Methods and compositions for treating inflammatory and autoimmune conditions

Abstract

The present disclosure relates to the manipulation of IL-35 cytokine polypeptides to achieve improved therapies for inflammatory diseases and conditions. Thus, aspects of the present disclosure relate to polypeptides comprising SEQ ID NO:2 and comprising 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 combinations thereof. Further aspects relate to polypeptides comprising SEQ ID NO:4 and comprising one or more amino acid substitutions relative to SEQ ID NO:4, wherein the one or more amino acid substitutions include C74. Further aspects relate to polypeptides comprising SEQ ID NO:6 and comprising one or more amino acid substitutions relative to SEQ ID NO:6, wherein the one or more amino acid substitutions include C34.

Description

Technical Field

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

[0002] Sequence Listing This application includes a sequence listing submitted in XML format, which is hereby incorporated by reference in its entirety. The XML copy created on Jun. 24, 2023, is named "ARCDP0778WO.xml" and is 122,913 bytes in size.

[0003] I. Field of the Invention The present invention broadly relates to the field of medicine. More specifically, the present invention relates to compositions and methods involving anti-inflammatory agents engineered to treat autoimmune and inflammatory conditions.

Background Art

[0004] II. Background Multiple sclerosis (MS) is a disabling autoimmune disease that affects millions of people worldwide. Auto-reactive immune cells target the central nervous system (CNS), causing demyelination and resulting in local damage to the white matter. Lymphocytes and macrophages infiltrating the CNS cause axonal damage. Recent studies have shown that Th17 cells activated in secondary lymphoid organs (SLOs) migrate to the spinal cord and brain and play an important role in MS disease onset and severity. Therefore, inhibition of lymphocyte migration to the CNS and induction of an immunosuppressive microenvironment in SLOs would provide an effective treatment for MS. FTY720 (fingolimod) and anti-integrin α4 antibody (natalizumab) are clinically used to treat MS by sequestering lymphocytes in LNs and preventing them from reacting with self-antigens in the target tissue. Experimental autoimmune encephalomyelitis (EAE) is a widely accepted mouse model of MS that reflects many features of the disease progression and pathogenesis, including lymphocyte migration to the CNS and demyelination.

[0005] Rheumatoid arthritis (RA) is an autoimmune disease that is currently controlled by treatment with inhibitors of inflammatory pathways. The pathological features of RA are synovitis and joint destruction, which cause severe pain and joint dysfunction. The causative antigen of RA has not been fully elucidated, but collagen recognition by immune cells plays an important role. During the progression of RA, self-antigen-specific T cells, particularly Th17 cells, are activated and produce inflammatory cytokines, including IL-17. Inflammatory cytokines such as TNF-a and IL-6 within the joint 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 bone within the joint. Current RA treatment strategies are symptomatic, and considering that many inflammatory cytokines are involved in the progression of RA, various biological therapeutic agents, such as antibodies or soluble receptors against TNF-a, have been developed and approved for clinical use.

[0006] Various strategies for the treatment of autoimmune disorders such as MS and RA, including the use of anti-inflammatory cytokines, have been explored. So far, such strategies have not been translated into clinical use. Compositions and methods for the treatment of autoimmune disorders, including MS and RA, including effective delivery of anti-inflammatory cytokines, are still needed. Summary of the Invention

[0007] The present disclosure relates to the manipulation of IL-35 cytokine polypeptides to achieve improved therapies for inflammatory diseases and conditions. Accordingly, the present disclosure describes a polypeptide comprising 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 include K23, R74, R195, R197, or combinations thereof. Substitutions of K23, R74, R195, and / or R197 with respect to SEQ ID NO:2 may be excluded in the polypeptides of the present disclosure. Similarly disclosed is a polypeptide comprising the 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. The polypeptide may exclude the polypeptide of SEQ ID NO:4 having a C74 substitution. Similarly provided is a polypeptide comprising the 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. The polypeptide may exclude the polypeptide of SEQ ID NO:6 having a C34 substitution. Similarly described is a polypeptide comprising the 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 combinations thereof. Substitutions of K2, R53, R174, and / or R176 with respect to SEQ ID NO:90 may be excluded in the polypeptides of the present disclosure. Similarly described is 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 combinations thereof.Substitutions of K23, R74, R195, R197, C320 and / or C497 with respect to SEQ ID NO:1 may be excluded in the polypeptides of the present disclosure. Similarly described are polypeptides 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. Similarly described are polypeptides comprising the amino acid sequence of SEQ ID NO:91 having one or more amino acid substitutions with respect to SEQ ID NO:91, wherein the one or more amino acid substitutions comprise K2, R53, R174, R176, C299, C476 or combinations thereof. Substitutions of K2, R53, R174, R176, C299 and / or C476 with respect to SEQ ID NO:91 may be excluded in the polypeptides of the present disclosure.

[0008] Also described are chimeric or multimeric polypeptides comprising: (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 combinations 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 substitutions relative to SEQ ID NO:4. Also described are chimeric or multimeric polypeptides 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 the one or more amino acid substitutions relative to SEQ ID NO:90 include K2, R53, R174, R176, or combinations 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 substitutions relative to SEQ ID NO:4. Also provided are chimeric or multimeric polypeptides comprising: (i) the EBI3 subunit of IL-35, comprising less than 200 amino acids and comprising 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 substitutions relative to SEQ ID NO:4.

