplary amino acid sequence of the CD200 peptide is shown below:

[0159] The extracellular domain of mouse CD200 is represented by the following sequence:

[0160] Mouse CD200-MSA fusion protein (linker underlined):

[0161] The human CD200 extracellular domain (UniProt identifier P41217) is represented by the following sequence:

[0162]

[0163] An exemplary human CD200 (lowercase) - human serum albumin (uppercase) fusion protein (linker in uppercase and underlined) is represented as follows:

[0164]

[0165] Mouse CD200 (lowercase)-CBD fusion protein (uppercase) (linker is uppercase and underlined) is represented as follows:

[0166]

[0167] The human CD200 (lowercase)-CBD fusion protein (uppercase) (linker is uppercase and underlined) is represented as follows:

[0168]

[0169] The mouse CD200 (uppercase) - mouse serum albumin (lowercase) - CBD (italicized, underlined, uppercase) fusion protein is represented as follows (connector is uppercase and underlined):

[0170]

[0171] The fusion protein of human CD200 (uppercase) - human serum albumin (lowercase) - CBD (italicized, underlined, uppercase) is represented as follows (the linker is uppercase and underlined):

[0172]

[0173] IV. ECM affinity peptide

[0174] Collagen is an extracellular matrix (ECM) protein that regulates a variety of cellular 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, existing in almost all tissues in one or more of 28 isoforms (Ricard-Blum, Cold Spring Harb Perspect Biol 3:a004978, 2011). The vascular subendothelial space is rich in collagen. Due to its insolubility under physiological conditions, collagen is almost entirely absent in the blood (Dubois et al., Blood 107:3902-06, 2006; Bergmeier and Hynes, Cold Spring Harb Perspect Biol 4:a005132, 2012). It has been reported that tumor vascular systems are permeable due to their anomalous structure (Nagy et al., British journal of cancer 100:865, 2009). Therefore, due to their leaky vascular system, collagen is exposed within the tumor (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 an increased amount of collagen compared to normal tissue (Zhou et al., J Cancer 8:1466-76, 2017; Provenzano et al., BMC Med 6:11, 2008).

[0175] Vonoblastic hemophilia factor (vWF) is a clotting factor that binds to type I and type III collagen, as well as the adhesion receptor GPIb on platelets (Lenting et al., Journal of thrombosis and haemostasis: JTH10:2428-37, 2012; Shahidi Advances in experimental medicine and biology 906:285-306, 2017). Upon injury, collagen beneath endothelial cells is exposed to plasma, and vWF-collagen binding triggers a thrombotic cascade (Shahidi Advances in experimental medicine and biology 906:285-306, 2017; Wu et al., Blood99:3623-28, 2002). Among reported non-bacterial proteins / peptides, the vWFA domain exhibits the highest affinity for collagen (Addi et al., Tissue Engineering Part B: Reviews, 2016). Specifically within the A domain, the A3 domain of vWF has been reported as a collagen-binding domain (CBD) (Ribba et al. Thrombosis and Haemostasis 86:848-54, 2001). As described above, the inventors envision that fusion proteins possessing vWF A3 CBD can achieve targeted cytokine immunotherapy even upon systemic injection due to the exposure of collagen through the leaking tumor vascular system.

[0176] In some embodiments, the ECM affinity peptide comprises a collagen-binding domain derived from the core proteoglycan. In some embodiments, the ECM affinity peptide comprises a core proteoglycan peptide, such as bovine LRELHLNNNC (SEQ ID NO:1) or human LRELHLDNNC (SEQ ID NO:2).

[0177] In some embodiments, the ECM peptide comprises a peptide fragment derived from human core proteoglycan, represented by the following amino acid sequence:

[0178]

[0179] In some embodiments, the ECM peptide comprises a peptide fragment derived from vWF. In some embodiments, the ECM peptide comprises vWF A1, residues 1237-1458 (474-695 of mature VWF) or fragments thereof derived from a human sequence, represented by the following amino acid sequence:

[0180]

[0181] In some embodiments, the ECM peptide comprises all or a fragment of vWF A3, which is represented by the following amino acid sequence:

[0182]

[0183] In some embodiments, the ECM peptide comprises all or a fragment of vWF A3, which is represented by the following amino acid sequence:

[0184]

[0185] In some implementations, the ECM affinity peptide is a peptide derived from von Willebrand factor (vWF). The sequence of human vWF includes the following:

[0186]

[0187]

[0188] In some implementations, the peptide originates from the vWF A3 domain. The vWF A3 domain is derived from the human sequence, residues 1670-1874 (907-1111 of mature vWF), and has the following sequence:

[0189]

[0190] In some implementations, the ECM affinity peptide comprises a peptide derived from PlGF-2. PlGF-2 has the following sequence:

[0191]

[0192] Exemplary PlGF-2 ECM affinity peptides include: RRRPKGRGKRRREKQRPTDCHLCGDAVPRR (SEQ ID NO:9), RRRPKGRGKRRREKQRPTDCHL (SEQ ID NO:10), RRPKGRGKRRREKQRPTD (SEQ ID NO:11), RRRPKGRGKRRREKQ (SEQ ID NO:12), GKRRREKQ (SEQ ID NO:12) NO:13), RRRPKGRG (SEQ ID NO:14) and RRKTKGKRKRSRNSQTEEPHP (SEQ ID NO:15).

[0193] In some embodiments, the ECM affinity peptide is a peptide derived from CXCL-12γ. The sequence of CXCL-12γ is as follows: CXCL-12γ:KPVSLSYRCPCRFFESHVARANVKHLKILNTPNCALQIVARLKNNNRQVCIDPKLKWIQEYLEKALNKGRREEKVGKKEKIGKKKRQKKRKAAQKRKN (SEQ ID NO:16). Exemplary peptides include all or part of SEQ ID NO:12 and the following peptide: GRREEKVGKKEKIGKKKRQKKRKAAQKRKN (SEQ ID NO:17).

[0194] ECM affinity peptides can 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% identity (or any of these derivable ranges) with ECM or CBD peptides or fragments of the aforementioned peptides.

[0195] The linker sequence can be included in the anti-inflammatory agent-peptide construct. For example, a linker having at least, at most, or exactly the following amino acids can separate the antibody and the peptide: 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 or more (or any of these derivative ranges).

[0196] The disclosed ECM affinity peptides can bind to one or more components of the extracellular matrix, such as fibronectin, collagen (type I, type III, and / or type IV collagen), tendinin C, fibrinogen, and fibrin. In some respects, the ECM affinity peptides have an affinity for collagen. And in other respects, the ECM affinity peptides do not bind to fibronectin.

[0197] In some embodiments, the ECM affinity peptide and / or anti-inflammatory agent of this disclosure are further linked to serum proteins. Serum proteins include, for example, albumin, globulins, and fibrinogen. Globulins include α1-globulin, α2-globulin, β-globulin, and γ-globulin. Albumin can be mouse, human, bovine, or any other homologous albumin. In some embodiments, albumin includes human serum albumin, encoded by the ALB gene, and an example of the following amino acid sequence:

[0198]

[0199] In some embodiments, serum albumin comprises a polypeptide having the following sequence:

[0200]

[0201] In some implementations, the albumin comprises mouse albumin having the following sequence:

[0202]

[0203] The relevant implementation scheme includes vWF A3 (uppercase) linked to mouse serum albumin (MSA) (lowercase MSA) via a glycine-serine peptide linker (italic and underlined):

[0204]

[0205] Further related implementation schemes include vWF A3 (uppercase) linked to mouse serum albumin (MSA) (lowercase MSA) via a glycine-serine peptide linker (italicized uppercase and underlined): vWF A3 (uppercase) linked to human serum albumin (HSA) (lowercase HSA) via a glycine-serine peptide linker (italicized uppercase and underlined):

[0206]

[0207] V. Protein Composition

[0208] The polypeptides or polynucleotides disclosed herein, such as ECM affinity peptides, serum protein or cytokine polypeptides, may include 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 of the variant amino acids or nucleic acid substitutions.Or with at least or at most 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, of SEQ ID NO:1 to 66. 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, 14 3, 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, 300, 400, 500, 550, 1000 or more consecutive amino acids or nucleic acids,Or any range derived therefrom that is 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.

[0209] The polypeptides or polynucleotides disclosed herein, such as ECM affinity peptides, serum protein or cytokine polypeptides, may include SEQ ID NO:1 to SEQ ID NO:1. NO: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, 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, 300, 400, 500, 550, 1000 or more than 1000 consecutive amino acids, or any range thereof from which they can be derived.

[0210] In some embodiments, the polypeptide of this disclosure may comprise SEQ ID NO:1 to SEQ ID NO:1 The amino acids in NO:66 are 1 to 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, 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, 265266、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, 56 7, 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 (or any of the ranges from which they can be derived).

[0211] In some embodiments, the polypeptides disclosed herein, such as ECM affinity peptides, serum protein or cytokine polypeptides, may comprise SEQ ID NO:1 to SEQ ID NO:1. NO: 66 at least, at most, 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, 7 8, 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, 14 2, 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, 2 01, 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, 259260、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 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 (or any derivable range thereof) consecutive amino acids.

[0212] In some embodiments, the polypeptide, such as an ECM affinity peptide, serum protein, or cytokine polypeptide, may comprise SEQ ID NO:1 to SEQ ID NO:1. NO: 66 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, 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, 260261、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, 5 66, 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 (or any of the derivable ranges thereof) consecutive amino acids, which are related to SEQ 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 to SEQ ID NO:66.

[0213] The polypeptides disclosed herein, such as ECM affinity peptides, serum protein or cytokine polypeptides, may be 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% (or any range from which they may be derived) similar to, identical to or homologous to any one of SEQ ID NO:1 to SEQ ID NO:66.

[0214] In some respects, there are nucleic acid molecules or polypeptides that originate from the following positions: SEQ ID NO:1 to SEQ ID The 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 in NO:66 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, 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, 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, 2 04, 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, 263264、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, 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, and containing SEQ ID NO:1 to SEQ ID NO: Any one of the following numbers: 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, 13 2, 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, 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, 5 18, 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, 56 8, 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 nucleotides or amino acids.

[0215] The polypeptides and nucleic acids disclosed herein may include 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, 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, 1 79, 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, 21 0, 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, 272273、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 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 permutations (or any range derived therefrom).

[0216] The substitution can be performed on SEQ ID NO:1 to SEQ ID The amino acid position or nucleic acid position of NO:66 is 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, 7 9, 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, 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, 2 37, 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, 267268、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, 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 implementation plan.

[0217] Peptides, polypeptides, and proteins of this disclosure, such as ECM affinity peptides, serum proteins, or cytokine polypeptides, having at least, or having 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 to SEQ ID NO:66, comprising fragments or segments beginning with the following amino acids: 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, 2 ...0, 21, 22, 23, 24, 25, 26, 27, 28, 29, 20, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 20, 20, 21, 22, 23, 24 9, 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 or 200 (or any range derived therefrom),And ending with the following amino acids: 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, 11 9, 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, 1 64, 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 derived therefrom).

[0218] Substitution variants typically involve the exchange of one amino acid for another at one or more sites within a protein and can be engineered to modulate one or more properties of the polypeptide, with or without the loss of other functions or properties. Substitutions can be conserved, i.e., replacing one amino acid with an amino acid of similar shape and charge. Conservative substitutions are well known in the art and include, for example, variations such as: alanine to serine; arginine to lysine; asparagine to glutamine or histidine; aspartic acid to glutamate; cysteine ​​to serine; glutamine to asparagine; glutamate to aspartic acid; glycine to proline; histidine to asparagine or glutamine; isoleucine to leucine or valine; leucine to valine or isoleucine; lysine to arginine; methionine to leucine or isoleucine; phenylalanine to tyrosine, leucine, or methionine; serine to threonine; threonine to serine; tryptophan to tyrosine; tyrosine to tryptophan or phenylalanine; and valine to isoleucine or leucine. Alternatively, the substitution can be non-conservative, affecting the function or activity of the peptide. Non-conservative changes typically involve replacing residues with chemically different residues, such as replacing a non-polar or uncharged amino acid with a polar or charged amino acid, or vice versa. One or more of these substitutions may be specifically excluded from the embodiments.

[0219] The protein can be recombinant or synthesized in vitro. Alternatively, non-recombinant or recombinant proteins can be isolated from bacteria. It is also envisioned that bacteria containing such variants can be incorporated into the composition and method. Therefore, protein isolation is not required.

[0220] The term “functionally equivalent codon” is used in this document to refer to a codon that encodes the same amino acid, such as the six codons for arginine or serine, and also to a codon that encodes a biologically equivalent amino acid.

[0221] It will also be understood that amino acid and nucleic acid sequences may include additional residues, such as additional N-terminal or C-terminal amino acids, or 5' or 3' sequences, respectively, but will still be substantially as described in one of the sequences disclosed herein, provided that the sequence meets the above criteria, including maintaining the biological protein activity in the context of protein expression. The addition of terminal sequences is particularly applicable to nucleic acid sequences, which may, for example, include various non-coding sequences flanking the 5' or 3' portions of the coding region.

[0222] The following discussion focuses on altering the amino acids in proteins to create equivalent or even improved second-generation molecules. For example, certain amino acids can substitute for other amino acids in a protein structure without significantly losing their binding ability to interact. Structures such as enzyme catalytic domains or interacting components can have substituted amino acids to maintain this function. Since the interacting ability and properties of a protein determine its biological activity, certain amino acids can be substituted in the protein sequence and its underlying DNA coding sequence, yet still produce proteins with similar properties. Therefore, the inventors envision making various alterations to the DNA sequence of genes without significant loss of their biological utility or activity.

[0223] In other embodiments, the alteration of peptide function is intended by introducing one or more substitutions. For example, certain amino acids may substitute for other amino acids in the protein structure to alter the binding affinity of the interacting components. Structures such as, for example, protein-interacting domains, nucleic acid-interacting domains, and catalytic sites may have substituted amino acids to modify this function. Since the interacting affinity and properties of a protein determine its biological activity, certain amino acids can be substituted in the protein sequence and its underlying DNA coding sequence, yet still produce proteins with different properties. Therefore, the inventors contemplate the possibility of making various alterations to the DNA sequence of genes, thereby significantly changing their biological utility or activity.

[0224] When making such changes, the hydrophilicity index of the amino acids can be considered. The importance of the hydrophilic amino acid index in conferring biological functions of protein-protein interactions is widely recognized in the field (Kyte and Doolittle, 1982). The relative hydrophilicity of amino acids is considered to contribute to the secondary structure of the resulting protein, which in turn defines the protein's interactions with other molecules such as enzymes, substrates, receptors, DNA, antibodies, and antigens.

[0225] It should also be understood in the art that similar amino acid substitutions can be efficiently performed based on hydrophilicity. U.S. Patent No. 4,554,101 (incorporated herein by reference) states that the maximum local average hydrophilicity of a protein (controlled by the hydrophilicity of its adjacent amino acids) is related to the protein's biological properties. It should be understood that one amino acid can be substituted by another amino acid with a similar hydrophilicity value, and still produce a biologically and immunologically equivalent protein.

[0226] As mentioned above, amino acid substitutions are generally based on the relative similarity of the substituents in the amino acid side chains, such as their hydrophobicity, hydrophilicity, charge, and size. Exemplary substitutions taking into account the various properties mentioned above are well known and include: arginine and lysine; glutamic acid and aspartic acid; serine and threonine; glutamine and asparagine; valine, leucine, and isoleucine.

[0227] In specific implementations, all or part of the proteins described herein may also be synthesized in solution or on a solid support using conventional techniques. Various automated synthesizers are commercially available and can be used according to known protocols. See, for example, Stewart and Young, (1984); Tam et al., (1983); Merrifield, (1986); and Barany and Merrifield (1979), each incorporated herein by reference. Alternatively, recombinant DNA techniques may be employed, in which a nucleotide sequence encoding a peptide or polypeptide is inserted into an expression vector, transformed or transfected into a suitable host cell, and cultured under suitable expression conditions.

[0228] One implementation involves using gene transfer into cells, including microorganisms, to produce and / or present proteins. The gene for the protein of interest can be transferred into a suitable host cell, and the cell can then be cultured under suitable conditions. Nucleic acids encoding virtually any polypeptide can be used. This document discusses recombinant expression vectors and the generation of the elements contained therein. Alternatively, the protein to be produced can be an endogenous protein typically synthesized by cells used for protein production.

[0229] VI. Nucleic Acids

[0230] In some embodiments, this disclosure relates to recombinant polynucleotides encoding proteins, polypeptides, and peptides of the present invention, such as ECM affinity peptides operatively linked to anti-inflammatory agents and / or other molecules. Therefore, some embodiments relate to nucleotides encoding ECM affinity polypeptides and / or fragments of ECM affinity polypeptides fused to an anti-inflammatory agent or a fragment thereof.

[0231] As used in this application, the term "polynucleotide" refers to a recombinant or isolated nucleic acid molecule that does not contain total genomic nucleic acid. The term "polynucleotide" includes oligonucleotides (nucleic acids of 100 residues or less in length) and recombinant vectors, including, for example, plasmids, granules, bacteriophages, and viruses. In some respects, a polynucleotide includes a regulatory sequence substantially separate from its naturally occurring gene or protein-coding sequence. Polynucleotides can be single-stranded (coding or antisense) or double-stranded and can be RNA, DNA (genomic, cDNA, or synthetic), analogues thereof, or combinations thereof. Additional coding or non-coding sequences may, but do not necessarily, be present in the polynucleotide.

[0232] In this regard, the terms “gene,” “polynucleotide,” or “nucleic acid” are used to refer to nucleic acids that encode proteins, polypeptides, or peptides (including any sequences required for proper transcription, post-translational modification, or localization). As those skilled in the art will understand, the term includes genomic sequences, expression cassettes, cDNA sequences, and smaller engineered nucleic acid segments whose expression or may be adapted to express proteins, polypeptides, domains, peptides, fusion proteins, and mutants. Nucleic acids encoding all or part of a polypeptide may comprise 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 31 amino acid sequences encoding a polynucleotide sequence of one or more amino acids described or mentioned herein. 0, 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, 650, 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, 1060 A continuous nucleic acid sequence of 1070, 1080, 1090, 1095, 1100, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 9000, 10000, or more than 10000 nucleotides, nucleosides, or base pairs (or any range from which they can be derived), including all values ​​and ranges in between. It is also envisioned that a particular polypeptide could be encoded by a nucleic acid variant containing a slightly different nucleic acid sequence but still encoding the same or substantially similar protein.

[0233] In certain embodiments, the present invention relates to isolated nucleic acid segments and recombinant vectors incorporating nucleic acid sequences encoding polypeptides or peptides disclosed herein. The term "recombinant" may be used in conjunction with polynucleotides or polypeptides, and generally refers to polypeptides or polynucleotides generated and / or manipulated in vitro, or replicas of such molecules.

[0234] In other embodiments, the present invention relates to isolated nucleic acid segments and recombinant vectors incorporating nucleic acid sequences encoding polypeptides or peptides disclosed herein.

