Methods and compositions for treating inflammation and autoimmune diseases

Abstract

The present disclosure relates to engineered IL-35 cytokine polypeptides to achieve improved treatment for inflammatory diseases and inflammatory disorders. Accordingly, aspects of the disclosure relate to a polypeptide of SEQ ID NO: 2 wherein the polypeptide comprises one or more amino acid substitutions relative to SEQ ID NO: 2 wherein the one or more amino acid substitutions comprise K23, R74, R195, R197, or a combination thereof. Other aspects related to the polypeptide include SEQ ID NO: 4 wherein the polypeptide comprises one or more amino acid substitutions relative to SEQ ID NO: 4 wherein the one or more amino acid substitutions comprise C74. Other aspects related to the polypeptide include SEQ ID NO: 6 wherein the polypeptide comprises one or more amino acid substitutions relative to SEQ ID NO: 6 wherein the one or more amino acid substitutions comprise C34.

Description

[0001] This application claims the priority benefit of U.S. Provisional Patent Application No. 63 / 355,446, filed on Jun. 24, 2022, which is incorporated herein by reference in its entirety.

[0002] Sequence Listing

[0003] This application contains a Sequence Listing submitted in XML format, which is incorporated herein by reference in its entirety. The XML copy was created on Jun. 24, 2023, named "ARCDP0778WO.xml", and is 122,913 bytes in size. Background of the Invention

[0004] I. Field of the Technology

[0005] The present invention generally relates to the field of medicine. More specifically, it relates to engineered anti-inflammatory agent compositions and methods for treating autoinflammatory and inflammatory diseases.

[0006] II. Background Art

[0007] Multiple sclerosis (MS) is a potentially disabling autoimmune disease that affects millions of people worldwide. Auto-reactive immune cells are located in the central nervous system (CNS) and cause demyelination, resulting in focal lesions in the white matter. Lymphocytes and macrophages infiltrating into 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 key role in the disease development and severity of MS. Therefore, inhibiting lymphocyte migration into the CNS and inducing an immunosuppressive microenvironment in SLOs will provide an effective treatment for MS. Clinically, FTY720 (fingolimod) and anti-integrin α4 antibody (natalizumab monoclonal antibody) are used to treat MS by sequestering lymphocytes in LNs and preventing them from reacting with autoantigens in target tissues. Experimental autoimmune encephalomyelitis (EAE) is a widely accepted mouse model of MS that reflects many features of disease progression and pathogenesis, including lymphocyte migration into the CNS and demyelination.

[0008] Rheumatoid arthritis (RA) is an autoimmune disease currently controlled by treatment with inflammatory pathway inhibitors. The pathological features of RA are synovitis and joint destruction, which lead to severe pain and joint dysfunction. Although the pathogenic antigens of RA have not been fully elucidated, the recognition of collagen by immune cells plays a key role. During the progression of RA, autoantigen-specific T cells, especially Th17 cells, are activated and produce inflammatory cytokines including IL-17. Inflammatory cytokines in the joint, such as TNF-α and IL-6, induce the activation of macrophages and neutrophils as mediators of the inflammatory response. These inflammatory cells infiltrate the joint and cause various inflammatory responses, including the activation of osteoclasts that destroy joint bones. Current RA treatment strategies are symptomatic treatments, and considering that the progression of RA involves multiple inflammatory cytokines, a variety of biotherapies for clinical application have been developed and approved, such as antibodies or soluble receptors against TNF-α.

[0009] A variety of treatment strategies for autoimmune diseases such as MS and RA have been explored, all of which involve the use of anti-inflammatory cytokines. However, currently, this strategy has not been translated into clinical application. There is still a need for compositions and methods involving the effective delivery of anti-inflammatory cytokines for the treatment of autoimmune diseases including MS and RA. Summary of the Invention

[0010] The present disclosure relates to engineering IL-35 cytokine polypeptides for improved treatment of inflammatory diseases and inflammatory conditions. Accordingly, the present disclosure describes polypeptides having an amino acid sequence of SEQ ID NO: 2 with one or more amino acid substitutions relative to SEQ ID NO: 2, wherein the one or more amino acid substitutions include K23, R74, R195, R197, or a combination thereof. Substitutions of K23, R74, R195, and / or R197 with respect to SEQ ID NO: 2 may also be excluded from the polypeptides of the present disclosure. Also disclosed are polypeptides having an amino acid sequence of SEQ ID NO: 4 with one or more amino acid substitutions relative to SEQ ID NO: 4, wherein the one or more amino acid substitutions include C74. The polypeptides may exclude polypeptides of SEQ ID NO: 4 having a C74 substitution. Also provided are polypeptides having an amino acid sequence of SEQ ID NO: 6 with one or more amino acid substitutions relative to SEQ ID NO: 6, wherein the one or more amino acid substitutions include C34. The polypeptides may exclude polypeptides of SEQ ID NO: 6 having a C34 substitution. Also described are polypeptides having an amino acid sequence of SEQ ID NO: 90 with one or more amino acid substitutions relative to SEQ ID NO: 90, wherein the one or more amino acid substitutions include K2, R53, R174, R176, or a combination thereof. Substitutions of K2, R53, R174, and / or R176 with respect to SEQ ID NO: 90 may also be excluded from the polypeptides of the present disclosure. Also described are polypeptides having an amino acid sequence of SEQ ID NO: 1 with one or more amino acid substitutions relative to SEQ ID NO: 1, wherein the one or more amino acid substitutions include K23, R74, R195, R197, C320, C497, or a combination thereof. Substitutions of K23, R74, R195, R197, C320, and / or C497 with respect to SEQ ID NO: 1 may also be excluded from the polypeptides of the present disclosure. Also described are polypeptides comprising the EBI3 subunit of IL-35, wherein the EBI3 subunit comprises fewer than 200 amino acids and comprises the amino acid sequence of SEQ ID NO: 92 or an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 92. Also described are polypeptides having an amino acid sequence of SEQ ID NO: 91 with one or more amino acid substitutions relative to SEQ ID NO: 91, wherein the one or more amino acid substitutions include K2, R53, R174, R176, C299, C476, or a combination thereof. Substitutions of K2, R53, R174, R176, C299, and / or C476 with respect to SEQ ID NO: 91 may also be excluded from the polypeptides of the present disclosure.

[0011] Chimeric or multimeric polypeptides are also described, which comprise (i) an amino acid sequence of SEQ ID NO: 2 having one or more amino acid substitutions relative to SEQ ID NO: 2, wherein the one or more amino acid substitutions relative to SEQ ID NO: 2 include K23, R74, R195, R197, or a combination thereof; and (ii) a polypeptide of SEQ ID NO: 4, a polypeptide having at least 70% sequence identity with SEQ ID NO: 4; or a polypeptide comprising SEQ ID NO: 4 having one or more amino acid substitutions relative to SEQ ID NO: 4. Chimeric or multimeric polypeptides are also described, which comprise: (i) an amino acid sequence of SEQ ID NO: 90 having one or more amino acid substitutions relative to SEQ ID NO: 90, wherein the one or more amino acid substitutions relative to SEQ ID NO: 90 include K2, R53, R174, R176, or a combination thereof; and (ii) a polypeptide of SEQ ID NO: 4, a polypeptide having at least 70% sequence identity with SEQ ID NO: 4; or a polypeptide comprising SEQ ID NO: 4 having one or more amino acid substitutions relative to SEQ ID NO: 4. Chimeric or multimeric polypeptides are also provided, which comprise: (i) the EBI3 subunit of IL-35, wherein the EBI3 subunit comprises less than 200 amino acids and comprises the amino acid sequence of SEQ ID NO: 92 or an amino acid sequence having at least 70% sequence identity with SEQ ID NO: 92 and (ii) a polypeptide of SEQ ID NO: 4, a polypeptide having at least 70% sequence identity with SEQ ID NO: 4; or a polypeptide comprising SEQ ID NO: 4 having one or more amino acid substitutions relative to SEQ ID NO: 4.

[0012] Pharmaceutical compositions and host cells comprising the polypeptides of the present disclosure are also provided. The present disclosure also provides nucleic acids encoding the polypeptides of the present disclosure, expression vectors comprising the nucleic acids, and host cells comprising the nucleic acids and / or expression vectors of the present disclosure. The compositions may also comprise the nucleic acids, expression vectors, and / or host cells of the present disclosure.

[0013] The present method relates to a method for preparing cells, the method comprising transferring one or more nucleic acids or one or more expression vectors of the present disclosure into cells. Also provided is a method for preparing a polypeptide, the method comprising transferring one or more expression vectors of the present disclosure into cells and incubating the cells under conditions sufficient to express the polypeptide encoded on the expression vector. Also described is a method for treating an autoimmune or inflammatory disease in a subject, comprising administering to the subject a polypeptide or pharmaceutical composition of the present disclosure. Also provided is a method for reducing inflammation in a subject, comprising administering to the subject a polypeptide or pharmaceutical composition of the present disclosure. The method also comprises a method for delivering cytokine therapy to lymph nodes and / or for increasing tolerogenic dendritic cells in lymph nodes in a subject, comprising administering to the subject a polypeptide or pharmaceutical composition of the present disclosure.

[0014] The EBI3 polypeptide may have or have 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% sequence identity with SEQ ID No: 92. The EBI3 subunit may comprise one or more amino acid substitutions relative to SEQ ID NO: 92, and one or more of the amino acid substitutions include R33, R154, R156 or a combination thereof. Substitutions of R33, R154 and / or R156 with respect to SEQ ID NO: 92 may also be excluded from the polypeptides of the present disclosure. The EBI3 subunit may consist of 189 amino acids. The EBI3 subunit may comprise, at most comprise, at least comprise or consist of 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 or 209 amino acids, or any derivable range therein. The polypeptide may comprise substitutions of C6, C39, C49 or a combination thereof relative to SEQ ID NO: 92. Substitutions of C6, C39 and / or C49 with respect to SEQ ID NO: 92 may also be excluded from the polypeptides of the present disclosure. The substitution may be with serine. The polypeptide may comprise 1, 2 or 3 substitutions of C6, C39 and C49. The EBI3 subunit may be a polypeptide having the amino acid sequence of SEQ ID NO: 4, a polypeptide having an amino acid sequence with at least 70% sequence identity with SEQ ID NO: 4 or the amino proximal of a polypeptide comprising a substituted amino acid sequence of SEQ ID NO: 4. The EBI3 subunit may be a polypeptide having the amino acid sequence of SEQ ID NO: 4, a polypeptide having an amino acid sequence with at least 70% sequence identity with SEQ ID NO: 4 or the carboxyl proximal of a polypeptide comprising a substituted amino acid sequence of SEQ ID NO: 4. When the first region is linked to the carboxyl terminus of the second region, the first region is the carboxyl proximal of the second region. Other amino acid residues may be present between the first region and the second region. Thus, these regions need not be adjacent, unless specifically stated that there are no intervening amino acid residues.The term "amino-proximal" is similarly defined such that when a first region is linked to the amino terminus of a second region, the first region is amino-proximal to the second region. Similarly, there may be additional amino acid residues between the first region and the second region unless otherwise specified.

[0015] The polypeptide and / or a polypeptide comprising a substituted amino acid sequence of SEQ ID NO: 2 may comprise an amino acid sequence having at least 70% sequence identity with SEQ ID NO: 2. The polypeptide may comprise an amino acid sequence having or having 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% (or any range derivable therefrom) sequence identity with SEQ ID NO: 2. The polypeptide may comprise one or more substitutions relative to C36, C47, C80 and / or C90 of SEQ ID NO: 2. Substitutions with respect to C36, C47, C80 and C90 of SEQ ID NO: 2 may also be excluded from the polypeptides of the present disclosure. The substitution may be with serine. The polypeptide may comprise 1, 2, 3 or 4 substitutions located at C36, C47, C80 and C90.

[0016] The polypeptide and / or a polypeptide comprising a substituted amino acid sequence of SEQ ID NO: 90 may comprise an amino acid sequence having at least 70% sequence identity with SEQ ID NO: 90. The polypeptide may comprise an amino acid sequence having or having 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% (or any range derivable therefrom) sequence identity with SEQ ID NO: 90. The polypeptide may comprise one or more substitutions relative to C15, C26, C59 and C69 of SEQ ID NO: 90. Substitutions with respect to C15, C26, C59 and / or C69 of SEQ ID NO: 90 may also be excluded from the polypeptides of the present disclosure. The substitution may be with serine. The polypeptide may comprise 1, 2, 3 or 4 substitutions located at C15, C26, C59 and C69.

[0017] The polypeptide may comprise an amino acid sequence having at least 70% sequence identity with SEQ ID NO: 4. The polypeptide may comprise an amino acid sequence having or having 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% (or any range derivable therefrom) sequence identity with SEQ ID NO: 4. The polypeptide comprising one or more amino acid substitutions relative to SEQ ID NO: 4 may comprise a substitution of C74. The substitution of C74 with respect to SEQ ID NO: 4 may also not be included in the polypeptides of the present disclosure.

[0018] The polypeptide may comprise an amino acid sequence having at least 70% sequence identity with SEQ ID NO: 6. The polypeptide may comprise an amino acid sequence having or having 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% (or any range derivable therefrom) sequence identity with SEQ ID NO: 6.

[0019] The polypeptide may comprise an amino acid sequence having at least 70% sequence identity with SEQ ID NO: 1. The polypeptide may comprise an amino acid sequence having or having 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% (or any range derivable therefrom) sequence identity with SEQ ID NO: 1. One or more amino acid substitutions relative to SEQ ID NO: 1 may comprise K23, C320 and / or C497.

[0020] The polypeptide may comprise an amino acid sequence having at least 70% sequence identity with SEQ ID NO: 91. The polypeptide may comprise or have sequence identity with SEQ ID NO: 91 of 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% (or any range derivable therefrom). One or more amino acid substitutions relative to SEQ ID NO: 91 may comprise K2, C299 and / or C476. The polypeptide may comprise substitutions relative to C15, C26, C59, C69 or combinations thereof of SEQ ID NO: 91. Substitutions with respect to C15, C26, C59 and / or C69 of SEQ ID NO: 91 may also be excluded from the polypeptides of the present disclosure. The substitution may be with serine. The polypeptide may comprise 1, 2, 3 or 4 substitutions at C15, C26, C59 and C69.

[0021] The polypeptide may also include or may not include an ECM affinity peptide, a serum protein, an albumin binding protein, and / or an Fc peptide. ECM affinity peptides are described throughout this disclosure. The ECM affinity peptide may include or may not include the amino acid sequences of SEQ ID NO: 9 to SEQ ID NO: 27 or SEQ ID NO: 88, or an amino acid sequence having at least 70% sequence identity with SEQ ID NO: 9 to SEQ ID NO: 27 or SEQ ID NO: 88. The ECM affinity peptide may comprise an amino acid sequence having or having 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% (or any range derivable therefrom) sequence identity with SEQ ID NO: 9 to SEQ ID NO: 27 or SEQ ID NO: 88. The Fc peptide may include or may not include the amino acid sequences of SEQ ID NO: 86 or SEQ ID NO: 87, or an amino acid sequence having at least 70% sequence identity with SEQ ID NO: 86 or SEQ ID NO: 87. The Fc peptide may comprise an amino acid sequence having or having 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% (or any range derivable therefrom) sequence identity with SEQ ID NO: 86 or SEQ ID NO: 87. The ABP may include or may not include the amino acid sequence of SEQ ID NO: 93, or an amino acid sequence having at least 70% sequence identity with SEQ ID NO: 93.ABP may comprise an amino acid sequence having or having 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% (or any range derivable therefrom) sequence identity with SEQ ID NO: 93. The ECM affinity peptide, serum protein, ABP and / or Fc peptide may be the amino terminus of a polypeptide comprising a substituted amino acid sequence of any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 90, SEQ ID NO: 91 or the EBI3 subunit. The ECM affinity peptide, serum protein and / or Fc peptide may be the carboxy terminus of a polypeptide comprising a substituted amino acid sequence of any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 90, SEQ ID NO: 91 or the EBI3 subunit. As defined herein, the ECM affinity peptide, serum protein, ABP and / or Fc peptide may be the amino terminus of (i) and / or (ii).

[0022] The serum protein may comprise a polypeptide having the amino acid sequence of SEQ ID NO: 6 or a polypeptide having at least 70% sequence identity with a polypeptide having the amino acid sequence of SEQ ID NO: 6. The serum protein may comprise an amino acid sequence having or having 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% (or any range derivable therefrom) sequence identity with SEQ ID NO: 6. The polypeptide may comprise one or more amino acid substitutions relative to SEQ ID NO: 6, wherein one or more of the amino acid substitutions comprise C34. The substitution at C34 may be a substitution with an amino acid residue of alanine or serine.

[0023] One or more amino acid substitutions relative to SEQ ID NO: 2 may include K23, and one or more amino acid substitutions relative to SEQ ID NO: 4 may include C74. Substitutions of K23 and / or C74 may be substitutions with amino acid residues of alanine or serine. Substitutions of K23 and C74 may include one or more substitutions of K23A and / or C74S.

[0024] A polypeptide comprising a substituted amino acid sequence of SEQ ID NO: 2 may be a polypeptide having the amino acid sequence of SEQ ID NO: 4, a polypeptide having an amino acid sequence with at least 70% sequence identity to SEQ ID NO: 4, or the amino terminus of a polypeptide comprising a substituted amino acid sequence of SEQ ID NO: 4. A polypeptide comprising a substituted amino acid sequence of SEQ ID NO: 2 may be a polypeptide having the amino acid sequence of SEQ ID NO: 4, a polypeptide having an amino acid sequence with at least 70% sequence identity to SEQ ID NO: 4, or the carboxyl terminus of a polypeptide comprising a substituted amino acid sequence of SEQ ID NO: 4.

[0025] A polypeptide comprising a substituted amino acid sequence of SEQ ID NO: 90 may be a polypeptide having the amino acid sequence of SEQ ID NO: 4, a polypeptide having an amino acid sequence with at least 70% sequence identity to SEQ ID NO: 4, or the amino terminus of a polypeptide comprising a substituted amino acid sequence of SEQ ID NO: 4. A polypeptide comprising a substituted amino acid sequence of SEQ ID NO: 90 may be a polypeptide having the amino acid sequence of SEQ ID NO: 4, a polypeptide having an amino acid sequence with at least 70% sequence identity to SEQ ID NO: 4, or the carboxyl terminus of a polypeptide comprising a substituted amino acid sequence of SEQ ID NO: 4.

[0026] One or more amino acid substitutions relative to SEQ ID NO: 90 may include or consist of K2. One or more amino acid substitutions relative to SEQ ID NO: 4 may include or consist of C74. The polypeptide may include or consist of substitutions at K2 and C74 of SEQ ID NO: 90 and SEQ ID NO: 4. The substitutions may include K2A and C74S.