[0009] Also provided are pharmaceutical compositions and host cells comprising the polypeptides of the present disclosure. 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 comprise the nucleic acids, expression vectors and / or host cells of the present disclosure.

[0010] The method relates to a method of generating cells, comprising the step of introducing a nucleic acid or expression vector of the present disclosure into a cell. Also provided is a method for producing a polypeptide, comprising the step of introducing an expression vector of the present disclosure into a cell and incubating the cell under conditions sufficient for expression of the polypeptide encoded on the expression vector. Also described is a method for treating an autoimmune condition or an inflammatory condition in a subject, comprising the step of administering a polypeptide or pharmaceutical composition of the present disclosure to the subject. Also provided is a method for reducing inflammation in a subject, comprising the step of administering a polypeptide or pharmaceutical composition of the present disclosure to the subject. The method also comprises a method for delivering cytokine therapy to lymph nodes in a subject and / or a method for increasing tolerogenic dendritic cells in lymph nodes in a subject, comprising the step of administering a polypeptide or pharmaceutical composition of the present disclosure to the subject.

[0011] The EBI3 polypeptide may have 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, 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% sequence identity with SEQ ID NO:92. The EBI3 subunit may contain one or more amino acid substitutions, and the one or more amino acid substitutions include R33, R154, R156 or a combination thereof with respect to SEQ ID NO:92. Substitutions of R33, R154 and / or R156 with respect to SEQ ID NO:92 may be excluded in the polypeptides of the present disclosure. The EBI3 subunit may consist of 189 amino acids.The EBI3 subunit may contain 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 range of amino acids derivable therein, or may contain at most 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 range of amino acids derivable therein, or may contain at least 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 range of amino acids derivable therein, or may 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 range of amino acids derivable therein. The polypeptide may contain substitutions of C6, C39, C49 or combinations thereof with respect to SEQ ID NO:92. Substitutions of C6, C39 and / or C49 with respect to SEQ ID NO:92 may be excluded in the polypeptides of the present disclosure. The substitution may be a substitution with serine. The polypeptide may contain one, two or three of the substitutions C6, C39 and C49.The EBI3 subunit can be amino-proximal to 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 a polypeptide comprising a substituted amino acid sequence of SEQ ID NO:4. The EBI3 subunit can be carboxy-proximal to 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 a polypeptide comprising a substituted amino acid sequence of SEQ ID NO:4. When the first region is bound to the carboxy terminus of the second region, the first region is carboxy-proximal to the second region. There may be additional intervening amino acid residues between the first region and the second region. Thus, these regions do not necessarily have to be directly adjacent unless specifically specified as having no intervening amino acid residues. The term "amino-proximal" is similarly defined in that when the first region is bound to the amino terminus of the second region, the first region is amino-proximal to the second region. Similarly, there may be additional intervening amino acid residues between the first region and the second region unless otherwise specified.

[0012] A 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 have a sequence identity 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 therein) with SEQ ID NO:2, or may comprise an amino acid sequence having a sequence identity of 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 therein) with SEQ ID NO:2. The polypeptide may comprise one or more substitutions of C36, C47, C80 and / or C90 with respect to SEQ ID NO:2. Substitutions of C36, C47, C80 and C90 with respect to SEQ ID NO:2 may be excluded in the polypeptides of the present disclosure. The substitution may be a substitution with serine. The polypeptide may comprise one, two, three or four of the substitutions at C36, C47, C80 and C90.

[0013] A polypeptide and / or 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 have 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% (or any range derivable therein) sequence identity with SEQ ID NO:90, or may comprise an amino acid sequence 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 therein) sequence identity with SEQ ID NO:90. The polypeptide may comprise one or more substitutions of C15, C26, C59 and C69 relative to SEQ ID NO:90. Substitutions of C15, C26, C59 and / or C69 with respect to SEQ ID NO:90 may be excluded in the polypeptides of the present disclosure. The substitution may be a substitution with serine. The polypeptide may comprise one, two, three or four of the substitutions at C15, C26, C59 and C69.

[0014] The polypeptide may comprise an amino acid sequence having at least 70% sequence identity with SEQ ID NO:4. The polypeptide may have a sequence identity 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 therein) with SEQ ID NO:4, or may comprise an amino acid sequence having a sequence identity of 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 therein) with SEQ ID NO:4. The polypeptide comprising one or more amino acid substitutions relative to SEQ ID NO:4 may include a substitution at C74. The substitution at C74 with respect to SEQ ID NO:4 may be excluded in the polypeptides of the present disclosure.

[0015] The polypeptide may comprise an amino acid sequence having at least 70% sequence identity with SEQ ID NO:6. The polypeptide may have a sequence identity 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 therein) with SEQ ID NO:6, or may comprise an amino acid sequence having a sequence identity of 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 therein) with SEQ ID NO:6.

[0016] The polypeptide may comprise an amino acid sequence having at least 70% sequence identity with SEQ ID NO:1. The polypeptide may have 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% (or any range derivable therein) sequence identity with SEQ ID NO:1, or may comprise an amino acid sequence 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 therein) sequence identity with SEQ ID NO:1. One or more amino acid substitutions may include K23, C320 and / or C497 relative to SEQ ID NO:1.