[0235] The nucleic acid segments used in this disclosure can be combined with other nucleic acid sequences, such as promoters, polyadenylation signals, additional restriction enzyme sites, multiple cloning sites, and other coding segments, so that their total length can vary significantly. Therefore, it is contemplated that nucleic acid fragments of almost any length can be used, wherein the total length is preferably limited by the ease of preparation and use in the intended recombinant nucleic acid protocol. In some cases, the nucleic acid sequence can encode a polypeptide sequence with additional heterologous coding sequences, for example, to allow for the purification, transport, secretion, post-translational modification, or therapeutic benefits of the polypeptide, such as targeting or efficacy. As described above, tags or other heterologous polypeptides can be added to the modified polypeptide coding sequence, wherein "heterologous" means a polypeptide different from the modified polypeptide.

[0236] In some embodiments, this disclosure provides polynucleotide variants having substantial sequence identity with those disclosed herein; those of this disclosure obtained using the methods described herein (e.g., BLAST analysis using standard parameters) containing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or higher than 99% of the polynucleotide, including all values ​​and ranges therein.

[0237] This disclosure also envisions the use of polynucleotides complementary to all of the above-described polynucleotides.

[0238] A. Carrier

[0239] The polypeptides disclosed herein may be encoded by nucleic acid molecules contained in a vector. The term "vector" is used to refer to a vector nucleic acid molecule into which a heterologous nucleic acid sequence can be inserted for introduction into a cell in which it can be replicated and expressed. The nucleic acid sequence may be "heterologous," meaning that it is foreign in the cell into which the vector is introduced or in the environment of the nucleic acid incorporated therein, including sequences homologous to sequences in the cell or nucleic acid but located at locations not normally present in the host cell or nucleic acid. Vectors include DNA, RNA, plasmids, granules, viruses (bacteriophages, animal viruses, and plant viruses), and artificial chromosomes (e.g., YAC). Those skilled in the art will be able to construct vectors using standard recombination techniques (e.g., Sambrook et al., 2001; Ausubel et al., 1996, both of which are incorporated herein by reference). In addition to encoding the polypeptides of this disclosure, the vector 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 pIN vectors (Inouye et al., 1985), vectors encoding a histidine residue, and pGEX vectors, used to generate glutathione S-transferase (GST) soluble fusion proteins for later purification, isolation, or cleavage.

[0240] The term "expression vector" refers to a vector containing a nucleic acid sequence encoding at least a portion of a gene product capable of transcription. In some cases, the RNA molecule is subsequently translated into a protein, polypeptide, or peptide. Expression vectors may contain various "control sequences," which are nucleic acid sequences essential for transcription and potentially translation of coding sequences that are operablely linked in a particular host organism. In addition to control sequences that control transcription and translation, vectors and expression vectors may also contain nucleic acid sequences with other functions, as described herein.

[0241] B. Promoters and enhancers

[0242] A promoter is a control sequence. A promoter is typically a region of a nucleic acid sequence that controls the initiation and rate of transcription. It may contain genetic elements that regulate proteins and molecules that can bind, such as RNA polymerases and other transcription factors. The phrases "operably positioned," "operably linked," "under control," and "under transcriptional control" refer to the promoter being in the correct functional position and / or orientation relative to the nucleic acid sequence to control the initiation and expression of that sequence. Promoters may or may not be used in conjunction with "enhancers," which are cis-regulatory sequences involved in the transcriptional activation of a nucleic acid sequence.

[0243] Naturally, the use of promoters and / or enhancers that effectively guide the expression of DNA fragments in selected cell types or organisms for expression can be important. Those skilled in the art of molecular biology are generally familiar with the uses of combinations of promoters, enhancers, and cell types for protein expression (see Sambrook et al., 2001, incorporated herein by reference). The promoters employed can be constitutive, tissue-specific, or inducible, and in some embodiments can guide the high-level expression of the introduced DNA fragment under specific conditions, such as the large-scale production of recombinant proteins or peptides.

[0244] The specific promoter used to control the expression of the peptide or protein encoding the polynucleotide of the present invention is not considered critical, as long as it enables the expression of the polynucleotide in the target cell, preferably a bacterial cell. When targeting human cells, it is preferable to place the polynucleotide coding region near and under the control of a promoter capable of expression in human cells. Generally, such promoters may include bacterial, human, or viral promoters.

[0245] C. Initiation signal and internal ribosome binding site (IRES)

[0246] Effective translation of the encoded sequence may also require specific start signals. These signals include the ATG start codon or adjacent sequences. External translation control signals, including the ATG start codon, may need to be provided. Those skilled in the art will be able to readily determine this and provide the necessary signals.

[0247] In some embodiments of the invention, internal ribosome entry site (IRES) elements are used to generate multigene or polycistronic information. IRES elements are able to bypass ribosome scanning models of 5' methylation-dependent translation and initiate translation at an internal site (Pelletier and Sonenberg, 1988; Macejak and Sarnow, 1991). IRES elements can be linked to heterologous open reading frames (ORFs). Multiple ORFs can be transcribed together, each separated by an IRES, creating polycistronic information. Transcription of a single information using a single promoter / enhancer can efficiently express multiple genes (see U.S. Patents 5,925,565 and 5,935,819, incorporated herein by reference).

[0248] D. Selectable and filterable tags

[0249] In some embodiments of the invention, cells containing the nucleic acid constructs of the present disclosure can be identified in vitro or in vivo by encoding selectable or selective markers in an expression vector. During transcription and translation, the markers confer recognizable changes to the cells, allowing easy identification of cells containing the expression vector. Typically, a selective marker is a marker that confers the property of allowing selection. A positive selection marker is one in which the presence of the marker allows selection, while a negative selection marker is one in which the presence of the marker prevents selection. An example of a positive selection marker is an antibiotic resistance marker.

[0250] E. Host cell

[0251] As used herein, the terms “cell,” “cell line,” and “cell culture” are used interchangeably. All these terms also include their progeny, i.e., any and all progeny. It should be understood that all progeny may differ due to intentional or unintentional mutations. In the context of expressing a heterologous nucleic acid sequence, “host cell” refers to a prokaryotic or eukaryotic cell, and includes any transformable organism capable of replicating a vector or expressing a heterologous gene encoded by a vector. Host cells can and have been used as recipients of vectors or viruses. Host cells can be “transfected” or “transformed,” which refers to the process of transferring or introducing a foreign nucleic acid, such as a recombinant protein-coding sequence, into a host cell. Transformed cells include the primary target cell and its progeny.

[0252] Host cells can be derived from prokaryotes or eukaryotes, including bacterial, yeast, insect, and mammalian cells, and are used for vector replication or the expression of partial or complete nucleic acid sequences. Many cell lines and cultures can be used as host cells and are available from the American Type Culture Collection (ATCC), an organization that serves as an archive of living cultures and genetic material (www.atcc.org).

[0253] F. Expression System

[0254] Various expression systems exist that comprise at least some or all of the above-described compositions. Prokaryotic and / or eukaryotic-based systems can be used in conjunction with this invention to generate nucleic acid sequences, or their homologous polypeptides, proteins, and peptides. Many such systems are commercially available.

[0255] Insect cell / baculovirus systems can produce high levels of protein expression of heterologous nucleic acid fragments, such as those described in U.S. Patent Nos. 5,871,986 and 4,879,236, both of which are incorporated herein by reference and are available for purchase, for example, from [source missing]. Name 2.0 and from BACPACK TM Baculovirus expression system.

[0256] In addition to the expression system disclosed in this invention, other examples of expression systems include... A fully controlled inducible mammalian expression system involving a synthetic ecdysone inducible receptor, or its pET expression system, is described, specifically an E. coli expression system. Another example of an inducible expression system can be found from... It was acquired, and it carries T-REX. TM (Tetracycline-regulated expression) system, an inducible mammalian expression system using a full-length CMV promoter. A yeast expression system, known as the Pichia pastoris expression system, is also provided. This system is designed for high-level production of recombinant proteins in the methyltrophic yeast Pichia pastoris. Those skilled in the art will know how to use expression vectors, such as expression constructs, to produce nucleic acid sequences or their homologous polypeptides, proteins, or peptides.

[0257] VII. Combination Therapy

[0258] The compositions and related methods disclosed herein, particularly the administration of ECM affinity peptides operably linked to anti-inflammatory agents and / or other molecules, may also be used in combination with the administration of other therapies (such as those described herein) or other conventional therapies known in the art for the treatment of autoimmune or inflammatory conditions.

[0259] The therapeutic compositions and treatments disclosed herein can be administered at intervals ranging from minutes to weeks before, simultaneously with, and / or after another treatment or agent. In embodiments where an agent is applied alone to cells, tissues, or organisms, it is generally ensured that there is not a long time interval between each delivery so that the therapeutic agent will still be able to exert a beneficial combined effect on the cells, tissues, or organisms. For example, in such cases, it is contemplated that cells, tissues, or organisms can be contacted substantially simultaneously (i.e., within less than about one minute) with two, three, four, or more than four agents or treatments. In other respects, before and / or after the administration of another therapeutic agent or treatment, one or more therapeutic agents or treatments may be administered or provided within the following timeframes: 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, 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 more than 8 weeks, and any range derived therefrom.

[0260] Various combinations of therapeutic agents and treatments can be employed. Non-limiting examples of such combinations are shown below, wherein the therapeutic agent, such as the composition disclosed herein, is "A," and the second agent, such as other agents or therapies described herein or known in the art, is "B":

[0261]

[0262] In some implementation schemes, more than one treatment course may be used. Multiple treatment courses are envisioned.

[0263] VIII. Treatment Methods

[0264] The compositions disclosed herein can be used for in vivo, in vitro, or ex vivo administration. Routes of administration may include, for example, intradermal, subcutaneous, intravenous, local, topical, and intraperitoneal administration.

[0265] Suitable autoimmune or inflammatory conditions for treatment may include, but are not limited to, the following: diabetes (e.g., type 1 diabetes), transplant rejection, arthritis (rheumatoid arthritis, such as acute arthritis, chronic rheumatoid arthritis, gout or gouty arthritis, acute gouty arthritis, acute immune arthritis, chronic inflammatory arthritis, degenerative arthritis, type II collagen-induced arthritis, infectious arthritis, Lyme arthritis, proliferative arthritis, psoriatic arthritis, Still's disease, vertebral arthritis and systemic juvenile rheumatoid arthritis, osteoarthritis, chronic progressive arthritis, deforming arthritis, chronic primary polyarthritis, reactive arthritis and ankylosing spondylitis), inflammatory hyperproliferative skin diseases, psoriasis such as plaque psoriasis, guttate psoriasis, pustular psoriasis and nail psoriasis, atopic diseases, including atopic disorders such as hay fever and Job's syndrome, dermatitis, including contact dermatitis, chronic contact dermatitis, exfoliative dermatitis Allergic dermatitis, allergic contact dermatitis, herpetic dermatitis, nummular dermatitis, seborrheic dermatitis, nonspecific dermatitis, primary irritant contact dermatitis, and atopic dermatitis, X-linked hyper-IgM syndrome, allergic intraocular inflammatory diseases, urticaria such as chronic allergic urticaria and chronic idiopathic urticaria, including chronic autoimmune urticaria, myositis, polymyositis / dermatomyositis, juvenile dermatomyositis, toxic epidermal necrolysis, scleroderma (including systemic scleroderma). ), sclerosis such as systemic sclerosis, multiple sclerosis (MS) such as spinal-ocular MS, primary progressive MS (PPMS) and relapsing-remitting MS (RRMS), progressive systemic sclerosis, atherosclerosis, arteriosclerosis, disseminated sclerosis, ataxia sclerosis, neuromyelitis optica (NMO), inflammatory bowel disease (IBD) (e.g. Crohn's disease), autoimmune-mediated gastrointestinal diseases, colitis such as ulcerative colitis, ulcerative colitis Ulcers, microscopic colitis, collagenous colitis, polyposis colitis, necrotizing enterocolitis, transmural colitis, and autoimmune inflammatory bowel disease, intestinal inflammation, pyoderma gangrenosa, erythema nodosum, primary sclerosing cholangitis, respiratory distress syndrome, including adult or acute respiratory distress syndrome (ARDS), meningitis, total or partial uveitis, iritis, choroiditis, autoimmune blood disorders, rheumatoid spondylitis, and rheumatoid synovitis. Inflammation, hereditary angioedema, meningitis and other cranial nerve damage, herpes gestationis, pemphigoid gestationis, scrotal pruritus, autoimmune premature ovarian failure, sudden hearing loss caused by autoimmune diseases, IgE-mediated diseases such as allergic reactions 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, nongranulomatous uveitis, lens antigen uveitis,Posterior uveitis or autoimmune uveitis, glomerulonephritis (GN) with or without nephrotic syndrome, such as chronic or acute glomerulonephritis such as primary GN, immune-mediated GN, membranous GN (membranous nephropathy), idiopathic membranous GN or idiopathic membranous nephropathy, membranous or membranous proliferative GN (MPGN), including type I and II, and rapidly progressive GN, proliferative glomerulonephritis, autoimmune polyglandular endocrine failure, balanitis, including plasma cell-related localized balanitis, balanoposthitis, centrifugal annular erythema, persistent erythema pigmentosum, erythema multiforme, annular granuloma, lichen sclerosus, lichen sclerosus, chronic simple lichen, lichen microspinipes, lichen planus, lamellar ichthyosis, epidermolytic hyperkeratosis, precancerous keratosis, pyoderma gangrenosa, allergic conditions and reactions, allergic reactions, eczema, including allergic Or atopic eczema, sebaceous abscess eczema, sweating disorder eczema and vesicular palmoplantar eczema, asthma such as bronchial asthma, allergic asthma and autoimmune asthma, conditions involving T cell infiltration and chronic inflammatory response, immune response to foreign antigens such as fetal ABO blood type during pregnancy, chronic inflammatory lung disease, autoimmune myocarditis, leukocyte adhesion defects, lupus, including lupus nephritis, lupus encephalitis, childhood lupus, non-renal lupus, extrarenal lupus, discoid lupus and discoid lupus erythematosus, lupus alopecia, systemic lupus erythematosus (SLE), such as cutaneous SLE or subacute cutaneous SLE, neonatal lupus syndrome (NLE) and disseminated lupus erythematosus, juvenile (type I) diabetes, including childhood insulin-dependent diabetes mellitus (IDDM) and adult-onset diabetes mellitus (type II) and autoimmune diabetes. Also considered are anticipated immune responses associated with acute and delayed hypersensitivity reactions mediated by cytokines and T lymphocytes, sarcoidosis, granulomatous diseases including lymphomatoid granulomatosis, Wegener's granulomatosis, agranulocytosis, vasculitis including vasculitis, large vessel vasculitis (including polymyalgia rheumatica and giant cell (Gauan) arteritis), medium vessel vasculitis (including Kawasaki disease and polyarteritis nodosa / periarteritis nodosa), microscopic polyarteritis, immune vasculitis, CNS vasculitis, cutaneous vasculitis, allergic vasculitis, necrotizing vasculitis such as systemic necrotizing vasculitis, and ANCA-associated vasculitis such as allergic granulomatous vasculitis. Or syndromes (CSS) and ANCA-associated small vessel vasculitis, 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 involving leukocyte exudation, CNS inflammatory diseases, Alzheimer's disease, Parkinson's disease, multiple organ injury syndromes such as those secondary to sepsis, trauma or hemorrhage, and antigen-antibody complex-mediated diseases.Antiglomerular basement membrane disease, antiphospholipid antibody syndrome, allergic neuritis, Behçet's disease / syndrome, Castleman syndrome, Goodpasture syndrome, Raynaud's syndrome, Sjögren's syndrome, Stevens-Johnson syndrome, bullous pemphigoid such as bullous pemphigoid and cutaneous pemphigoid, pemphigus (including chronic pemphigus, foliaceous pemphigus, mucocele, membranous pemphigoid, and erythematous pemphigus), autoimmune polyendocrine disorders, Reiter's disease or syndrome, heat injury, preeclampsia, immune complex diseases such as immune complex nephritis, antibody-mediated nephritis, polyneuropathy, chronic neuropathy such as IgM polyneuropathy or IgM-mediated neuropathy, autoimmune or immune-mediated diseases. Mediated thrombocytopenic purpura, such as idiopathic thrombocytopenic purpura (ITP), including chronic or acute ITP; scleritis, such as idiopathic keratoscleritis, superficial scleritis; autoimmune diseases of the testes and ovaries, including autoimmune orchitis and oophoritis; primary hypothyroidism; hypoparathyroidism; autoimmune endocrine disorders, including thyroiditis such as autoimmune thyroiditis, Hashimoto's disease, chronic thyroiditis (Hashimoto's thyroiditis) or subacute thyroiditis, autoimmune thyroid diseases, idiopathic hypothyroidism, Graves' disease; polyglandular syndromes such as autoimmune polyglandular syndrome (or polyglandular endocrine syndrome); paraneoplastic syndromes, including neuroparaneoplastic syndromes such as... Lambert-Eton myasthenia gravis or Eaton-Lambert syndrome, stiff person 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, neurogenic myotonia, oculoclonus or oculoclonus-myoclonus syndrome (OMS) and sensory neuropathy, multifocal motor neuropathy, Sheehan's syndrome, autoimmune hepatitis, chronic hepatitis, lupus-like hepatitis, giant cell hepatitis, chronic active hepatitis or autoimmune chronic active hepatitis, lymphocytic interstitial pneumonia (LIP), obliterative bronchiolitis (non-transplant) and NSIP, Guillain-Barré syndrome, Burger's disease IgA nephropathy, idiopathic IgA nephropathy, linear IgA dermatosis, acute febrile neutrophilic dermatosis, subcorneal pustular dermatosis, transient acantholytic dermatosis, cirrhosis such as primary biliary cirrhosis and pulmonary cirrhosis, autoimmune enteropathic syndrome, celiac disease, celiac disease, celiac disease, unresponsive spreh, idiopathic stomatitis, cryoglobulinemia, migraine-related lateral sclerosis (ALS; Glenk's disease), coronary artery disease, autoimmune ear diseases such as autoimmune inner ear disease (AIED), autoimmune hearing loss, polychondritis such as refractory or relapsing polychondritis, pulmonary alveolar proteinosis, Cogan syndrome / non-syphilitic interstitial keratitis, Bell's palsy,Sweet's disease / syndrome, autoimmune rosacea, herpes zoster-related pain, amyloidosis, non-cancerous lymphocytosis, essential lymphocytosis, including monoclonal B-cell lymphoblastic diseases (e.g., benign monoclonal gammopathy and monoclonal gammopathy of indeterminate significance, MGUS), peripheral neuropathy, paraneoplastic syndromes, ion channelopathies such as epilepsy, migraine, arrhythmia, muscle diseases, deafness, blindness, periodic paralysis, and CN. S-channel disease, autism, inflammatory myopathy, focal or segmental or focal segmental glomerulosclerosis (FSGS), endocrine eye disease, uveitis, choroiditis, autoimmune liver disease, fibromyalgia, polyendocrine failure, Schmidt's syndrome, adrenalitis, gastric atrophy, Alzheimer's disease, demyelinating diseases such as autoimmune demyelinating diseases and chronic inflammatory demyelinating polyneuropathy, post-myocardial infarction syndrome, alopecia areata. Greata, alopecia totalis, CREST syndrome (calcification, Raynaud's phenomenon, esophageal motility disorder, scleroderma, and telangiectasia), male and female autoimmune infertility (e.g., due to antisperm antibodies), mixed connective tissue disease, Chagas disease, rheumatic fever, recurrent miscarriage, farmer's lung, erythema multiforme, postcardiotomy syndrome, Cushing's syndrome, birdkeeper's lung, allergic granulomatous vasculitis, benign lymphocytic vasculitis, Alport syndrome, alveolitis such as allergic alveolitis and fibrotic alveolitis, interstitial pneumonia, transfusion reactions, leprosy, malaria, Parasitic diseases such as leishmaniasis, trypanosomiasis, schistosomiasis, ascariasis, aspergillosis, Sampter's syndrome, Kaplan syndrome, dengue fever, endocarditis, endocardial myocardial fibrosis, diffuse interstitial pulmonary fibrosis, pulmonary fibrosis, idiopathic pulmonary fibrosis, cystic fibrosis, endophthalmitis, persistent erythema elevatedis, erythroblastosis of fetus, eosinophilic fasciitis, Shulman syndrome, Felty's syndrome, filariasis, and cyclitis such as chronic cyclitis and heterochronic cyclitis. Cycitis, iridocyclitis (acute or chronic) or Fuch's cyclitis, allergic purpura, human immunodeficiency virus (HIV) infection, SCID, acquired immunodeficiency syndrome (AIDS), echovirus 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 failure, Siddenham's chorea, post-streptococcal nephritis, thromboangiitis obliterans, thyrotoxicosis, tabes dorsalis, choroiditis, polymyalgia giant cells, chronic allergic pneumonia, keratoconjunctivitis sicca, epidemic keratoconjunctivitis, idiopathic nephrotic syndrome.Minimal change disease, benign familial and ischemia-reperfusion injury, organ transplant reperfusion, retinal autoimmunity, joint inflammation, bronchitis, chronic obstructive airway / lung disease, silicosis, aphthous stomatitis, aphthous stomatitis, arteriosclerotic diseases, azoospermia, autoimmune hemolysis, Burke's disease, cryoglobulinemia, Dupuytren's contracture, endophthalmia phacoanaphylactica, allergic colitis, erythema nodosum leprosy, idiopathic facial nerve palsy, chronic fatigue syndrome, rheumatic fever, Harman-Ritchie disease, sensorineural hearing loss, paroxysmal hemoglobinuria, hypogonadism, regional ileitis, leukopenia, infectious mononucleosis, transverse myelitis, primary idiopathic myxedema, nephrotic disorders, ophthalmia Symphatica, orchitis granulomatosa, pancreatitis, acute polyradiculitis acuta, pyoderma gangrenosa, Quervain's thyroiditis, acquired spenic atrophy, non-malignant thymoma, leukoplakia, toxic shock syndrome, food poisoning, diseases involving T-cell infiltration, leukocyte adhesion defects, immune responses related to acute and delayed hypersensitivity reactions mediated by cytokines and T lymphocytes, diseases involving leukocyte exudation, multiple organ injury syndrome, antigen-antibody complex-mediated diseases, anti-glomerular basement membrane disease, allergic neuritis, autoimmune polyendocrine disorders, oophoritis, primary myxedema, autoimmune atrophic gastritis, sympathetic ophthalmia, rheumatic diseases, mixed connective tissue disorders. Diseases such as nephrotic syndrome, pancreatitis, polyendocrine failure, autoimmune polyglandular syndrome type I, adult-onset idiopathic hypoparathyroidism (AOIH), cardiomyopathy such as dilated cardiomyopathy, acquired epidermolysis bullosa (EBA), hemochromatosis, myocarditis, nephrotic syndrome, primary sclerosing cholangitis, purulent or non-purulent sinusitis, acute or chronic sinusitis, ethmoid sinusitis, frontal sinusitis, maxillary sinusitis, or sphenoid sinusitis, eosinophilic disorders such as eosinophilia, and pulmonary eosinophilic infiltration. Infiltration eosinophilia, eosinophilic-myalgia syndrome, Löffler's syndrome, chronic eosinophilic pneumonia, tropical pulmonary eosinophilia, bronchopneumonia with aspergillosis, aspergilloma or granulomas containing eosinophils, allergic reactions, seronegative spondyloarthritides, polyendocrine autoimmune diseases, sclerosing cholangitis, sclera,Spectral outer layer, chronic mucocutaneous candidiasis, Bruton's syndrome, transient hypogammaglobulinemia in infancy, Wiskott-Aldrich syndrome, ataxia-telangiectasia syndrome, vasodilation, autoimmune diseases associated with collagen disorders, rheumatism, neurological disorders, lymphadenitis, hypotensive response, vascular dysfunction, tissue damage, cardiovascular ischemia, hyperalgesia, renal ischemia, cerebral ischemia, diseases associated with angiogenesis, allergic hypersensitivity, glomerulonephritis, reperfusion injury, ischemic-reperfusion disorder, heart disease. Reperfusion injury of muscle or other tissues, lymphomatous tracheobronchitis, inflammatory skin diseases, skin diseases with acute inflammatory components, multiple organ failure, bullous diseases, renal cortical necrosis, acute purulent meningitis or other inflammatory diseases of the central nervous system, orbital inflammatory diseases, granulocyte transfusion-related syndrome, cytokine-induced toxicity, narcolepsy, acute severe inflammation, chronic refractory inflammation, pyelonephritis, arterial intimal hyperplasia, peptic ulcers, valvular heart disease, graft-versus-host disease, contact hypersensitivity reactions, asthmatic airway hyperresponsiveness, and endometriosis.