[0027] Linkers such as amino acid or peptide mimetic sequences can be inserted into the polypeptides of the present disclosure. The linker can have one or more than one property, including a flexible conformation, an inability to form an ordered secondary structure, or hydrophobic or charged properties, which can facilitate or interact with any domain. Examples of amino acids commonly found in flexible protein regions can include Gly, Asn, and Ser. For example, suitable peptide linkers can be GGGSGGGS (SEQ ID NO: 7) or (GGGS)n (SEQ ID NO: 8), where n = 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 (or any range derivable 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). The peptide and the antibody heavy chain or antibody light chain can be linked by a peptide sequence having from about 1 to 25 amino acid residues. Examples of linkers can also include chemical moieties and coupling agents, such as sulfo-succinimide derivatives (sulfo-SMCC, sulfo-SMPB), disuccinimidyl suberate (DSS), disuccinimidyl glutarate (DSG), and disuccinimidyl tartrate (DST). Examples of linkers also include linear carbon chains, such as C N (where N = 1 to 100 carbon atoms, such as N = 2, 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms). The linker can be a dipeptide linker, such as valine-citrulline (val-cit), phenylalanine-lysine (phe-lys) linker, or maleimidocaproyl-valine-citrulline-p-aminobenzyloxycarbonyl (vc) linker. The linker can be sulfo-succinimidyl 4-[N-maleimidomethyl] cyclohexane-1-carboxylate (smcc). Sulfo-smcc coupling occurs through the maleimide group, which reacts with a thiol group (mercaptan, --SH), while its sulfo-NHS ester reacts with primary amines (such as lysine and the N-terminus of a protein or peptide). In addition, the linker can be maleimidocaproyl (mc). One or more than one of these linkers can be specifically excluded in some respects.

[0028] As defined herein, a chimeric polypeptide can comprise a linker between (i) and (ii). The linker can also be between (i) and (ii), and in serum proteins, Fc peptides, ABP, HSA, and / or ECM affinity peptides. The linker can comprise or consist of any one of the amino acid sequences of SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NOs: 75 to 85. The linker can comprise glycine residues and serine residues. The linker can comprise (G3S) nA linker, where n is an integer from 1 to 10. The linker can comprise or consist of the amino acid sequence of SEQ ID NO: 3. The linker can comprise or consist of the amino acid sequence of SEQ ID NO: 5. The linker can comprise or consist of the amino acid sequence of SEQ ID NO: 75. The linker between one or more of (i), (ii), serum proteins, Fc peptides, ABP, and / or ECM affinity peptides, at its amino terminus, its carboxy terminus, or its flanks may not be included in the polypeptides of the present disclosure.

[0029] The polypeptide can comprise or can further comprise a serum albumin (SA) polypeptide. The SA polypeptide can further be defined as a human SA polypeptide. The SA polypeptide can comprise a polypeptide having the amino acid sequence of SEQ ID NO: 6 or a polypeptide having at least 70% sequence identity with the polypeptide having the amino acid sequence of SEQ ID NO: 6. The SA polypeptide can comprise an amino acid sequence having or having 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% (or any range derivable therefrom) sequence identity with SEQ ID NO: 6. The polypeptide can comprise one or more amino acid substitutions relative to SEQ ID NO: 6, where one or more of the amino acid substitutions comprise C34. The substitution at C34 can be a substitution of cysteine for any other amino acid. The substitution at C34 can be a substitution of an amino acid residue with alanine or serine. As defined herein, the SA polypeptide can be the carboxy terminus of (i) and / or (ii). As defined herein, the SA polypeptide can be the amino terminus of (i) and / or (ii).

[0030] The substitution can be a substitution with a non-polar amino acid. The substitutions at K23, R74, R195, R197 relative to SEQ ID NO: 2, the substitution at C74 relative to SEQ ID NO: 4, the substitution at C34 relative to SEQ ID NO: 6, or the substitutions at K23, R74, R195, R197, C320, C497 relative to SEQ ID NO: 1 can be substitutions with an amino acid residue of alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine or valine. The substitution can be a substitution with alanine, leucine, isoleucine, proline, tryptophan, valine, phenylalanine or methionine. The substitutions described herein can be without or with a substitution with an amino acid residue of alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine or valine. The substitution can be a substitution with alanine, leucine, isoleucine, proline, tryptophan, valine, phenylalanine or methionine. The substitutions at K23, R74, R195, R197 relative to SEQ ID NO: 2, the substitution at C74 relative to SEQ ID NO: 4, the substitution at C34 relative to SEQ ID NO: 6, or the substitutions at K23, R74, R195, R197, C320, C497 relative to SEQ ID NO: 1 can be substitutions with an amino acid residue of alanine, leucine, isoleucine, proline, tryptophan, valine, phenylalanine or methionine. The substitution can be a substitution with alanine. The substitution can be a substitution with serine or glycine. In the polypeptides of the present disclosure, the substitution with lysine or arginine can be absent. In any other polypeptides of the present disclosure, the substitution with any other amino acid can also be absent.

[0031] The polypeptides of the present disclosure may comprise a signal peptide. The signal peptide may comprise SEQ ID NO: 89. The polypeptides of the present disclosure may comprise or consist of a polypeptide comprising one or more amino acid sequences of SEQ ID NO: 1 to SEQ ID NO: 93, or an amino acid sequence having or having 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% (or any range derivable therefrom) sequence identity to one or more of SEQ ID NO: 1 to SEQ ID NO: 93. Any of SEQ ID NO: 1 to SEQ ID NO: 93 may not be included in the polypeptides of the present disclosure.

[0032] The inflammation treated by the polypeptides and compositions of the present disclosure may be inflammation associated with an autoimmune disease or an inflammatory disease. The autoimmune disease or inflammatory disease may include or not include cytokine release syndrome, cytokine storm, arthritis, inflammatory bowel disease, scleroderma, inflammatory bowel disease, idiopathic pulmonary fibrosis, multiple sclerosis, type 1 diabetes, Crohn's disease, psoriasis, acute inflammation, chronic inflammation, neuroinflammation, acute respiratory distress syndrome, rheumatoid arthritis, fibrosis, infection, allergy, adverse events related to inflammation treatment, ischemic stroke, pulmonary hypertension, ischemia, cardiac stroke, stroke, atherosclerosis, wound healing, peripheral artery disease, cerebral ischemia reperfusion (I / R) injury, myocardial infarction and related inflammatory diseases. The inflammation treated may be inflammation associated with a viral infection, such as influenza, coronavirus or SARS-CoV-2 infection. The inflammation treated may be inflammation associated with the administration of immunotherapy, such as checkpoint inhibitor treatment, administration of CAR-T cells or engineered TCR-T cells. The method may be used to treat cytokine release syndrome or cytokine storm. The autoimmune disease or inflammatory disease may include multiple sclerosis. The autoimmune disease or inflammatory disease may include rheumatoid arthritis.

[0033] The composition or polypeptide may be administered by any route of administration described throughout the present disclosure. The composition or polypeptide may be administered systemically. The composition or polypeptide may be administered by intravenous injection. The composition may be administered topically. The composition may be administered to the site of inflammation or a site adjacent to the site of inflammation.

[0034] The subject can be a subject previously treated with an anti-inflammatory agent, anti-inflammatory treatment, or autoimmune treatment. The subject can be a subject determined to be non-responsive to the previous treatment. The subject can be a human who has not been previously treated for an inflammatory disease or autoimmune disease. The method can further include administering additional anti-inflammatory treatment or autoimmune treatment. The additional anti-inflammatory treatment or autoimmune treatment can include or not include anti-cytokine drugs or steroids. The additional anti-inflammatory treatment or autoimmune treatment can include or not include one or more than one of the following drugs: steroids, corticosteroids, anti-TNF-α treatment, anti-integrin treatment, infliximab, mesalazine, and vedolizumab, fingolimod, interferon-β, dimethyl fumarate, teriflunomide, integrin α4β1, anti-αLβ2 antibody, anti-IL-6R agent, anti-IL-6 agent, and Janus kinase inhibitors (such as tofacitinib, baricitinib, upadacitinib). The steroids can include or not include dexamethasone, methylprednisolone, and / or prednisolone. The polypeptide or composition can be a substance that reduces the number of Th17 cells in the subject. The polypeptide or composition can be a substance that inhibits the function of Th17 cells in the subject. The polypeptide or composition can be administered to the subject by a drug-loaded syringe.

[0035] The polypeptide or composition can be administered at a dose of 0.1 mg / kg to 50 mg / kg. The polypeptide or composition can be administered at, or at least at, or at most at 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, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 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,administered at a dose of 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, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265, 270, 275, 280, 285, 290, 295, 300, 305, 310, 315, 320, 325, 330, 335, 340, 345, 350, 355, 360, 365, 370, 375, 380, 385, 390, 395, 400, 405, 410, 415, 420, 425, 430, 435, 440, 445, 450, 455, 460, 465, 470, 475, 480, 485, 490, 495, 500, 505, 510, 515, 520, 525, 530, 535, 540, 545, 550, 555, 560, 565, 570, 575, 580, 585, 590, 595, 600, 605, 610, 615, 620, 625, 630, 635, 640, 645, 650, 655, 660, 665, 670, 675, 680, 685, 690, 695, 700, 705, 710, 715, 720, 725, 730, 735, 740, 745, 750, 755, 760, 765, 770, 775, 780, 785, 790, 795, 800, 805, 810, 815, 820, 825, 830, 835, 840, 845, 850, 855, 860, 865, 870, 875, 880, 885, 890, 895, 900, 905, 910, 915, 920, 925, 930, 935, 940, 945, 950, 955, 960, 965, 970, 975, 980, 985, 990, 995 or 1000 pg, ng, μg, mg, g, pg / kg, ng / kg, μg / kg, mg / kg or g / kg (or any range derivable therefrom), where " / kg" refers to the dose administered per kg of the subject.,

[0036] The method may further include detecting anti - inflammatory cytokines in the lymph nodes of the subject. The method may include obtaining a lymph sample from the subject. The detection may include detecting the presence of the polypeptide of the present disclosure in the lymph sample.

[0037] The terms "protein", "polypeptide", and "peptide" are used interchangeably herein when referring to gene products that comprise amino acid polymers.

[0038] The terms "subject", "mammal", and "patient" are used interchangeably. A subject can be a mammal. A subject can be a human. A subject can be a mouse, rat, rabbit, dog, donkey, or laboratory experimental animal such as a fruit fly, zebrafish, etc.

[0039] It is contemplated that the methods and compositions include the exclusion of any aspects described herein.

[0040] Throughout this application, the term "about" is used in its ordinary and customary sense in the field of cell and molecular biology to indicate a value that includes the standard deviation of the error of the device or method used to determine the value.

[0041] When used without a quantifier before the term in conjunction with the term "comprising / including", it can mean "one / a", but is also consistent with the meanings of "one or more / a or more", "at least one / at least one", and "one or more than one / a or more than one".

[0042] As used herein, the terms "or" and "and / or" are used to describe multiple components that are combined with or exclusive of each other. For example, "x, y, and / or z" can refer to "x" alone, "y" alone, "z" alone, "x, y, and z", "(x and y) or z", "x or (y and z)", or "x or y or z". It is specifically contemplated that x, y, or z can be specifically excluded from one or more than one embodiment or aspect.

[0043] The words "comprising", "including", "having", "characterized by", or "containing" and their like are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

[0044] Compositions and methods of using them can "comprise", "consist essentially of", or "consist of" any of the ingredients or steps disclosed throughout this specification. The phrase "consist of" does not include any unspecified elements, steps, or ingredients. The phrase "consist essentially of" limits the scope of the subject matter to the specific materials or steps, and those materials or steps that do not materially affect its essential and novel characteristics. It is contemplated that the embodiments and aspects described in the context of the term "comprising / including" can also be implemented in the context of the term "consist of" or "consist essentially of".

[0045] Specifically contemplated is that any limitation discussed with respect to one embodiment or aspect of the present invention can be applied to any other embodiment or aspect of the present invention. Additionally, any composition of the present invention can be used in any method of the present invention, and any method of the present invention can be used to produce or use any composition of the present invention. Aspects of the embodiments shown in the examples are also embodiments that can be implemented in the context of the embodiments or aspects discussed elsewhere in this application, such as in the invention overview, the detailed description of the invention, the specific implementation manner, the claims, and the description of the drawings.

[0046] Other objects, features, and advantages of the present invention will become apparent from the following detailed description. However, it should be understood that although the detailed description and specific examples illustrate specific embodiments or specific aspects of the present invention, they are given by way of illustration only, because various changes and modifications within the spirit and scope of the present invention will become apparent to those skilled in the art in light of this detailed description. Description of the Drawings

[0047] The following drawings form a part of this specification and are included to further illustrate certain aspects of the present invention. The present invention can be better understood by referring to one or more of these drawings and in conjunction with the detailed description of the specific embodiments provided herein.

[0048] Figure 1 , the secretion of recombinant proteins expressed by CHO cells. Four particularly effective sites (K22A, R194A, R73A, and R196A) were selected.

[0049] Figure 2 , combinatorial studies of the mutation sites.

[0050] Figure 3 , the formation of mutant aggregates cultured under conditions of increased antioxidant stress tolerance was significantly reduced, and the secretion of IL-35-HSA was significantly increased.

[0051] Figure 4 , optimization of the linker sequence. We constructed a variety of IL-35-HSA molecules that have different linker domains between IL-35 and HSA. Linker ID: 1 to Linker ID: 12 are shown in detail in Table 6. After protein production, we performed Western blot analysis to determine the presence of IL35-HSA protein in the culture medium.

[0052] Figure 5, an expression vector of a mutant in which four cysteines in EBI3 are replaced with serine was produced by CHO cells. IL-35 was detected by Western blot analysis of the culture medium, and ImageJ software was used to perform image analysis on the obtained signal intensity to evaluate the amount of secreted protein. The relative amount of secreted protein was calculated with IL-35-HSA / WT as 1. After replacing cysteine with serine at four positions of EBI3, the secretion of IL-35-HSA increased slightly.

[0053] Figure 6 , it was observed that IL-35-HSA underwent polymerization during culture medium and purification processes. To monomerize the polymerized IL-35-HSA, additives were investigated. The culture supernatant of CHO cells stably expressing IL-35-HSA was concentrated through an ultrafiltration membrane and replaced with 30 mM phosphate buffer (pH 7) and 50 mM Tris buffer (pH 9) respectively. After equal aliquots were dispensed into each tube, additives were added respectively. The additives used were ascorbic acid (final 1 mM), methionine (final 1 mM), glutathione (final 1 mM), and cysteine (final 1 mM, 10 mM, 50 mM respectively). Then the samples were incubated at 37 °C for 15 minutes. Then each secretion profile of IL-35-HSA was analyzed by Western blot.

[0054] Figure 7 , analyzed by SDS-PAGE, HEK239-derived IL-35-HSA could become monomeric after incubation with 5 mM DTT for 15 minutes.

[0055] Figure 8 , the inventors prepared N-terminal deletion variants of IL-35-HSA and analyzed them by SDS-PAGE (10 amino acid deletion: delta10 and 20 amino acid deletion: delta20). The culture supernatant of HEK293 cells transfected with His6-tagged IL-35-HSA was purified using a His-tag purification Ni2+ column, and then further purified using a size exclusion column in PBS. The non-reducing conditions of SDS-PAGE showed that deletion of 20 amino acids rather than 10 amino acids could resolve the over-large aggregation. ( Figure 8 )

[0056] Figure 9 , 40 μg of IL-35-HSA was subcutaneously injected into the back skin of mice. At 4 hours and 8 days after injection, the inventors collected lymph nodes. After homogenizing the lymph nodes, they used an ELISA kit to detect human EBI3 protein to detect the presence of human IL-35. The signal at 450 nm showed that IL-35-HSA was detectable and persisted in the lymph nodes for a long time.

[0057] Figure 10A to 1 0D, (A) On day 0 and day 7, C57B6 mice were subcutaneously injected twice with 10 μg of IL-35-HSA protein. On day 9, 2.5 μg of TNF-α (TNFa) was intraperitoneally injected into the mice. Two hours after the injection of TNFa, the inventors collected plasma from the mice. IL-6 in the plasma was detected by ELISA. (B-D) On day 0 and day 7, C57B6 mice were subcutaneously injected twice with 10 μg of IL-35 protein. On day 7, the inventors collected lymph nodes from the mice and analyzed the immune cell population using flow cytometry.

[0058] Figures 11A to 11C: Evaluation of prophylactic treatment with SA IL-35 in collagen antibody-induced arthritis. (A) Timeline of the collagen antibody-induced arthritis experiment. (B) Results of the arthritis clinical score from day 3 to day 12. (C) Arthritis clinical score at the endpoint. Treatment with mutant SA IL-35 prevented the onset of severe disease.

[0059] Figure 12A to 12F , (A) T cell immunophenotype in the popliteal lymph nodes. Treatment with mutant SA IL-35 increased the expression of PD-1 in CD4 + T cells and CD8 + T cells. (B) T cell immunophenotype in the spleen. Treatment with mutant SA IL-35 increased the expression of PD-1 in CD4 + T cells. (C) Myeloid cell immunophenotype in the popliteal lymph nodes. Treatment with mutant SA IL-35 decreased the expression of CD40 and CD86 on CD11b + myeloid cells. The treatment also decreased the percentage of CD11b + F4 / 80 + macrophages and the expression of CD86 on macrophages, while increasing the ratio of M2 macrophages to M1 macrophages. (D) Myeloid cell immunophenotype in the spleen. Treatment with mutant SA IL-35 decreased the percentage of CD11b+ myeloid cells in the spleen and the expression of CD86 on CD11b+ cells. The treatment also decreased the percentage of CD11b + F4 / 80 + macrophages, while increasing the percentage of Arg1 + M2 macrophages. (E) Dendritic cell immunophenotype in the lymph nodes. Treatment with mutant SA IL-35 decreased the percentage of CD11c + dendritic cells and the expression of CD86 and CD40 on CD11c + dendritic cells. The treatment also decreased the expression of CD86 and CD40 on CD11c + CD11b+ The percentage of dendritic cells and the expression of CD86, CD40, and MHC class II on these cells. (F) Immunophenotype of myeloid-derived suppressor cells in lymph nodes. Treatment with mutant SA IL-35 reduced CD11b + Ly6C hi Ly6G - The expression of CD86 and CD40 on MDSCs. Detailed implementation manners

[0060] I. ECM Affinity Peptides

[0061] Collagen is an extracellular matrix (ECM) protein responsible for regulating multiple cell biological functions in normal and tumor tissues, such as proliferation, differentiation, and adhesion (Ricard-Blum, Cold Spring Harb Perspect Biol 3:a004978, 2011). Collagen is the most abundant protein in mammals and exists in almost all tissues in one or more of 28 isoforms (Ricard-Blum, Cold Spring Harb Perspect Biol 3:a004978, 2011). The subendothelial space of blood vessels is rich in collagen. Due to its insolubility under physiological conditions, collagen is almost absent in 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 blood vessels are permeable due to structural abnormalities (Nagy et al., British journal of cancer 100:865, 2009). Therefore, due to their leaky blood vessels, collagen is exposed in tumors (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). In addition, compared with normal tissues, tumor tissues contain more collagen (Zhou et al., J Cancer 8:1466-76, 2017; Provenzano et al., BMC Med 6:11, 2008).

[0062] Von Willebrand factor (vWF) is a blood coagulation factor that binds to type I and type III collagens as well as the adhesion receptor GPIb on platelets (Lenting et al., Journal of thrombosis and haemostasis: JTH 10:2428-37, 2012; Shahidi Advances in experimental medicine and biology 906:285-306, 2017). When endothelial cells are damaged, the underlying collagen is exposed to plasma, and the binding of vWF to collagen initiates the thrombin formation cascade (Shahidi Advances in experimental medicine and biology 906:285-306, 2017; Wu et al., Blood 99:3623-28, 2002). Among the reported non-bacterial-derived proteins / peptides, the A domain of vWF has the highest affinity for collagen (Addi et al., Tissue Engineering Part B: Reviews, 2016). In particular, within the A domain, the A3 domain of vWF has been reported as the collagen-binding domain (CBD) (Ribba et al., Thrombosis and Haemostasis 86:848-54, 2001). As described above, the inventors envisioned that since collagen would be exposed through the leaky tumor vasculature system, targeted cytokine immunotherapy could be achieved even with systemic injection of a fusion protein containing vWF A3CBD.