[0017] The polypeptide may comprise an amino acid sequence having at least 70% sequence identity with SEQ ID NO:91. The polypeptide may have 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% (or any range derivable therein) sequence identity with SEQ ID NO:91, or may comprise an amino acid sequence having 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% (or any range derivable therein) sequence identity with SEQ ID NO:91. One or more amino acid substitutions may include K2, C299 and / or C476 relative to SEQ ID NO:91. The polypeptide may include substitutions of C15, C26, C59, C69 or combinations thereof relative to SEQ ID NO:91. Substitutions of C15, C26, C59 and / or C69 with respect to SEQ ID NO:91 may be excluded in the polypeptides of the present disclosure. The substitution may be a substitution with serine. The polypeptide may include one, two, three or four of the substitutions at C15, C26, C59 and C69.

[0018] The polypeptide may further comprise an ECM affinity peptide, a serum protein, an albumin-binding protein, and / or an Fc peptide, or may exclude an ECM affinity peptide, a serum protein, an albumin-binding protein, and / or an Fc peptide. The ECM affinity peptides are described throughout this disclosure. The ECM affinity peptide may comprise an amino acid sequence of one of SEQ ID NOs: 9-27 or 88, or an amino acid sequence having at least 70% sequence identity with one of SEQ ID NOs: 9-27 or 88, or may exclude an amino acid sequence of one of SEQ ID NOs: 9-27 or 88, or an amino acid sequence having at least 70% sequence identity with one of SEQ ID NOs: 9-27 or 88. The ECM affinity peptide may have a sequence identity 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 therein) with one of SEQ ID NOs: 9-27 or 88, or may include an amino acid sequence having a sequence identity of 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 therein) with one of SEQ ID NOs: 9-27 or 88. The Fc peptide may comprise an amino acid sequence of SEQ ID NO: 86 or 87, or an amino acid sequence having at least 70% sequence identity with SEQ ID NO: 86 or 87, or may exclude an amino acid sequence of SEQ ID NO: 86 or 87, or an amino acid sequence having at least 70% sequence identity with SEQ ID NO: 86 or 87.The Fc peptide may have a sequence identity 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 therein) with SEQ ID NO:86 or 87, or may contain an amino acid sequence having a sequence identity of 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 therein). The ABP may comprise 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, or may exclude 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. The ABP may have a sequence identity 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 therein) with SEQ ID NO:93, or may contain an amino acid sequence having a sequence identity of 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 therein). The ECM affinity peptide, serum protein, ABP and / or Fc peptide may be amino-proximal to a polypeptide comprising a substituted amino acid sequence of one of SEQ ID NO:1, 2, 4, 6, 90, 91 or an EBI3 subunit.The ECM affinity peptide, serum protein and / or Fc peptide can be carboxy-proximal to a polypeptide comprising a substituted amino acid sequence of one of SEQ ID NO: 1, 2, 4, 6, 90, 91 or the EBI3 subunit. The ECM affinity peptide, serum protein, ABP and / or Fc peptide can be amino-proximal to (i) and / or (ii) as defined herein.

[0019] The serum protein can include 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 can have 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% (or any range derivable therein) sequence identity with SEQ ID NO: 6, or can include an amino acid sequence having 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% (or any range derivable therein) sequence identity with SEQ ID NO: 6. The polypeptide can include one or more amino acid substitutions relative to SEQ ID NO: 6, including C34. The substitution in C34 can be a substitution by an alanine amino acid residue or a serine amino acid residue.

[0020] One or more amino acid substitutions relative to SEQ ID NO: 2 can include K23, and one or more amino acid substitutions relative to SEQ ID NO: 4 can include C74. The K23 and / or C74 substitutions can be substitutions by an alanine amino acid residue or a serine amino acid residue. The K23 and C74 substitutions can include K23A and / or C74S substitutions.

[0021] A polypeptide comprising the substituted amino acid sequence of SEQ ID NO:2 may be amino-proximal to 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 to SEQ ID NO:4, or a polypeptide comprising the substituted amino acid sequence of SEQ ID NO:4. A polypeptide comprising the substituted amino acid sequence of SEQ ID NO:2 may be carboxy-proximal to 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 to SEQ ID NO:4, or a polypeptide comprising the substituted amino acid sequence of SEQ ID NO:4.

[0022] A polypeptide comprising the substituted amino acid sequence of SEQ ID NO:90 may be amino-proximal to 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 to SEQ ID NO:4, or a polypeptide comprising the substituted amino acid sequence of SEQ ID NO:4. A polypeptide comprising the substituted amino acid sequence of SEQ ID NO:90 may be carboxy-proximal to 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 to SEQ ID NO:4, or a polypeptide comprising the substituted amino acid sequence of SEQ ID NO:4.

[0023] One or more amino acid substitutions relative to SEQ ID NO:90 may include K2 or may consist of K2. One or more amino acid substitutions relative to SEQ ID NO:4 may include C74 or may consist of C74. The polypeptide may include substitutions at K2 and C74 of SEQ ID NO:90 and 4, respectively, or may consist of substitutions at K2 and C74 of SEQ ID NO:90 and 4, respectively. The substitutions may include K2A and C74S.