[0266] IX. Pharmaceutical Compositions and Methods

[0267] In some embodiments, the pharmaceutical composition is administered to a subject. Different aspects involve administering an effective amount of the composition to the subject. In some embodiments, a composition containing an anti-inflammatory agent may be administered to a subject or patient to treat inflammation and / or autoimmunity. Furthermore, this compound may be administered in combination with other treatments.

[0268] The composition can be formulated for parenteral administration, for example, for injection via intravenous, catheter-based, intra-arterial, intramuscular, subcutaneous, or even intraperitoneal routes. Typically, such compositions can be formulated as injectable preparations, such as liquid solutions or suspensions; they can also be prepared in a solid form suitable for adding liquid before injection to prepare a solution or suspension; and the formulation can be emulsified. The preparation of such formulations according to this disclosure will be known to those skilled in the art. Other routes of administration include intratumoral, peritumoral, intralymphatic, inflamed tissue, or lymph node injection. In some embodiments, administration is systemic.

[0269] Other application routes are also envisioned. For example, the construct and the agent can be applied in combination with a carrier. In some embodiments, the carrier is a nanoparticle or microparticle.

[0270] Particles can have structures of variable size and are referred to in various ways as microspheres, microparticles, nanoparticles, nanospheres, or liposomes. Such particulate formulations can be formed by covalent or non-covalent coupling of a construct with the particles. The terms “particle,” “microparticle,” “bead,” “microsphere,” and grammatical equivalents herein refer to small, discrete particles applicable to an object. In some embodiments, the shape of the particles is substantially spherical. As used herein, the term “substantially spherical” means that the shape of the particle deviates from a sphere by no more than about 10%. Particles typically consist of a substantially spherical core and optionally one or more layers. The size and composition of the core can vary. In addition to the core, the particle may have one or more layers to provide functionality suitable for the application of interest. If present, the thickness of the layers can vary depending on the needs of the specific application. For example, layers can impart useful optical properties.

[0271] Suitable drug forms for injection include sterile aqueous solutions or dispersions; preparations containing sesame oil, peanut oil, or aqueous propylene glycol; and sterile powders for the ad hoc preparation of sterile injections or dispersions. In all cases, the form must be sterile and must be an easily injectable fluid. It should also be stable under manufacturing and storage conditions and must be protected against contamination by microorganisms such as bacteria and fungi.

[0272] The carrier can also be a solvent or dispersion medium comprising, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), suitable mixtures thereof, and vegetable oils. Suitable flowability can be maintained, for example, by using a coating such as lecithin, by maintaining the desired particle size in the dispersed case, and by using surfactants. Various antimicrobial and antifungal agents can prevent microbial action, such as parabens, chlorobutanol, phenol, sorbic acid, thimerosal, etc. In many cases, isotonic agents, such as sugars or sodium chloride, will be preferred. The absorption of injectable compositions can be prolonged by using delayed absorption agents, such as aluminum monostearate and gelatin.

[0273] Sterile injection solutions are prepared by adding the desired amount of the active compound along with various other ingredients listed above to a suitable solvent, followed by filtration and sterilization as needed. Typically, dispersions are prepared by incorporating various sterilized active ingredients into a sterile carrier containing a basic dispersion medium and the other desired ingredients listed above. In the case of sterile powders used to prepare sterile injection solutions, preferred preparation methods include vacuum drying and freeze-drying techniques, which produce powders of the active ingredient, plus any other desired ingredients from a previously sterile filtered solution.

[0274] As used herein, the term "pharmaceutically acceptable" refers to compounds, materials, compositions, and / or dosage forms that, to a reasonable extent of medical judgment, are suitable for contact with human and animal tissues without excessive toxicity, irritation, allergic reactions, or other problematic complications in proportion to a reasonable benefit / risk ratio. The term "pharmaceutically acceptable carrier" refers to pharmaceutically acceptable materials, compositions, or media involved in carrying or transporting chemical reagents, such as liquid or solid fillers, diluents, excipients, solvents, or encapsulating materials.

[0275] As used herein, a "pharmaceutically acceptable salt" refers to a derivative of the disclosed compound in which the parent compound is modified by converting an existing acid or base moiety into its salt form. Examples of pharmaceutically acceptable salts include, but are not limited to, inorganic or organic acid salts of basic residues such as amines; alkali metal or organic salts of acidic residues such as carboxylic acids; and so on. Pharmaceutically acceptable salts include conventional non-toxic salts or quaternary ammonium salts of parent compounds formed from, for example, non-toxic inorganic or organic acids. Pharmaceutically acceptable salts can be synthesized from parent compounds containing basic or acidic moieties using conventional chemical methods.

[0276] Dosage will inevitably vary depending on the individual's condition. In any case, the person responsible for administration will determine the appropriate dose for the individual. The effective amount of the therapeutic or preventative composition is determined based on the intended outcome. The term "unit dose" or "dose" refers to a physically discrete unit applicable to the individual, each unit containing a predetermined amount of the composition, calculated to produce the desired response discussed above in relation to its administration, i.e., the appropriate route and regimen. Depending on the number of treatments and the unit dose, the amount administered depends on the desired effect. The precise amount of the composition also depends on the practitioner's judgment and varies from person to person. Factors affecting dosage include the individual's physical and clinical condition, route of administration, intended therapeutic outcome (symptom relief vs. cure), and the potency, stability, and toxicity of the specific composition.

[0277] After preparation, the solution will be administered in a manner compatible with the dosage form and at a therapeutic or preventative dose. The formulation is readily available in various dosage forms, such as the injectable type described above.

[0278] Typically, for an adult (weighing approximately 70 kg), the following amounts of the compound are administered: approximately 0.1 mg to approximately 3000 mg (inclusive), or approximately 5 mg to approximately 1000 mg (inclusive), or approximately 10 mg to 100 mg (inclusive). It should be understood that these dosage ranges are for illustrative purposes only and may be adjusted for dosage based on factors known to the physician.

[0279] In some embodiments, the amount applied to the target is about, at least about, or at most about 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, 9.6, 9.7, 9.8, 9.9, 10.0, 10.5, 11.0, 11.5, 12.0, 12.5, 13.0, 1 3.5, 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, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 6 4, 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, 12 0, 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, 275280, 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, 880, 890, 900, 9 10, 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 The dosages of the pharmaceuticals discussed herein are 3600, 3700, 3800, 3900, 4000, 4100, 4200, 4300, 4400, 4500, 4600, 4700, 4800, 4900, 5000, 6000, 7000, 8000, 9000, and 10000 mg (mg) or mcg (mcg) or μg / kg or mcg / kg / min or mg / kg / min or mcg / kg / hour or mg / kg / hour, or μM or mM. Any ranges that may be derived from these are contemplated.

[0280] One dose may be administered as needed, or every 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 18 hours, or 24 hours (or any range derived therefrom), or once, twice, three times, four times, five times, six times, seven times, eight times, or nine times daily (or any range derived therefrom). The first dose may be administered before or after the onset of symptoms. In some implementations, the first dose of the treatment regimen is administered to the patient 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, or 12 hours (or any range derived therefrom) or 1 day, 2 days, 3 days, 4 days, or 5 days (or any range derived therefrom) after the patient experiences or exhibits signs or symptoms of the condition. Patients may continue treatment for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 days or more (or any range derived therefrom) or until symptoms disappear or lessen, or 6, 12, 18 or 24 hours or 1, 2, 3, 4 or 5 days after symptoms of infection disappear or lessen.

[0281] X. Example

[0282] The following embodiments are included to illustrate preferred embodiments of this disclosure. Those skilled in the art will understand that the techniques disclosed in the following embodiments represent techniques that the inventors have discovered that work well in the practice of this disclosure, and therefore can be considered as constituting a preferred mode of practice. However, those skilled in the art will understand from the content of this disclosure that many changes can be made to the specific embodiments disclosed without departing from the spirit and scope of this disclosure and still obtaining the same or similar results.

[0283] Example 1: Collagen-binding modification engineering enhances the efficacy of anti-inflammatory agents

[0284] A. Result

[0285] 1. CBP conjugation provides collagen affinity for anti-TNFα antibodies (αTNF).

[0286] CBPs were covalently cross-linked with the anti-TNFα antibody (αTNF) after being mixed with sulfosuccinimide-4-(N-maleimidemethyl)cyclohexane-1-carboxylic acid ester (sulfon-SMCC). Up to five CBPs bound to the antibody were counted by matrix-assisted laser desorption / ionization-time-of-flight (MALDI-TOF) mass spectrometry (Figure 1A). To examine the ability of CBP-conjugated αTNF (CBP-αTNF) to bind 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 tested collagen types, while the binding signal of WT-αTNF to collagen was undetectable (Figure 1B).

[0287] 2. CBP conjugation enables αTNF to be localized in the inflamed claw in an arthritis model.

[0288] CBP-αTNF was identified and located in the inflamed paw of a collagen antibody-induced arthritis (CAIA) model by binding to endogenous collagen through in vivo biodistribution analysis. 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 pad. Local LPS injection induced severe arthritis in the right hind paw compared to other paws. On the day after LPS injection, fluorescently labeled CBP-αTNF and WT-αTNF were intravenously injected into CAIA and juvenile mice. Systemic fluorescence levels were measured before antibody injection and at 0.5 h, 1 h, 2 h, 4 h, 6 h, 24 h, and 48 h after injection. Fluorescence levels in the arthritic paw of the right hind paw of CAIA mice injected with CBP-αTNF and WT-αTNF increased immediately after injection, while fluorescence levels in the arthritic and non-arthritic paws of juvenile mice were almost identical (Fig. 2A and Fig. 2B). The ratio of CBP-αTNF levels in the arthritic paw to the non-arthritic paw was higher in mice injected with CBP-αTNF than in those injected with WT-αTNF (Fig. 2C). Injected CBP-αTNF was detected by immunohistochemistry in the synovium and pannus, the main inflammatory areas of arthritis (Figure 2D). These data indicate that CBP-αTNF preferentially localizes to inflamed tissue (i.e., the arthritic claw) after systemic injection, rather than its unmodified form.

[0289] 3. CBP conjugation enhances the efficacy of αTNF in an arthritis model.

[0290] The inventors then examined the anti-inflammatory efficacy of CBP-αTNF in a CAIA model. Arthritis was induced in all paws 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. Control mice showed increased arthritis scores, while WT-αTNF and CBP-αTNF scores decreased. Figure 3 A). Mice treated with CBP-αTNF showed a significantly greater reduction in joint scores than mice treated with WT-αTNF. Histological observation revealed that CBP-αTNF significantly inhibited joint destruction. Figure 3 B). The inventors further investigated whether CBP-αTNF could demonstrate therapeutic efficacy via subcutaneous injection. Similar accumulation trends and inhibitory efficacy were observed even in subcutaneous CBP-αTNF injection (Figure 4). These data suggest that CBP modification of αTNF provides better anti-inflammatory efficacy than its unmodified form.

[0291] 4. Local injection of ECM combined with αTNF has a strong effect on the development of arthritis.

[0292] To evaluate the therapeutic efficacy of local treatment, the inventors combined αTNF with a hybrid ECM-binding peptide derived from placental growth factor 2 (specifically PlGF-2) in the same manner as the previously described CBP conjugation. 123-144) Combined with (32, 33). PlGF-2 123-144 The peptide binds to a variety of ECM proteins with high affinity, thus remaining at the injection site. PlGF-2 injected into the left hind paw pad of CAIA mice... 123-144 -Conjugated αTNF(PlGF-2) 123-144 WT-αTNF was retained at the injection site, while WT-αTNF signaling rapidly decreased after injection (Figure 5A). To compare efficacy, control IgG, WT-αTNF, or PlGF-2 were subcutaneously injected into the left hind paw pad of CAIA mice. 123-144 -αTNF. In mice treated with control IgG, arthritis scores in both the right and left hind limbs increased. WT-αTNF did not inhibit scores in this treatment regimen. However, even at doses 100 times lower than WT-αTNF, PlGF-2... 123-144 -αTNF also almost completely inhibited the development of arthritis in the treated claw (left). Interestingly, PlGF-2 123-144 -αTNF did not inhibit the development of arthritis in the untreated claw (right), indicating its local efficacy (Figure 5B). These data suggest that drug accumulation at the site of inflammation is crucial for suppressing inflammation and maximizing the efficacy of anti-inflammatory drugs.

[0293] 5. CBD proteins derived from the vWF A3 domain can target the inflamed spinal cord in an experimental autoimmune encephalomyelitis (EAE) model.

[0294] IL-4 is a cytokine that induces the differentiation of naive helper T cells (Th0) into Th2 cells. Intrathecal or intranasal administration of IL-4 to the central nervous system has been reported to improve clinical symptoms and axonal morphology in an experimental autoimmune encephalomyelitis (EAE) model (a mouse model of multiple sclerosis). Therefore, to achieve EAE-targeting of IL-4 via intravenous injection, a clinically relevant route of administration, the inventors synthesized a vWF A3 domain-fused IL-4 protein (A3-IL4). A3-IL4 binds to collagen III with a dissociation constant (Kd) of 28.6 nM (Figure 6A). The fusion of the vWF A3 domain with IL-4 did not eliminate its binding affinity to its receptor IL-4Rα (Figure 6B). Subsequently, the localization of the vWF A3 domain protein in the inflamed spinal cord of the EAE model was determined by fluorescence imaging via binding to endogenous collagen. On day 14 post-immunization, when target tissues were inflamed, fluorescently labeled A3 or A3-IL4 was intravenously injected into naive and EAE mice. A3 and A3-IL4 were detected in the spinal cord of EAE mice, but not in the spinal cord of naive mice (Figure 6C). The inventors then examined the therapeutic effect of A3-IL4 on EAE symptoms. Starting from the first appearance of EAE symptoms on day 14 post-immunization, PBS, normal IL-4, and A3-IL4 were administered intravenously every other day. A3-IL4 reduced the mean disease score, while normal IL-4 showed no therapeutic effect (Figure 6E).