[0063] The ECM affinity peptide may comprise a collagen-binding domain from decorin. The ECM affinity peptide may comprise a decorin peptide such as LRELHLNNNC (SEQ ID NO: 9) derived from bovine or LRELHLDNNC (SEQ ID NO: 10) derived from human.

[0064] The ECM peptide may comprise a peptide segment from human decorin, the amino acid sequence of which is shown as:

[0065]

[0066] The ECM peptide may comprise a peptide segment from vWF. The ECM peptide may comprise vWF A1, residues 1237 to 1458 (474 to 695 of mature VWF) or a fragment thereof derived from the human sequence, the amino acid sequence of which is shown as:

[0067]

[0068] The ECM peptide may comprise all of vWF A3 or a fragment of vWF A3, the amino acid sequence of which is shown as:

[0069]

[0070] The ECM peptide may comprise all of vWF A3 or a fragment of vWF A3, the amino acid sequence of which is shown as:

[0071]

[0072] The ECM affinity peptide may be a peptide from von Willebrand factor (vWF). The sequence of human vWF includes the following:

[0073]

[0074]

[0075] The peptide may be from the vWF A3 domain. The vWF A3 domain is derived from the human sequence, i.e., residues 1670 to residue 1874 (residues 907 to residue 1111 of mature vWF), and has the following sequence:

[0076]

[0077] The ECM affinity peptide may comprise a peptide from PlGF-2. PlGF-2 has the following sequence:

[0078]

[0079] Exemplary PlGF-2 ECM affinity peptides include:

[0080]

[0081] The ECM affinity peptide may be a peptide from CXCL-12γ. The sequence of CXCL-12γ is as follows: CXCL-12γ:

[0082] KPVSLSYRCPCRFFESHVARANVKHLKILNTPNCALQIVARLKNNNRQVCIDPKLKWIQEYLEKALNKGRREEKVGKKEKIGKKKRQKKRKAAQKRKN (SEQ ID NO:26). Exemplary peptides include all or part of SEQ ID NO:12, and the following peptide: GRREEKVGKKEKIGKKKRQKKRKAAQKRKN (SEQ ID NO:27).

[0083] The ECM affinity peptide can be a peptide having 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% (or any range derivable therefrom) identity to the ECM or CBD peptide or peptide fragment described above.

[0084] The linker sequence can be included in the anti-inflammatory agent-peptide construct. For example, a linker having at least, at most, or exactly 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 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 than 100 amino acids (or any range derivable therefrom) can separate the antibody from the peptide.

[0085] The ECM affinity peptides of the present disclosure can have an affinity for one or more components of the extracellular matrix, such as fibronectin, collagen (type I collagen, type III collagen, and / or type IV collagen), tenascin C, fibrinogen, and fibrin. The ECM affinity peptide can have an affinity for collagen. The ECM affinity peptide can be a peptide that does not bind fibronectin.

[0086] II. Protein Component

[0087] The polypeptide or polynucleotide of the present disclosure may comprise, may at least comprise, or may at most comprise 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 amino acid variations or nucleic acid substitutions, or 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% identical to 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, 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 of SEQ ID NOs: 1 to SEQ ID NO: 27,137, 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, or any continuous range of amino acids or nucleic acids derivable therefrom that are similar, identical or homologous.,

[0088] The polypeptide or polynucleotide of the present disclosure may comprise, may at least comprise, or may at most comprise 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 of SEQ ID NO: 1 to SEQ ID NO: 27214, 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, or any continuous range of amino acids or nucleic acids derivable therefrom.,

[0089] The polypeptides of the present disclosure may comprise 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

[0090] to 172, 173, 174, 175, 176, 177, 178, 179, 180

[0091] to 181, 182, 183, 184, 185, 186, 187, 188, 189

[0092] to 190, 191, 192, 193, 194, 195, 196, 197, 198

[0093] pieces, 199 pieces, 200 pieces, 201 pieces, 202 pieces, 203 pieces, 204 pieces, 205 pieces, 206 pieces, 207

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[0118] pieces, 424, 425, 426, 427, 428, 429, 430, 431, 432

[0119] pieces, 433, 434, 435, 436, 437, 438, 439, 440, 441

[0120] from 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 (or any range derivable therefrom) amino acids.

[0121] The polypeptides of the present disclosure may comprise, consist essentially of, or consist of 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 of SEQ ID NO: 1 to SEQ ID NO: 27214, 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, 413414, 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, 613614 or 615 (or any range derivable therefrom) consecutive amino acids.

[0122] The polypeptides of the present disclosure may comprise, consist essentially of, or consist of 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 of SEQ ID NO: 1 to SEQ ID NO: 27.213, 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, 412413, 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, 612613, 614, or 615 (or any range derivable therefrom) consecutive amino acids that are at least, at most, or exactly 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% similar, identical, or homologous to any one of the sequences of SEQ ID NO: 1 to SEQ ID NO: 27.

[0123] The polypeptides described herein may have, have at least, or have at most 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% (or any range derivable therefrom) sequence identity or homology to any one of the sequences of SEQ ID NO: 1 to SEQ ID NO: 27.

[0124] This disclosure includes starting from position 1, position 2, position 3, position 4, position 5, position 6, position 7, position 8, position 9, position 10, position 11, position 12, position 13, position 14, position 15, position 16, position 17, position 18, position 19, position 20, position 21, position 22, position 23, position 24, position 25, position 26, position 27, position 28, position 29, position 30, position 31, position 32, position 33, position 34, position 35, position 36, position 37, position 38, position 39, position 40, position 41, position 42, position 43, position 44, position 45, position 46, position 47, position 48, position 49, position 50, position 51, position 52, position 53, position 54, position 55, position 56, position 57, position 58, position 59, position 60, position 61, position 62, position 63, position 64, position 65, position 66, position 67, position 68, position 69, position 70, position 71, position 72, position 73, position 74, position 75, position 76, position 77, position 78, position 79, position 80, position 81, position 82, position 83, position 84, position 85, position 86, position 87, position 88, position 89, position 90, position 91, position 92, position 93, position 94, position 95, position 96, position 97, position 98, position 99, position 100, position 101, position 102, position 103, position 104, position 105, position 106, position 107, position 108, position 109, position 110, position 111, position 112, position 113, position 114, position 115, position 116, position 117, position 118, position 119, position 120, position 121, position 122, position 123, position 124, position 125, position 126, position 127, position 128, position 129, position 130, position 131, position 132, position 133, position 134, position 135, position 136, position 137, position 138, position 139, position 140, position 141, position 142, position 143, position 144, position 145, position 146, position 147, position 148, position 149, position 150, position 151, position 152, position 153, position 154, position 155, position 156, position 157, position 158, position 159, position 160, position 161, position 162, position 163, position 164, position 165, position 166, position 167, position 168, position 169, position 170, position 171, position 172, position 173, position 174, position 175, position 176, position 177, position 178 of any one of the sequences from SEQ ID NO: 1 to SEQ ID NOS: 27Position 179, Position 180, Position 181, Position 182, Position 183, Position 184, Position 185, Position 186, Position 187, Position 188, Position 189, Position 190, Position 191, Position 192, Position 193, Position 194, Position 195, Position 196, Position 197, Position 198, Position 199, Position 200, Position 201, Position 202, Position 203, Position 204, Position 205, Position 206, Position 207, Position 208, Position 209, Position 210, Position 211, Position 212, Position 213, Position 214, Position 215, Position 216, Position 217, Position 218, Position 219, Position 220, Position 221, Position 222, Position 223, Position 224, Position 225, Position 226, Position 227, Position 228, Position 229, Position 230, Position 231, Position 232, Position 233, Position 234, Position 235, Position 236, Position 237, Position 238, Position 239, Position 240, Position 241, Position 242, Position 243, Position 244, Position 245, Position 246, Position 247, Position 248, Position 249, Position 250, Position 251, Position 252, Position 253, Position 254, Position 255, Position 256, Position 257, Position 258, Position 259, Position 260, Position 261, Position 262, Position 263, Position 264, Position 265, Position 266, Position 267, Position 268, Position 269, Position 270, Position 271, Position 272, Position 273, Position 274, Position 275, Position 276, Position 277, Position 278, Position 279, Position 280, Position 281, Position 282, Position 283, Position 284, Position 285, Position 286, Position 287, Position 288, Position 289, Position 290, Position 291, Position 292, Position 293, Position 294, Position 295, Position 296, Position 297, Position 298, Position 299, Position 300, Position 301, Position 302, Position 303, Position 304, Position 305, Position 306, Position 307, Position 308, Position 309, Position 310, Position 311, Position 312, Position 313, Position 314, Position 315, Position 316, Position 317, Position 318, Position 319, Position 320, Position 321, Position 322, Position 323, Position 324, Position 325, Position 326, Position 327, Position 328, Position 329, Position 330, Position 331, Position 332, Position 333, Position 334, Position 335, Position 336, Position 337, Position 338, Position 339, Position 340, Position 341, Position 342, Position 343, Position 344Position 345, Position 346, Position 347, Position 348, Position 349, Position 350, Position 351, Position 352, Position 353, Position 354, Position 355, Position 356, Position 357, Position 358, Position 359, Position 360, Position 361, Position 362, Position 363, Position 364, Position 365, Position 366, Position 367, Position 368, Position 369, Position 370, Position 371, Position 372, Position 373, Position 374, Position 375, Position 376, Position 377, Position 378, Position 379, Position 380, Position 381, Position 382, Position 383, Position 384, Position 385, Position 386, Position 387, Position 388, Position 389, Position 390, Position 391, Position 392, Position 393, Position 394, Position 395, Position 396, Position 397, Position 398, Position 399, Position 400, Position 401, Position 402, Position 403, Position 404, Position 405, Position 406, Position 407, Position 408, Position 409, Position 410, Position 411, Position 412, Position 413, Position 414, Position 415, Position 416, Position 417, Position 418, Position 419, Position 420, Position 421, Position 422, Position 423, Position 424, Position 425, Position 426, Position 427, Position 428, Position 429, Position 430, Position 431, Position 432, Position 433, Position 434, Position 435, Position 436, Position 437, Position 438, Position 439, Position 440, Position 441, Position 442, Position 443, Position 444, Position 445, Position 446, Position 447, Position 448, Position 449, Position 450, Position 451, Position 452, Position 453, Position 454, Position 455, Position 456, Position 457, Position 458, Position 459, Position 460, Position 461, Position 462, Position 463, Position 464, Position 465, Position 466, Position 467, Position 468, Position 469, Position 470, Position 471, Position 472, Position 473, Position 474, Position 475, Position 476, Position 477, Position 478, Position 479, Position 480, Position 481, Position 482, Position 483, Position 484, Position 485, Position 486, Position 487, Position 488, Position 489, Position 490, Position 491, Position 492, Position 493, Position 494, Position 495, Position 496, Position 497, Position 498, Position 499, Position 500, Position 501, Position 502, Position 503, Position 504, Position 505, Position 506, Position 507, Position 508, Position 509, Position 510A nucleic acid molecule or polypeptide at position 511, position 512, position 513, position 514, position 515, position 516, position 517, position 518, position 519, position 520, position 521, position 522, position 523, position 524, position 525, position 526, position 527, position 528, position 529, position 530, position 531, position 532, position 533, position 534, position 535, position 536, position 537, position 538, position 539, position 540, position 541, position 542, position 543, position 544, position 545, position 546, position 547, position 548, position 549, position 550, position 551, position 552, position 553, position 554, position 555, position 556, position 557, position 558, position 559, position 560, position 561, position 562, position 563, position 564, position 565, position 566, position 567, position 568, position 569, position 570, position 571, position 572, position 573, position 574, position 575, position 576, position 577, position 578, position 579, position 580, position 581, position 582, position 583, position 584, position 585, position 586, position 587, position 588, position 589, position 590, position 591, position 592, position 593, position 594, position 595, position 596, position 597, position 598, position 599, position 600, position 601, position 602, position 603, position 604, position 605, position 606, position 607, position 608, position 609, position 610, position 611, position 612, position 613, position 614 or position 615, and comprising 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 of any one of the sequences from SEQ ID NO: 1 to SEQ ID NOS: 2785, 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, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287288, 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, 487488, 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 consecutive nucleotides or amino acids.

[0125] The polypeptides and nucleic acids of the present disclosure may comprise, may at least comprise, or may at most comprise 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, 216217, 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, 416417, 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, 613614 or 615 (or any range derivable therefrom) permutations.

[0126] The substitution can be at the amino acid position or nucleic acid position 1, position 2, position 3, position 4, position 5, position 6, position 7, position 8, position 9, position 10, position 11, position 12, position 13, position 14, position 15, position 16, position 17, position 18, position 19, position 20, position 21, position 22, position 23, position 24, position 25, position 26, position 27, position 28, position 29, position 30, position 31, position 32, position 33, position 34, position 35, position 36, position 37, position 38, position 39, position 40, position 41, position 42, position 43, position 44, position 45, position 46, position 47, position 48, position 49, position 50, position 51, position 52, position 53, position 54, position 55, position 56, position 57, position 58, position 59, position 60, position 61, position 62, position 63, position 64, position 65, position 66, position 67, position 68, position 69, position 70, position 71, position 72, position 73, position 74, position 75, position 76, position 77, position 78, position 79, position 80, position 81, position 82, position 83, position 84, position 85, position 86, position 87, position 88, position 89, position 90, position 91, position 92, position 93, position 94, position 95, position 96, position 97, position 98, position 99, position 100, position 101, position 102, position 103, position 104, position 105, position 106, position 107, position 108, position 109, position 110, position 111, position 112, position 113, position 114, position 115, position 116, position 117, position 118, position 119, position 120, position 121, position 122, position 123, position 124, position 125, position 126, position 127, position 128, position 129, position 130, position 131, position 132, position 133, position 134, position 135, position 136, position 137, position 138, position 139, position 140, position 141, position 142, position 143, position 144, position 145, position 146, position 147, position 148, position 149, position 150, position 151, position 152, position 153, position 154, position 155, position 156, position 157, position 158, position 159, position 160, position 161, position 162, position 163, position 164, position 165, position 166, position 167, position 168, position 169, position 170, position 171, position 172, position 173, position 174, position 175, position 176, position 177 of any one of the sequences from SEQ ID NO: 1 to SEQ ID NO: 27.Position 178, Position 179, Position 180, Position 181, Position 182, Position 183, Position 184, Position 185, Position 186, Position 187, Position 188, Position 189, Position 190, Position 191, Position 192, Position 193, Position 194, Position 195, Position 196, Position 197, Position 198, Position 199, Position 200, Position 201, Position 202, Position 203, Position 204, Position 205, Position 206, Position 207, Position 208, Position 209, Position 210, Position 211, Position 212, Position 213, Position 214, Position 215, Position 216, Position 217, Position 218, Position 219, Position 220, Position 221, Position 222, Position 223, Position 224, Position 225, Position 226, Position 227, Position 228, Position 229, Position 230, Position 231, Position 232, Position 233, Position 234, Position 235, Position 236, Position 237, Position 238, Position 239, Position 240, Position 241, Position 242, Position 243, Position 244, Position 245, Position 246, Position 247, Position 248, Position 249, Position 250, Position 251, Position 252, Position 253, Position 254, Position 255, Position 256, Position 257, Position 258, Position 259, Position 260, Position 261, Position 262, Position 263, Position 264, Position 265, Position 266, Position 267, Position 268, Position 269, Position 270, Position 271, Position 272, Position 273, Position 274, Position 275, Position 276, Position 277, Position 278, Position 279, Position 280, Position 281, Position 282, Position 283, Position 284, Position 285, Position 286, Position 287, Position 288, Position 289, Position 290, Position 291, Position 292, Position 293, Position 294, Position 295, Position 296, Position 297, Position 298, Position 299, Position 300, Position 301, Position 302, Position 303, Position 304, Position 305, Position 306, Position 307, Position 308, Position 309, Position 310, Position 311, Position 312, Position 313, Position 314, Position 315, Position 316, Position 317, Position 318, Position 319, Position 320, Position 321, Position 322, Position 323, Position 324, Position 325, Position 326, Position 327, Position 328, Position 329, Position 330, Position 331, Position 332, Position 333, Position 334, Position 335, Position 336, Position 337, Position 338, Position 339, Position 340, Position 341, Position 342, Position 343Position 344, Position 345, Position 346, Position 347, Position 348, Position 349, Position 350, Position 351, Position 352, Position 353, Position 354, Position 355, Position 356, Position 357, Position 358, Position 359, Position 360, Position 361, Position 362, Position 363, Position 364, Position 365, Position 366, Position 367, Position 368, Position 369, Position 370, Position 371, Position 372, Position 373, Position 374, Position 375, Position 376, Position 377, Position 378, Position 379, Position 380, Position 381, Position 382, Position 383, Position 384, Position 385, Position 386, Position 387, Position 388, Position 389, Position 390, Position 391, Position 392, Position 393, Position 394, Position 395, Position 396, Position 397, Position 398, Position 399, Position 400, Position 401, Position 402, Position 403, Position 404, Position 405, Position 406, Position 407, Position 408, Position 409, Position 410, Position 411, Position 412, Position 413, Position 414, Position 415, Position 416, Position 417, Position 418, Position 419, Position 420, Position 421, Position 422, Position 423, Position 424, Position 425, Position 426, Position 427, Position 428, Position 429, Position 430, Position 431, Position 432, Position 433, Position 434, Position 435, Position 436, Position 437, Position 438, Position 439, Position 440, Position 441, Position 442, Position 443, Position 444, Position 445, Position 446, Position 447, Position 448, Position 449, Position 450, Position 451, Position 452, Position 453, Position 454, Position 455, Position 456, Position 457, Position 458, Position 459, Position 460, Position 461, Position 462, Position 463, Position 464, Position 465, Position 466, Position 467, Position 468, Position 469, Position 470, Position 471, Position 472, Position 473, Position 474, Position 475, Position 476, Position 477, Position 478, Position 479, Position 480, Position 481, Position 482, Position 483, Position 484, Position 485, Position 486, Position 487, Position 488, Position 489, Position 490, Position 491, Position 492, Position 493, Position 494, Position 495, Position 496, Position 497, Position 498, Position 499, Position 500, Position 501, Position 502, Position 503, Position 504, Position 505, Position 506, Position 507, Position 508, Position 509Position 510, Position 511, Position 512, Position 513, Position 514, Position 515, Position 516, Position 517, Position 518, Position 519, Position 520, Position 521, Position 522, Position 523, Position 524, Position 525, Position 526, Position 527, Position 528, Position 529, Position 530, Position 531, Position 532, Position 533, Position 534, Position 535, Position 536, Position 537, Position 538, Position 539, Position 540, Position 541, Position 542, Position 543, Position 544, Position 545, Position 546, Position 547, Position 548, Position 549, Position 550, Position 551, Position 552, Position 553, Position 554, Position 555, Position 556, Position 557, Position 558, Position 559, Position 560, Position 561, Position 562, Position 563, Position 564, Position 565, Position 566, Position 567, Position 568, Position 569, Position 570, Position 571, Position 572, Position 573, Position 574, Position 575, Position 576, Position 577, Position 578, Position 579, Position 580, Position 581, Position 582, Position 583, Position 584, Position 585, Position 586, Position 587, Position 588, Position 589, Position 590, Position 591, Position 592, Position 593, Position 594, Position 595, Position 596, Position 597, Position 598, Position 599, Position 600, Position 601, Position 602, Position 603, Position 604, Position 605, Position 606, Position 607, Position 608, Position 609, Position 610, Position 611, Position 612, Position 613, Position 614 or Position 615. One or more than one of these substitutions may be specifically excluded in some respects.