[0024] Linkers such as amino acid sequences or peptide mimetic sequences can be inserted into the polypeptides of the present disclosure. The linker can have one or more properties including mobility in three-dimensional structure, inability to form a regular secondary structure, or hydrophobicity or charge that can promote or interact with any domain. Examples of amino acids commonly found in mobile 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 therein). Other nearly neutral amino acids such as Thr and Ala can also be used in linker sequences. 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). Peptides and the heavy or light chains of antibodies can be joined by peptide sequences having about 1 to 25 amino acid residues. Examples of linkers can also include chemical moieties and conjugating agents such as sulfo-succinimidyl derivatives (sulfo-SMCC, sulfo-SMPB), disuccinimidyl suberate (DSS), disuccinimidyl glutarate (DSG), and disuccinimidyl tartrate (DST). Examples of linkers can include C N(Here, linear carbon chains with N = 1 to 100 carbon atoms, for example, N = 2, 3, 4, 5, 6, 7, 8, 9, or 10) etc. can be further mentioned. 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 sulfosuccinimidyl-4-[N-maleimidomethyl]cyclohexane-1-carboxylate (smcc). The sulfo-smcc conjugation is carried out via a maleimide group that reacts with sulfhydryl (thiol, --SH), and its sulfo-NHS ester is reactive towards primary amines (found at the lysine and N-terminus of proteins or peptides). Further, the linker can be maleimidocaproyl (mc). One or more of these linkers can be specifically excluded from the aspect.

[0025] The chimeric polypeptide may contain a linker between (i) and (ii) as defined herein. A linker may be present between (i) and (ii) and a serum protein, Fc peptide, ABP, HSA and / or ECM affinity peptide. The linker may comprise or consist of one amino acid sequence of SEQ ID NO: 3, 5, 7, 8 or 75 - 85. The linker may contain glycine residues and serine residues. The linker is (G3S) where n is an integer from 1 to 10 nIt may contain a linker. The linker may comprise or consist of the amino acid sequence of SEQ ID NO:3, or may comprise or consist of the amino acid sequence of SEQ ID NO:5, or may comprise or consist of the amino acid sequence of SEQ ID NO:75. The linker between one or more of (i), (ii), serum protein, Fc peptide, ABP and / or ECM affinity peptide, the linker proximal to amino to one or more of (i), (ii), serum protein, Fc peptide, ABP and / or ECM affinity peptide, the linker proximal to carboxy to one or more of (i), (ii), serum protein, Fc peptide, ABP and / or ECM affinity peptide, or the linker adjacent to one or more of (i), (ii), serum protein, Fc peptide, ABP and / or ECM affinity peptide may be excluded in the polypeptide of the present disclosure.

[0026] The polypeptide may contain or may further contain a serum albumin (SA) polypeptide. The SA polypeptide may be further defined as a human SA polypeptide. The SA polypeptide may include 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 may have a sequence identity 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 therein) with SEQ ID NO:6, or may include an amino acid sequence having a sequence identity of 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 therein) with SEQ ID NO:6. The polypeptide may include one or more amino acid substitutions relative to SEQ ID NO:6, including C34. The substitution at C34 may be a substitution of cysteine with any other amino acid. The substitution at C34 may be a substitution with an alanine amino acid residue or a serine amino acid residue. The SA polypeptide may be carboxy-proximal to (i) and / or (ii) as defined herein. The SA polypeptide may be amino-proximal to (i) and / or (ii) as defined herein.

[0027] The substitution can be a substitution with a non-polar amino acid. Substitutions at positions K23, R74, R195, R197 with respect to SEQ ID NO:2, substitution at position C74 with respect to SEQ ID NO:4, substitution at position C34 with respect to SEQ ID NO:6, or substitutions at positions K23, R74, R195, R197, C320, C497 with respect to SEQ ID NO:1 can be substitutions with alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine or valine amino acid residues. The substitution can be a substitution with alanine, leucine, isoleucine, proline, tryptophan, valine, phenylalanine or methionine. The substitutions described herein may or may not include substitutions with alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine or valine amino acid residues. The substitution can be a substitution with alanine, leucine, isoleucine, proline, tryptophan, valine, phenylalanine or methionine. Substitutions at positions K23, R74, R195, R197 with respect to SEQ ID NO:2, substitution at position C74 with respect to SEQ ID NO:4, substitution at position C34 with respect to SEQ ID NO:6, or substitutions at positions K23, R74, R195, R197, C320, C497 with respect to SEQ ID NO:1 can be substitutions with 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. Substitutions with lysine or arginine may be excluded in the polypeptides of the present disclosure. Substitutions with any other amino acid may also be excluded in any polypeptide of the present disclosure.

[0028] The polypeptide of the present disclosure may include a signal peptide. The signal peptide may include SEQ ID NO:89. The polypeptide of the present disclosure has one or more amino acid sequences among SEQ ID NOs: 1 to 93, or has a sequence identity 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) with one or more of SEQ ID NOs: 1 to 93, or may include a polypeptide having an amino acid sequence having a sequence identity 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) with one or more of SEQ ID NOs: 1 to 93, or may consist of a polypeptide having an amino acid sequence having a sequence identity 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) with one or more of SEQ ID NOs: 1 to 93. Any one of SEQ ID NOs: 1 to 93 may be excluded in the polypeptide of the present disclosure.