[0295] 6. A3 protein and CBP conjugates can also target inflamed tissues in other inflammatory disease models.

[0296] To explore the potential applications of collagen-binding engineering in inflammatory diseases, the localization of A3 protein and CBP-conjugated antibodies was determined by fluorescence imaging in inflamed tissues of spontaneous inflammatory bowel disease (IBD), bleomycin-induced idiopathic pulmonary fibrosis (IPF), and type 1 diabetes mellitus (T1D) models. Fluorescently labeled A3, CBP-αTNF, or CBP-conjugated anti-TGF-β antibody (CBP-αTGF) were intravenously injected into EAE, IBD, IPF, and T1D models when the target tissues were inflamed, and the fluorescence levels in the target tissues were determined. A3 was detected in the colon of IBD-developing mice, the pancreas of spontaneous T1D and cyclophosphamide-induced T1D mice, but not in healthy mice (Figs. 7A and 7C). Similarly, CBP-αTNF was detected in the spinal cord of EAE models and the colon of IBD-developing mice, but not in the colon of healthy mice (Figs. 6D and 7A). Histopathological analysis showed that CBP-αTNF was localized in the lamina propria of the colon in the IBD model, where infiltrating cells were present (Fig. 7A). Furthermore, CBP-αTGF was detected in the lungs of the IPF model, while it was not present in the unmodified antibody (Fig. 7B). These results suggest that the affinity of collagen for antibodies and cytokines enables them to target inflamed tissues.

[0297] B. Discussion

[0298] This study demonstrates that conjugating collagen affinity with anti-inflammatory antibodies enhances its retention in inflamed tissues and its therapeutic efficacy. Intravenous and subcutaneous CBP-αTNF accumulated in the inflamed paws of a CAIA model. Furthermore, the conjugation of CBP with antibodies enabled its 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 ubiquitous and abundant around the vascular system, but is only exposed to the bloodstream when high vascular permeability occurs in inflamed tissues. Therefore, collagen affinity as a drug delivery method is neither a tissue-, molecular expression, nor a disease-specific approach, but rather a general inflammation-specific approach. More importantly, in the CAIA model, CBP-αTNF reduced arthritis scores more effectively than unmodified αTNF. αTNF treatment for inflammatory diseases such as RA and IBD does not show complete response in most patients and can have significant side effects (6-10). Therefore, CBP-αTNF has the potential to translate into advanced treatments for inflammatory and autoimmune diseases.

[0299] To assess whether other collagen-binding proteins could also target inflammatory sites after systemic injection, the inventors used a recombinant protein with the vWF A3 domain. A3-IL4 accumulated in the spinal cord and reduced disease scores in the EAE model, while the normal form of IL-4 did not. Targeting neuronal IL-4 signaling is expected to be a novel therapeutic strategy to halt the progression of disability in multiple sclerosis (34). While intrathecal or intranasal routes have been proposed for IL-4 therapy to cross the blood-brain barrier, this embodiment demonstrates that providing collagen-binding capacity for IL-4 can reduce clinical symptoms in the EAE model, even via an intravenous route. It also shows accumulation of A3 protein in inflamed tissues 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 fibrosis with inflammation, and the T1D model for spontaneous T cell-mediated autoimmune diseases as primary inflammation models. These data suggest that collagen-bound antibodies and cytokines can achieve effective antibody and cytokine therapy in a variety of inflammatory diseases by accumulating at sites of inflammation.

[0300] This example demonstrates local injection of PlGF-2. 123-144 -αTNF is retained at the injection site and shows strong therapeutic effects at low doses. However, PlGF-2 123-144 -αTNF therapy is only effective when administered via local injection due to its mixed ECM affinity. A major translational advantage of collagen-bound methods for targeted inflammation therapy is their ability to target the site of inflammation via systemic delivery. For the clinical translation of collagen-bound anti-inflammatory drugs, the advantage of using the A3 domain from vWF or CBP from core proteoglycans lies in their natural presence in the human body, limiting the likelihood of recognition by the immune system. Furthermore, CBP can be conjugated to antibodies via a simple chemical reaction. This feature is advantageous because it simplifies production, as optimized antibodies can be used. In this embodiment, the inventors have demonstrated that CBP can conjugate both anti-TNFα and anti-TGFβ antibodies. The CBP conjugation synthesis reaction of the antibody can be completed in just 90 minutes using a chemical reaction similar to protein PEGylation. The same reaction is used for antibody-drug conjugates, for example, in the production of trastuzumab-metazidine conjugates (35, 36). Regarding A3-IL4, given that cytokines are small molecules and generally readily produced, the inventors opted for recombinant fusion rather than conjugating A3 to IL-4. These characteristics could facilitate the development of collagen-based drug therapies to overcome barriers to clinical translation.

[0301] In summary, the discovery that collagen's affinity for antibodies and cytokines enables them to target sites of inflammation has been demonstrated. Furthermore, anti-inflammatory drugs conjugated with collagen exhibit higher therapeutic efficacy compared to their unmodified form. This simple approach to engineering collagen-conjugated drugs holds clinical translational potential as targeted anti-inflammatory agents.

[0302] C. Materials and Methods

[0303] 1. Synthesize CBP-conjugated antibodies

[0304] Rat anti-mouse TNF-α antibody (clone XT3.11, BioXcell) was reacted with 20 equivalents of sulfosuccinimide-4-(N-maleimidemethyl)cyclohexane-1-carboxylic acid ester (sulfon-SMCC) at room temperature for 30 min. Excess sulfon-SMCC was removed using a Zeba rotary desalting column (Thermo Fisher Scientific). Then, 30 equivalents of a collagen-binding sequence peptide derived from decorion (CBP, LRELHLNNNC) were added and reacted at room temperature for 1 h to conjugate the thiol moiety on the C residue. The peptide was synthesized by Genscript with a purity >95%.

[0305] 2. Production and purification of recombinant vWF A3 domain and A3 fusion IL-4 protein

[0306] The sequence encoding the human vWF A3 domain residues Cys1670-Gly1874 (907-1111 of mature vWF), the human vWF A3 domain, and the mouse IL-4 fusion protein was synthesized and subcloned into the mammalian expression vector pcDNA3.1(+) provided by Genscript. To further purify the recombinant protein, a sequence encoding 6His was added to the N-terminus. Suspension-adapted HEK-293F cells were routinely maintained in serum-free FreeStyle 293 expression medium (Gibco). On the day of transfection, cells were fed at a rate of 1 × 10⁶ cells / year. 6 Cells were seeded at a density of [number] cells / m² into fresh culture medium. 2 μg / ml plasmid DNA, 2 μg / ml linear 25 kDa polyethyleneimine (Polysciences), and OptiPRO SFM medium (4% final concentration, Thermo Fisher) were added sequentially. The culture flask was incubated at 37°C with a stirring motion of 135 rpm in the presence of 5% CO₂. Six days post-transfection, the cell culture medium was collected by centrifugation and filtered through a 0.22 μm filter. The medium was then loaded into a HisTrap HP 5 ml column (GE Healthcare), which uses [specific technology / method / process]. Pure 25 (GE Healthcare). After washing the column with wash buffer (20 mM imidazole, 20 mM NaH₂PO₄, 0.5 M NaCl, pH 7.4), the protein was eluted with a gradient of 500 mM imidazole (20 mM NaH₂PO₄, 0.5 M NaCl, pH 7.4). The eluent was further purified by size exclusion chromatography using a HiLoadSuperdex 200PG column (GE Healthcare). All purification steps were performed at 4°C. A3 and A3-IL-4 expression was determined by Western blotting using an anti-His-tagged antibody (BioLegend), and the protein purity was confirmed as >90% by SDS-PAGE.

[0307] 3. Detect the binding of CBP-αTNF or A3-IL4 to collagen.

[0308] Measurements were performed as described previously (32). 96-well ELISA plates (Greiner Bio One) were coated overnight at 37°C with human collagen types I, II, and III (PBS, Millipore Sigma, 10 μg / mL each), then blocked for 1 hour at room temperature with PBS containing 1% BSA and 0.05% Tween 20 (PBS-T). The wells were then washed with PBS-T and further incubated for 1 hour at room temperature with 1 μM CBP-αTNF, 1 μM unmodified αTNF, or 0-740 nM A3-IL4. After washing three times with PBS-T, antibodies were detected using HRP-conjugated anti-rat IgG antibody and incubated for 1 hour at room temperature (Jackson Immuno Research). A3-IL4 was detected using a specific antibody against mouse IL-4 (R&D Systems). After washing, the binding protein was detected using tetramethylbenzidine substrate by measuring absorbance at 450 nm and subtracting absorbance at 570 nm.

[0309] 4. Detect the binding of A3-IL4 to its receptor.

[0310] Measurements were performed as described previously (32). 96-well ELISA plates (Greiner Bio One) were coated overnight at 37°C with recombinant mouse IL-4Rα protein (10 μg / mL each in PBS and R&D Systems), then blocked for 1 hour at room temperature with PBS and PBS-T containing 1% BSA. The wells were then washed with PBS-T and further incubated with 0–740 nM A3-IL4 or IL-4 for 1 hour at room temperature. After washing three times with PBS-T, IL-4 was detected using a specific antibody against mouse IL-4 (R&D Systems). After washing, the binding protein was detected using a tetramethylbenzidine substrate by measuring absorbance at 450 nm and subtracting absorbance at 570 nm.

[0311] 5. MALDI-TOF MS

[0312] Antibodies were analyzed using MALDI-TOF MS (Bruker Ultraflextreme MALDI TOF / TOF). All spectra were acquired using Bruker FlexControl software. TM Data was collected and analyzed using the Bruker FlexAnalysis software. TM Processing. First, a saturated solution of matrix, α-cyano-4-hydroxycinnamic acid (Sigma-Aldrich), was prepared in a 50:50 acetonitrile:1% TFA solvent. Then, the analyte (5 μL, 0.1 mg / mL) in PBS and the matrix solution (25 μL) were mixed, and 1 μL of this mixture was deposited onto an MTP 384 abrasive steel target plate. The droplet was dried in a nitrogen stream to form a homogeneous sample / matrix coprecipitate. All samples were analyzed using a high-quality linear positive mode method with 2500 laser emissions at 75% laser intensity. Measurements were externally calibrated at three points using a mixture of carbonic anhydrase, phosphorylase B, and bovine serum albumin.

[0313] 6. In vivo biodistribution studies

[0314] TNFα antibody (clone XT3.11, BioXcell) and anti-TGF-β antibody (clone 1D11.16.8, BioXcell) were incubated with 8 equivalents of SM(PEG)24 (Thermo Fisher Scientific) at room temperature for 30 minutes. Excess SM(PEG) was removed using a Zeba rotary desalting column (Thermo Fisher Scientific). 24Then, 30 equivalents of Cy7-labeled CBP ([Cy7]LRELHLNNNC[COOH]) were added and reacted at room temperature for 30 minutes to conjugate the thiol moiety on the C residue. The peptide was synthesized by Genscript with a purity >95%. Unreacted dye was removed by dialysis with PBS. For CBP-unconjugated antibodies, αTNF and αTGF were labeled with sulfonated Cy7 NHS ester (Lumiprobe) according to the manufacturer's instructions. A3 and A3-IL4 were labeled with DyLight 800NHS ester (Thermo Fisher Sientific) according to the manufacturer's instructions. When inflammation occurred in the target tissues of model mice, 10 μg to 100 μg of Cy7-labeled WT-αTNF, WT-αTGF, CBP-αTNF, and CBP-αTGF or DyLight 800-labeled A3 and A3-IL4 were administered intravenously. Mouse organs were harvested and imaged using the Xenogen IVIS Imaging System 100 (Xenogen) under the following conditions: f / stop: 2; excitation wavelength 745 nm; excitation wavelength 800 nm; exposure time: 5 seconds; small resolution.

[0315] 7. Mouse model of collagen antibody-induced arthritis (CAIA)

[0316] Arthritis was induced in female Balb / c mice (7 weeks old) by intraperitoneal injection of an anti-collagen antibody mixture (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, mice were intravenously or subcutaneously injected with control IgG (200 μg / mouse), WT-αTNF (200 μg / mouse), or CBP-αTNF (200 μg / mouse) intravenously or subcutaneously in the back; or subcutaneously injected with control IgG (100 μg / mouse), WT-αTNF (100 μg / mouse), or PlGF-2 in the left hind paw pad. 123-144 -αTNF (1 μg / mouse). Joint swelling was scored daily as described elsewhere (31). On day 8, the hind paws were fixed in 10% neutral formalin (Sigma-Aldrich), decalcified in Decalcifer II (Leica), and then provided for histological analysis.

[0317] 8. Experimental autoimmune encephalomyelitis (EAE) model

[0318] By administering MOG on day 0 with complete Freund's adjuvant (200 μg / mouse, Hooke Laboratories) 35-55Evasive immunization was performed in female C57BL / 6 mice (13 weeks old), followed by administration of pertussis toxin (100 ng / mouse) in PBS on the day of immunization and the following day. When EAE symptoms appeared on day 14 post-immunization, recombinant mice were treated with either IL-4 (Peprotech) 1 μg / mouse or A3-IL4 1 μg / mouse (equivalent to 0.4 μg / mouse, molars), repeated every other day. Disease severity in individual mice was scored daily according to the following scales: 0, no clinical disease; 0.5, tail weakness; 1, tail paralysis; 2, hind limb weakness; 3, hind limb paralysis; 3.5, forelimb weakness; 4, forelimb paralysis; or 5, dying or dead.

[0319] 9. A spontaneous colitis model of inflammatory bowel disease (IBD)

[0320] IL-10 - / - ×TLR-4 - / - (DKO) mice were kindly provided by Cathyn Nagler (The University of Chicago). DKO mice spontaneously develop colitis and exhibit a high incidence of rectal prolapse. Mice were used for imaging analysis as IBD-developing mice at the first sign of rectal prolapse at 29 weeks of age. As controls that did not develop IBD, 16-week-old DKO mice and genetically predisposed (C57BL / 6) mice were used.

[0321] 10. Bleomycin-induced idiopathic pulmonary fibrosis (IPF) model

[0322] In 9-week-old female C57BL / 6 mice, inflammatory pulmonary fibrosis was induced by intranasal instillation of 100 μg / mouse bleomycin in saline (Sigma-Aldrich). Mice were used for imaging analysis on day 7 post-bleomycin administration when lung inflammation was observed.

[0323] 11. NOD mouse model of type 1 diabetes (T1D)

[0324] Non-obese diabetic (NOD) mice are considered a spontaneous model of T-cell-mediated autoimmune insulin-dependent diabetes mellitus (37, 38). Cyclophosphamide promotes the onset of diabetes in NOD mice (39). For imaging analysis, Balb / c mice were used as non-diabetic controls, and naturally developing diabetic NOD mice were induced by intraperitoneal injection of 300 mg / kg cyclophosphamide (Sigma-Aldrich). Blood glucose levels were used as an indicator of diabetes development.

[0325] 12. Histological analysis and immunohistochemistry

[0326] Paraffin-embedded joint tissue from CAIA mice and colon from mice developing IBD were cut into 5 μm thick sections and subjected to pathological analysis using H&E and / or PAS staining. The severity of synovial hyperplasia and bone resorption in the arthritis models was assessed using a three-tiered scale (0-2) based on previously reported criteria, with slight modifications as follows: 0, normal to minimal pannus in the cartilage and subchondral bone margins; 1, mild to moderate infiltration in the margins with mild cortical and medullary bone destruction; 2, severe infiltration with complete or near-complete destruction of the joint structure. The scores for both hind paws of each mouse were summed (total score per mouse, 0-4).

[0327] Immunohistochemical staining was performed according to standard procedures. In short, sections were incubated in 0.3% H2O2 for 20 minutes, blocked with 1% BSA buffered in PBS for 1 hour, and incubated overnight at 4°C with HRP-conjugated anti-rat IgG (Jackson Immuno Research), followed by 1 hour at room temperature and staining with diaminobenzidine.

[0328] 13. Statistical Analysis

[0329] Statistical analysis was performed using GraphPad Prism software, and p < 0.05 was considered statistically significant. Changes in arthritis scores over time were assessed using repeated measures two-way ANOVA. When interactions were considered significant, Dunnett's multiple comparison test was used to evaluate the data at each measurement time point. To compare the efficacy of CBP-αTNF versus WT-αTNF, data from day 8 were reanalyzed 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.

[0330] D. References

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[0371] Example 2: Engineered IL-10 enhances lymph node transport and inhibits rheumatoid arthritis in mouse models

[0372] Rheumatoid arthritis (RA) is a major autoimmune disease. While clinical trials using interleukin-10 (IL-10) have explored it as a potential treatment for RA, its efficacy has been limited, likely due to insufficient retention in lymphoid organs, where antigen recognition is primarily involved. Here, the inventors engineered IL-10 to fuse with serum albumin (SA) but not with the collagen-binding domain (CBD). Following intravenous injection, SA-IL-10 and CBD-SA-IL-10 exhibited longer circulation times than unmodified IL-10; furthermore, SA fusion resulted in enhanced lymph node (LN) accumulation compared to unmodified IL-10. Intravenous treatment with SA-IL-10 and CBD-SA-IL-10 restored the immune cell composition in the claw to a normal state, increased the frequency of suppressor M2 macrophages, and protected joint morphology. Intravenous SA-IL-10 and CBD-SA-IL-10 demonstrated efficacy similar to that of treatment using anti-TNF-α antibodies. The integration of SA and IL-10 is a simple yet effective engineering strategy for achieving LN accumulation and controlling RA.

[0373] Rheumatoid arthritis (RA) is an autoimmune disease currently managed through treatment using inhibitors of inflammatory pathways. The pathological features of RA are synovitis and joint destruction, causing severe pain and joint dysfunction (1, 2). Although the causative antigens of RA are not fully elucidated, immune cells play a crucial role in the recognition of collagen. During the progression of RA, autoantigen-specific T cells, particularly Th17 cells, are activated and produce inflammatory cytokines, including IL-17. Inflammatory cytokines in the joints, such as TNF-α and IL-6, induce macrophage and neutrophil activation, acting as mediators of the inflammatory response. These inflammatory cells infiltrate the joints and cause various inflammatory responses, including the activation of osteoclasts that destroy joint bone (3). Current RA treatment strategies are symptomatic, and given the involvement of many inflammatory cytokines in RA progression, various biological therapies, such as antibodies or soluble receptors for TNF-α, have been developed and approved for clinical use (4).

[0374] As another type of biological therapeutic agent, the administration of anti-inflammatory cytokines for the treatment of RA has been studied to induce systemic suppression of inflammation or tolerance. IL-10 is such an 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 efficacy of IL-10 in autoimmune diseases remains controversial, possibly due to its short circulating half-life and uncontrolled biodistribution after systemic administration (8).