[0127] At position 1, position 2, position 3, position 4, position 5, position 6, position 7, position 8, position 9, position 10, position 11, position 12, position 13, position 14, position 15, position 16, position 17, position 18, position 19, position 20, position 21, position 22, position 23, position 24, position 25, position 26, position 27, position 28, position 29, position 30, position 31, position 32, position 33, position 34, position 35, position 36, position 37, position 38, position 39, position 40, position 41, position 42, position 43, position 44, position 45, position 46, position 47, position 48, position 49, position 50, position 51, position 52, position 53, position 54, position 55, position 56, position 57, position 58, position 59, position 60, position 61, position 62, position 63, position 64, position 65, position 66, position 67, position 68, position 69, position 70, position 71, position 72, position 73, position 74, position 75, position 76, position 77, position 78, position 79, position 80, position 81, position 82, position 83, position 84, position 85, position 86, position 87, position 88, position 89, position 90, position 91, position 92, position 93, position 94, position 95, position 96, position 97, position 98, position 99, position 100, position 101, position 102, position 103, position 104, position 105, position 106, position 107, position 108, position 109, position 110, position 111, position 112, position 113, position 114, position 115, position 116, position 117, position 118, position 119, position 120, position 121, position 122, position 123, position 124, position 125, position 126, position 127, position 128, position 129, position 130, position 131, position 132, position 133, position 134, position 135, position 136, position 137, position 138, position 139, position 140, position 141, position 142, position 143, position 144, position 145, position 146, position 147, position 148, position 149, position 150, position 151, position 152, position 153, position 154, position 155, position 156, position 157, position 158, position 159, position 160, position 161, position 162, position 163, position 164, position 165, position 166, position 167, position 168, position 169, position 170, position 171, position 172, position 173, position 174, position 175, position 176, position 177, position 178, position 179 of any peptide or polypeptide in SEQ ID NO: 1 to SEQ ID NO: 27Position 180, Position 181, Position 182, Position 183, Position 184, Position 185, Position 186, Position 187, Position 188, Position 189, Position 190, Position 191, Position 192, Position 193, Position 194, Position 195, Position 196, Position 197, Position 198, Position 199, Position 200, Position 201, Position 202, Position 203, Position 204, Position 205, Position 206, Position 207, Position 208, Position 209, Position 210, Position 211, Position 212, Position 213, Position 214, Position 215, Position 216, Position 217, Position 218, Position 219, Position 220, Position 221, Position 222, Position 223, Position 224, Position 225, Position 226, Position 227, Position 228, Position 229, Position 230, Position 231, Position 232, Position 233, Position 234, Position 235, Position 236, Position 237, Position 238, Position 239, Position 240, Position 241, Position 242, Position 243, Position 244, Position 245, Position 246, Position 247, Position 248, Position 249, Position 250, Position 251, Position 252, Position 253, Position 254, Position 255, Position 256, Position 257, Position 258, Position 259, Position 260, Position 261, Position 262, Position 263, Position 264, Position 265, Position 266, Position 267, Position 268, Position 269, Position 270, Position 271, Position 272, Position 273, Position 274, Position 275, Position 276, Position 277, Position 278, Position 279, Position 280, Position 281, Position 282, Position 283, Position 284, Position 285, Position 286, Position 287, Position 288, Position 289, Position 290, Position 291, Position 292, Position 293, Position 294, Position 295, Position 296, Position 297, Position 298, Position 299, Position 300, Position 301, Position 302, Position 303, Position 304, Position 305, Position 306, Position 307, Position 308, Position 309, Position 310, Position 311, Position 312, Position 313, Position 314, Position 315, Position 316, Position 317, Position 318, Position 319, Position 320, Position 321, Position 322, Position 323, Position 324, Position 325, Position 326, Position 327, Position 328, Position 329, Position 330, Position 331, Position 332, Position 333, Position 334, Position 335, Position 336, Position 337, Position 338, Position 339, Position 340, Position 341, Position 342, Position 343, Position 344, Position 345The amino acid at position 346, position 347, position 348, position 349, position 350, position 351, position 352, position 353, position 354, position 355, position 356, position 357, position 358, position 359, position 360, position 361, position 362, position 363, position 364, position 365, position 366, position 367, position 368, position 369, position 370, position 371, position 372, position 373, position 374, position 375, position 376, position 377, position 378, position 379, position 380, position 381, position 382, position 383, position 384, position 385, position 386, position 387, position 388, position 389, position 390, position 391, position 392, position 393, position 394, position 395, position 396, position 397, position 398, position 399 or position 400 may be replaced with alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine or valine.

[0128] The peptides, polypeptides, and proteins of the present disclosure may comprise, may at least comprise, or may at most have 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 with any one of the sequences of SEQ ID NO: 1 to SEQ ID NO: 27, and may comprise from amino acid position 1, position 2, position 3, position 4, position 5, position 6, position 7, position 8, position 9, position 10, position 11, position 12, position 13, position 14, position 15, position 16, position 17, position 18, position 19, position 20, position 21, position 22, position 23, position 24, position 25, position 26, position 27, position 28, position 29, position 30, position 31, position 32, position 33, position 34, position 35, position 36, position 37, position 38, position 39, position 40, position 41, position 42, position 43, position 44, position 45, position 46, position 47, position 48, position 49, position 50, position 51, position 52, position 53, position 54, position 55, position 56, position 57, position 58, position 59, position 60, position 61, position 62, position 63, position 64, position 65, position 66, position 67, position 68, position 69, position 70, position 71, position 72, position 73, position 74, position 75, position 76, position 77, position 78, position 79, position 80, position 81, position 82, position 83, position 84, position 85, position 86, position 87, position 88, position 89, position 90, position 91, position 92, position 93, position 94, position 95, position 96, position 97, position 98, position 99, position 100, position 101, position 102, position 103, position 104, position 105, position 106, position 107, position 108, position 109, position 110, position 111, position 112, position 113, position 114, position 115, position 116, position 117, position 118, position 119, position 120, position 121, position 122, position 123, position 124, position 125, position 126, position 127, position 128, position 129, position 130, position 131, position 132, position 133, position 134, position 135, position 136, position 137, position 138, position 139, position 140, position 141, position 142, position 143, position 144, position 145, position 146, position 147, position 148, position 149, position 150, position 151starting from position 152, position 153, position 154, position 155, position 156, position 157, position 158, position 159, position 160, position 161, position 162, position 163, position 164, position 165, position 166, position 167, position 168, position 169, position 170, position 171, position 172, position 173, position 174, position 175, position 176, position 177, position 178, position 179, position 180, position 181, position 182, position 183, position 184, position 185, position 186, position 187, position 188, position 189, position 190, position 191, position 192, position 193, position 194, position 195, position 196, position 197, position 198, position 199 or position 200 (or any range derivable therefrom), and ending at amino acid position 10, position 11, position 12, position 13, position 14, position 15, position 16, position 17, position 18, position 19, position 20, position 21, position 22, position 23, position 24, position 25, position 26, position 27, position 28, position 29, position 30, position 31, position 32, position 33, position 34, position 35, position 36, position 37, position 38, position 39, position 40, position 41, position 42, position 43, position 44, position 45, position 46, position 47, position 48, position 49, position 50, position 51, position 52, position 53, position 54, position 55, position 56, position 57, position 58, position 59, position 60, position 61, position 62, position 63, position 64, position 65, position 66, position 67, position 68, position 69, position 70, position 71, position 72, position 73, position 74, position 75, position 76, position 77, position 78, position 79, position 80, position 81, position 82, position 83, position 84, position 85, position 86, position 87, position 88, position 89, position 90, position 91, position 92, position 93, position 94, position 95, position 96, position 97, position 98, position 99, position 100, position 101, position 102, position 103, position 104, position 105, position 106, position 107, position 108, position 109, position 110, position 111, position 112, position 113, position 114, position 115, position 116, position 117, position 118, position 119, position 120, position 121, position 122, position 123, position 124, position 125, position 126, position 127, position 128, position 129, position 130, position 131, position 132, position 133, position 134, position 135, position 136, position 137, position 138A segment or section that ends at position 139, position 140, position 141, position 142, position 143, position 144, position 145, position 146, position 147, position 148, position 149, position 150, position 151, position 152, position 153, position 154, position 155, position 156, position 157, position 158, position 159, position 160, position 161, position 162, position 163, position 164, position 165, position 166, position 167, position 168, position 169, position 170, position 171, position 172, position 173, position 174, position 175, position 176, position 177, position 178, position 179, position 180, position 181, position 182, position 183, position 184, position 185, position 186, position 187, position 188, position 189, position 190, position 191, position 192, position 193, position 194, position 195, position 196, position 197, position 198, position 199, position 200, position 201, position 202, position 203, position 204 or position 205 (or any range derivable therefrom).

[0129] Substitution variants generally involve substituting one amino acid for another at one or more sites of a protein and can be designed to modulate one or more properties of a polypeptide, either with or without loss of other functions or properties. The substitution can be conservative, i.e., one amino acid is replaced by an amino acid having a similar shape and charge. Conservative substitutions are well known in the art and include, for example, the following changes: alanine is substituted for serine; arginine is substituted for lysine; asparagine is substituted for glutamine or histidine; aspartic acid is substituted for glutamic acid; cysteine is substituted for serine; glutamine is substituted for asparagine; glutamic acid is substituted for aspartic acid; glycine is substituted for proline; histidine is substituted for asparagine or glutamine; isoleucine is substituted for leucine or valine; leucine is substituted for valine or isoleucine; lysine is substituted for arginine; methionine is substituted for leucine or isoleucine; phenylalanine is substituted for tyrosine, leucine or methionine; serine is substituted for threonine; threonine is substituted for serine; tryptophan is substituted for tyrosine; tyrosine is substituted for tryptophan or phenylalanine; and valine is substituted for isoleucine or leucine. Alternatively, the substitution can be non-conservative, thereby affecting the function or activity of the polypeptide. Non-conservative changes generally involve substituting with a residue having a different chemical nature, such as substituting a non-polar or uncharged amino acid with a polar or charged amino acid, and vice versa. One or more of these substitutions can be specifically excluded in some respects.

[0130] The protein can be recombinant or synthesized in vitro. Alternatively, non-recombinant or recombinant proteins can be isolated from bacteria. It is also contemplated that bacteria containing such mutations can be utilized in the compositions and methods. Thus, the protein need not be isolated.

[0131] As used herein, the term "functionally equivalent codon" refers to codons that encode the same amino acid, such as the six codons for arginine or serine, and also refers to codons that encode biologically equivalent amino acids.

[0132] It should also be understood that amino acid and nucleic acid sequences can contain additional residues, such as additional N-terminal or C-terminal amino acids, or additional 5' or 3' sequences, and still be substantially identical to one of the sequences disclosed herein so long as the sequence meets the criteria described above including maintaining biological protein activity in terms of protein expression. Adding terminal sequences is particularly applicable to nucleic acid sequences that may include various non-coding sequences, for example, flanking the 5' or 3' portions of the coding region.

[0133] The following discussion is based on altering the amino acids of a protein to create equivalent or even improved second-generation molecules. For example, in the protein structure, certain amino acids can be replaced by other amino acids without significantly reducing the interaction binding ability. Structures such as enzyme catalytic domains or interaction components can maintain their function through amino acid substitution. Since the interaction ability and properties of a protein determine its functional activity, certain amino acid substitutions can be made in the protein sequence and its corresponding DNA coding sequence, and still produce a protein with similar characteristics. Thus, the inventors contemplate that various changes can be made in the DNA sequence of a gene without loss of its biological utility or activity.

[0134] In other aspects, the function of a polypeptide is altered by introducing one or more substitutions. For example, certain amino acids in the protein structure can be replaced by other amino acids to change the interaction binding ability of the interaction components. Structures such as protein interaction domains, nucleic acid interaction domains, and catalytic sites can have their function changed by substituting amino acids. Since the interaction ability and properties of a protein determine its functional activity, certain amino acid substitutions can be made in the protein sequence and its corresponding DNA coding sequence to generate a protein with different characteristics. Thus, the inventors contemplate that various changes can be made in the DNA sequence of a gene to significantly alter its biological utility or activity.

[0135] When making such changes, the hydrophilicity index of the amino acids can be considered. It is generally understood in the art the importance of the hydrophilic amino acid index in conferring biological functions of protein interactions (Kyte and Doolittle, 1982). It is generally believed that the relative hydrophilic nature of the amino acids is related to the generation of the secondary structure of the protein, which in turn defines the interaction of the protein with other molecules such as enzymes, substrates, receptors, DNA, antibodies, antigens, etc.

[0136] The art also understands that substitutions of similar amino acids can be effectively made based on hydrophilicity. U.S. Patent No. 4,554,101, incorporated herein by reference, states that the maximum local average hydrophilicity of a protein is affected by the hydrophilicity of its neighboring amino acids and is related to the biological properties of the protein. It should be understood that an amino acid can be substituted with another amino acid having a similar hydrophilicity value and still produce a biologically and immunologically equivalent protein.

[0137] As mentioned above, amino acid substitutions are generally based on the relative similarity of the amino acid side chain substituents, such as their hydrophobicity, hydrophilicity, charge, size, etc. Exemplary substitutions considering the various properties mentioned above are well known and include: arginine and lysine; glutamic acid and aspartic acid; serine and threonine; glutamine and asparagine; and valine, leucine, and isoleucine.

[0138] In certain aspects, all or part of the proteins described herein can also be synthesized in solution or on a solid support according to conventional techniques. There are a variety of commercially available automated synthesizers and they 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), all incorporated herein by reference. Alternatively, recombinant DNA techniques can be employed, wherein 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 conditions suitable for expression.

[0139] In one aspect, it includes the use of gene transfer into cells, including microorganisms, to generate and / or present proteins. The gene of the target protein is transferred into a suitable host cell, and then the cell is cultured under appropriate conditions. Nucleic acids encoding almost any polypeptide can be used. The generation of recombinant expression vectors and the elements they contain are discussed herein. Alternatively, the protein to be generated can be an endogenous protein that is normally synthesized by the cells used for protein production.

[0140] III. Nucleic Acids

[0141] In some aspects, the present disclosure relates to recombinant polynucleotides encoding the polypeptides described herein. Accordingly, some aspects relate to nucleotides encoding the polypeptides, chimeric polypeptides, or multimeric polypeptides described herein.

[0142] As used herein, the term "polynucleotide" refers to a nucleic acid molecule that is either recombinant or isolated from total genomic nucleic acid. The term "polynucleotide" includes oligonucleotides (nucleic acids that are 100 residues or less in length), recombinant vectors such as plasmids, cosmids, phages, viruses, and the like. In some aspects, the polynucleotide includes regulatory sequences that are substantially isolated from their naturally occurring gene or protein coding sequences. The polynucleotide can be a single-stranded (coding or antisense) polynucleotide or a double-stranded polynucleotide, and can be RNA, DNA (genomic DNA, cDNA, or synthetic DNA), analogs thereof, or combinations thereof. Additional coding or non-coding sequences can be present, but are not required to be present, within the polynucleotide.

[0143] In this regard, the terms "gene", "polynucleotide", or "nucleic acid" are used to refer to nucleic acids encoding proteins, polypeptides, or peptides (including any sequences required for proper transcription, post-translational modification, or localization). As will be understood by those skilled in the art, the term encompasses genomic sequences, expression components, cDNA sequences, and smaller engineered nucleic acid fragments that can express or can be engineered to express proteins, polypeptides, domains, peptides, fusion proteins, and mutants. Nucleic acids encoding all or part of a polypeptide may comprise contiguous nucleic acid sequences that are 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, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 441, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 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, 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 derivable therefrom), including all values and ranges of polynucleotides encoding one or more amino acid sequences described or cited herein. It is also contemplated that a particular polypeptide may be encoded by nucleic acids comprising variant nucleic acid sequences that differ slightly but encode the same or substantially the same protein.

[0144] In certain aspects, the invention relates to isolated nucleic acid fragments and recombinant vectors that incorporate nucleic acid sequences encoding the polypeptides or peptides of the present disclosure. The term "recombinant" can be used in combination with polynucleotides or polypeptides and generally refers to polypeptides or polynucleotides that are generated and / or manipulated in vitro, or replication products of such molecules.

[0145] In other aspects, the invention relates to isolated nucleic acid fragments and recombinant vectors that incorporate nucleic acid sequences encoding the polypeptides or peptides of the present disclosure.

[0146] The nucleic acid fragments used in the present invention can be combined with other nucleic acid sequences, such as promoters, polyadenylation signals, additional restriction enzyme sites, multiple cloning sites, other coding fragments, etc., and thus their overall length may vary greatly. Thus, nucleic acid fragments of almost any length can be considered, and their total length is preferably limited by the convenience of preparation and use in a given recombinant nucleic acid protocol. In some cases, the nucleic acid sequence can encode a polypeptide sequence with additional heterologous coding sequences, such as polypeptide sequences for purifying the polypeptide, transporting, secreting, post-translationally modifying, or for therapeutic benefits such as targeting or efficacy. As described above, tags or other heterologous polypeptides can be added to the modified polypeptide coding sequence, where "heterologous" refers to a polypeptide different from the modified polypeptide.

[0147] In certain aspects, the present disclosure provides polynucleotide variants having substantial identity to the sequences of the present disclosure; these variants contain sequences having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or greater than 99% sequence identity compared to the polynucleotide sequences of the present disclosure, including all values and ranges therebetween, as determined using methods described herein (e.g., BLAST analysis using standard parameters).

[0148] The present disclosure also contemplates the use of polynucleotides complementary to all of the above polynucleotides.

[0149] A. Vectors

[0150] The polypeptides of the present disclosure can be encoded by nucleic acid molecules contained in a vector. The term "vector" refers to a vector nucleic acid molecule into which a heterologous nucleic acid sequence can be inserted for introduction into a cell for replication and expression therein. The nucleic acid sequence can be "heterologous", meaning that it is in an external environment of the cell into which the vector is introduced or the integrated nucleic acid, which contains a sequence homologous to a sequence in the cell or nucleic acid but is located at a position not normally present in the host cell or nucleic acid. Vectors include DNA, RNA, plasmids, cosmids, viruses (phages, animal viruses, and plant viruses), and artificial chromosomes (e.g., YACs). One of ordinary skill in the art will be able to construct vectors by standard recombinant techniques (e.g., Sambrook et al., 2001; Ausubel et al., 1996, both incorporated herein by reference). In addition to encoding the polypeptides of the present disclosure, the vector can also encode other polypeptide sequences, such as one or more other bacterial peptides, tags, or immunogenicity-enhancing peptides. Useful vectors for encoding such fusion proteins include pIN vectors (Inouye et al., 1985), vectors encoding a stretch of histidine sequence, and pGEX vectors for generating glutathione S-transferase (GST) soluble fusion proteins for subsequent purification, isolation, or cleavage.

[0151] The term "expression vector" refers to a vector containing a nucleic acid sequence encoding at least a portion of a gene product capable of being transcribed. In some cases, the RNA molecule is subsequently translated into a protein, polypeptide, or peptide. An expression vector can contain a variety of "control sequences", which refers to nucleic acid sequences necessary for the transcription and possibly translation of an operably linked coding sequence in a particular host organism. In addition to control sequences that control transcription and translation, vectors and expression vectors can also contain nucleic acid sequences with other functions, as described herein.