[0029] The inflammation treated by the polypeptides and compositions of the present disclosure can be inflammation associated with an autoimmune condition or an inflammatory condition. The autoimmune condition or inflammatory condition can 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, infectious disease, allergy, adverse events related to inflammatory therapy, ischemic stroke, pulmonary hypertension, ischemia, cardiac stroke, stroke, atherosclerosis, wound healing, peripheral arterial disease, cerebral ischemia-reperfusion (I / R) injury, myocardial infarction and related inflammatory diseases, or can exclude 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, infectious disease, allergy, adverse events related to inflammatory therapy, ischemic stroke, pulmonary hypertension, ischemia, cardiac stroke, stroke, atherosclerosis, wound healing, peripheral arterial disease, cerebral ischemia-reperfusion (I / R) injury, myocardial infarction and related inflammatory diseases. The inflammation treated can be inflammation associated with viral infections such as influenza, coronavirus or SARS-CoV-2 infection. The inflammation treated can be inflammation associated with the administration of immunotherapy such as the administration of checkpoint inhibitor therapy, the administration of CAR-T cells, or the administration of engineered TCR-T cells. The method can be for treating cytokine release syndrome or cytokine storm. The autoimmune condition or inflammatory condition can include multiple sclerosis. The autoimmune condition or inflammatory condition can include rheumatoid arthritis.

[0030] The composition or polypeptide can be administered by any route of administration described throughout the present disclosure. The composition or polypeptide can be administered systemically. The composition or polypeptide can be administered by intravenous injection. The composition can be administered locally. The composition can be administered at or adjacent to the site of inflammation.

[0031] The subject can be a subject previously treated with an anti-inflammatory agent, anti-inflammatory therapy or autoimmune therapy. The subject can be a subject determined to be non-responsive to a previous treatment. The subject can be a subject not previously treated for an inflammatory disease or autoimmune disease. The method may further comprise administration of additional anti-inflammatory therapy or autoimmune therapy. The additional anti-inflammatory therapy or autoimmune therapy may include an anti-cytokine agent or steroid, or may exclude an anti-cytokine agent or steroid. The additional anti-inflammatory therapy or autoimmune therapy may include one or more of a steroid, corticosteroid, anti-TNF-α therapy, anti-integrin therapy, infliximab, mesalamine, vedolizumab, fingolimod, interferon-β, dimethyl fumarate, teriflunomide, integrin α4β1, anti-αLβ2 antibody, anti-IL-6R agent, anti-IL-6 agent, and Janus kinase inhibitor (e.g., tofacitinib, baricitinib, upadacitinib), or may exclude one or more of a steroid, corticosteroid, anti-TNF-α therapy, anti-integrin therapy, infliximab, mesalamine, vedolizumab, fingolimod, interferon-β, dimethyl fumarate, teriflunomide, integrin α4β1, anti-αLβ2 antibody, anti-IL-6R agent, anti-IL-6 agent, and Janus kinase inhibitor (e.g., tofacitinib, baricitinib, upadacitinib). The steroid may include dexamethasone, methylprednisolone and / or prednisone, or may exclude dexamethasone, methylprednisolone and / or prednisone. The polypeptide or composition can be one that decreases the number of Th17 cells in the subject. The polypeptide or composition can be one that inhibits the function of Th17 cells in the subject. The polypeptide or composition can be administered to the subject via a pre-filled syringe.

[0032] 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 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, 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), at least 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、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), or at most 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、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、It may be administered at a dose of 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.,

[0033] The method may further include detecting an anti-inflammatory cytokine in the lymph nodes of the subject. The method may include obtaining a lymph sample from the subject. The detecting step may include detecting the presence of the polypeptide of the present disclosure in the lymph sample.

[0034] The terms “protein”, “polypeptide” and “peptide” are used interchangeably herein when referring to gene products that include polymers of amino acids.

[0035] The terms “subject”, “mammal” and “patient” are used interchangeably. The subject can be a mammal. The subject can be a human. The subject can be a mouse, rat, rabbit, dog, horse, or a research test animal such as a Drosophila or zebrafish.

[0036] The method and composition are intended to include any exclusion of aspects described herein.

[0037] Throughout this application, the term "about" is used in its plain and ordinary sense in the field of cell and molecular biology to indicate that for a given value, it includes the standard deviation of error for the device or method used to determine that value.

[0038] The use of the words "a" or "an" when used with the term "comprising" can mean "one", but is also consistent with the meanings of "one or more", "at least one", and "one or two or more".

[0039] As used herein, the terms "or" and "and / or" are used to describe combinations of multiple components either in combination or exclusively with each other. For example, "x, y, and / or z" can refer to "x" only, "y" only, "z" only, "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 may be specifically excluded from an aspect or situation.

[0040] The words "comprising" (and any form of "comprising", such as "comprise" and "comprises"), "having" (and any form of "having", such as "have" and "has"), "including" (and any form of "including", such as "includes" and "include"), "characterized by" (and any form of "including", such as "characterized as") or "containing" (and any form of "containing", such as "contains" and "contain") are inclusive or non-limiting and do not exclude additional elements or method steps not recited.

[0041] Compositions and methods for their use can "comprise", "consist essentially of", or "consist of" any of the components or steps disclosed throughout this application. The phrase "consist of" excludes any element, step, or component not specified. The phrase "consist essentially of" limits the scope of the described subject matter to those that do not materially affect the basic and novel characteristics of the specified materials or steps. Aspects or situations described in the context of the term "comprising" are also contemplated to be practicable in the context of the terms "consisting of" or "consisting essentially of".

[0042] It is particularly contemplated that any limitation discussed with respect to one aspect or aspect of the present invention may be applicable to any other aspect or aspect of the present invention. Further, any composition of the present invention may be used in any method of the present invention, and any method of the present invention may be used to make or utilize any composition of the present invention. Aspects of the embodiments described in the examples may also be implemented in other places in different examples or elsewhere in this application, for example, in the context of aspects or aspects discussed in the summary of the invention, the detailed description of the aspects, the claims, and the description of the legends of the figures.