[0375] In this study, the inventors engineered IL-10 by fusing serum albumin (SA) to provide prolonged blood circulation and by fusing a collagen-binding domain (CBD) to provide binding affinity to inflamed sites, thereby enhancing vascular permeability and exposing extracellular matrix proteins, including collagen, to blood-derived proteins (16, 17). Therefore, the inventors hypothesized that CBD-SA fusion would increase inflamed site targeting of the SA-fused cytokine. By doing so, the inventors sought to explore whether enhanced blood circulation and vascular targeting of disease sites synergistically improved the therapeutic effect of IL-10 on rheumatoid arthritis (RA). However, the inventors observed that SA-IL-10 fusion not only prolonged circulation time but also increased accumulation in lymph nodes (LNs). Here, the inhibitory effect of engineered IL-10 on arthritis was evaluated using a mouse model of passively induced collagen antibody-induced arthritis (CAIA) and an active CIA model. The inventors found that CBD fusion enhanced accumulation in inflamed claws, and that SA-IL-10 fusion enhanced the transport of IL-10 to LNs after intravenous injection. SA-fused IL-10 significantly improved the anti-inflammatory effect of IL-10 in two mouse RA models, and its function was similar to TNF-α blockade.

[0376] A. Albumin-fused IL-10 binds to FcRn and APC and accumulates in LN.

[0377] Recombinant expression of wild-type (wt) mouse IL-10, SA fusion mouse IL-10, and CBD-SA fusion IL-10 was performed, with the molecular weight of the fusion proteins correspondingly higher than that of wt IL-10, as determined by SDS-PAGE. Furthermore, most SA-IL-10 and CBD-SA-IL-10 existed as monomers under non-reducing conditions (Figure 8A and 8B). Figure 14A Surface plasmon resonance (SPR) analysis showed that SA-IL-10 and CBD-SA-IL-10 interact with neonatal Fc receptors (FcRn) at micromolar levels. d Combined (Figure 8B and) Figure 14B Furthermore, CBD-SA-IL-10 displays K at the nanomolar level. d Combining type I and type III collagen ( Figure 14B The binding affinity of these proteins to splenocytes and single cells isolated from the popliteal fossa LN was further assessed by flow cytometry (Fig. 8C). SA-fused IL-10 showed high binding affinity to both macrophages and dendritic cells in both splenocytes and LN-derived cells. Significantly higher fluorescence signal was observed in the popliteal fossa LN after intravenous injection of fluorescently labeled SA-IL-10 compared to wt IL-10 (Fig. 8D). Interestingly, the higher fluorescence signal was located around high endothelial venules (HEVs), where antigen-presenting cells (APCs) reside (18).

[0378] B. Albumin-fused IL-10 showed prolonged blood circulation, while CBD fusion led to accumulation in the inflamed claw.

[0379] SA is known to exhibit prolonged circulation via FcRn-mediated endothelial cell circulation (19, 20). As expected, SA-IL-10 showed significantly prolonged blood circulation compared to wtIL-10; CBD-SA-IL-10 also exhibited comparable circulation to SA-IL-10 (Fig. 9A). Fig. 9B represents the fluorescence signals of major organs in mice after intravenous injection of proteins labeled with DyLight800. SA-IL-10 and CBD-SA-IL-10 showed higher signals in the heart, lungs, and spleen than wt IL-10, reflecting their prolonged circulation characteristics. Furthermore, the signal detected in the inflamed paws of mice treated with CBD-SA-IL-10 was significantly higher than that of wt IL-10, while the fluorescence observed in the non-inflamed paws showed no significant difference between wt IL-10 and CBD-SA-IL-10, indicating the inflammatory targeting ability of CBD-SA-IL-10 via collagen affinity, as previously reported in studies under other conditions (16, 21).

[0380] C. Albumin-fused IL-10 inhibits the development of arthritis.

[0381] The therapeutic effect of engineered IL-10 in a passive collagen antibody-induced arthritis (CAIA) model was evaluated (Figure 10). Intravenous injection of SA-IL-10 or CBD-SA-IL-10 significantly inhibited the development of arthritis, while severe inflammation was observed in the paws of mice injected with PBS or wt IL-10 (Figure 10A). The therapeutic effect of CBD-SA-IL-10 was compared with that of treatment with an anti-TNF-α antibody (αTNF-α), a mouse model of an antibody drug clinically used to treat RA, where the CAIA inhibition induced by CBD-SA-IL-10 was comparable to that of αTNF-α (Figure 10B). Histological analysis showed that both CBD-SA-IL-10 and αTNF-α inhibited joint destruction compared with PBS- or wt IL-10 treatment (Figure 10C). Histological scores were also significantly reduced due to treatment with both CBD-SA-IL-10 and αTNF-α (Figure 10C). The effect of the route of administration on therapeutic efficacy was also investigated, comparing intravenous, local (plantar), and subcutaneous (distal, mid-back) administration (Fig. 10D). Compared with the intravenous injection results shown in Figs. 10A and 10B, plantar injection of CBD-SA-IL-10 showed a relatively high inhibitory effect on CAIA-induced lesions, indicating that CBD-SA-IL-10 is retained in the inflamed claw through collagen affinity (Fig. 8B). Notably, SA-IL-10 showed a fairly high inhibitory effect on CAIA across all tested routes of administration (Fig. 10D).

[0382] As a second arthritis model, the active collagen-induced arthritis (CIA) model was used to evaluate the therapeutic effects of CBD-SA-IL-10 and SA-IL-10 on RA. Compared with PBS-treated CIA mice, CBD-SA-IL-10 significantly inhibited the increase in clinical scores (Fig. 11A), with therapeutic efficacy comparable to αTNF-α treatment. Histological and histological scores of the joints also showed that CBD-SA-IL-10 inhibited arthritis formation (Fig. 11B). Importantly, 5 out of 10 mice treated with CBD-SA-IL-10 showed a score of 1 or lower, indicating a high therapeutic efficacy of CBD-SA-IL-10. In the absence of CBD domains, a single injection of SA-IL-10 into CIA mice induced a significant inhibition of arthritis formation compared to PBS (Fig. 11C). As shown by histological and histological examination, most mice treated with PBS exhibited severe inflammation in the paws (Fig. 11D). In contrast, mice treated with SA-IL-10 exhibited almost identical paw conditions to juvenile mice, and most mice showed a histological score of 1 or lower. In summary, these results demonstrate that local or intravenous injection of CBD-SA-IL-10, and even subcutaneous injection of SA-IL-10, has a highly inhibitory effect on inflammation.

[0383] D. Albumin-fused IL-10 accumulates in LN, reducing immune activity.

[0384] SA-fused IL-10 exhibits micromolar affinity for FcRn (Fig. 8B) and accumulates in the lymph nodes (LN) after intravenous injection (Fig. 8D). Next, the concentration and pharmacokinetics of IL-10 in the LN were quantitatively assessed (Figs. 12A to 12C). Following intravenous injection of wt IL-10, SA-IL-10, or CBD-SA-IL-10 in CAIA mice, the concentration of IL-10 in the LN at different time points was detected using ELISA. Compared to wt IL-10 and CBD-SA-IL-10, at 4 hours post-injection, SA-IL-10 showed significantly higher IL-10 signal in the joint drainage (popliteal) LN and mesenteric LN, and a relatively higher signal in the non-drained (cervical) LN. CBD-SA-IL-10 showed higher accumulation than wt IL-10 (Fig. 12A). Mice injected with SA-IL-10 also showed peak IL-10 concentrations in the lymph node (LN) approximately 1 hour after injection (Fig. 12B), with AUCs 5–10 times higher than wt IL-10 (Fig. 12C). These data indicate that SA-IL-10 accumulates in the LN immediately after intravenous injection and has higher retention in the LN compared to wt IL-10.

[0385] High concentrations and AUCs of SA-IL-10 in the lymph nodes (LN) can affect the phenotype of various immune cells in the LN and other secondary lymphoid organs. Therefore, the immune cell populations in the spleen and popliteal LN were analyzed by flow cytometry (Figure 15). Intravenous injection of SA-IL-10 induced CD3+ in the spleen. + T cells and CD45 + The frequency of lymphocytes was significantly reduced. Figure 15A Furthermore, compared to PBS or wt IL-10, CD86 levels were significantly lower after SA-IL-10 injection. + Dendritic cells, granulocyte myeloid-derived suppressor cells (G-MDSCs), and CD86 + The frequency of M1 macrophages decreased, CD206 + The frequency of M2 macrophages increased. A similar trend was observed in the popliteal fossa LN. Figure 15BThese data indicate that SA-IL-10 inhibits APC activity and simultaneously activates immunosuppressive M2 macrophages. APC inactivation and high IL-10 accumulation in the LN suppresses Th17 cell activity, which plays 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 LN in both drained (popliteal) and non-drained (cervical) LNs: SA-IL-10 treatment significantly reduced levels in the popliteal LN compared to treatment with wt IL-10 and IL-17, but not statistically significantly with CBD-SA-IL-10, and levels in the cervical LN were not statistically reduced by the IL-10 variant (Figs. 12D and 12E). Treatment with SA-IL-10 reduced GM-CSF concentrations in the popliteal LN, while wt IL-10 did not (Fig. 12F).

[0386] E. Albumin-fused IL-10 inhibits inflammatory responses in the paws.

[0387] Next, flow cytometry was used to analyze the immune cell population in the hind paw. Figure 13A Following intravenous administration of SA-IL-10 or CBD-SA-IL-10, CD45 levels were significantly lower compared to the PBS- or wt IL-10 treatment group. + The frequency of immune cells was significantly reduced. (In CD45) + Intracellularly, the frequency of B cells and dendritic cells became comparable to that of healthy mice, and CD11b... + The cell count was also significantly reduced to levels found in healthy mice. CD11b + In cells, the number of G-MDSCs decreased, and the macrophage frequency returned to the level of healthy mice. Furthermore, compared with PBS or wt IL-10 treatment, SA-IL-10 injection significantly increased CD206. + The frequency of M2 macrophages even exceeded that of healthy mice. Analysis of the T cell population in the paws showed that SA-IL-10 inhibited CD4+ in CAIA mice. + Cells and Foxp3 + Changes in Treg ( Figure 16A Furthermore, SA-IL-10 inhibits the decrease in the frequency of Tregs in the blood. Figure 16B Reflecting these changes in the immune cell population, intravenous administration of SA-IL-10 or CBD-SA-IL-10 significantly reduced various inflammatory cytokines in the paw, with levels comparable to those in healthy mice (Fig. 13B). Histological analysis showed that intravenous administration of SA-IL-10 significantly suppressed the inflammatory response in the paw and reduced joint pathology compared to PBS-treated mice (Fig. 13C).

[0388] F. Albumin-infused IL-10 showed no toxicity after injection.

[0389] Finally, a safety assessment was conducted to investigate whether the engineered IL-10 exhibited any adverse effects. Representative blood parameters and spleen weight measured by hematology analyzer showed no significant changes between the treatment groups. Figure 17A They also used a biochemical analyzer to study various biochemical markers in serum. Figure 17B In the engineered IL-10 treatment group, most biomarkers showed similar levels compared to the PBS treatment group, except for amylase (which did not increase but decreased slightly), indicating that engineered IL-10 has a high safety profile after systemic administration.

[0390] G. Discussion

[0391] Current treatment for RA is based on symptomatic relief, aiming to alleviate pain, control synovitis, and inhibit joint damage. Antibody drugs that neutralize inflammatory cytokines, particularly TNF-α, or competitive soluble receptors offer high therapeutic efficacy for RA patients (4). These biotherapeutic drugs primarily act on inflamed joints to capture inflammatory cytokines. However, these inhibitory drugs are known to increase the risk of infection because their targets are pleiotropic in terms of immune function, and these drugs are repeatedly administered to provide anti-inflammatory effects at the disease site (24-27). Furthermore, the administration of antibody drugs can induce neutralizing anti-drug antibodies, thereby reducing treatment efficacy (28). Therefore, there is a need to develop alternative approaches with structurally different molecular classes and different immunosuppressive molecular mechanisms, such as tolerance.

[0392] Here, the inventors explore a novel approach to treating RA by enhancing lymph node transport using engineered IL-10, a representative anti-inflammatory cytokine that modulates the phenotype of RA-associated immune cells to an immunosuppressive state. Clinical trials using recombinant IL-10 to treat autoimmune diseases, including RA, have been conducted (6-8, 29). One drawback of IL-10 is its short half-life in the blood (8). Here, the inventors fuse SA with the IL-10 gene to prolong its retention time in the blood and secondary lymphoid organs. Furthermore, to enhance binding to highly permeable inflammatory sites in the microvascular system, the inventors fuse the gene with CBD, a blood protein derived from von Willebrand factor (16, 17). The inventors evaluated both SA-IL-10 and CBD-SA-IL-10, comparing their improvement in arthritis in both models to wt% IL-10. CAIA is a macrophage- and neutrophil-mediated acute RA model, while CIA is a T-cell-mediated, particularly Th17-mediated, RA model. Given that RA is a clinically heterogeneous disease and the models are complementary, the demonstration that SA fusion with IL-10 inhibits disease severity in both models is encouraging. The SA-IL-10 fusion is crucial for achieving significant therapeutic effects, comparable to clinically common treatments using αTNF-α antibodies. Furthermore, to the inventors' knowledge, this study is the first to demonstrate the therapeutic effect of IL-10 in a CAIA model.

[0393] Compared to wt IL-10, SA-IL-10 fusion resulted in enhanced accumulation in the lymph node (LN) after intravenous injection and maintained a high IL-10 concentration in the LN for an extended period (Fig. 12A). To date, LN transport of SA or albumin-bound nanoparticles has primarily been achieved through intradermal or subcutaneous administration, where the LN enters via afferent lymphatic vessels downstream of the injection site (30-33). In the context of studies on the biodistribution of inflammatory cytokines, one paper showed that human SA-fused IL-2 was highly localized in the spleen, liver, and LN following intravenous injection into these organs, where T cells expressing the IL-2 receptor were present, but the exact mechanism of this high localization remains unclear (34). Here, the inventors reveal enhanced transport of SA-fused IL-10 into the LN after intravenous injection, where SA enters the LN via the vascular system. CBD-SA-IL-10 accumulation in the LN was less than that of SA-IL-10, likely due to binding to collagen in other tissues. Both SA-IL-10 and CBD-SA-IL-10 exhibited high binding affinity for FcRn (micromolar K). d (as expected) (Figure 8B and Figure 14BThe FcRn-mediated circulation of IL-10, expressed in vascular endothelial cells, provides prolonged blood circulation performance for both proteins (Fig. 9A). IgG endocytic transport via FcRn (from the basolateral to the luminal side) is a well-established phenomenon, and the same phenomenon has recently been reported in SA (19, 20). Here, in the LN, molecular transport appears to be in the opposite direction, from the lumen to the basolateral. Interestingly, histological analysis revealed the accumulation of SA-IL-10 around the HEV in the LN (Fig. 8D). Further experiments are needed to elucidate the more detailed mechanisms enhancing LN accumulation and its relationship with FcRn, for example, LN accumulation analysis using IL-10 fused to mutant SA to eliminate FcRn binding.

[0394] SA-IL-10 exhibits high binding to APCs (Fig. 8B). Upon accumulation in the LN, SA-IL-10 molecules are uptaken by APCs residing in the LN, leading to inhibition of dendritic cell and M1 macrophage activity and induction of M2 macrophages (Fig. 15). M2 macrophages can alter the differentiation fate of Th0 cells into Treg cells in the LN (35). Furthermore, the immunosuppressive environment of high concentrations of IL-10 in the LN can lead to further polarization of macrophages towards the M2 phenotype and inhibition of Th17 differentiation (36, 37), resulting in the reduction of IL-17, GM-CSF, or other cytokines in the LN observed by the inventors (Fig. 12D and Fig. 12F). GM-CSF is a cytokine that serves as a marker of pathogenic Th17, and its inhibitory antibodies are currently being tested in clinical trials (38). Therefore, the reduction of GM-CSF after SA-IL-10 treatment indicates decreased immune activation in the joint drainage LN. It has been reported that Th17 cells express the IL-10 receptor, and IL-10 binding inhibits the expression and secretion of IL-17 (14, 36). Because Th17 cell antigen recognition primarily occurs in lymphoid tissues, SA-IL-10 can directly bind to Th17 cells, inhibiting the IL-17 pathway. These changes in LN also suppress the infiltration of immune cells, particularly G-MDSCs and macrophages, into the claw (…). Figure 13A It also induces an increase in M2 macrophages. Figure 13A This leads to a reduction in inflammatory cytokines (Fig. 13B) and suppression of joint inflammation (Fig. 10, Fig. 11 and Fig. 13C).

[0395] SA-IL-10 induced a high anti-inflammatory response after administration via any of the tested routes—intravenous, subcutaneous (distal), or footpad (local) injection—indicating that SA-IL-10 can be taken up systemically into the lymphatic system via lymphatic drainage from the local injection site and return to the systemic circulation via the thoracic duct. The high therapeutic efficacy of subcutaneous injection suggests specific clinical benefits for SA-IL-10. Intravenous CBD-SA-IL-10 also showed inhibitory effects on the development of CAIA and CIA, comparable to anti-TNF-α antibodies, currently the standard biological treatment for RA. However, the therapeutic efficacy of CBD-SA-IL-10 was lower than that observed with SA-IL-10, corresponding to the lower LN transport of CBD-SA-IL-10 (Figure 12A). After CBD-SA-IL-10 penetrates into inflamed tissue, it can bind to collagen, leading to accumulation in the inflammatory claws (Fig. 9B). However, this collagen affinity can interfere with LN transport of CBD-SA-IL-10 (16, 39). Therefore, SA fusion is a simple yet effective method for preparing engineered cytokines to achieve enhanced LN transport.

[0396] In this study, the fusion of SA with IL-10 resulted in a persistent increase in the lymph node (LN), where autoimmune-related immune recognition developed and persisted. As a result, SA-IL-10 suppressed the major inflammatory pathways in RA progression but did not inhibit pleiotropic inflammatory cytokines such as TNF-α. Furthermore, SA-IL-10 did not exhibit any 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 to suppress RA in clinical applications, and the inventors' findings more broadly demonstrate the ability to modulate the immune system through systemic tolerogenic manipulation of the LN in other autoimmune and inflammatory diseases.

[0397] Amino acid sequences of wt IL-10, SA-IL-10 and CBD-SA-IL-10 from H. mice

[0398]

[0399] I. Materials and Methods

[0400] 1. Research Design

[0401] This study aimed to test a strategy of targeting anti-inflammatory cytokines to lymph nodes (LNs) through engineered affinity for FcRn. Specifically, the inventors tested in a mouse model of renal inflammatory disease (RA) whether LNs targeting the anti-inflammatory cytokine IL-10 via serum albumin fusion were superior to untargeted wt IL-10 and currently available anti-inflammatory antibody therapeutics (αTNF-α). To evaluate the efficacy of wt IL-10, SA-IL-10, and the anti-TNF-α antibody, the inventors scored arthritis symptoms and joint histology in passive CAIA and active CIA models. The inventors also measured various aspects of biodistribution and LN transport, immune responses in LNs and paws, and post-treatment toxicity. Statistical methods were not used to predetermine the necessary sample size, but the sample size was selected based on estimates from pilot trials to ensure that appropriate statistical trials would yield statistically significant results. 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. For animal studies, mice were randomly assigned to treatment groups within cages before the first drug injection and treated in the same manner. The n-value used to calculate the statistic is indicated in the legend. Drug administration and pathological analysis were performed in a blinded manner. The statistical methods are described in the "Statistical Analysis" section.