[0152] B. Promoters and Enhancers

[0153] A "promoter" is a control sequence. A promoter generally refers to a nucleic acid sequence region that controls the initiation and rate of transcription. It can contain regulatory proteins and molecules that can bind genetic elements such as RNA polymerase and other transcription factors. The phrases "operably positioned", "operably linked", "controlled", and "under transcriptional control" mean that the promoter is in the correct functional position and / or orientation relative to a nucleic acid sequence to control the initiation and expression of transcription of that sequence. A promoter can be used alone or in combination with an "enhancer", which refers to a cis-acting regulatory sequence involved in the transcriptional activation of a nucleic acid sequence.

[0154] Of course, it is important to employ promoters and / or enhancers that can effectively direct the expression of DNA fragments in the selected cell type or organism. Those skilled in the art of molecular biology are generally aware of using combinations of promoters, enhancers, and cell types to express proteins (see Sambrook et al., 2001, cited herein). The promoters used can be constitutive, tissue-specific, or inducible, and in some aspects can direct high-level expression of the introduced DNA fragment under specific conditions, such as for large-scale production of recombinant proteins or polypeptides.

[0155] It is believed that the specific promoter used to control the expression of the polypeptide or protein-encoding polynucleotide of the present invention is not critical as long as it can express the polynucleotide in the target cell, preferably a bacterial cell. When targeting human cells, the polynucleotide coding region is preferably placed adjacent to and under the control of a promoter capable of expressing in human cells. Generally, such promoters can include bacterial promoters, human promoters, or viral promoters.

[0156] C. Initiation Signals and Internal Ribosome Entry Sites (IRESs)

[0157] Specific initiation signals may also be required for efficient translation of the coding sequence. These signals include the ATG initiation codon or adjacent sequences. It may be necessary to provide exogenous translation control signals including the ATG initiation codon. One of ordinary skill in the art will be able to readily determine this and provide the necessary signals.

[0158] In certain aspects of the invention, internal ribosome entry site (IRES) elements are used to create polygenic or polycistronic messages. IRES elements are capable of bypassing the ribosome scanning model of 5'-methylated cap-dependent translation and initiating translation from an internal site (Pelletier and Sonenberg, 1988; Macejak and Sarnow, 1991). IRES elements can be linked to heterologous open reading frames. Multiple open reading frames can be transcribed together, separated by IRESs between each open reading frame, thereby forming a polycistronic message. The use of a single promoter / enhancer to transcribe a single message can effectively express multiple genes (see U.S. Pat. Nos. 5,925,565 and 5,935,819, which are incorporated herein by reference).

[0159] D. Selectable Markers and Screenable Markers

[0160] In certain aspects of the present invention, cells containing the nucleic acid constructs of the present disclosure can be identified in vitro or in vivo by encoding a selectable or screenable marker in an expression vector. When transcribed and translated, the marker confers an identifiable change to the cell, allowing cells containing the expression vector to be readily identified. Generally, a selectable marker refers to a property that confers a certain selectivity to the cell. A positive selectable marker is one whose presence enables it to be selected, while a negative selectable marker is one whose presence prevents it from being selected. An example of a positive selectable marker is a drug resistance marker.

[0161] E. Host cell

[0162] As used herein, the terms "cell", "cell line", and "cell culture" are used interchangeably. These terms also include their progeny, i.e., all subsequent generations. It should be understood that due to artificial mutagenesis or unintended mutations, all progeny may not be identical. In the case of expressing a heterologous nucleic acid sequence, a "host cell" refers to a prokaryotic or eukaryotic cell, which includes any transformable organism capable of replicating a vector or expressing a heterologous gene encoded by the vector. Host cells can, and have been, used as recipients for vectors or viruses. A host cell can be "transfected" or "transformed", which refers to the process of transferring or introducing exogenous nucleic acid (such as a sequence encoding a recombinant protein) into the host cell. Transformed cells include the original test cells and their progeny cells.

[0163] Host cells can be derived from prokaryotes or eukaryotes, including bacteria, yeast cells, insect cells, and mammalian cells for replicating vectors or expressing part or all of the nucleic acid sequences. Many cell lines and cell cultures can be used as host cells, and these can be obtained from the American Type Culture Collection (ATCC), which is an archive organization for live cultures and genetic materials (www.atcc.org).

[0164] F. Expression system

[0165] There are many expression systems that contain at least part or all of the above compositions. The present invention can utilize prokaryotic and / or eukaryotic systems to generate nucleic acid sequences, or their associated polypeptides, proteins, and peptides. Many such systems are widely commercially available.

[0166] The insect cell / baculovirus system can achieve high-level protein expression of heterologous nucleic acid fragments, as described in U.S. Patents 5,871,986 and 4,879,236, both of which are incorporated herein by reference, and can be obtained from, for example by purchasing a product named 2.0 and by purchasing BACPACK TM baculovirus expression system.

[0167] In addition to the expression systems disclosed by the present invention, examples of other expression systems include the COMPLETE CONTROL inducible mammalian expression system of, which involves a synthetic ecdysone-inducible receptor or its pET expression system, and the pET expression system is an E. coli expression system. Another example of an inducible expression system is available from obtained, and this system is equipped with T-REX TM (tetracycline-regulated expression) system, which is an inducible mammalian expression system using a full-length CMV promoter. A yeast expression system called the Pichia pastoris expression system is also provided, which is designed for high-yield production of recombinant proteins in the methanol-nutritional yeast Pichia pastoris. One of ordinary skill in the art will know how to express vectors, such as expression constructs, to generate nucleic acid sequences or their corresponding polypeptides, proteins, or polypeptide fragments.

[0168] IV. Combination Therapy

[0169] The components and related methods of the present disclosure, particularly the administration of the polypeptides in the present disclosure, can also be administered in combination with additional therapies, such as other therapeutic agents described herein, or with other conventional therapeutic agents known in the art for treating autoimmune or inflammatory diseases.

[0170] The therapeutic compositions and treatments of the present disclosure can precede, be concurrent with, and / or follow another treatment or drug at intervals ranging from a few minutes to a few weeks. In cases where drugs are administered separately to a cell, tissue, or organism, one typically ensures that the time between each administration is not too long so that the therapeutic drugs can still have a beneficial combined effect on the cell, tissue, or organism. For example, in such cases, it is contemplated that a cell, tissue, or organism can be contacted with two, three, four, or more than four reagents or treatments almost simultaneously (i.e., with a deviation of less than about one minute). In other aspects, one or more than one therapeutic reagent or treatment can be administered or provided within 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, 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, 8 weeks, or longer than 8 weeks before and / or after the administration of another therapeutic drug or treatment, and any range derivable therefrom.

[0171] In some aspects, multi-course treatments can be employed. It is contemplated that multi-courses can be implemented.

[0172] V. Methods of Treatment

[0173] The compositions of the present disclosure can be used for in vivo, in vitro, or ex vivo administration. The route of administration of the composition can be, for example, intradermal, subcutaneous, intravenous, intradermal, intramuscular, topical, external, and / or intraperitoneal administration. It is specifically contemplated that certain aspects of the present disclosure do not include one or more than one of these routes of administration.

[0174] In some aspects, the compositions of the present disclosure are provided by subcutaneous administration (i.e., by subcutaneous injection). In some aspects, the compositions of the present disclosure are provided by intradermal administration (i.e., by intradermal injection). In some aspects, the compositions of the present disclosure are provided by intramuscular administration (i.e., by intramuscular injection).

[0175] In some aspects, the therapeutic compositions of the present disclosure are administered during the cessation of one or more other therapies. For example, in some aspects, disclosed is a method that includes administering the compositions of the present disclosure to a subject during the discontinuation of additional anti-inflammatory therapies (e.g., fingolimod, interferon-β, dimethyl fumarate, teriflunomide, integrin α4β1, anti-αLβ2 antibody, anti-TNFα agent, anti-IL-6R agent, anti-IL-6 agent, or Janus kinase inhibitor) to the subject.

[0176] The compositions of the present disclosure can be used for in vivo, in vitro, or ex vivo administration. The route of administration of the compositions can be, for example, intradermal, subcutaneous, intravenous, topical, transdermal, and / or intraperitoneal administration.

[0177] Aspects of the present disclosure relate to methods for treating autoimmune or inflammatory diseases. In some aspects, disclosed herein are methods for treating autoimmune or inflammatory diseases that include administering the compositions of the present disclosure to a subject, where the subject has, is at risk of developing, or is suspected of having an autoimmune or inflammatory disease. Such methods can include administering one or more additional anti-inflammatory agents. Such methods can exclude administering one or more additional anti-inflammatory agents. Additional anti-inflammatory agents include, but are not limited to, fingolimod, interferon-β, dimethyl fumarate, teriflunomide, integrin α4β1, anti-αLβ2 antibody, anti-TNFα agent, anti-IL-6R agent, anti-IL-6 agent, and Janus kinase inhibitors (e.g., tofacitinib, baricitinib, upadacitinib).

[0178] Autoimmune or inflammatory diseases suitable for treatment may include, but are not limited to, the following conditions: 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, spondyloarthritis, and systemic juvenile rheumatoid arthritis, osteoarthritis, progressive chronic arthritis, deformans arthritis, primary chronic polyarthritis, reactive arthritis, and ankylosing spondylitis); inflammatory skin hyperplasia diseases; psoriasis such as plaque psoriasis, guttate psoriasis, pustular psoriasis, and nail psoriasis; atopic reactions including atopic diseases such as hay fever and Job syndrome; dermatitis, including contact dermatitis, chronic contact dermatitis, exfoliative dermatitis, allergic dermatitis, allergic contact dermatitis, dermatitis herpetiformis, 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-optic MS, primary progressive MS (PPMS), and relapsing-remitting MS (RRMS); progressive systemic sclerosis; atherosclerosis; arteriosclerosis; multiple sclerosis; ataxic sclerosis; neuromyelitis optica (NMO); inflammatory bowel disease (IBD) (e.g., Crohn's disease, autoimmune gastrointestinal diseases, colitis such as ulcerative colitis, colonic ulcer, microscopic colitis, collagenous colitis, polypoid colitis, necrotizing enterocolitis, and transmural colitis, and autoimmune inflammatory bowel disease); intestinal inflammation; pyoderma gangrenosum; erythema nodosum; primary sclerosing cholangitis; respiratory distress syndrome, including adult respiratory distress syndrome or acute respiratory distress syndrome (ARDS)); meningitis; inflammation of all or part of the uvea; iritis; choroiditis; autoimmune blood diseases; rheumatoid spondylitis; rheumatoid synovitis; hereditary angioedema; cranial nerve injury such as in meningitis; herpes gestationis; pemphigoid gestationis; pruritus scroti; autoimmune premature ovarian failure; sudden hearing loss due to an autoimmune condition; IgE-mediated diseases such as anaphylaxis and allergic and atopic rhinitis; encephalitis such as Rasmussen's encephalitis and marginal and / or brainstem encephalitis;Uveitis such as anterior uveitis, acute anterior uveitis, granulomatous uveitis, non-granulomatous uveitis, lens-induced uveitis, posterior uveitis or autoimmune uveitis; glomerulonephritis (GN) with or without nephrotic syndrome, such as chronic or acute glomerulonephritis, for example primary GN, immune-mediated GN, membranous GN (membranous nephropathy), idiopathic membranous GN or idiopathic membranous nephropathy, membranous or membranoproliferative GN (MPGN) including types I and II, and rapidly progressive GN; proliferative nephritis; autoimmune polyendocrine failure; balanitis, including plasmacytic balanitis circumscripta, balanoposthitis; erythema annulare centrifugum; erythema dyschromicum perstans; erythema multiforme; granuloma annulare; lichen nitidus; lichen sclerosus et atrophicus; lichen simplex chronicus; lichen spinulosus; lichen planus; ichthyosis lamellaris; epidermolytic hyperkeratosis; precancerous keratosis; pyoderma gangrenosum; allergic conditions and reactions; anaphylaxis; eczema, including allergic or atopic eczema, xerotic eczema, pompholyx eczema and vesicular palmar and plantar eczema; asthma, such as bronchial asthma, allergic asthma and autoimmune asthma; conditions involving T-cell infiltration and chronic inflammatory responses; immune responses to foreign antigens, such as immune responses to fetal A-B-O blood groups during pregnancy; chronic pulmonary inflammatory diseases; autoimmune myocarditis; leukocyte adhesion deficiency; lupus, including lupus nephritis, lupus encephalitis, juvenile 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-onset (type I) diabetes, including childhood insulin-dependent diabetes mellitus (IDDM) and adult-onset diabetes (type II diabetes) and autoimmune diabetes. In addition, immune responses mediated by cytokines and T lymphocytes associated with acute and delayed hypersensitivity reactions are contemplated; sarcoidosis; granulomatosis, including lymphomatoid granulomatosis, Wegener's granulomatosis; agranulocytosis; vasculitis, including angiitis, large-vessel vasculitis (including polymyalgia rheumatica and giant cell arteritis (Takayasu arteritis)); medium-vessel vasculitis (including Kawasaki disease and polyarteritis nodosa / periarteritis nodosa); microscopic polyarteritis; immune vasculitis; central nervous system vasculitis; cutaneous vasculitis; hypersensitivity vasculitis; necrotizing vasculitis, such as systemic necrotizing vasculitis and ANCA-associated vasculitis, such as allergic granulomatous angiitis (CSS) and ANCA-associated small-vessel vasculitis; temporal arteritis, aplastic anemia; autoimmune aplastic anemia; Coombs'-positive anemia; congenital aplastic anemia; hemolytic anemia or immune hemolytic anemia, including autoimmune hemolytic anemia (AIHA); Addison's disease; autoimmune neutropenia; pancytopenia; leukopenia; diseases involving leukocyte extravasation;Inflammatory diseases of the central nervous system; dementia; mental health disorders; depression; Alzheimer's disease; Parkinson's disease; multiple organ damage syndromes such as secondary injuries caused by sepsis, trauma or hemorrhage; antigen-antibody complex-mediated diseases; anti-glomerular basement membrane disease; antiphospholipid antibody syndrome; allergic neuritis; Behçet's disease / Behçet's syndrome; Castleman disease, Goodpasture syndrome; Raynaud's syndrome; Sjogren's syndrome; Stevens-Johnson syndrome; pemphigoid such as bullous pemphigoid and cutaneous pemphigoid; pemphigus (including pemphigus vulgaris, pemphigus foliaceus, pemphigus mucosae and pemphigus erythematosus); autoimmune polyendocrinopathy; Reiter's disease or Reiter's syndrome; thermal injury; preeclampsia; immune complex disorders such as immune complex nephritis; antibody-mediated nephritis; polyneuropathy; chronic neuropathy such as IgM polyneuropathy or IgM-mediated neuropathy; autoimmune or immune-mediated thrombocytopenia such as idiopathic thrombocytopenic purpura (ITP) including chronic or acute ITP; scleritis such as idiopathic keratitis scleritis, episcleritis; autoimmune diseases of the testis and ovary, including autoimmune orchitis and oophoritis; primary hypothyroidism; hypoparathyroidism; autoimmune endocrine diseases, including thyroiditis such as autoimmune thyroiditis, Hashimoto's disease, chronic thyroiditis (Hashimoto's thyroiditis) or subacute thyroiditis, autoimmune thyroid disease, idiopathic hypothyroidism, Graves' disease, polyglandular syndrome such as autoimmune polyendocrine syndrome (or polyendocrine endocrine disease syndrome), paraneoplastic syndrome, including neurological paraneoplastic syndrome such as Lambert-Eaton myasthenic syndrome or myasthenic syndrome; stiff-person syndrome; encephalomyelitis such as allergic encephalomyelitis and experimental allergic encephalomyelitis (EAE); experimental autoimmune encephalomyelitis; myasthenia gravis such as thymoma-associated myasthenia gravis; cerebellar degeneration; neuromyotonia; opsoclonus or opsoclonus-myoclonus syndrome (OMS) and sensory neuropathy; multifocal motor neuropathy; Sheehan syndrome; autoimmune hepatitis; chronic hepatitis; lupus-like hepatitis; giant cell hepatitis; chronic active hepatitis or autoimmune chronic active hepatitis; lymphocytic interstitial pneumonia (LIP); bronchiolitis obliterans (non-transplant) with NSIP; Guillain-Barré syndrome; Berger's disease (IgA nephropathy); idiopathic IgA nephropathy; linear IgA dermatosis; acute febrile neutrophilic dermatosis; subepidermal pustular dermatosis; transient acantholytic dermatosis; cirrhosis such as primary biliary cirrhosis and pulmonary cirrhosis; autoimmune enteropathy syndrome; celiac disease or celiac-like sprue; celiac disease (gluten enteropathy); refractory celiac disease; idiopathic celiac disease; cryoglobulinemia; amyotrophic lateral sclerosis (ALS; Lou Gehrig's disease); coronary artery disease; autoimmune ear diseases such as autoimmune inner ear disease (AIED); autoimmune hearing loss; polychondritis such as refractory or recurrent polychondritis;Pulmonary alveolar proteinosis; Cogan's syndrome / non-syphilitic interstitial keratitis; Bell's palsy; Sweet's disease / Sweet's syndrome; Rosacea autoimmune; Herpes zoster-related pain; Amyloidosis; Non-cancerous lymphocytosis; Primary lymphocytosis, including monoclonal B lymphocytosis (e.g., benign monoclonal gammopathy and monoclonal gammopathy of undetermined significance, MGUS); Peripheral neuropathy; Paraneoplastic syndrome; Ion channelopathies, such as epilepsy, migraine, arrhythmia, muscle diseases, deafness, blindness, periodic paralysis, and ion channelopathies of the central nervous system; Autism; Inflammatory myopathy; Focal or segmental or focal segmental glomerulosclerosis (FSGS); Endocrine ophthalmopathy; Uveitis; Choroiditis; Autoimmune liver disease; Fibromyalgia; Multiple endocrine 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; Alopecia totalis; Acrosclerosis syndrome (calcinosis, Raynaud's phenomenon, esophageal dysmotility, sclerodactyly, and telangiectasia) and telangiectasia); Male and female autoimmune infertility, e.g., due to anti-sperm antibodies; Mixed connective tissue disease; Chagas' disease; Rheumatic fever; Recurrent miscarriage; Farmer's lung; Erythema multiforme; Post-cardiac surgery syndrome; Cushing's syndrome; Avian breeder's lung; Allergic granulomatous angiitis; Benign lymphocytic angiitis; Alport syndrome; Alveolitis, such as allergic alveolitis and fibrotic alveolitis; Interstitial lung disease; Transfusion reaction; Leprosy; Malaria; Parasitic diseases, such as leishmaniasis, trypanosomiasis (kypanosomiasis), schistosomiasis, ascariasis, aspergillosis; Sampter syndrome; Caplan syndrome; Dengue fever; Endocarditis; Endomyocardial fibrosis; Diffuse interstitial pulmonary fibrosis; Interstitial pulmonary fibrosis; Pulmonary fibrosis; Idiopathic pulmonary fibrosis; Cystic fibrosis; Hepatic fibrosis; Renal fibrosis; Endophthalmitis; Persistent nodular erythema; Fetal erythrocytosis; Eosinophilic fasciitis; Shulman's syndrome; Felty's syndrome; Filariasis; Cyclitis, such as chronic cyclitis, heterochronic cyclitis, iridocyclitis (acute or chronic), or Fuchs' cyclitis; Henoch-Schönlein purpura; Human immunodeficiency virus (HIV) infection; Severe combined immunodeficiency disease; Acquired immunodeficiency syndrome (AIDS); Echovirus infection; Septicemia; Endotoxemia; Pancreatitis; Thyrotoxicosis; Parvovirus infection; Rubella virus infection; Post-vaccination syndrome; Congenital rubella infection; Epstein-Barr virus infection; Mumps; Evans syndrome; Autoimmune gonadal failure; Rheumatic chorea; Post-streptococcal glomerulonephritis; Thromboangiitis obliterans; Thyrotoxicosis; Tabes dorsalis; Choroidoretinitis; Giant cell polymyalgia; Chronic hypersensitivity pneumonitis; Keratoconjunctivitis sicca; Epidemic keratoconjunctivitis; Idiopathic nephrotic syndrome; Minimal change nephropathy;Benign familial and ischemic reperfusion injury; reperfusion of transplanted organs; retinal autoimmunity; joint inflammation; bronchitis; chronic obstructive pulmonary disease / airway diseases; silicosis; aphthae; aphthous stomatitis; arteriosclerosis; aspirin-insensitive asthma (asperniogenese); autoimmune hemolysis; Behcet's disease; cryoglobulinemia; Dupuytren's contracture; phacoanaphylactic endophthalmitis; allergic enteritis; erythema nodosum leprosum; idiopathic facial paralysis; chronic fatigue syndrome; rheumatic fever; Hamman-Rich disease; sensorineural deafness; paroxysmal nocturnal hemoglobinuria; hypogonadism; regional enteritis; leukopenia; infectious mononucleosis; transverse myelitis; primary idiopathic myxedema; kidney disease; sympathetic ophthalmia; granulomatous orchitis; pancreatitis; acute polyneuritis; pyoderma gangrenosum; Hashimoto's thyroiditis; acquired splenic atrophy; non-malignant thymoma; vitiligo; toxic shock syndrome; food poisoning; disease conditions involving T cell infiltration; leukocyte adhesion deficiency; immune responses related to acute and delayed hypersensitivity mediated by cytokines and T lymphocytes; diseases involving leukocyte extravasation; multiple organ injury syndrome; antigen-antibody complex-mediated diseases; anti-glomerular basement membrane disease; allergic neuritis; autoimmune polyendocrinopathy; oophoritis; primary myxedema; autoimmune atrophic gastritis; sympathetic ophthalmia; rheumatic diseases; mixed connective tissue disease; nephrotic syndrome; insulitis; multiple endocrine failure; type I autoimmune polyglandular syndrome; adult-onset idiopathic hypoparathyroidism (AOIH); cardiomyopathy, such as dilated cardiomyopathy; acquired epidermolysis bullosa (EBA); hemochromatosis; myocarditis; nephrotic syndrome; primary sclerosing cholangitis; suppurative or non-suppurative sinusitis; acute or chronic sinusitis; ethmoiditis, frontal sinusitis, maxillary sinusitis or sphenoid sinusitis; diseases related to eosinophils, such as eosinophilia, pulmonary eosinophilia, eosinophilic myalgia syndrome, Löffler's syndrome, chronic eosinophilic pneumonia, tropical pulmonary eosinophilia; bronchopulmonary aspergillosis; aspergilloma or granuloma containing eosinophils; allergic reactions; seronegative spondyloarthropathies; polyendocrine autoimmune diseases; sclerosing cholangitis; sclera, episclera, chronic mucocutaneous candidiasis; Bruton syndrome; infantile transient hypogammaglobulinemia; Wiskott-Aldrich syndrome; ataxia telangiectasia syndrome; vasodilation; autoimmune diseases associated with collagen diseases; rheumatism; nervous system diseases; lymphadenitis; reduced blood pressure response; vascular dysfunction; tissue damage; cardiovascular ischemia; hyperalgesia; renal ischemia; cerebral ischemia and diseases accompanied by vascularization; allergic hypersensitivity diseases; glomerulonephritis; reperfusion injury; ischemic reperfusion disorders; myocardial or other tissue reperfusion injury;Lymphatic tracheobronchitis; inflammatory skin diseases; skin diseases with an acute inflammatory component; multiple organ failure; bullous diseases; renal cortical necrosis; acute suppurative meningitis or other central nervous system inflammatory diseases; ocular and orbital inflammatory diseases; granulocyte transfusion-related syndrome; cytokine-induced toxicity; narcolepsy; acute severe inflammation; chronic refractory inflammation; pyelonephritis; intimal hyperplasia; peptic ulcer; endocarditis; graft-versus-host disease; contact hypersensitivity; asthmatic airway hyperresponsiveness; ischemic stroke; pulmonary hypertension; adverse reactions to immunotherapy; atherosclerosis; wound healing; peripheral arterial disease; cerebral ischemia-reperfusion (I / R) injury; myocardial infarction and endometriosis. The compositions and polypeptides can also be used to treat inflammation associated with stroke, cerebrovascular accident, cardiogenic stroke, and / or acute respiratory distress syndrome. The compositions and polypeptides can also be used to treat inflammation and post-infection symptoms associated with viral infections such as COVID, SARS, and MERS, and / or inflammation and symptoms after vaccination.;