[0043] Other objects, features, and advantages of the present invention will become apparent from the following detailed description. However, since various changes and modifications within the spirit and scope of the present invention will become apparent to those skilled in the art from this detailed description, it should be understood that the detailed description and specific examples are illustrative only while showing specific aspects or aspects of the present invention.

Brief Description of the Drawings

[0044] The following drawings form part of this specification and are included to further demonstrate certain aspects of the present invention. The present invention may be better understood by reference to these drawings in conjunction with the detailed description of the specific embodiments presented herein.

[0045]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Figure 10-1

Figure 10-2

Figure 11

Figure 12A

Figure 12B

Figure 12C

Figure 12D

Figure 12E

Figure 12F

Mode for Carrying Out the Invention

[0046] Detailed Description I. ECM Affinity Peptide Collagen is an extracellular matrix (ECM) protein that regulates various cell biological functions such as proliferation, differentiation, and adhesion in both normal and tumor tissues (Ricard-Blum, Cold Spring Harb Perspect Biol 3:a004978, 2011). Collagen is the most abundant protein in the mammalian body and is present as one or more of 28 isoforms in almost all tissues (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 rarely present in the blood (Dubois et al., Blood 107:3902-06, 2006; Bergmeier and Hynes, Cold Spring Harb Perspect Biol 4:a005132, 2012). Tumor vasculature has been reported to be permeable due to its abnormal structure (Nagy et al., British journal of cancer 100:865, 2009). Therefore, due to its leaky vasculature, 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). Additionally, tumor tissue contains an increased amount of collagen compared to normal tissue (Zhou et al. J Cancer 8:1466-76, 2017; Provenzano et al. BMC Med 6:11, 2008).

[0047] Von Willebrand factor (vWF) is a blood coagulation factor that binds to both type I and type III collagens, as well as to the adhesion receptor GPIb on platelets (Lenting et al., Journal of thrombosis and haemostasis: JTH 10:2428 - 37, 2012; Shahidi Advances in experimental medicine and biology 906:285 - 306, 2017). Upon injury, subendothelial collagen is exposed to plasma, and vWF - collagen binding initiates the thrombotic cascade (Shahidi Advances in experimental medicine and biology 906:285 - 306, 2017; Wu et al. Blood 99:3623 - 28, 2002). Among the reported proteins / peptides of non - bacterial origin, the vWF A domain 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 to be the collagen - binding domain (CBD) (Ribba et al. Thrombosis and Haemostasis 86:848 - 54, 2001). As described above, the inventors considered that a fusion protein with vWF A3 CBD could enable targeted cytokine immunotherapy even in the case of systemic injection due to the exposure of collagen by the leaky vasculature of tumors.

[0048] The ECM - affinity peptide may contain a collagen - binding domain derived from decorin. The ECM - affinity peptide may contain a decorin peptide such as LRELHLNNNC (SEQ ID NO:9) derived from bovine or LRELHLDNNC (SEQ ID NO:10) derived from human.

[0049] The ECM peptide has the following amino acid sequence: It may contain a peptide fragment derived from human decorin represented by TIFF2025521602000001.tif48159.

[0050] The ECM peptide may contain a peptide fragment derived from vWF. The ECM peptide has an amino acid sequence and may contain vWF A1 derived from the human sequence represented by TIFF2025521602000002.tif33160, residues 1237 to 1458 (residues 474 to 695 of mature VWF) or a fragment thereof.

[0051] The ECM peptide has the following amino acid sequence: and may contain all or a fragment of vWF A3 represented by TIFF2025521602000003.tif55160.

[0052] The ECM peptide has the following amino acid sequence: and may contain all or a fragment of vWF A3 represented by TIFF2025521602000004.tif26157.

[0053] The ECM affinity peptide may be a peptide derived from von Willebrand factor (vWF). The sequence of human vWF is as follows: including TIFF2025521602000005.tif187160TIFF2025521602000006.tif172160.

[0054] The peptide may be derived from the vWF A3 domain. The vWF A3 domain is derived from the human sequence, residues 1670 to 1874 (residues 907 to 1111 of mature vWF), and has the following sequence: TIFF2025521602000007.tif26160.

[0055] The ECM affinity peptide may contain a peptide derived from PlGF-2. PlGF-2 has the following sequence: TIFF2025521602000008.tif26159.

[0056] Exemplary PlGF-2 ECM affinity peptides are as follows: It includes TIFF2025521602000009.tif26160.

[0057] The ECM - affinity peptide can be a peptide derived from CXCL - 12γ. The sequence of CXCL - 12γ is as follows: CXCL - 12γ: TIFF2025521602000010.tif11158. Exemplary peptides include all or part of SEQ ID NO:12 and the following peptides: It includes TIFF2025521602000011.tif4128.

[0058] 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 with the above - mentioned ECM or CBD peptide or fragments of those peptides.

[0059] A linker array can be included in the anti-inflammatory agent-peptide construct. For example, at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or more (or any range derivable therefrom) amino acids, 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 or more (or any range derivable therefrom) amino acids, 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,A linker having 100 or more (or any range derivable therefrom) amino acids can separate the antibody and the peptide.

[0060] 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 one that does not bind to fibronectin.