[0402] 2. Production and purification of recombinant proteins

[0403] The sequences encoding mouse serum albumin (25 to 608 amino acids of whole serum albumin) without propeptide, mouse IL-10, human VWF A3 domain residues Cys1670-Gly1874 (907-1111 of mature VWF, referred to as CBD in this paper), and the (GGGS)2 linker were synthesized and subcloned into the mammalian expression vector pcDNA3.1(+) provided by Genscript. A sequence encoding 6His was added to the C-terminus for further purification of the recombinant protein. Suspension-adapted HEK-293F cells were routinely maintained in serum-free FreeStyle293 expression medium (Gibco). On the day of transfection, cells were loaded at 1 × 10⁶ cells / day. 6 Cells were seeded at a density of [number] cells / mL into fresh culture medium. 2 μg / mL plasmid DNA, 2 μg / mL linear 25 kDa polyethyleneimine (Polysciences), and OptiPRO SFM medium (4% final concentration, Thermo Fisher) were added sequentially. The culture flask was stirred at 135 rpm in the presence of 5% CO2 at 37°C. Seven days post-transfection, the cell culture medium was collected by centrifugation and filtered through a 0.22 μm filter. The medium was then loaded into a HisTrap HP 5 mL column (GE Healthcare), which uses [specific technology / method / process]. The column was washed with wash buffer (20 mM NaH2PO4, 0.5 M NaCl, pH 8.0) and then eluted with a gradient of 500 mM imidazole (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 elution buffer. All purification steps were performed at 4 °C. The purity of the expressed protein was confirmed as >90% by SDS-PAGE. Endotoxin levels of the purified protein were tested using the HEK-Blue TLR4 reporter cell line, confirming endotoxin levels of less than 0.01 EU / mL. Protein concentration was determined by absorbance at 280 nm using NanoDrop (Thermo Scientific).

[0404] 3. Detection of binding with collagen and FcRn

[0405] SPR measurements were performed using a Biacore X100 instrument. In the collagen binding assay, recombinant human type I or type III collagen (Millipore Sigma) was immobilized on a CM5 sensor chip using a standard amine coupling method (approximately 1500 resonance units (RU)) and blocked with ethanolamine. Reference cells were also blocked with ethanolamine. The binding assay was performed at room temperature, and the K+ of CBD-SA-IL-10 was measured. d Values ​​were determined by fitting a 1:1 Langmuir binding model to the data using BIAevaluation software (GE Healthcare). In the FcRn binding assay, approximately 200 RU of recombinant mouse FcRn (Acro Biosystems) was immobilized on a C1 chip (GE Healthcare) via amine coupling according to the manufacturer's instructions. SA-IL-10 or CBD-SA-IL-10 was flowed at a decreasing concentration at 30 μL / min in run buffer (0.01 M anhydrous sodium dihydrogen phosphate, pH 5.8, 0.15 M NaCl) at room temperature. For each cycle, the sensor chip was regenerated with PBS, pH 7.4. The specific binding of the SA fusion protein to FcRn was calculated by comparison with a non-functionalized channel used as a reference. Ka values ​​for SA-IL-10 and CBD-SA-IL-10 were also calculated. d The values ​​were determined by fitting the 1:1 Langmuir combination model to the data using BIAevaluation software (GE Healthcare).

[0406] 4. Mice

[0407] Seven-week-old female BALB / c mice and eight-week-old male DBA / 1J mice were obtained from the Jackson Laboratory. The experiment was conducted with the approval of the University of Chicago Institutional Animal Care and Use Committee.

[0408] 5. Protein binding to spleen cells or LN-derived cells

[0409] Spleen and popliteal lymph nodes were gently disrupted through a 70 μm cell filter to obtain a single-cell suspension. Red blood cells were lysed using ACK lysis buffer (Quality Biological) for spleen cells. Cells were counted and resuspended in RPMI-1640 supplemented with 10% FBS and 1% penicillin / streptomycin (both from Life Technologies). 1 × 10⁻⁶ 5 Cells were seeded per well in 96-well microplates and incubated on ice for 30 minutes with 2 μg / 100 μL SA, SA-IL-10, or CBD-SA-IL-10. After washing four times with PBS, the cells were further incubated on ice for 20 minutes with anti-mouse albumin antibody (abcam). After washing three times with PBS, cells were incubated on ice for 20 minutes with 1 μg / mL Alexa Fluor 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.

[0410] 6. Plasma pharmacokinetics of proteins

[0411] IL-10, SA-IL-10, or CBD-SA-IL-10 (equivalent to 35 μg IL-10) were intravenously injected into female BALB / c mice. Blood samples were collected in low-protein-binding tubes at 1, 5, 10, and 30 minutes, and at 1, 4, 8, and 24 hours post-injection, and then incubated overnight at 4°C. Serum IL-10 concentrations were measured using an uncoated mouse ELISA kit (Invitrogen) according to the manufacturer's protocol. Exponential biphasic decay (Y = Ae0) was used. -αt +Be -βt Fitting is used to calculate the half-life. Fast elimination half-life, t 1 / 2,αSlow elimination half-life, t 1 / 2,β The data was analyzed using Prism software (v8, GraphPad).

[0412] 7. CAIA Model

[0413] In female BALB / c mice, arthritis was induced by intraperitoneal injection of an anti-collagen antibody mixture (1.0 mg / mouse, Chondrex) on day 0, followed by intraperitoneal injection of LPS (25 μg / mouse, Chondrex) on day 3. (For use only) Figure 3 BC, mice were injected with a mixture of 1.5 mg / mouse anti-collagen antibody. On day 3, mice were injected intravenously, subcutaneously (mid-back), or into the paw pads with PBS, wt IL-10, SA-IL-10, CBD-SA-IL-10 (each equivalent to 43.5 μg IL-10), or 200 μg rat anti-mouse TNF-α antibody (clone XT3.11, Bio X Cell), followed by LPS. Joint swelling was scored daily according to the manufacturer's protocol (Chondrex). On the last day of scoring, the hind paw was fixed in 10% neutral formalin (Sigma-Aldrich), decalcified in Decalcifer II (Leica), and then provided for histological analysis. Paraffin-embedded paws were cut into 5 μm thick sections and stained with H&E. Images were scanned using a panoramic digital slide scanner and analyzed using panoramic viewer software. The severity of synovial hyperplasia and bone resorption in the arthritis model was assessed using a three-tiered scale (0-2) based on previously reported criteria, with slight modifications as follows: 0, normal to minimal pannus in the cartilage and subchondral bone margins; 1, mild to moderate infiltration in the margins with mild cortical and medullary bone destruction; 2, severe infiltration with complete or near-complete destruction of the joint structure. The scores of both hind paws for each mouse were summed (total score per mouse, 0-4). Histopathological analysis was performed in a blinded manner.

[0414] 8. CIA Model

[0415] 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 booster injection of bovine collagen / incomplete Freund's adjuvant (IFA) emulsion (Hooke Kit, Hooke Laboratories) was given. Following the booster, mice were examined daily, and joint swelling was scored according to the manufacturer's protocol (Hooke Laboratories). When the total score was 2–4 (defined as day 0), mice were intravenously injected with PBS, SA-IL-10, CBD-SA-IL-10 (each equivalent to 43.5 μg IL-10), or 200 μg of rat anti-mouse TNF-α antibody (clone XT3.11, Bio X Cell). On the last day of scoring, the hind paws were collected and histologically analyzed as described above.

[0416] 9. In vivo biodistribution studies

[0417] To prepare the fluorescently labeled protein, wt% IL-10, SA-IL-10, and CBD-SA-IL-10 were incubated with an 8-fold molar excess of DyLight 800NHS ester (Thermo Fisher) at room temperature for 1 hour, and unreacted dye was removed by a Zebaspin rotating column (Thermo Fisher) according to the manufacturer's instructions. BALB / c mice were intraperitoneally injected with an anti-collagen antibody mixture (1.0 mg / mouse) on day 0, followed by an injection of 10 μg LPS into the right hind paw on day 3. On day 2, 20 μg of DyLight 80-labeled protein was intravenously injected. Four hours later, organs harvested from the disease model were imaged using a Xenogen IVIS Imaging System 100 (Xenogen) under the following conditions: f / stop: 2; filter excitation wavelength 745 nm; excitation wavelength 800 nm; exposure time: 5 seconds; small aperture. Each organ was weighed to normalize the fluorescence signal from each organ.

[0418] 10. LN microscope

[0419] BALB / c mice were intravenously injected with either 43.5 μg of wt IL-10 labeled with DyLight594 or an equimolar amount of SA-IL-10 labeled with the dye. Twenty-four hours post-injection, the popliteal lymph nodes (LNs) were harvested and frozen on dry ice at the optimal cutting temperature (OCT). Tissue sections (10 μm) were obtained by cryosectioning. The tissues were fixed for 15 minutes at room temperature with PBS containing 2% paraformaldehyde. After washing with PBS-T, the tissues were blocked with 2% BSA in PBS-T for 1 hour at room temperature. The tissues were stained with anti-mouse CD3 antibody (1:100, 145-2C11, BioLegend) or anti-mouse peripheral lymph node addressing protein (PNAd) antibody (1:200, MECA79, BioLegend) and Alexa Fluor 488 donkey anti-rat (1:400, Jackson ImmunoResearch). The tissue was washed three times and then covered with ProLong gold anti-fading mounting medium for 4′,6-diamidinyl-2-phenylindole (DAPI; ThermoFisher Scientific). CD3 staining was imaged at 10x magnification using an IX83 microscope (Olympus), and PNAd staining was imaged at 20x magnification using a Leica SP8 3D laser scanning confocal microscope. Images were processed using ImageJ software (NIH).

[0420] 11. LN Pharmacokinetics

[0421] wt IL-10, SA-IL-10, or CBD-SA-IL-10 (each equivalent to 35 μg IL-10) were intravenously injected into CAIA mice. Popliteal fossa, mesentery, and cervical lymph nodes were collected at 30 min, 1 h, 4 h, 8 h, and 24 h post-injection. The samples were then homogenized for 40 sec using Lysing Matrix D and FastPrep-24 5G (MP Biomedical) at 5000 rpm in a mixture of T-PER tissue protein extraction reagent (Thermo Scientific) and cOmplete™ protease inhibitors (Roche). After homogenization, the samples were incubated overnight at 4°C. The 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). Simultaneously, mouse uncoated ELISA kits (Invitrogen) or Ready-SET-Go were also performed. The ELISA kit (eBioscience) measured cytokine levels in LN extracts according to the manufacturer's protocol. To detect GM-CSF, wt IL-10 or SA-IL-10 (each equivalent to 35 μg IL-10) was administered intravenously twice to CAIA mice at 3-day intervals. Popliteal lymph nodes were collected the day after the last injection for GM-CSF detection.

[0422] 12. Flow cytometry

[0423] CAIA mice were intravenously injected with PBS, wt IL-10, SA-IL-10, or CBD-SA-IL-10 (each equivalent to 43.5 μg IL-10). Eight days later, blood and hind paws were collected. Red blood cells in the blood were lysed with ACK lysis buffer (Quality Biological) and then stained with antibodies for flow cytometry. Paws were digested in Dalberg modified Eagle medium (DMEM) supplemented with 2% FBS, 2 mg / mL collagenase D, and 40 μg / mL DNase I (Roche) at 37°C for 60 min. Single-cell suspensions were obtained by gently lysing the cells through a 70 μm cell filter. 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), and MHC. Cell types included II (M5 / 114.15.2, BioLegend), CD206 (C068C2, BioLegend), Ly6G (1A8, BioLegend), Ly6C (HK1.4, BioLegend), CD11b (M1 / 70, BioLegend), CD11c (HL3, BD Biosciences), and B220 (RA3-6B2, BioLegend). Live / dead cell differentiation was performed using a FixableViability Dye eFluor455 (eBioscience) according to the manufacturer's instructions. Unless otherwise specified, staining was performed on ice for 20 minutes. Intracellular staining was performed using a Foxp3 staining kit according to the manufacturer's instructions (BioLegend). After washing, cells were stained on ice with specific antibodies for 20 minutes and then fixed. All flow cytometry analyses were performed using a Fortessa (BD Biosciences) flow cytometer and analyzed using FlowJo software (Tree Star).

[0424] 13. Security Assessment

[0425] 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 IL-10). Two days after injection, blood samples were collected from the mice and analyzed using a COULTER Ac·T 5diff CP hematology analyzer (Beckman Coulter) according to the manufacturer's instructions. Spleen weight was also measured. Serum samples collected from the protein-injected mice were analyzed using a biochemical analyzer (Alfa Wassermann Diagnostic Technologies) according to the manufacturer's instructions.

[0426] 14. Statistical Analysis

[0427] Statistically significant differences between experimental groups were determined using Prism software (v8, GraphPad). Similarities between groups were found using Brown-Forsythe test after one-way ANOVA followed by Tukey's HSD post-hoc test. For nonparametric data, the Kruskal-Wallis test was used, followed by Dunn's multiple comparison test. For individual comparisons, a two-tailed Student's t-test was used. The symbols *, **, ***, and **** indicate p-values ​​less than 0.05, 0.01, 0.001, and 0.0001, respectively; ns, no significance.

[0428] J. References

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[0469] Example 3: Albumin-fused IL-4 improves experimental autoimmune encephalomyelitis by long-term FcRn residence in secondary lymphoid organs.

[0470] Multiple sclerosis (MS) is a common and severe demyelinating autoimmune disease of the central nervous system. Although interleukin (IL)-4 inhibits the development of pathology in a mouse model of MS and experimental autoimmune encephalomyelitis (EAE), IL-4 has not yet been translated into clinical use. Here, the inventors designed a fusion protein of serum albumin (SA) and IL-4 (SA-IL-4) to target secondary lymphoid organs (SLOs), where antigen-specific T cell initiation is primarily observed. Compared to wild-type (wt) IL-4, SA-IL-4 showed longer accumulation and residence time in lymph nodes (LNs) and spleen via neonatal Fc receptor (FcRn). Subcutaneous administration of SA-IL-4 prevented EAE disease development in all mice and showed higher therapeutic efficacy compared to FTY720 and wt IL-4. SA-IL-4 prevented immune cell infiltration into the spinal cord, promoting the maintenance of spinal cord structure and the resulting neurological function. SA-IL-4 reduced antigen-reactive CD4+. +Integrin expression in T cells indicates impaired cell migration. SA-IL-4 increases the number of granulocyte-like myeloid-derived suppressor cells (a key inhibitor of EAE disease) and the expression of programmed death-ligand-1 in the spinal cord draining lung (dLN). SA-IL-4 reduces the number of Th17 cells, the pathogenic cell population in EAE disease. In the chronic phase of EAE, SA-IL-4 also showed significant therapeutic effects while inhibiting immune cell infiltration into the spinal cord and completely eliminating the immune response to spleen myelin antigens. Engineered SA-IL-4 demonstrates translational potential for both preventative and therapeutic treatment of MS through accumulation in the spinal cord.

[0471] Multiple sclerosis (MS) is a potentially disabling autoimmune disease affecting millions worldwide. Autoreactive immune cells reside in the central nervous system (CNS), leading to demyelination and thus focal damage to 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 and play a crucial role in the development and severity of MS (20). Therefore, inhibiting lymphocyte migration to the CNS and inducing an immunosuppressive microenvironment in the SLOs could provide an effective treatment for MS. FTY720 and anti-integrin α4 antibodies have been used clinically to inhibit lymphocyte migration (3,4). Experimental autoimmune encephalomyelitis (EAE) is a widely accepted mouse model of MS, reflecting many features of the mechanisms of disease progression and development, including lymphocyte migration to the CNS and demyelination.

[0472] A.SA-IL-4 binds to immune cells and inhibits Th17 differentiation.

[0473] The inventors recombinantly expressed wild-type (wt) mouse IL-4 and mouse SA-fusion mouse IL-4 (Figure 18A). SDS-PAGE revealed that SA fusion with IL-4 increased the molecular size. When added to freshly isolated immune cells from the lungs and spleen, SA-IL4 preferentially bound antigen-presenting cells (APCs), such as macrophages and dendritic cells (DCs), in vitro compared to other immune cells (Figure 18B).

[0474] IL-4 receptors are expressed on T cells upon stimulation (12). SA-IL-4 induces downstream phosphorylation of STAT6 in T cells, with an EC50 32-fold higher than that of wt IL-4. This indicates that wt IL-4 is more active than SA-IL-4 in vitro (Fig. 18C). The inventors found that both wt IL-4 and SA-IL-4 inhibit naive CD4 cells cultured in Th17 cell differentiation medium. +Th17 differentiation of T cells (Figure 18D). In summary, the results indicate that the inventors have successfully prepared a functionally active SA-IL-4 fusion protein.

[0475] SA-IL-4 increases blood half-life and SLO persistence in LN and spleen.

[0476] Surface plasmon resonance (SPR) analysis showed that the dissociation constant (Ki) of SA-IL-4 binding with FcRn was... D The plasma half-life of SA-IL-4 was 385 nM (Fig. 19A). Intravenous (iv) injection of SA-IL-4 significantly prolonged the plasma half-life, in stark contrast to the rapid clearance of wt IL-4 from the plasma within minutes (Fig. 19B). The inventors then tested whether intravenously injected SA-IL-4 accumulated in the spleen and lumbar nerve nucleus (LN) using naïve mice. SA-IL-4 significantly increased the amount of IL-4 in the lumbar and brachial artery LNs and the spleen after intravenous injection (Fig. 2cd). Fluorescence-based biodistribution analysis also showed enhanced accumulation of SA-IL-4 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, the inventors mutated the P573K point mutation in SA in its fusion with IL-4, which eliminated FcRn binding (Fig. 25A) (13). Compared to SA-IL-4, the SA(P573K) mutation reduced the amount of IL-4 in the LN to a level similar to wt IL-4 (Fig. 19E). SA(P573K)-IL-4 has a longer blood half-life than wt IL-4 due to its increased molecular size, but a shorter blood half-life due to impaired FcRn binding (Fig. 25B). In summary, these data indicate that SA transport to the LN requires FcRn binding.

[0477] Following intravenous injection of SA-IL-4, histological examination of the lumbar lymph nodes (LN) showed increased SA-IL-4 signal intensity compared to wt% IL-4. SA-IL-4 was specifically localized in the subcapsular space and did not interact with CD3. + T cell colocalization (Fig. 19G). Higher magnification shows that the SA-IL-4 moiety colocalizes with high endothelial sites, which is caused by peripheral node addressing proteins. + (PNAd + Cellular indicators (19H). The amount of SA(P573K)-IL-4 in the spleen was lower than that of SA-IL-4 (Fig. 19F). In summary, the inventors have demonstrated that SA-IL-4 fusion increases the persistence of IL-4 in the LN and spleen via FcRn binding.

[0478] B.SA-IL-4 treatment significantly inhibits the development of EAE in preventive treatment.