[0179] VI. Pharmaceutical Compositions and Methods

[0180] In some aspects, the pharmaceutical composition is administered to a subject. Different aspects relate to administering an effective amount of the composition to a subject. In some aspects, a composition comprising an anti-inflammatory agent can be administered to a subject or patient to treat inflammation and / or autoimmunity. Additionally, such compounds can be administered in combination with other therapies.

[0181] The composition can be formulated for parenteral administration, for example, formulated into dosage forms that can be injected via intravenous injection, catheter injection, intra-arterial injection, intramuscular injection, subcutaneous injection, or even intraperitoneal injection. Generally, such compositions can be prepared as injections, which can be either liquid solutions or suspensions; they can also be prepared in solid form and formulated into solutions or suspensions by adding a liquid before injection; in addition, these preparations can also be emulsified. Those skilled in the art will know the preparation of such formulations based on the present disclosure. In some aspects, the administration is systemic administration.

[0182] Other administration routes are also contemplated. For example, these structures and drugs can be administered with a carrier. In some aspects, the carrier is nanoparticles or microparticles.

[0183] Particles can have a structure of variable size and are referred to as microspheres, microparticles, nanoparticles, nanospheres or liposomes. Such microparticle formulations can be formed by covalently or non-covalently coupling the structure to the particle. In this article, the terms "particle", "microparticle", "bead", "microsphere" and their grammatical equivalents all refer to tiny discrete particles that can be administered to an object. In some aspects, these particles are substantially spherical. As used herein, the term "substantially spherical" means that the shape of the particle deviates from a spherical shape by no more than about 10%. These particles typically consist of a substantially spherical core and optionally one or more than one layer. The size and composition of the core may vary. In addition to the core part, the microparticle may also have one or more than one layer of structure to provide functions suitable for specific applications. If present, the thickness of each layer may vary according to the requirements of the specific application. For example, these layers can impart useful optical properties.

[0184] Drug forms suitable for injection include sterile aqueous solutions or dispersions; preparations containing sesame oil, peanut oil or aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the preparation must be sterile and must be of a fluidity that allows for easy injection. It should also be stable under the conditions of manufacture and storage and must protect against the contaminating action of microorganisms such as bacteria and fungi.

[0185] The carrier can also be a solvent or dispersion medium, which contains, for example, water, ethanol, polyols (such as glycerol, propylene glycol and liquid polyethylene glycol, etc.), suitable mixtures thereof and vegetable oils. For example, by using a coating of lecithin, the desired particle size can be maintained in the case of dispersion, and by using surfactants, appropriate fluidity can be maintained. The action of microorganisms can be prevented by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, etc. In many cases, it is preferred to add isotonic agents such as sugars or sodium chloride. The extended absorption of injectable compositions can be achieved by using delayed absorption agents such as aluminum stearate and gelatin in the composition.

[0186] A sterile injectable solution is prepared by dissolving the active compound in a suitable solvent in the required amount and adding the other ingredients mentioned above as needed, followed by filtration sterilization. Generally, a dispersion is prepared by adding various sterilized active ingredients to a sterile medium containing a basic dispersion medium and the other required ingredients mentioned above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred preparation methods are vacuum drying and freeze-drying techniques, which produce a powder of the active ingredient and any other required ingredients from a previously sterile filtered solution.

[0187] As used herein, the term "pharmaceutically acceptable" refers to compounds, materials, compositions, and / or dosage forms that, within the scope of reasonable medical judgment, do not produce excessive toxicity, irritation, allergic response, or other complications commensurate with a reasonable risk / benefit ratio when in contact with human and animal tissues. The term "pharmaceutically acceptable carrier" refers to a pharmaceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, solvent, or encapsulating material, which participates in carrying or transporting a chemical agent.

[0188] As used herein, the term "pharmaceutically acceptable salt" refers to derivatives of the disclosed compounds in which the parent compound is modified by converting an existing acidic or basic 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 salts or organic salts of acidic residues such as carboxylic acids, etc. Pharmaceutically acceptable salts also include conventional non-toxic salts formed from non-toxic inorganic or organic acids or quaternary ammonium salts of the parent compound. Pharmaceutically acceptable salts can be synthesized from the parent compound containing a basic or acidic moiety by conventional chemical methods.

[0189] Depending on the condition of the subject, the dosage will necessarily vary. In any case, the person responsible for administration will determine the appropriate dose for an individual subject. The effective amount of the therapeutic or prophylactic composition is determined according to the intended goal. The term "unit dose" refers to a physically discrete unit suitable for use by the subject, each unit containing a predetermined quantity of the composition calculated to produce the desired response associated with its mode of administration, i.e., the appropriate route and regimen. The dosage administered, whether according to the number of treatments or unit dose, depends on the desired effect. The precise amount of the composition also depends on the judgment of the professional and varies from individual to individual. Factors affecting the dose include the physical condition and clinical status of the subject, the route of administration, the purpose of treatment (alleviating symptoms or curing), and the potency, stability, and toxicity of the particular ingredient.

[0190] Once the formulation is determined, it will be administered in a manner compatible with the dosage formulation, and the dosage should achieve a therapeutic or prophylactic effect. These formulations can be readily administered in various dosage forms, such as the injectable solution type described above.

[0191] Generally, for an adult (weighing approximately 70 kg), the dosage of the compound is from about 0.1 mg to about 3000 mg (including all values and ranges therebetween), or from about 5 mg to about 1000 mg (including all values and ranges therebetween), or from about 10 mg to about 100 mg (including all values and ranges therebetween). It should be understood that these dosage ranges are only examples and can be adjusted according to factors known to those skilled in the art.

[0192] In some aspects, the subject is administered 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, 13.5, 14.0, 14.5, 15.0, 15.5, 16.0, 16.5, 17.0, 17.5, 18.0, 18.5, 19.0, 19.5, 20.0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 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, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265, 270, 275,280, 285, 290, 295, 300, 305, 310, 315, 320, 325, 330, 335, 340, 345, 350, 355, 360, 365, 370, 375, 380, 385, 390, 395, 400, 410, 420, 425, 430, 440, 441, 450, 460, 470, 475, 480, 490, 500, 510, 520, 525, 530, 540, 550, 560, 570, 575, 580, 590, 600, 610, 620, 625, 630, 640, 650, 660, 670, 675, 680, 690, 700, 710, 720, 725, 730, 740, 750, 760, 770, 775, 780, 790, 800, 810, 820, 825, 830, 840, 850, 860, 870, 875, 880, 890, 900, 910, 920, 925, 930, 940, 950, 960, 970, 975, 980, 990, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100, 3200, 3300, 3400, 3500, 3600, 3700, 3800, 3900, 4000, 4100, 4200, 4300, 4400, 4500, 4600, 4700, 4800, 4900, 5000, 6000, 7000, 8000, 9000 or 10000 milligrams (mg) or micrograms (mcg) or μg / kg or micrograms / kg / minute or mg / kg / minute or micrograms / kg / hour or mg / kg / hour, or μM or mM of the drugs discussed herein. Envision any ranges derivable therefrom.,

[0193] Administration may be carried out 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 derivable therefrom) as needed, or 1 time, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times or more than 9 times per day (or any range derivable therefrom). The first administration may be carried out before or after the appearance of signs of the disease. In some aspects, the patient is administered the first dose of the treatment regimen 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours (or any range derivable therefrom) after experiencing or showing signs or symptoms, or 1 day, 2 days, 3 days, 4 days or 5 days (or any range derivable therefrom) later. The patient may receive treatment for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days or more than 10 days (or any range derivable therefrom), or until the disease disappears or reduces, or until 6 hours, 12 hours, 18 hours or 24 hours, or 1 day, 2 days, 3 days, 4 days or 5 days after the symptoms of the infection disappear or reduce.

[0194] VII. Sequence

[0195]

[0196]

[0197]

[0198]

[0199]

[0200]

[0201]

[0202]

[0203]

[0204]

[0205]

[0206]

[0207]

[0208]

[0209]

[0210]

[0211]

[0212]

[0213]

[0214]

[0215]

[0216] VIII. Examples

[0217] The following examples are used to illustrate the preferred embodiments of the present disclosure. Those skilled in the art should understand that the technologies disclosed in the following examples represent the technologies discovered by the inventors that function well in the practice of the present disclosure, and thus can be considered to constitute a preferred mode of practicing the present disclosure. However, those skilled in the art should understand according to the present disclosure that many changes can be made to the specific embodiments disclosed and still obtain the same or similar results without departing from the spirit and scope of the present disclosure.

[0218] In the examples, drawings, and description of the drawings, the amino acids K22, R196, R73, and R194 refer to the 23rd, 197th, 74th, and 194th amino acids of the IL-35 amino acid sequence shown in SEQ ID NO: 1, respectively, and the EBI3 subunit sequence is shown in SEQ ID NO: 2.

[0219] Example 1: Secretion Assay of IL-35-HSA Mutants

[0220] The expression plasmid was constructed based on pcDNA 3.1 (Thermo Fisher Scientific, US). The full-length cDNA fragment (SEQ ID NO: 1) was synthesized by GeneArt Gene Synthesis Service (Thermo Fisher Scientific, US). To generate the expression plasmid pcDNA-IL35-HSA / WT, the cDNA fragment was digested with NheI and EcoRV and then cloned into pcDNA 3.1 digested with the same restriction enzymes. To construct the expression plasmids of each IL-35-HSA mutant, site-directed mutagenesis was performed using the standard method of inverse PCR with pcDNA-IL35-HSA / WT as the template. The sequencing results confirmed that the mutations were correctly introduced. The constructed mutants are shown in Table 1.

[0221] The expression plasmid was linearized with the restriction endonuclease ScaI and transfected into CHO cells according to the instructions of Lipofectamine 2000 (ThermoFisher Scientific, US). 24 hours after transfection, antibiotic selection was carried out with 500 μg / mL of G418 (Thermo Fisher Scientific, US) to construct a stable expression pool.

[0222] The constructed stable expression pool was seeded into 125 mL culture flasks at an inoculation density of 2x10 6 cells / mL and incubated for 72 hours. The medium was centrifuged at 10,000 g for 10 minutes, and the supernatant was collected.

[0223] Subsequently, the secretion levels of IL-35-HSA WT and IL-35-HSA mutants were analyzed by SDS-PAGE, and the signal intensities obtained were analyzed by ImageJ software (online access: / / imagej.nih.gov / ij / ) to evaluate the amount of secreted protein. The results showed that the secretion levels of the K22A and R196A mutants increased (Table 1, Figures 1 to 3 ).

[0224] Table 1

[0225]

[0226] Example 2: Comprehensive evaluation of the secretion of IL-35-HSA, IL-35-Fc and IL-35 mutants

[0227] The inventors performed substitutions of all 19 different amino acids to compare the expression of IL-35 protein.

[0228] Using the same method described in Example 1, expression plasmids with 19 different amino acid substitutions were constructed for each of the 4 sites (K22, R196, R73, R194) of EBI3 (Table 2). The constructed mutants are shown in Table 2. According to the method of the Lipofectamine 2000 instruction manual, the expression plasmids were transfected into CHO cells (5x10 4Cells / well, 96-well plate, DNA 1 μg / well). The transfection method followed the instructions of Lipofectamine 2000. For IL-35, the p35 / WT plasmid was co-transfected. The transfectants were incubated for 72 hours. The medium was centrifuged at 10,000 g for 10 minutes, and the supernatant was collected. The proteins secreted into the culture supernatant were quantified using Human EBI3DuoSet ELISA (R&D SYSTEMS, product number: DY6456-05) (n = 3). According to the secreted protein concentration, with the corresponding control as 1, the relative secreted protein amount was calculated (see Tables 3 to 5). Among the mutations at the four sites, the mutations of K22 and R196 significantly increased the secretion of IL-35-HSA, IL-35-Fc, and IL-35. For the mutations of R73 and P194, their effects on the secretion of IL-35-HSA, IL-35-Fc, and IL-35 were slightly increased. Among the 19 amino acids, the most secreted were A, G > V, I > L > H, and S > T. It was found that non-polar amino acids were more preferred. The inventors also prepared different versions of Fc in order to achieve different blood half-lives (SEQ ID NOs: 70 to 74).

[0229] Table 2

[0230]

[0231] Table 3

[0232] IL-35-HAS secretion amount (relative ratio, sorted)

[0233]

[0234] Table 4

[0235] IL-35-Fc secretion amount (relative ratio, sorted)

[0236]

[0237] Table 5

[0238] IL-35 secretion amount (relative ratio, sorted)

[0239]

[0240] Example 3: Verification of the Linker Peptide Connecting IL-35 and Human Serum Albumin (HSA)

[0241] The inventors designed different linker peptide amino acid sequences between IL35 and HSA to compare the expression of IL-35 protein.

[0242] Connect IL-35 and HSA using the linker shown in Table 6, and evaluate the amount of the secreted IL-35-HSA fusion protein. Construct expression vectors connecting IL-35, HSA, and each linker peptide using the PCR method. According to the method in the Lipofectamine 2000 instruction manual, transfect the expression plasmid into CHO cells (5x10 4 cells / well, 96-well plate, 1 μg / well of DNA). The transfection method follows the Lipofectamine 2000 instruction manual. Incubate the transfectants for 24 hours. Centrifuge the medium at 10,000 g for 10 minutes and collect the supernatant. Subsequently, analyze the secretion level of each IL-35-HSA by Western blotting, and perform image analysis on the obtained signal intensity using ImageJ software (online access: imagej.nih.gov / ij / ) to evaluate the amount of the secreted protein. Calculate the relative secretion amount of IL-35-HSA with linker-12 as 1 (Table 6). Compared with linker-12, the secretion of IL-35-HSA with linker-1, linker-10, and linker-11 was significantly increased.

[0243] Table 6

[0244]

[0245] Table 6 shows SEQ ID NO: 75 to SEQ ID NO: 85 and SEQ ID NO: 3, respectively.

[0246] Example 4: Evaluate mutants in which four cysteines in EBI3 are replaced with serine

[0247] The inventors replaced the cysteines in the EBI3 portion with serine to compare the expression of the IL-35 protein.