[0061] II. Proteinaceous compositions The polypeptide or polynucleotide of the present disclosure is at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 300, 400, 500, 550, 1000 or more consecutive amino acids or nucleic acids, or any range derivable therefrom, of SEQ ID NOs: 1 to 27Alternatively, 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, 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 or any range of consecutive amino acids or nucleic acids derivable therefrom, and 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 or more variant amino acid or nucleic acid substitutions, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 or more variant amino acid or nucleic acid substitutions, or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 or more variant amino acid or nucleic acid substitutions may be included, or may be at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% similar, identical, or homologous.

[0062] The polypeptide or polynucleotide of the present disclosure is any continuous amino acid sequence of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 300, 400, 500, 550, 1000 or more, or any range of continuous amino acids derivable therefrom, at least 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 300, 400, 500, 550, 1000 or more, or any range of consecutive amino acids derivable therefrom, or at most 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24It may contain 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 300, 400, 500, 550, 1000 or more or any range of consecutive amino acids derivable therefrom.,

[0063] The polypeptides of the present disclosure are amino acids 1 to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269,270、271、272、273、274、275、276、277、278、279、280、281、282、283、284、285、286、287、288、289、290、291、292、293、294、295、296、297、298、299、300、301、302、303、304、305、306、307、308、309、310、311、312、313、314、315、316、317、318、319、320、321、322、323、324、325、326、327、328、329、330、331、332、333、334、335、336、337、338、339、340、341、342、343、344、345、346、347、348、349、350、351、352、353、354、355、356、357、358、359、360、361、362、363、364、365、366、367、368、369、370、371、372、373、374、375、376、377、378、379、380、381、382、383、384、385、386、387、388、389、390、391、392、393、394、395、396、397、398、399、400、401、402、403、404、405、406、407、408、409、410、411、412、413、414、415、416、417、418、419、420、421、422、423、424、425、426、427、428、429、430、431、432、433、434、435、436、437、438、439、440、441、442、443、444、445、446、447、448、449、450、451、452、453、454、455、456、457、458、459、460、461、462、463、464、465、466、467、468、469、470、471、472、473、474、475、476、477、478、479、480、481、482、483、484、485、486、487、488、489、490、491、492、493、494、495、496、497、498、499、500、501、502、503、504、505、506、507、508、509、510、511、512、513、514、515、516、517、518、519、It may include up to 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 therein).

[0064] The polypeptides of the present disclosure are SEQ ID NO: 1 to 27, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270,271、272、273、274、275、276、277、278、279、280、281、282、283、284、285、286、287、288、289、290、291、292、293、294、295、296、297、298、299、300、301、302、303、304、305、306、307、308、309、310、311、312、313、314、315、316、317、318、319、320、321、322、323、324、325、326、327、328、329、330、331、332、333、334、335、336、337、338、339、340、341、342、343、344、345、346、347、348、349、350、351、352、353、354、355、356、357、358、359、360、361、362、363、364、365、366、367、368、369、370、371、372、373、374、375、376、377、378、379、380、381、382、383、384、385、386、387、388、389、390、391、392、393、394、395、396、397、398、399、400、401、402、403、404、405、406、407、408、409、410、411、412、413、414、415、416、417、418、419、420、421、422、423、424、425、426、427、428、429、430、431、432、433、434、435、436、437、438、439、440、441、442、443、444、445、446、447、448、449、450、451、452、453、454、455、456、457、458、459、460、461、462、463、464、465、466、467、468、469、470、471、472、473、474、475、476、477、478、479、480、481、482、483、484、485、486、487、488、489、490、491、492、493、494、495、496、497、498、499、500、501、502、503、504、505、506、507、508、509、510、511、512、513、514、515、516、517、518、519、520、521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 537, 538, 539, 540, 541, 542, 543, 544, 545, 546, 547, 548, 549, 550, 551, 552, 553, 554, 555, 556, 557, 558, 559, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569, 570, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 596, 597, 598, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 612, 613, 614, or 615 consecutive amino acids (or any range derivable therefrom), at least 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, 173174、175、176、177、178、179、180、181、182、183、184、185、186、187、188、189、190、191、192、193、194、195、196、197、198、199、200、201、202、203、204、205、206、207、208、209、210、211、212、213、214、215、216、217、218、219、220、221、222、223、224、225、226、227、228、229、230、231、232、233、234、235、236、237、238、239、240、241、242、243、244、245、246、247、248、249、250、251、252、253、254、255、256、257、258、259、260、261、262、263、264、265、266、267、268、269、270、271、272、273、274、275、276、277、278、279、280、281、282、283、284、285、286、287、288、289、290、291、292、293、294、295、296、297、298、299、300、301、302、303、304、305、306、307、308、309、310、311、312、313、314、315、316、317、318、319、320、321、322、323、324、325、326、327、328、329、330、331、332、333、334、335、336、337、338、339、340、341、342、343、344、345、346、347、348、349、350、351、352、353、354、355、356、357、358、359、360、361、362、363、364、365、366、367、368、369、370、371、372、373、374、375、376、377、378、379、380、381、382、383、384、385、386、387、388、389、390、391、392、393、394、395、396、397、398、399、400、401、402、403、404、405、406、407、408、409、410、411、412、413、414、415、416、417、418、419、420、421、422、423、424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, 500, 501, 502, 503, 504, 505, 506, 507, 508, 509, 510, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 537, 538, 539, 540, 541, 542, 543, 544, 545, 546, 547, 548, 549, 550, 551, 552, 553, 554, 555, 556, 557, 558, 559, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569, 570, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 596, 597, 598, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 612, 613, 614, or 615 consecutive amino acids (or any range derivable therefrom), or at most 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67,68、69、70、71、72、73、74、75、76、77、78、79、80、81、82、83、84、85、86、87、88、89、90、91、92、93、94、95、96、97、98、99、100、101、102、103、104、105、106、107、108、109、110、111、112、113、114、115、116、117、118、119、120、121、122、123、124、125、126、127、128、129、130、131、132、133、134、135、136、137、138、139、140、141、142、143、144、145、146、147、148、149、150、151、152、153、154、155、156、157、158、159、160、161、162、163、164、165、166、167、168、169、170、171、172、173、174、175、176、177、178、179、180、181、182、183、184、185、186、187、188、189、190、191、192、193、194、195、196、197、198、199、200、201、202、203、204、205、206、207、208、209、210、211、212、213、214、215、216、217、218、219、220、221、222、223、224、225、226、227、228、229、230、231、232、233、234、235、236、237、238、239、240、241、242、243、244、245、246、247、248、249、250、251、252、253、254、255、256、257、258、259、260、261、262、263、264、265、266、267、268、269、270、271、272、273、274、275、276、277、278、279、280、281、282、283、284、285、286、287、288、289、290、291、292、293、294、295、296、297、298、299、300、301、302、303、304、305、306、307、308、309、310、311、312、313、314、315、316、317、318、319、320、321、322、323、324、325、326、327、328、329、330、331、332、333、334、335、336、337、338、339、340、341、342、343、344、345、346、347、348、349、350、351、352、353、354、355、356、357、358、359、360、361、362、363、364、365、366、367、368、369、370、371、372、373、374、375、376、377、378、379、380、381、382、383、384、385、386、387、388、389、390、391、392、393、394、395、396、397、398、399、400、401、402、403、404、405、406、407、408、409、410、411、412、413、414、415、416、417、418、419、420、421、422、423、424、425、426、427、428、429、430、431、432、433、434、435、436、437、438、439、440、441、442、443、444、445、446、447、448、449、450、451、452、453、454、455、456、457、458、459、460、461、462、463、464、465、466、467、468、469、470、471、472、473、474、475、476、477、478、479、480、481、482、483、484、485、486、487、488、489、490、491、492、493、494、495、496、497、498、499、500、501、502、503、504、505、506、507、508、509、510、511、512、513、514、515、516、517、518、519、520、521、522、523、524、525、526、527、528、529、530、531、532、533、534、535、536、537、538、539、540、541、542、543、544、545、546、547、548、549、550、551、552、553、554、555、556、557、558、559、560、561、562、563、564、565、566、567、568、569、570、571、572、573、574、575、It may contain 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 596, 597, 598, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 612, 613, 614, or 615 consecutive amino acids (or any range derivable therefrom).