[0479] The inventors then treated myelin oligodendrocyte glycoprotein (MOG) antigen-induced EAE with SA-IL-4 during the acute phase of EAE (Fig. 20). SA-IL-4 was administered subcutaneously (sc) or intraperitoneally (ip). The inventors chose subcutaneous injection because it is clinically convenient and the drug is absorbed slowly from the injection site. Intraperitoneal injection was performed as an alternative to intravenous injection because the tail vein becomes loose in mice that have developed EAE, making tail vein injection difficult. The inventors further compared the therapeutic effects of SA-IL-4 with FTY720, a clinically approved drug for the treatment of MS (fingolimod), which isolates lymphocytes in the lymph nodes and prevents them from reacting with autoantigens in the target tissue (3). Subcutaneous injection of SA-IL-4 completely inhibited disease development in all mice (Fig. 20A, Fig. 20B). Intraperitoneal injection of SA-IL-4 and FTY720 prevented the development of EAE in 4 out of 7 mice and inhibited the disease severity in the remaining mice. Compared to the PBS treatment group, Wt IL-4 treatment did not show suppression of EAE clinical scores, and all of these mice developed the disease. Using body weight change as a clinical indicator of health, mice treated with PBS and wt IL-4 showed significant weight loss (Figure 20C). Mice subcutaneously injected with SA-IL-4 gained more weight than all other groups, indicating good health. The intraperitoneally injected SA-IL-4 group showed a mean weight gain, while mice treated with FTY720 maintained their weight. The inventors then analyzed spinal cord demyelination, a major morphological manifestation of EAE disease (…). Figure 20D Importantly, mice subcutaneously injected with SA-IL-4 showed no detectable demyelination, indicating protection against spinal cord injury. All mice treated with wt IL-4 exhibited demyelination. The inventors then monitored the mice long-term, up to day 24, where intraperitoneal injection of SA-IL-4 inhibited disease development and progression. Figure 26 Importantly, SA(P573K)-IL-4 did not suppress the disease score. Figure 27 These data indicate that the fusion of SA and IL-4 significantly enhances the therapeutic efficacy of IL-4 in suppressing EAE disease.

[0480] C.SA-IL-4 treatment inhibits immune cell infiltration into the spinal cord and induces an immunosuppressive environment in the dLN.

[0481] The inventors then analyzed immune cells in the spinal cord and dLN after treatment. Notably, subcutaneous injection of SA-IL-4 significantly inhibited immune cell infiltration into the CNS; very few CD45 cells were detected in the spinal cord. +Immune cells (Fig. 21A). Therefore, Th17 cells in the spinal cord were almost undetectable in the subcutaneous SA-IL4 treatment group (Fig. 21B). Intraperitoneal injection of SA-IL-4 suppressed immune cell infiltration in 4 out of 7 mice, corresponding to the incidence of EAE disease in this group. As expected, administration of FTY720 also suppressed immune cell infiltration into the spinal cord. Compared with PBS treatment, wt IL-4 had no effect on the infiltration of immune cells, including Th17, into the spinal cord.

[0482] Next, the inventors analyzed immune cells in the lumbar dLN. SA-IL-4 increased granulocyte-like myeloid-derived suppressor cells (G-MDSCs) but decreased monocyte-like MDSCs (M-MDSCs) (Figures 21C and 21D). SA-IL-4 treatment (intraperitoneal and subcutaneous injections) also reduced CD4 counts in the dLN compared to FTY720 treatment. + The frequency of Th17 cells in T cells (Fig. 21E). Compared with the PBS group, FTY720 treatment tended to increase the frequency of Th17 cells in the dLN, possibly because FTY720 inhibited the outflow of suppressor lymphocytes from the LN. SA-IL-4 treatment decreased the frequency of M1 macrophages and increased the frequency of M2 macrophages in the dLN (Fig. 21F). Wt IL-4 did not decrease the frequency of M1 macrophages but increased the frequency of M2 macrophages. Maintaining CD11b + Intracellular macrophage frequency (Fig. 28A), and CD45 + Intracellular DC frequency (Supplementary Fig. 4b). B cells have been reported to promote induced EAE by facilitating T cell reactivation (14). SA-IL-4 (subcutaneous injection) reduced B cell frequency compared to both PBS and FTY720 treatment groups (Fig. 21H). In summary, these data suggest that SA-IL-4 treatment creates an immunosuppressive environment in the dLN and prevents immune cell infiltration into the spinal cord.

[0483] D.SA-IL-4 treatment reduces antigenic reactivity CD4 + IL-17-related cytokines and integrins expressed on T cells

[0484] The inventors then analyzed the molecular mechanisms of reduced immune cell infiltration in the spinal cord and complete prevention of EAE disease through subcutaneous injection of SA-IL-4. The inventors discovered that MOGs in the dLN... 35-55The number of reactive T cells remained constant across all treatment groups, indicating that SA-IL-4 does not alter antigen recognition (Figure 22A). Therefore, the inventors hypothesized that SA-IL-4 alters T cell function. The inventors first tested T cell migration ability. IL-4 has been reported to reduce the expression levels of (16) αLβ2 and α4β1 integrins, key adhesion molecules (15) for lymphocyte migration. The inventors found that SA-IL-4 treatment significantly reduced the expression of αLβ2 integrin in MOG cells. 35-55 Reactive CD4 + Expression on T cells, but in total CD8 + CD4 is not expressed on T cells (Figure 22B-E). + T cells, regardless of total CD8 + On T cells. Since αLβ2 integrin is essential for Th17 cell infiltration into the spinal cord (15), this suggests that downregulating integrin expression is one of the mechanisms by which SA-IL-4 reduces lymphocyte migration to the spinal cord.

[0485] The inventors then tested PD-1 expression on T cells and PD-L1 expression on MDSCs (Figs. 22F to 22K), since PD-1 and PD-L1 together inhibit T cell activation (17). Notably, SA-IL-4, rather than wt IL-4, increased central memory CD4. + T cells and central memory CD8 + PD-1 expression on T cells (Fig. 22F and Fig. 22G). Furthermore, SA-IL-4, but not wt IL-4, increased PD-1 expression levels and the frequency of PD-L1-expressing cells on both M-MDSCs and G-MDSCs (Fig. 22H to Fig. 22K). These data suggest that T cell suppression can be induced via MDSCs and the PD-1 / PD-L1 axis.

[0486] The inventors then analyzed the expression of Th17-related proteins. IL-23 is a key cytokine for Th17 function. It has been reported that IL-4 binds to APCs and silences IL-23 and co-occurring Th17 differentiation (18). The inventors found that SA-IL-4 treatment reduces MOG. 35-55 IL-23R in a reactive T cell library + Cell frequency (Fig. 22L).

[0487] The inventors then restimulated splenocytes with MOG protein (Fig. 22M,N). ELISA of the culture supernatant showed that treatment with SA-IL-4 instead of wt IL-4 reduced IL-17A expression compared to PBS (Fig. 22M). The reduction in IL-17 expression indicates that MOG expression was lower in the SA-IL-4 treatment group. 35-55The number and / or activity level of reactive Th17 cells decreased. IFNγ concentration remained unchanged, indicating that SA-IL-4 had little effect on Th1 cells (Fig. 22N). SA-IL-4 tended to decrease GM-CSF levels, GM-CSF being reported as a pathogenic cytokine of EAE (19) (Fig. 22O). The inventors then investigated MOG in spleen cells... 35-55 Cytokine expression in T cells was detected by flow cytometry after peptide restimulation (Figure 22P). The inventors analyzed GM-CSF, IL-17, IFNγ, and TNFα, all of which are pathogenic cytokines of EAE. Compared with other treatments, SA-IL-4 reduced CD4... + The frequency of cytokine expression in the T cell compartment. These results clearly demonstrate that Th17 cells in SA-IL-4-treated mice exhibited significantly reduced pathogenicity compared to other treatment groups. In summary, these data suggest that SA-IL-4 modulates multiple immune cellular responses in SLO and inhibits the development of EAE disease by preventing immune cell infiltration, particularly T cell infiltration into the spinal cord.

[0488] E.SA-IL-4 recovery in chronic paralysis caused by EAE

[0489] 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 reached a stage of severe paralysis. Intraperitoneal injections of IL-4 were initiated from day 21 post-induction (Figures 23A and 23B). Notably, even at this later time point, SA-IL-4, rather than wt IL-4, showed therapeutic efficacy. Mice treated with SA-IL-4 instead of wt IL-4 showed weight gain, indicating disease recovery. The inventors then tested the effect of SA-IL-4 in the chronic phase by subcutaneous injection and compared it with oral FTY720 treatment (Figures 23C and 23D). As a result, mice treated with SA-IL-4 tended to have lower clinical scores compared to other treatment groups. Mice treated with SA-IL-4 also showed weight gain compared to other groups. Mice treated with both FTY720 and wt IL-4 did not show weight gain compared to mice treated with PBS.

[0490] The inventors then used flow cytometry to test the infiltration of immune cells into the spinal cord on day 34 post-induction (Figures 23E to 23G). Compared with PBS and wt IL-4 treatment, SA-IL-4 and FTY720 treatment reduced the number of infiltrating immune cells in the spinal cord, including CD4+. + T cells and MOG 35-55 Reactive Th17 cells. SA-IL-4 reduced MOGs in the spleen compared to other treatment groups. 35-55 Reactive CD4+ T cells expressing IL-23R (Fig. 23H). Finally, spleen cells were restimulated with MOG protein. ELISA of the culture supernatant showed that IL-17A and GM-CSF concentrations were decreased in the SA-IL-4 treatment group compared with PBS, but not after treatment with wt IL-4 and FTY720 (Fig. 23I and Fig. 23J). MOG 35-55 Flow cytometry analysis following peptide restimulation showed that, compared to other treatments, SA-IL-4 reduced CD4 levels. + The frequency of cytokine expression in T cell compartments (Figure 23K). In summary, these results indicate that SA-IL-4 treatment is effective in treating the chronic phase of EAE.

[0491] F.SA-IL-4 did not show significant toxicity after systemic injection.

[0492] To test whether SA-IL-4 exhibited any side effects, the inventors analyzed serum using a biochemical analyzer and blood using a hematology analyzer (Figure 29). SA-IL-4 treatment did not increase organ damage markers or alter blood cell counts (Figures 29A to 29M). SA-IL-4 and wt IL-4 induced splenomegaly (Figure 29N). wt IL-4 induced pulmonary edema, manifested as increased water content in the lungs, while SA-IL-4 did not (Figure 29N). This evidence suggests that SA-IL-4 is safe after systemic administration.

[0493] Despite the availability of various clinical treatments for MS, the disease remains largely unresolved. Some effective therapies, such as those using FTY720 (fingolimod) and anti-α4 integrin (natezumab), suppress effector lymphocyte infiltration into diseased tissues, which is associated with an increased risk of immune-related adverse events (20). IFNβ works through multiple mechanisms, including reducing lymphocyte migration to the CNS (21). Although IFNβ is less effective than fingolimod (22), it is used clinically to modulate the pathology of MS (23). Here, the inventors seek to explore a therapy that can shift the immune response from the Th17 pathway, known to be involved in the disease pathology, to a more tolerable phenotype using IL-4 without side effects. Here, the inventors utilize molecular engineering methods to target SLO with IL-4 to improve the underlying autoimmune response to myelin antigens.

[0494] In this study, subcutaneous injection of SA-IL-4 prevented the development of acute exacerbation encephalopathy (EAE) in all test mice. Notably, SA-IL-4 significantly reduced lymphocyte infiltration into the spinal cord at both early and late time points. dLN analysis showed that SA-IL-4 increased M2 macrophages and G-MDSCs, while decreasing Th17 cells. However, wt IL-4 also increased the number of M2 macrophages, indicating that IL-4-mediated inflammatory macrophage suppression was insufficient to control EAE under these experimental conditions.

[0495] IL-4 has been reported to maintain and enhance the immunosuppressive properties of MDSCs (24). G-MDSCs are a key population for suppressing EAE development (17). G-MDSCs express PD-L1 to induce T cell functional suppression. On the other hand, SA-IL-4 treatment reduces M-MDSCs. The role of M-MDSCs in EAE is controversial, and pathogenic effects have been reported (25). Interestingly, SA-IL-4, rather than wt IL-4, enhances PD-L1 expression on both G-MDSCs and M-MDSCs in SLO. Simultaneously, SA-IL-4, rather than wt IL-4, enhances PD-L1 expression on CD4+ in SLO. + and CD8 + Expression on central memory T cells. The inventors hypothesize that this PD-1 / PD-L1 induction is the mechanism by which MDSCs suppress pathogenic T cells in SA-IL-4-treated mice, and that this phenomenon observed in SLO demonstrates the value of the SLO-targeting ability conferred by SA fusion.

[0496] Th17 cells play a crucial role in the severity and progression of EAE (26). SA-IL-4 treatment reduced the frequency of Th17 cells in dLN compared to FTY720 treatment. IL-4 directly inhibits the differentiation of naive T cells into Th17 cells, as illustrated by the inventors in Figure 18D. Furthermore, there may be a Th17 inhibition pathway mediated by APCs. IL-23 is expressed by APCs (26) and generates pathogenic Th17 cells and induces IL-17 expression (27). IL-4 has been reported to reduce IL-17 expression by silencing IL-23 in APCs (18). Therefore, it is possible that SA-IL-4 acts on APCs to reduce IL-23 expression, thereby preventing the generation of pathogenic Th17 cells in SLO. This model is consistent with the inventors' data that SA-IL-4 reduces GM-CSF. + T-cell pathogenicity mediated by GM-CSF + It is a marker of pathogenic Th17 cells.

[0497] Furthermore, SA-IL-4 treatment leads to MOG-responsive CD4. +Decreased integrin expression in T cell compartments. Although immunized mice had developed an anti-MOG response prior to treatment, SA-IL-4 was able to prevent autoimmune cells from migrating into the CNS. Therefore, SA-IL-4 treatment did not alter the number of antigen-reactive T cells, but rather inactivated T cell function. Overall, these data suggest that SA-IL-4 induces an immunosuppressive environment in SLO through multiple immune pathways.

[0498] FTY720 is one of the FDA-approved anti-MS drugs that works by inducing lymphopenia, thereby limiting the migration of effector lymphocytes to CNS disease sites. In this study, subcutaneous injection of SA-IL-4 showed higher efficacy than FTY720. This improvement in SA-IL-4 is strong evidence for its clinical translational potential. A drawback of FTY720 is that it cannot be administered to infected patients. Furthermore, in approved MS therapies that modulate lymphocyte migration, such as FTY720 and integrin α4 blockade, the induction of John Cunningham virus (JCV) activity can lead to progressive multifocal leukoencephalopathy (PML), which can ultimately result in serious adverse events, including death. SA-IL-4 only reduces antigen-reactive CD4. + Integrin expression on T cells, but without reducing CD8 expression. + Integrin expression on T cells. This indicates that SA-IL-4 responds to antigen-responsive CD4+. + T cells also exhibit migration-inhibiting activity, which could help suppress inflammation in the spinal cord. Hematological studies have shown that SA-IL-4 does not induce lymphopenia. This could be another advantage over approved drugs, as SA-IL-4 is not expected to strongly suppress immune responses to infectious diseases, thus potentially benefiting patients who are not suitable for FTY720 treatment. SA-IL-4 may suppress autoimmunity through various mechanisms independent of current therapies. In this study, FTY720 did not inhibit Th17 development in the dLN, while SA-IL-4 did. Therefore, SA-IL-4 could be useful for patients who have not achieved adequate therapeutic efficacy with current treatments.

[0499] Intradermal or subcutaneous injection of SA has been well studied for accumulation in the injection site-draining lymph nodes (LN) (28). Here, the inventors demonstrate that SA-IL-4 accumulates in the LN after intravenous injection, i.e., from the blood rather than via afferent lymphatic vessels. Although previous studies on the biodistribution of SA-fused IL-2 have shown that IL-2 accumulates in the spleen, liver, and LN after intravenous injection, the molecular mechanisms and localization in the LN remain unclear. Histological analysis has shown that intravenously injected SA-IL-4 accumulates and co-localizes around high endothelial venules (HEVs) (30). Although IgG transcytosis via FcRn has been well studied, albumin transcytosis via FcRn binding has only recently been reported by several research groups (31-33). Because FcRn is highly expressed in the LN (34), it is likely that intravenously injected SA-IL-4 is transported to the LN via endocytosis. In this study, SA(P573K)-IL-4 achieved a lower level of IL-4 in the LN compared to SA-IL-4, similar to wt IL-4. This suggests that FcRn binding plays an important role in the transport of SA-IL-4 from the blood to the LN. Based on the inventors' observations of SA-IL-4 concentration around the medullary cavity where APCs are located, the inventors hypothesize that SA-IL-4 enters via HEVs and then binds to DCs and macrophages, as illustrated in Figure 18. SA(P573K)-IL-4 in Figure 27 The absence of a disease-suppressing score suggests that FcRn binding and the subsequent increased SLO persistence are crucial for suppressing EAE disease symptoms. Therefore, the inventors believe that the role of SA-cytokine fusion immunosuppressive molecules is primarily not through prolonging blood circulation time, but rather through prolonging the circulation time of SLOs such as LN and in the spleen. Thus, SA-fusion cytokines can be collected by one immune cell and circulated through that cell to stimulate another immune cell. Therefore, blood circulation lifetime is not directly related to the lifetime of SLO presence. The inventors believe that this biological discovery opens up new avenues of research into LN molecular transport, and further investigation will elucidate the detailed mechanisms.

[0500] SA-IL-4 has demonstrated therapeutic efficacy in the chronic phase of EAE. Nasal or lumbar administration of IL-4 has previously been reported to lead to direct binding of IL-4 to neurons in an attempt to regenerate the nervous system in EAE (9). Intraperitoneal injection of SA-IL-4 showed comparable efficacy to previously reported nasal administration of wt IL-4. This suggests that SA-IL-4 can also bind to neurons and induce regeneration. Because the blood-brain barrier is disrupted in the EAE model, SA-IL-4 can enter neurons within the spinal cord. It would be interesting to investigate the direct effects of SA-IL-4 on neural regeneration in future studies.

[0501] Subcutaneous administration of SA-IL-4 has shown higher prophylactic efficacy than intraperitoneal injection. This route is easier to translate clinically and typically demonstrates slow release from tissues. The inventors hypothesize that this slow release is a further contributing factor to the inhibition of EAE development.

[0502] Systemic injection of SA-IL-4 did not demonstrate significant toxicity in blood biochemistry and hematology analyses. Although splenomegaly was observed by the inventors, this was generally transient and not considered a serious toxicity. wt IL-4 induces pulmonary edema, which would be more concerning. However, pulmonary edema was not observed with SA-IL-4; this difference could be due to the reduced activity of SA-IL-4 in STAT6 activation. These results suggest a low risk of inducing adverse events via SA-IL-4 administration in clinical settings.

[0503] In summary, engineered SA-IL-4 demonstrated persistence in SLO via FcRn binding. SA-IL-4 exhibited significant therapeutic effects on multiple phases of EAE following systemic injection, and it modulates key immune pathways in EAE, such as reducing Th17 cells and increasing G-MDSC and PD-L1 expression in MDSCs in SLO. SA-IL-4 has translational potential for preventative and therapeutic uses through a novel biological approach utilizing mechanisms different from currently approved therapies.