[0248] Generate an expression vector for the mutant in the manner described in Example 1, in which four cysteines in EBI3 are replaced with serine. According to the method in the Lipofectamine 2000 instruction manual, transfect the expression plasmid into CHO cells (5x10 4cells / well, 96-well plate, 1 μg / well of DNA). The transfection method followed the instructions of Lipofectamine 2000. The transfectants were incubated for 24 hours. The medium was centrifuged at 10,000 g for 10 minutes, and the supernatant was collected. Subsequently, the secretion level of each IL-35-HSA was analyzed by Western blotting, and the signal intensity obtained was analyzed by ImageJ software (online access: imagej.nih.gov / ij / ) to evaluate the amount of secreted protein. The relative amount of secreted protein was calculated with IL-35-HSA / WT as 1. After replacing cysteine with serine at four positions of EBI3, the secretion of IL-35-HSA increased slightly ( Figure 5 ).

[0249] Example 5: Monomerization of IL-35-HSA using additives

[0250] The inventors tested additives for preventing IL35 dimerization.

[0251] It was observed that IL-35-HSA was polymerized in the medium and during the purification process. To monomerize the polymerized IL-35-HSA, additives were studied. The culture supernatant of CHO cells stably expressing IL-35-HSA was concentrated by an ultrafiltration membrane and replaced with 30 mM phosphate buffer (pH 7) and 50 mM Tris buffer (pH 9) respectively. After equal aliquots were dispensed into each tube, additives were added respectively. The additives used were ascorbic acid (final 1 mM), methionine (final 1 mM), glutathione (final 1 mM), and cysteine (final 1 mM, 10 mM, 50 mM respectively). Then the samples were incubated at 37 °C for 15 minutes. Then each secretion profile of IL-35-HSA was analyzed by Western blotting ( Figure 6 ). The results showed that at pH 7 and 9, after adding methionine, glutathione, and cysteine, the polymers composed of IL-35-HSA completely disappeared and all were converted into monomers. The inventors also tested anion cyclodextrin (0.001 mM to 1000 mM), reducing agents (mercaptoethanol and DTT) (0.001 mM to 1000 mM), cationic agents (arginine 0.001 mM to 1000 mM), and PEG polymers (0.001 mM to 1000 mM).

[0252] Example 6

[0253] The reducing conditions can prevent the aggregation and dimerization of His6-tagged IL-35-HSA. The culture supernatant of HEK293 cells transfected with His6-tagged IL-35-HSA was purified using a His-tag purification Ni2+ column, and then further purified using a size exclusion column in PBS. Under the reducing conditions of SDS-PAGE after reaction with 50 mM DTT, the results showed that the multimers composed of IL-35-HSA completely disappeared and were all converted into monomers (see Figure 7 ).

[0254] Example 7: Deletion of 20 amino acids in the N-terminal Ebi3

[0255] Analysis by AlphaFold2 showed that the N-terminal of the Ebi3 protein did not contribute to the protein structure. Therefore, the inventors prepared N-terminal deletion variants (10 amino acid deletion: delta10 and 20 amino acid deletion: delta20). Aggregation and dimerization of His6-tagged IL-35-HSA. The culture supernatant of HEK293 cells transfected with His6-tagged IL-35-HSA was purified using a His-tag purification Ni2+ column, and then further purified using a size exclusion column in PBS. The non-reducing conditions of SDS-PAGE showed that the deletion of 20 amino acids rather than 10 amino acids could resolve the ultra-large aggregation ( Figure 8 ).

[0256] Example 8 Eight days after subcutaneous injection of IL-35-HSA, IL-35-HSA was detected in the lymph nodes.

[0257] Forty micrograms of IL-35-HSA was subcutaneously injected into the back skin of mice. Four hours and eight days after injection, the inventors collected the lymph nodes ( Figure 9 ). After homogenizing the lymph nodes, they used an ELISA kit (R&D Systems, DY6456-05) to detect the human EBI3 protein to detect the presence of human IL-35. This kit does not detect the mouse endogenous EBI3 protein. The signal at 450 nm showed that IL-35-HSA was detectable and persisted in the lymph nodes for a long time.

[0258] Example 9 Injection of IL-35-HSA reduces cytokine release syndrome

[0259] Ten micrograms of IL-35-HSA was subcutaneously injected twice on day 0 and day 7. On day 9, 2.5 μg of TNFα protein was intraperitoneally injected. Blood was collected 2 hours after TNFα injection, and IL-6 in the serum was analyzed by ELISA ( Figure 10A)。The results showed that IL-35-HSA injection reduced the TNFα-induced serum IL-6 concentration. Flow cytometry analysis indicated that in the lymph nodes, the number of dendritic cells (DCs) increased and exhibited a tolerogenic CD206+ phenotype rather than a pro-inflammatory phenotype (Figures 10B to 10D).

[0260] Example 10 Fusion of the collagen-binding domain with IL-35 for localization

[0261] The inventors previously found that the collagen-binding domain derived from the von Willebrand factor A3 domain prolongs the post-injection tissue retention time through collagen affinity in tissues. Prolonging the retention time of IL-35 at the lesion site may contribute to prolonged efficacy. The inventors have prepared a collagen-binding domain-fused IL-35 protein (SEQ ID NOs: 53 to 55). Collagen-binding IL35 can be injected via various injection routes, including intravenous injection, intradermal injection, and subcutaneous injection.

[0262] Example 11 Evaluation of the efficacy of SA IL-35 in a collagen antibody-induced arthritis model

[0263] A. Results:

[0264] The CHO cell-derived mutant SA IL-35 supernatant was purified by cation exchange in 50 mM acetate buffer (pH 5) and eluted with 50 mM acetic acid (pH 5) containing 1 M NaCl. The resulting protein was further purified by size exclusion chromatography, as Figure 1As shown. Subsequently, the effect of mutant SA IL-35 as a prophylactic treatment was evaluated to prevent the development of collagen antibody-induced arthritis (CAIA). As shown in Figure 11A, in 8-week-old female BALB / c mice (Jackson Laboratory), the CAIA model was induced by intraperitoneal injection of a collagen-II antibody mixture. The antibody mixture consisted of four arthritis-inducing monoclonal antibodies specific for epitopes located on type II collagen [1]. Antibody binding to the epitope induces the formation of immune complexes, which are subsequently deposited in cartilage and synovium and trigger a complement-dependent inflammatory response, resulting in joint swelling [1]. On the 3rd day after immunization with collagen II antibody, CAIA mice were treated by subcutaneous injection of 40 μg of mutant SA IL-35 (equimolar to wild-type IL-35) through the dorsal side, followed by intraperitoneal injection of 25 μg of LPS. From the 3rd day to the 12th day, the clinical scores of the forepaws and hind paws of the mice were recorded daily. The severity score of joint inflammation ranged from 0 to 4, where 0 points indicated healthy paws, 1 point indicated swelling and / or redness of one joint, 2 points indicated swelling and / or redness of multiple joints, 3 points indicated swelling and / or redness of the entire paw, and 4 points indicated maximum swelling. The clinical score results of the experiment are shown in Figures 11 to 11C. In the CAIA mouse model of arthritis, a single dose of mutant SA IL-35 prophylactic treatment prevented the onset of severe disease.

[0265] To evaluate the effect of mutant SA IL-35 on immune cell populations in secondary lymphoid organs, single-cell suspensions of draining lymph nodes (popliteal) and spleens were collected at the end of the experiment, and these cells were stained for T cell and myeloid phenotypes and activation markers as previously described [2]. In the T cell area, mutant SA IL-35 treatment increased the expression of PD-1 on CD4+ T cells and CD8+ T cells ( Figure 12A ) in the lymph nodes and CD4+ T cells ( Figure 12B ) in the spleen. In the myeloid area, mutant SA IL-35 treatment decreased the expression of CD40 and CD86 on CD11b+ myeloid cells in the lymph nodes ( Figure 12C ). The treatment also decreased the percentage of CD11b+F4 / 80+ macrophages in the lymph nodes and the expression of CD86 on macrophages, while increasing the ratio of M2 macrophages to M1 macrophages ( Figure 12C ). In the spleen, after treatment with mutant SA IL-35, the percentage of CD11b+ myeloid cells and the expression of CD86 on CD11b+ cells were both decreased ( Figure 12D ). The treatment also decreased the percentage of CD11b+F4 / 80+ macrophages, while increasing the percentage of Arg1+ M2 macrophages ( Figure 12D)。In the dendritic cell compartment, after treatment with mutant SA IL-35, the percentage of CD11c+ dendritic cells in the lymph nodes and the expression of CD86 and CD40 on CD11c+ dendritic cells were both decreased ( Figure 12E )。This treatment also decreased the percentage of CD11c+CD11b+ dendritic cells and the expression of CD86, CD40, and MHC class II molecules on these cells ( Figure 12E )。In the myeloid-derived suppressor cell compartment, after treatment with mutant SA IL-35, the expression of CD86 and CD40 on CD11b+Ly6Chi Ly6G-MDSC was decreased ( Figure 12F )。

[0266] B. Methods:

[0267] 1. Collagen-induced arthritis model: (Adapted from Yuba et al., 2021)

[0268] As previously described, arthritis was induced in 8-week-old female BALB / c mice (purchased from Jackson Laboratory) [2]. On day 0, a mixture of anti-collagen antibodies (1 mg / mouse) (Chondrex) was injected intraperitoneally into the mice, followed by an injection of 25 μg lipopolysaccharide (Chondrex). On the 3rd day after immunization with collagen II antibody, CAIA mice were treated with 40 μg of mutant SA IL-35 (equimolar to wild-type IL-35) by subcutaneous injection on the dorsal side, followed by an intraperitoneal injection of 25 μg of LPS. According to the manufacturer's protocol (Chondrex), from day 3 to day 12, the clinical scores of the forepaws and hind paws of the mice were recorded daily.

[0269] 2. Flow cytometry analysis:

[0270] Popliteal lymph node and spleen tissues were digested in DMEM medium containing 2% FBS and 2 mg / ml collagenase D (Sigma-Aldrich) at 37 °C for 45 minutes. Single cell suspensions were obtained by gently passing through a 70 μm cell strainer. For the spleen, red blood cells in the blood were lysed using ACK lysis buffer (Quality Biological), followed by antibody staining for flow cytometry. According to the manufacturer's instructions, cells were first blocked with CD16 / 32 antibody and stained with a fixable live / dead cell dye (Live / dead fixable aqua) (eBioscience) on ice for 20 minutes. After washing, the cells were stained with surface antibodies on ice for 20 minutes. Intracellular staining was performed using a FoxP3 staining kit according to the manufacturer's instructions (BioLegend). After washing once, the cells were stained with intracellular antibodies overnight at room temperature. Flow cytometry analysis was performed using a Fortessa flow cytometer (BD Biosciences), and analysis was carried out using FlowJo software (Tree Star).

[0271] *********

[0272] Although certain embodiments have been described above to a certain degree of particularity, or with reference to one or more than one independent embodiment, those skilled in the art can make numerous modifications to the disclosed embodiments without departing from the scope of the present invention. Additionally, in appropriate cases, aspects of any of the above embodiments can be combined with aspects of any other described embodiment to form more embodiments with comparable or different characteristics and to solve the same or different problems. Similarly, it can be understood that the above benefits and advantages can relate to one embodiment or several embodiments. Any reference to a patent disclosure or other disclosure constitutes a specific reference to the disclosed content of that disclosure. Unless the phrases "means for..." or "step for..." are used separately in a given claim, a claim shall not be construed as including a "means-plus-function" or "step-plus-function" limitation.

[0273] References

[0274] [1]Chondrex, Inc. (2023). Collagen antibody induced arthritis. Chondrex. Retrieved from https: / / www.chondrex.com / collagen-antibody-arthritis

[0275] [2]Yuba, E., Budina, E., Katsumata, K., Ishihara, A., Mansurov, A., Alpar, A. T., Watkins, E. A., Hosseinchi, P., Reda, J. W., Lauterbach, A. L., Nguyen, M., Solanki, A., Kageyama, T., Swartz, M. A., Ishihara, J., & Hubbell, J. A. (2021). Suppression of Rheumatoid Arthritis by Enhanced Lymph Node Trafficking of Engineered Interleukin-10 in Murine Models. Arthritis and Rheumatology, 73(5), pp. 769 - 778.

Claims

1. A polypeptide comprising an amino acid sequence of SEQ ID NO: 2 having one or more amino acid substitutions relative to SEQ ID NO: 2, wherein the one or more amino acid substitutions include K23, R74, R195, R197, or a combination thereof.

2. The polypeptide according to claim 1, wherein the polypeptide comprises an amino acid sequence having at least 70% sequence identity with SEQ ID NO:

2.

3. The polypeptide according to claim 1 or 2, wherein the polypeptide comprises one or more substitutions relative to C36, C47, C80, and C90 of SEQ ID NO:

2.

4. The polypeptide according to claim 3, wherein the one or more substitutions are substitutions with serine.

5. A polypeptide comprising an amino acid sequence of SEQ ID NO: 4 having one or more amino acid substitutions relative to SEQ ID NO: 4, wherein the one or more amino acid substitutions include C74.

6. The polypeptide according to claim 5, wherein the polypeptide comprises an amino acid sequence having at least 70% sequence identity with SEQ ID NO:

4.

7. A polypeptide comprising an amino acid sequence of SEQ ID NO: 6 having one or more amino acid substitutions relative to SEQ ID NO: 6, wherein the one or more amino acid substitutions include C34.

8. The polypeptide according to claim 7, wherein the polypeptide comprises an amino acid sequence having at least 70% sequence identity with SEQ ID NO:

6.

9. A polypeptide comprising an amino acid sequence of SEQ ID NO: 90 having one or more amino acid substitutions relative to SEQ ID NO: 90, wherein the one or more amino acid substitutions include K2, R53, R174, R176, or a combination thereof.

10. The polypeptide according to claim 9, wherein the polypeptide comprises an amino acid sequence having at least 70% sequence identity with SEQ ID NO:

90.

11. The polypeptide according to claim 9 or 10, wherein the polypeptide comprises one or more substitutions relative to C15, C26, C59, and C69 of SEQ ID NO:

90.

12. The polypeptide according to claim 11, wherein the one or more substitutions are substitutions with serine.

13. A polypeptide comprising the EBI3 subunit of IL-35, wherein the EBI3 subunit comprises less than 200 amino acids and comprises the amino acid sequence of SEQ ID NO: 92 or an amino acid sequence having at least 70% sequence identity with SEQ ID NO:

92.

14. The polypeptide according to claim 13, wherein the EBI3 subunit comprises one or more amino acid substitutions relative to SEQ ID NO: 92, and wherein the one or more amino acid substitutions include R33, R154, R156, or a combination thereof.

15. The polypeptide according to claim 13 or 14, wherein the EBI3 subunit consists of 189 amino acids.

16. The polypeptide according to any one of claims 13 to 15, wherein the polypeptide comprises a substitution of C6, C39, C49, or a combination thereof relative to SEQ ID NO:

92.

17. The polypeptide according to claim 16, wherein one or more of the substitutions are substitutions with serine.

18. A polypeptide comprising the amino acid sequence of SEQ ID NO: 1 having one or more amino acid substitutions relative to SEQ ID NO: 1, wherein the one or more amino acid substitutions include K23, R74, R195, R197, C320, C497, or a combination thereof.

19. The polypeptide according to claim 18, wherein the polypeptide comprises an amino acid sequence having at least 70% sequence identity with SEQ ID NO:

1.

20. The polypeptide according to claim 18 or 19, wherein the one or more amino acid substitutions comprise K23, C320, and C497 relative to SEQ ID NO:

1.

21. A polypeptide comprising the amino acid sequence of SEQ ID NO: 91 having one or more amino acid substitutions relative to SEQ ID NO: 91, wherein the one or more amino acid substitutions include K2, R53, R174, R176, C299, C476, or a combination thereof.

22. The polypeptide according to claim 21, wherein the polypeptide comprises an amino acid sequence having at least 70% sequence identity with SEQ ID NO:

91.

23. The polypeptide according to claim 21 or 22, wherein the one or more amino acid substitutions comprise K2, C299, and C476 relative to SEQ ID NO:

91.

24. The polypeptide according to any one of claims 21 to 23, wherein the polypeptide further comprises an amino acid substitution relative to C15, C26, C59, C69 of SEQ ID NO: 91 or a combination thereof.

25. The polypeptide according to claim 24, wherein the amino acid substitution is a substitution with serine.

26. The polypeptide according to any one of claims 1 to 25, wherein the polypeptide further comprises an ECM affinity peptide, a serum protein, an albumin binding protein (ABP) and / or an Fc peptide.

27. The polypeptide according to claim 26, wherein the ECM affinity peptide comprises any one of the amino acid sequences of SEQ ID NO: 9 to SEQ ID NO: 27 or SEQ ID NO: 88, or an amino acid sequence having at least 70% sequence identity with any one of the amino acid sequences of SEQ ID NO: 9 to SEQ ID NO: 27 or SEQ ID NO:

88.

28. The polypeptide according to claim 26 or 27, wherein the Fc peptide comprises the amino acid sequence of SEQ ID NO: 86 or SEQ ID NO: 87, or an amino acid sequence having at least 70% sequence identity with SEQ ID NO: 86 or SEQ ID NO:

87.

29. The polypeptide according to any one of claims 26 to 28, wherein the ABP comprises the amino acid sequence of SEQ ID NO: 93, or an amino acid sequence having at least 70% sequence identity with SEQ ID NO:

93.

30. The polypeptide according to any one of claims 26 to 29, wherein the ECM affinity peptide, the serum protein, the ABP and / or the Fc peptide is at the amino terminus of the polypeptide, and the polypeptide comprises any one of the substituted amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 90, SEQ ID NO: 91 or the EBI3 subunit.

31. The polypeptide according to any one of claims 26 to 28, wherein the ECM affinity peptide, the serum protein and / or the Fc peptide is at the carboxyl terminus of the polypeptide, and the polypeptide comprises any one of the substituted amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 90, SEQ ID NO: 91 or the EBI3 subunit.

32. The polypeptide according to any one of claims 26 to 31, wherein the serum protein comprises a polypeptide having the amino acid sequence of SEQ ID NO: 6 or a polypeptide having at least 70% sequence identity with the polypeptide having the amino acid sequence of SEQ ID NO:

6.

33. The polypeptide according to claim 32, wherein the polypeptide comprises one or more amino acid substitutions relative to SEQ ID NO: 6, and wherein the one or more amino acid substitutions comprise C34.

34. The polypeptide according to claim 33, wherein the amino acid substitution at C34 is a substitution with an alanine amino acid residue or a serine amino acid residue.

35. A chimeric polypeptide or a multimeric polypeptide, comprising: (i) the amino acid sequence of SEQ ID NO: 2 having one or more amino acid substitutions relative to SEQ ID NO: 2, wherein the one or more amino acid substitutions relative to SEQ ID NO: 2 include K23, R74, R195, R197, or a combination thereof; and (ii) the polypeptide of SEQ ID NO: 4, a polypeptide having at least 70% sequence identity with SEQ ID NO: 4; or a polypeptide comprising SEQ ID NO: 4 having one or more amino acid substitutions relative to SEQ ID NO:

4.

36. The chimeric polypeptide or multimeric polypeptide according to claim 35, wherein the polypeptide comprising the substituted amino acid sequence of SEQ ID NO: 2 has at least 70% sequence identity with SEQ ID NO:

2.

37. The chimeric polypeptide or multimeric polypeptide according to claim 35 or 36, wherein the polypeptide comprises an amino acid substitution at C36, C47, C80, C90, or a combination thereof relative to the amino acid sequence of SEQ ID NO:

2.

38. The polypeptide according to claim 37, wherein the one or more amino acid substitutions are substitutions with serine.

39. The chimeric polypeptide or multimeric polypeptide according to any one of claims 35 to 38, wherein the polypeptide comprises a polypeptide having one or more amino acid substitutions relative to SEQ ID NO: 4, and wherein the one or more amino acid substitutions include C74.