[0504] The amino acid sequences of G.wt IL-4 and SA-IL-4

[0505]

[0506] H. Materials and Methods

[0507] 1. Production and purification of recombinant proteins

[0508] Sequences encoding mouse SA (25 to 608 amino acids of whole serum albumin) without propeptide, mouse IL-4, and (GGGS)2 linkers were synthesized and subcloned into the mammalian expression vector pcDNA3.1(+) provided by Genscript. A sequence encoding 6His was added to the C-terminus for further purification of the recombinant protein. The amino acid sequence of the protein is shown in Supplementary Table 1. Suspension-adapted HEK-293F cells were routinely maintained in serum-free FreeStyle 293 expression medium (Gibco). On the day of transfection, cells were sputtered at 1 × 10⁶ cells / day. 6Cells were seeded at a density of [number] cells / ml into fresh culture medium. 2 μg / ml plasmid DNA, 2 μg / ml linear 25 kDa polyethyleneimine (Polysciences), and OptiPRO SFM medium (4% final concentration, Thermo Fisher) were added sequentially. The culture flask was stirred at 135 rpm in the presence of 5% CO2 at 37°C. Seven days post-transfection, the cell culture medium was collected by centrifugation and filtered through a 0.22 μm filter. The medium was loaded into a HisTrap HP 5 ml column (GE Healthcare), which uses [further processing steps]. 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 500 mM imidazole gradient (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 elution buffer. All purification steps were performed at 4 °C. The purity of the expressed protein was confirmed as >90% by SDS-PAGE. Endotoxin levels of the purified protein were detected using the HEK-Blue TLR4 reporter cell line, confirming endotoxin levels below 0.01 EU / ml. Protein concentration was determined by absorbance at 280 nm using NanoDrop (Thermo Scientific).

[0509] 2. Mice

[0510] Eight-week-old female C57BL / 6 mice were obtained from Charles River Laboratories. Mice were held in captivity at the University of Chicago animal facility for at least one week prior to immunization. All experiments were approved by the University of Chicago Institutional Animal Care and Use Committee.

[0511] 3. Protein binding to spleen cells or LN-derived cells

[0512] Single-cell suspensions were obtained by gently disrupting the spleen or popliteal lymph nodes through a 70 μm cell filter. Red blood cells were lysed using ACK lysis buffer (Quality Biological) for spleen cells. Cells were counted and resuspended in RPMI-1640 supplemented with 10% FBS and 1% penicillin / streptomycin (both from Life Technologies). 1 × 10⁻⁶ cells were then added to the lysate. 5Cells were seeded per well in 96-well microplates and incubated on ice for 30 min with 2 μg / 100 μl SA and SA-IL4. After washing four times with PBS, the cells were further incubated on ice for 20 min with rabbit monoclonal anti-mouse serum albumin antibody (clone EPR20195 abcam). After washing three times with PBS, the cells were incubated on ice for 20 min with 1 μg / ml AlexaFluor 647-labeled anti-rabbit IgG, anti-B220, anti-CD3, anti-CD4, anti-CD8, anti-CD11c, anti-CD45, and anti-F4 / 80 antibodies. Cells were analyzed by flow cytometry as described below.

[0513] 4. Flow cytometry analysis of STAT6 phosphorylation

[0514] Using EasySep mouse CD4 + T-cell isolation kit (stem cells) for purifying mouse CD4 from the spleen of C57BL / 6 mice. + T cells. Purified CD4 + T cells (10⁶ cells / ml) were activated in six-well plates pre-coated with 5 μg / ml anti-CD3 antibody (clone 17A2, Bioxcell) and supplemented with soluble 2 μg / ml anti-CD28 antibody (clone 37.51, BioLegend) for 2 days. The culture 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 cells were stimulated with 50 ng / ml recombinant mouse IL-2. + T cells (Peprotech) were used for 3 hours to induce IL-4Rα expression. After stimulation with IL-2, cells were washed and incubated in fresh culture medium for 3 hours. Cells were then transferred to 96-well plates (50,000 cells / well). The specified amount of wt IL-4 or SA-IL-4 was applied to CD4+ cells at 37°C. + T cells were immobilized for 15 minutes to induce STAT6 phosphorylation. Cells were immediately fixed at 37°C for 10 minutes with BD Phosflow Lyse / Fix buffer, then permeabilized on ice with BD Phosflow Perm Buffer III for 30 minutes. Cells were stained with Alexa Fluor 647 anti-pSTAT6 antibody (clone J71-773.58.11, BD) to recognize Tyr641 phosphorylation. Staining was performed in the dark at room temperature (RT) for 1 hour. Cells were obtained on BD LSR and data were analyzed using FlowJo (Treestar). The mean fluorescence intensity (MFI) of the pSTAT6+ population was plotted against cytokine concentration. A dose-response curve was fitted using Prism (v8, GraphPad).

[0515] 5. Surface Plasmon Resonance (SPR)

[0516] 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) at approximately 200 resonance units (RUs) via amine coupling, according to the manufacturer's instructions. SA-IL4 was flowed at a reduced concentration of 30 μl / min in run buffer (0.01 M anhydrous sodium dihydrogen phosphate, pH 5.8, 0.15 M NaCl). For each cycle, the sensor chip was regenerated with PBS, pH 7.4. Specific binding of the SA fusion protein to FcRn was calculated by comparison with a non-functionalized channel used as a reference. Experimental results were fitted to Langmuir binding kinetics using BIAevaluation software (GE Healthcare).

[0517] 6. Differentiation of Th17 cells in vitro in culture

[0518] According to the manufacturer's instructions, use EasySep TM young mouse CD4 + T-cell isolation kit (STEMCELL Technologies) for isolating immature CD4 cells from spleen cells + T cells. 10 5 Cells were seeded in 96-well plates and cultured for 3 days. This was used as the Th17 induction medium.

[0519] 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. Data were analyzed using Prism software (v6, GraphPad).

[0520] 7. Plasma pharmacokinetics of proteins

[0521] Wt IL-4 or SA-IL-4 (equivalent to 10 μg IL-4) was intravenously injected into female C57BL / 6 mice. Blood samples were collected in protein-low binding tubes at 1 minute, 10 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, and 24 hours post-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.

[0522] 8. Pharmacokinetics of Proteins in Lymph Nodes and Spleen

[0523] Wt IL-4, SA-IL-4, or SA(P573K)-IL-4 (equivalent to 40 μg IL-4) were intravenously injected into healthy C57BL / 6 mice. Lumbar vertebral and brachial artery lymph nodes and spleen were collected at 1, 4, and 24 hours post-injection and subsequently analyzed using T-PER tissue protein extraction reagent (Thermo Scientific) with COmplete. TM The protease inhibitor mixture (Roche) was homogenized for 40 seconds at 5000 bpm using Lysing Matrix D and FastPrep-24 5G (MP Biomedical). After homogenization, the samples were incubated overnight at 4°C. The samples were centrifuged (5000g, 5 min), and the total protein concentration and IL-4 concentration were analyzed using the BCA assay kit (Thermo Fisher) and the 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.

[0524] 9. Fluorescence-based detection of IL-4 in LN

[0525] To prepare fluorescently labeled wt IL-4 and SA-IL-4, the protein was incubated with an 8-fold molar excess of DyLight 800NHS ester (Thermo Fisher) at room temperature for 1 hour, and unreacted dye was removed using a Zebaspin spinning column (Thermo Fisher) according to the manufacturer's instructions. Naïve C57BL / 6 mice were intravenously injected with 10 μg of DyLight 800-labeled wt IL-4 and SA-IL-4 with equivalent fluorescence. Four hours later, the iliac bone LN was imaged using a Xenogen IVIS imaging system 100 (Xenogen) under the following conditions: f / stop: 2; filter excitation wavelength 745 nm; excitation wavelength 800 nm; exposure time: 5 seconds; small aperture.

[0526] 10. Immunofluorescence

[0527] As described above, wt IL-4 and SA-IL-4 were fluorescently labeled using DyLight 594NHS ester (Thermo Fisher). One hour after intravenous injection of fluorescently labeled IL-4 (40 μg wt IL-4, with the same fluorescence intensity for SA-IL-4), mice were sacrificed. Mouse LNs were harvested and fixed overnight in 2% PFA in PBS, followed by washing with PBS. After incubation overnight in 30% sucrose solution, the LNs were embedded in compounds at the optimal cutting temperature. Then, 5 μm frozen sections were cut using a cryostat. The sections were then blocked at room temperature with 2% BSA in PBS and incubated for 2 hours at room temperature 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 antibody (clone: ​​MECA-79, BioLegend). After washing with PBS-T, tissues were stained at room temperature for 1 hour using the following fluorescently labeled secondary antibodies: Alexa Fluor 647 goat anti-hamster (1:400, Jackson Immuno Research) and Alexa Fluor 488 donkey anti-rat (1:400, Jackson Immuno Research). The tissues were washed three times and then covered with ProLong gold anti-fading mounting medium and 4′,6-diamidindo-2-phenylindole (DAPI; Thermo Fisher Scientific). CD3 staining was imaged at 10X magnification using an IX83 microscope (Olympus), while PNAd staining was imaged at 20X magnification using a Leica SP8 3D laser scanning confocal microscope. Images were processed using ImageJ software (NIH).

[0528] 11. EAE Model

[0529] MOG in complete Freund's adjuvant (CFA) 35-55 The emulsion was administered subcutaneously on the dorsal side to immunize young female C57BL / 6 mice, followed by a first intraperitoneal injection of pertussis toxin (PTX) in PBS on the same day and a second injection the following day. 35-55 CFA emulsion and PTX were purchased from Hooke Laboratories. EAE severity was monitored after the first immunization, and clinical scores were measured daily starting on day 8 post-immunization. Clinical scores were determined by AI, MN, or AS based on Hooke Laboratories' blinding criteria for treatment grouping. IL-4, SA-IL-4, and PBS were administered intraperitoneally or subcutaneously (on the back of the mouse) every other day in 100 μl PBS. FTY720 (1 mg / kg body weight) was administered orally daily.

[0530] 12. Spinal cord histology

[0531] Thoracic and lumbar vertebrae were harvested from EAE mice and excised at the thoracolumbar junction. Tissue was fixed overnight in 2% PFA. After washing with PBS, the tissue was decalcified overnight using decalcifying agent II (Leica Biosystem). The tissue was then embedded in paraffin. After paraffin embedding, the blocks were cut into 5 mm sections. After dewaxing and rehydration, the tissue sections were treated with targeted repair solution (S1699, DAKO) and heated in an autoclave at >95°C for 20 minutes. The tissue sections were incubated with anti-mouse aMBP (abcam ab40390) at room temperature in a humidified chamber for 1 hour. After washing with TBS, the tissue sections were incubated with biotinylated anti-rat IgG (10 mg / mL, Vector Laboratories) at room temperature for 30 minutes. Antigen-antibody binding was detected using an Elite kit (PK-6100, Vector Laboratories) and a DAB (DAKO, K3468) system. Slides were imaged using an EVOS FL Auto (LifeTechnologies) camera.

[0532] 13. Flow cytometry

[0533] EAE mice were treated with PBS, wt IL-4, or SA-IL-4 (equivalent to 10 μg IL-4) every other day, starting 8 days post-immunization. Spinal cord, spleen, and lumbar vertebral nuclei were harvested on days 13, 17, or 34 post-immunization. Spinal cord tissue was digested for 30 minutes at 37°C in Dalberg 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 gentle lysis through a 70 μm cell filter. For the spleen, erythrocytes were lysed with ACK lysis buffer (Quality Biological) and then stained with antibodies by flow cytometry. Antibodies against the following molecules were used: 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), and CD86 (GL1, BD Biosciences). 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) 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 T cells that recognize MOG, T-SelectI-Ab MOG is used. 35-55 Tetramer - PE (MBL International Corporation) or MOG 38-49 Tetramer-PE (NIHTetramer Core Facility). Fixable live / dead cells were distinguished 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 on ice with a specific antibody 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).

[0534] 14. Restimulation of spleen cells

[0535] Single-cell suspensions were generated from dLN and spleen. To analyze cytokine production, 5 x 10-1 cells were used. 5 One lymphocyte and 2x10 6 One splenocyte was seeded in a 96-well round-bottom plate. Using 10 μM MOG... 35-55 Cells were stimulated with a peptide (Genscript). Two hours later, GolgiPlug (brefeldin A) and GolgiStop (Monensin) were added according to the manufacturer's protocol to block the secretion of intracellular cytokines. Four hours after adding GolgiPlug and GolgiStop, cells were stained for flow cytometry. For fixation, Cytofix / Cytoperm (BD Bioscience) was used for 20 minutes at 4°C. For permeabilization, perm / wash buffer (BD Bioscience) was used, and cells were stained in perm / wash buffer at 4°C for 30 minutes. For restimulation after 3 days, 2.5 x 10⁻⁶ cells were used. 5 One lymphocyte or 1x10 6 One splenocyte was seeded in a 96-well round-bottom plate. Using 10 μM MOG... 35-55Cells were cultured for 6 hours, followed by flow cytometry, or stimulated with 100 μg / ml MOG protein (cultured for 72 hours) (Anaspec). After 72 hours, the supernatant was collected and analyzed by ELISA using the Ready-Set-Go! kit (Invitrogen) or the LEGEND MAX mouse GM-CSF ELISA kit (BioLegend).

[0536] 15. Safety assessment of SA-IL-4

[0537] C57BL / 6 mice were intravenously injected with PBS, wt IL-4, or SA-IL-4 (equivalent to 10 μg IL-4). Two days later, blood samples collected from the mice were analyzed using a COULTER Ac·T 5diff CP hematology analyzer (Beckman Coulter) according to the manufacturer's instructions. Lungs and spleens were harvested and weighed. The water content in the lungs 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 biochemical analyzer (Alfa Wassermann Diagnostic Technologies) according to the manufacturer's instructions.

[0538] 16. Statistical Analysis

[0539] Statistically significant differences between experimental groups were determined using Prism software (v6, GraphPad). Similarity between groups was found by Brown-Forsythe test using one-way ANOVA and Tukey's HSD post-hoc test. Two-tailed Student's t-tests were used for individual comparisons. The symbols * and ** indicate p-values ​​less than 0.05 and 0.01, respectively.

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Claims

1. A composition comprising an anti-inflammatory agent operatively linked to a peptide comprising a collagen binding domain (CBD) from von Willebrand Factor (VWF), wherein the anti-inflammatory agent is an IL-4 or IL-10 polypeptide, and wherein the operative linkage is selected from the group consisting of covalent linkage, cross-linking through a bifunctional linker, and linkage through a peptide bond, wherein the anti-inflammatory agent operatively linked to the CBD-vWF peptide further comprises a serum protein operatively linked to the peptide or anti-inflammatory agent.

2. The composition of claim 1, wherein the CBD-vWF peptide comprises a peptide having an amino acid sequence that is at least 85% identical to one of SEQ ID NO: 4 to SEQ ID NO: 7, SEQ ID NO: 47, or SEQ ID NO: 52, or a peptide having an amino acid sequence that is at least 85% identical to a fragment of one of SEQ ID NO: 4 to SEQ ID NO: 7, SEQ ID NO: 47, or SEQ ID NO:

52.

3. The composition of claim 1, wherein the serum protein is operatively linked to the CBD-vWF peptide.

4. The composition of claim 3, wherein the serum protein is operatively linked to the CBD-vWF peptide through a peptide bond.

5. The composition of claim 1, wherein the serum protein comprises albumin.

6. The composition of any one of claims 1 to 5, wherein the ratio of CBD-vWF peptide to the anti-inflammatory agent is 1: 1 to 5:

1.

7. The composition of any one of claims 1 to 5, wherein the composition further comprises a second anti-inflammatory agent operatively linked to an extracellular matrix (ECM) affinity peptide.

8. The composition of claim 1, wherein the composition comprises a peptide of SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 47, or SEQ ID NO: 52 conjugated to the amino terminus of an IL-4 polypeptide.

9. The composition of claim 1, wherein the composition comprises a peptide of SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 47, or SEQ ID NO: 52 conjugated to the amino terminus of an IL-10 polypeptide.

10. Use of a composition according to any one of claims 1 to 9 in the manufacture of a medicament for treating an autoimmune or inflammatory disorder in a subject, wherein the composition is administered to the subject.

11. The use of claim 10, wherein the autoimmune or inflammatory disorder comprises inflammatory bowel disease, multiple sclerosis, type 1 diabetes, Crohn’s disease, psoriasis, acute inflammation, chronic inflammation, neuroinflammation, arthritis, fibrosis, infection, allergy.

12. The use of claim 11, wherein the autoimmune or inflammatory disorder comprises fibrosis, and wherein the fibrosis is idiopathic pulmonary fibrosis.

13. The use of claim 11, wherein the autoimmune or inflammatory disorder comprises multiple sclerosis.

14. The use of claim 11, wherein the autoimmune or inflammatory disorder comprises rheumatoid arthritis.

15. The use of any one of claims 10-14, wherein the composition is administered systemically.

16. The use of claim 15, wherein the composition is administered by intravenous injection.

17. The use of any one of claims 10-14, wherein the composition is administered topically.

18. The use of claim 17, wherein the composition is administered to an inflammation site or adjacent to an inflammation site.

19. The use of any one of claims 10-14, wherein the administered dose of the composition comprising an anti-inflammatory agent operatively linked to the peptide is less than the minimum effective dose of the anti-inflammatory agent administered without the peptide.

20. The use of any one of claims 10-14, wherein the administered dose of the composition comprising an anti-inflammatory agent operatively linked to the peptide is less than the minimum effective dose of the anti-inflammatory agent administered by the same route of administration without the peptide.

21. The use of claim 20, wherein the administered dose of the anti-inflammatory agent operatively linked to the peptide is at least 10% less than the minimum effective dose of the anti-inflammatory agent administered without the peptide.

22. The use of any one of claims 10-14, wherein the subject has been previously treated with an anti-inflammatory agent, an anti-inflammatory therapy, or an autoimmune therapy.

23. The use of claim 22, wherein the subject is determined to have not responded to the previous treatment.

24. The use of any one of claims 10-14, wherein the subject has not been previously treated for an inflammatory or autoimmune disease.

25. The use of any one of claims 10-14, wherein the method further comprises administering an additional inflammatory or autoimmune therapy.

26. The use of any one of claims 10-14, wherein the method further comprises administering a second anti-inflammatory agent operatively linked to an extracellular matrix (ECM)-affinity peptide.

27. Use of the composition of any one of claims 1-9 in the manufacture of a medicament for reducing inflammation in a subject, wherein the composition is administered to the subject.

28. The use of claim 27, wherein the inflammation is due to an autoimmune or inflammatory disorder, and wherein the autoimmune or inflammatory disorder comprises inflammatory bowel disease, idiopathic pulmonary fibrosis, multiple sclerosis, type 1 diabetes, arthritis, or rheumatoid arthritis.

29. The use of claim 28, wherein the autoimmune or inflammatory disorder comprises multiple sclerosis.

30. The use of claim 28, wherein the autoimmune or inflammatory disorder comprises rheumatoid arthritis.

31. The use of any one of claims 28-30, wherein the composition is administered systemically.

32. The use of any one of claims 28-30, wherein the composition is administered topically.

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