40. A chimeric polypeptide or multimeric polypeptide according to any one of claims 35 to 39, wherein the polypeptide comprising the substituted amino acid sequence of SEQ ID NO: 2 is a polypeptide having the amino acid sequence of SEQ ID NO: 4, a polypeptide having an amino acid sequence having at least 70% sequence identity with SEQ ID NO: 4, or the amino terminus of a polypeptide comprising the substituted amino acid sequence of SEQ ID NO:

4.

41. A chimeric polypeptide or multimeric polypeptide according to any one of claims 35 to 39, wherein the polypeptide comprising the substituted amino acid sequence of SEQ ID NO: 2 is a polypeptide having the amino acid sequence of SEQ ID NO: 4, a polypeptide having an amino acid sequence having at least 70% sequence identity with SEQ ID NO: 4, or the carboxy terminus of a polypeptide comprising the substituted amino acid sequence of SEQ ID NO:

4.

42. A chimeric polypeptide or multimeric polypeptide according to any one of claims 35 to 41, wherein one or more amino acid substitutions relative to SEQ ID NO: 2 include K23, and wherein one or more amino acid substitutions relative to SEQ ID NO: 4 include C74.

43. The chimeric polypeptide or multimeric polypeptide according to claim 42, wherein the amino acid substitution at K23 and / or C74 is a substitution with an alanine amino acid residue and / or a serine amino acid residue.

44. The chimeric polypeptide or multimeric polypeptide according to claim 43, wherein the substitution comprises K23A and C74S.

45. A chimeric polypeptide or multimeric polypeptide comprising: (i) an amino acid sequence of SEQ ID NO: 90 having one or more amino acid substitutions relative to SEQ ID NO: 90, wherein one or more amino acid substitutions relative to SEQ ID NO: 90 include K2, R53, R174, R176, or a combination thereof; and (ii) a polypeptide of SEQ ID NO: 4, a polypeptide having at least 70% sequence identity with SEQ ID NO: 4; or a polypeptide comprising SEQ ID NO: 4 having one or more amino acid substitutions relative to SEQ ID NO:

4.

46. The chimeric polypeptide or multimeric polypeptide according to claim 45, wherein the polypeptide comprising the substituted amino acid sequence of SEQ ID NO: 90 comprises at least 70% sequence identity with SEQ ID NO:

90.

47. The chimeric polypeptide or multimeric polypeptide according to claim 45 or 46, wherein the polypeptide comprises an amino acid substitution relative to C15, C26, C59, C69 of SEQ ID NO: 90 or a combination thereof.

48. The chimeric polypeptide or multimeric polypeptide according to claim 47, wherein one or more than one amino acid substitution is a substitution with serine.

49. The chimeric polypeptide or multimeric polypeptide according to any one of claims 45 to 48, wherein the polypeptide comprises a polypeptide having one or more than one amino acid substitution relative to SEQ ID NO: 4, and wherein the one or more than one amino acid substitution comprises C74.

50. The chimeric polypeptide or multimeric polypeptide according to any one of claims 45 to 49, wherein the polypeptide comprising the substituted amino acid sequence of SEQ ID NO: 90 is a polypeptide having the amino acid sequence of SEQ ID NO: 4, a polypeptide having an amino acid sequence having at least 70% sequence identity with SEQ ID NO: 4, or the amino proximal end of a polypeptide comprising the substituted amino acid sequence of SEQ ID NO:

4.

51. The chimeric polypeptide or multimeric polypeptide according to any one of claims 45 to 49, wherein the polypeptide comprising the substituted amino acid sequence of SEQ ID NO: 90 is a polypeptide having the amino acid sequence of SEQ ID NO: 4, a polypeptide having an amino acid sequence having at least 70% sequence identity with SEQ ID NO: 4, or the carboxyl proximal end of a polypeptide comprising the substituted amino acid sequence of SEQ ID NO:

4.

52. The chimeric polypeptide or multimeric polypeptide according to any one of claims 45 to 51, wherein one or more than one amino acid substitution relative to SEQ ID NO: 90 comprises K2, and wherein one or more than one amino acid substitution relative to SEQ ID NO: 4 comprises C74.

53. The chimeric polypeptide or multimeric polypeptide according to claim 52, wherein the amino acid substitution at K2 and / or C74 is an amino acid substitution with an alanine amino acid residue and / or a serine amino acid residue.

54. The chimeric polypeptide or multimeric polypeptide according to claim 53, wherein the substitution comprises K2A and C74S.

55. A chimeric polypeptide or multimeric polypeptide, comprising: (i) The EBI3 subunit of IL-35, wherein the EBI3 subunit comprises less than 200 amino acids and comprises the amino acid sequence of SEQ ID NO: 92 or an amino acid sequence having at least 70% sequence identity with SEQ ID NO: 92, and (ii) The polypeptide of SEQ ID NO: 4, a polypeptide having at least 70% sequence identity with SEQ ID NO: 4; or a polypeptide comprising SEQ ID NO: 4 with one or more amino acid substitutions relative to SEQ ID NO:

4.

56. The chimeric polypeptide or multimeric polypeptide according to claim 55, wherein the EBI3 subunit comprises one or more amino acid substitutions relative to SEQ ID NO: 92, and wherein the one or more amino acid substitutions include R33, R154, R156, or a combination thereof.

57. The chimeric polypeptide or multimeric polypeptide according to claim 55 or 56, wherein the EBI3 subunit consists of 189 amino acids.

58. The chimeric polypeptide or multimeric polypeptide according to any one of claims 55 to 57, wherein the EBI3 subunit comprises a substitution of C6, C39, C49, or a combination thereof relative to SEQ ID NO:

92.

59. The chimeric polypeptide or multimeric polypeptide according to claim 58, wherein one or more amino acid substitutions are substitutions with serine.

60. The chimeric polypeptide or multimeric polypeptide according to any one of claims 55 to 59, wherein the polypeptide comprises a polypeptide having one or more amino acid substitutions relative to SEQ ID NO: 4, and wherein the one or more amino acid substitutions include C74.

61. The chimeric polypeptide or multimeric polypeptide according to claim 60, wherein the amino acid substitution includes C74S.

62. The chimeric polypeptide or multimeric polypeptide according to any one of claims 55 to 61, wherein the EBI3 subunit is the amino terminus of a polypeptide having the amino acid sequence of SEQ ID NO: 4, a polypeptide having an amino acid sequence with at least 70% sequence identity with SEQ ID NO: 4, or a polypeptide comprising a substituted amino acid sequence of SEQ ID NO:

4.

63. The chimeric polypeptide or multimeric polypeptide according to any one of claims 55 to 60, wherein the EBI3 subunit is the carboxyl terminus of a polypeptide having the amino acid sequence of SEQ ID NO: 4, a polypeptide having an amino acid sequence with at least 70% sequence identity with SEQ ID NO: 4, or a polypeptide comprising a substituted amino acid sequence of SEQ ID NO:

4.

64. The chimeric polypeptide or multimeric polypeptide according to any one of claims 35 to 63, wherein the chimeric polypeptide comprises a linker between (i) and (ii).

65. The chimeric polypeptide or multimeric polypeptide according to claim 64, wherein the linker comprises or consists of any one of the amino acid sequences of SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NOs: 75 to 85.

66. The chimeric polypeptide or multimeric polypeptide according to claim 64 or 65, wherein the linker comprises glycine residues and serine residues.

67. The chimeric polypeptide or multimeric polypeptide according to claim 66, wherein the linker comprises (G3S) n linker, where n is an integer from 1 to 10.

68. The chimeric polypeptide or multimeric polypeptide according to claim 67, wherein the linker comprises or consists of the amino acid sequence of SEQ ID NO:

3.

69. The chimeric polypeptide or multimeric polypeptide according to any one of claims 35 to 68, wherein the chimeric polypeptide further comprises a serum albumin (SA) polypeptide.

70. The chimeric polypeptide or multimeric polypeptide according to claim 69, wherein the SA polypeptide comprises a human SA (HSA) polypeptide.

71. The chimeric polypeptide or multimeric polypeptide according to claim 69 or 70, wherein the SA polypeptide comprises a polypeptide having the amino acid sequence of SEQ ID NO: 6 or a polypeptide having at least 70% sequence identity with the polypeptide having the amino acid sequence of SEQ ID NO:

6.

72. The chimeric polypeptide or multimeric polypeptide according to claim 71, wherein the polypeptide comprises one or more amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 6, wherein the one or more amino acid substitutions include C34.

73. The chimeric polypeptide or multimeric polypeptide according to claim 72, wherein the amino acid substitution at C34 is a substitution with an alanine amino acid residue or a serine amino acid residue.

74. The chimeric polypeptide or multimeric polypeptide according to any one of claims 69 to 72, wherein the SA polypeptide is carboxy - proximal to (i) and / or (ii).

75. The chimeric polypeptide or multimeric polypeptide according to any one of claims 69 to 72, wherein the SA polypeptide is amino - proximal to (i) and / or (ii).

76. The chimeric polypeptide or multimeric polypeptide according to any one of claims 69 to 75, wherein the chimeric polypeptide comprises a linker between the SA polypeptide and the polypeptide of (i) and / or (ii).

77. The chimeric polypeptide or multimeric polypeptide according to claim 76, wherein the linker comprises or consists of any one amino acid sequence of SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NOs: 75 to 85.

78. The chimeric polypeptide or multimeric polypeptide according to claim 76 or 77, wherein the linker comprises glycine residues and serine residues.

79. The chimeric polypeptide or multimeric polypeptide according to claim 78, wherein the linker comprises a (G3S) n linker, where n is an integer from 1 to 10 or the linker comprises the linker of SEQ ID NO:

75.

80. The chimeric polypeptide or multimeric polypeptide according to any one of claims 76 to 79, wherein the linker comprises SEQ ID NO: 5 or SEQ ID NO:

75.

81. The chimeric polypeptide or multimeric polypeptide according to any one of claims 76 to 80, wherein the linker consists of SEQ ID NO: 5 or SEQ ID NO:

75.

82. The chimeric polypeptide or multimeric polypeptide according to any one of claims 35 to 81, wherein the chimeric polypeptide comprises an amino acid sequence having at least 70% sequence identity with SEQ ID NO: 1 or SEQ ID NO:

91.

83. The chimeric polypeptide or multimeric polypeptide according to any one of claims 35 to 82, wherein the chimeric polypeptide or multimeric polypeptide further comprises an ECM affinity peptide.

84. The chimeric polypeptide or multimeric polypeptide according to claim 83, wherein the ECM affinity peptide is carboxy - proximal to (i) and / or (ii).

85. The chimeric polypeptide or multimeric polypeptide according to claim 83, wherein the ECM affinity peptide is amino - proximal to (i) and / or (ii).

86. The chimeric polypeptide or multimeric polypeptide according to any one of claims 83 to 85, wherein the ECM affinity peptide comprises any one amino acid sequence of SEQ ID NOs: 9 to 27 or SEQ ID NO: 88, or an amino acid sequence having at least 70% sequence identity with any one amino acid sequence of SEQ ID NOs: 9 to 27 or SEQ ID NO:

88.

87. The chimeric polypeptide or multimeric polypeptide according to any one of claims 83 to 86, wherein the chimeric polypeptide comprises a linker between the ECM affinity peptide and the polypeptide of (i) and / or (ii).

88. The chimeric polypeptide or multimeric polypeptide according to claim 87, wherein the linker comprises or consists of any one of the amino acid sequences of SEQ ID NO: 3, SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NOs: 75 to 85.

89. The chimeric polypeptide or multimeric polypeptide according to any one of claims 35 to 88, wherein the chimeric polypeptide or multimeric polypeptide further comprises an Fc peptide.

90. The chimeric polypeptide or multimeric polypeptide according to claim 89, wherein the Fc peptide is carboxy-proximal to (i) and / or (ii).

91. The chimeric polypeptide or multimeric polypeptide according to claim 89, wherein the Fc peptide is amino-proximal to (i) and / or (ii).

92. The chimeric polypeptide or multimeric polypeptide according to any one of claims 89 to 91, wherein the Fc peptide comprises the amino acid sequence of SEQ ID NO: 86 or SEQ ID NO: 87, or an amino acid sequence having at least 70% sequence identity with SEQ ID NO: 86 or SEQ ID NO:

87.

93. The chimeric polypeptide or multimeric polypeptide according to any one of claims 89 to 92, wherein the chimeric polypeptide comprises a linker between the Fc peptide and (i) and / or (ii).

94. The chimeric polypeptide or multimeric polypeptide according to claim 93, wherein the linker comprises or consists of any one of the amino acid sequences of SEQ ID NO: 3, SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NOs: 75 to 85.

95. The chimeric polypeptide or multimeric polypeptide according to any one of claims 35 to 94, wherein the chimeric polypeptide or multimeric polypeptide further comprises an albumin binding protein (ABP).

96. The chimeric polypeptide or multimeric polypeptide according to claim 89, wherein the ABP is carboxy-proximal to (i) and / or (ii).

97. The chimeric polypeptide or multimeric polypeptide according to claim 89, wherein the ABP is amino-proximal to (i) and / or (ii).

98. The chimeric polypeptide or multimeric polypeptide according to any one of claims 95 to 97, wherein the ABP comprises the amino acid sequence of SEQ ID NO: 93, or an amino acid sequence having at least 70% sequence identity with SEQ ID NO:

93.

99. The chimeric polypeptide or multimeric polypeptide according to any one of claims 95 to 98, wherein the chimeric polypeptide comprises a linker between the ABP and (i) and / or (ii).

100. The chimeric polypeptide or multimeric polypeptide according to claim 99, wherein the linker comprises or consists of any one of the amino acid sequences of SEQ ID NO: 3, SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NOs: 75 to 85.

101. The polypeptide according to any one of claims 1 to 100, wherein the polypeptide comprises any one of the amino acid sequences of SEQ ID NOs: 1 to 93, or an amino acid sequence having at least 70% sequence identity with any one of the amino acid sequences of SEQ ID NOs: 1 to 93.

102. The polypeptide according to any one of claims 1 to 101, wherein the substitution is a substitution with a non-polar amino acid.

103. The polypeptide according to any one of claims 1 to 102, wherein the substitution is a substitution with alanine, leucine, isoleucine, proline, tryptophan, valine, phenylalanine, or methionine.

104. The polypeptide according to claim 103, wherein the substitution is an amino acid substitution with alanine.

105. The polypeptide according to any one of claims 1 to 102, wherein the amino acid substitution is a substitution with serine or glycine.

106. The polypeptide according to any one of claims 1 to 102, wherein the amino acid substitution does not include a substitution with lysine or arginine.

107. A nucleic acid encoding the polypeptide according to any one of claims 1 to 106.

108. An expression vector comprising the nucleic acid according to claim 107.

109. A host cell comprising the polypeptide according to any one of claims 1 to 103, the nucleic acid according to claim 107, or the expression vector according to claim 108.

110. A pharmaceutical composition comprising the polypeptide according to any one of claims 1 to 106, the nucleic acid according to claim 107, the expression vector according to claim 108, or the host cell according to claim 109.

111. A method for preparing a cell, the method comprising transferring the nucleic acid according to claim 107 or the expression vector according to claim 108 into a cell.

112. A method for preparing a polypeptide, the method comprising transferring the expression vector according to claim 108 into a cell and incubating the cell under conditions sufficient to express the polypeptide encoded on the expression vector.

113. A method for treating an autoimmune or inflammatory disease in a subject, the method comprising administering to the subject a polypeptide according to any one of claims 1 to 106 or a pharmaceutical composition according to claim 110.

114. A method for reducing inflammation in a subject, for delivering cytokine therapy to lymph nodes and / or increasing tolerogenic dendritic cells in lymph nodes, which comprises administering to the subject a polypeptide according to any one of claims 1 to 106 or a pharmaceutical composition according to claim 110.

115. The method according to claim 114, wherein the inflammation is associated with an autoimmune or inflammatory disease.

116. The method according to claim 113 or 115, wherein the autoimmune or inflammatory disease includes cytokine release syndrome, cytokine storm, arthritis, inflammatory bowel disease, scleroderma, inflammatory bowel disease, idiopathic pulmonary fibrosis, multiple sclerosis, type 1 diabetes, Crohn's disease, psoriasis, acute inflammation, chronic inflammation, neuroinflammation, acute respiratory distress syndrome, rheumatoid arthritis, fibrosis, infection, allergy, inflammation treatment-related adverse events, ischemic stroke, pulmonary hypertension, ischemia, cardiogenic stroke, stroke, atherosclerosis, wound healing, peripheral artery disease, cerebral ischemia reperfusion (I / R) injury, myocardial infarction and related inflammatory diseases.

117. The method according to claim 116, wherein the autoimmune or inflammatory disease includes multiple sclerosis.

118. The method according to claim 116, wherein the autoimmune or inflammatory disease includes rheumatoid arthritis.

119. The method according to claim 116, wherein the autoimmune or inflammatory disease includes cytokine release syndrome.

120. The method according to any one of claims 113 to 118, wherein the composition is administered systemically.

121. The method according to claim 120, wherein the composition is administered by intravenous injection.

122. The method according to any one of claims 113 to 118, wherein the composition is administered locally.

123. The method according to claim 122, wherein the composition is administered to the site of inflammation or a site adjacent to the site of inflammation.

124. The method according to any one of claims 113 to 123, wherein the subject is a subject previously treated with an anti-inflammatory agent, anti-inflammatory treatment or autoimmune treatment.

125. The method according to claim 124, wherein the subject is a subject determined to be non-responsive to the previous treatment.

126. The method according to any one of claims 113 to 123, wherein the subject has not been previously treated for an inflammatory disease or an autoimmune disease.

127. The method according to any one of claims 113 to 126, wherein the method further comprises administering an additional inflammatory treatment or autoimmune treatment.

128. The method according to claim 127, wherein the additional inflammatory treatment or autoimmune treatment comprises an anti-cytokine reagent or a steroid.

129. The method according to claim 128, wherein the additional inflammatory treatment or autoimmune treatment comprises one or more than one of the following drugs: steroids, corticosteroids, anti-TNF-α treatment, anti-integrin treatment, infliximab, mesalazine, and vedolizumab, fingolimod, interferon-β, dimethyl fumarate, teriflunomide, integrin α4β1, anti-αLβ2 antibody, anti-IL-6R agent, anti-IL-6 agent, and Janus kinase inhibitors (such as tofacitinib, baricitinib, upadacitinib).

130. The method according to claim 129, wherein the steroids include dexamethasone, methylprednisolone, and / or prednisolone.

131. The method according to any one of claims 113 to 130, wherein the subject is a human subject.

132. The method according to any one of claims 113 to 131, wherein the polypeptide or composition reduces the number of Th17 cells in the subject.

133. The method according to any one of claims 113 to 132, wherein the polypeptide or composition inhibits the function of Th17 cells in the subject.

134. The method according to any one of claims 113 to 133, wherein the polypeptide or composition is administered to the subject by a prefilled syringe.

135. The method according to any one of claims 113 to 134, wherein the polypeptide or composition is administered at a dose of 0.1 mg / kg to 50 mg / kg.

136. The method according to any one of claims 113 to 135, the method further comprises detecting the anti-inflammatory cytokine in the lymph nodes of the subject.

137. The method according to claim 136, wherein the detection comprises obtaining a lymph sample from the subject.

138. The method according to claim 137, wherein the detection comprises detecting the presence of an anti-inflammatory cytokine in the lymph sample.

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