[0065] The polypeptides of the present disclosure are any one of SEQ ID NO: 1 to 27 and are 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%, 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 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 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 of SEQ ID NO: 1 to 27132、133、134、135、136、137、138、139、140、141、142、143、144、145、146、147、148、149、150、151、152、153、154、155、156、157、158、159、160、161、162、163、164、165、166、167、168、169、170、171、172、173、174、175、176、177、178、179、180、181、182、183、184、185、186、187、188、189、190、191、192、193、194、195、196、197、198、199、200、201、202、203、204、205、206、207、208、209、210、211、212、213、214、215、216、217、218、219、220、221、222、223、224、225、226、227、228、229、230、231、232、233、234、235、236、237、238、239、240、241、242、243、244、245、246、247、248、249、250、251、252、253、254、255、256、257、258、259、260、261、262、263、264、265、266、267、268、269、270、271、272、273、274、275、276、277、278、279、280、281、282、283、284、285、286、287、288、289、290、291、292、293、294、295、296、297、298、299、300、301、302、303、304、305、306、307、308、309、310、311、312、313、314、315、316、317、318、319、320、321、322、323、324、325、326、327、328、329、330、331、332、333、334、335、336、337、338、339、340、341、342、343、344、345、346、347、348、349、350、351、352、353、354、355、356、357、358、359、360、361、362、363、364、365、366、367、368、369、370、371、372、373、374、375、376、377、378、379、380、381、382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, 500, 501, 502, 503, 504, 505, 506, 507, 508, 509, 510, 511, 512, 513, 514, 515, 516, 517, 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) consecutive amino acids, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50、51、52、53、54、55、56、57、58、59、60、61、62、63、64、65、66、67、68、69、70、71、72、73、74、75、76、77、78、79、80、81、82、83、84、85、86、87、88、89、90、91、92、93、94、95、96、97、98、99、100、101、102、103、104、105、106、107、108、109、110、111、112、113、114、115、116、117、118、119、120、121、122、123、124、125、126、127、128、129、130、131、132、133、134、135、136、137、138、139、140、141、142、143、144、145、146、147、148、149、150、151、152、153、154、155、156、157、158、159、160、161、162、163、164、165、166、167、168、169、170、171、172、173、174、175、176、177、178、179、180、181、182、183、184、185、186、187、188、189、190、191、192、193、194、195、196、197、198、199、200、201、202、203、204、205、206、207、208、209、210、211、212、213、214、215、216、217、218、219、220、221、222、223、224、225、226、227、228、229、230、231、232、233、234、235、236、237、238、239、240、241、242、243、244、245、246、247、248、249、250、251、252、253、254、255、256、257、258、259、260、261、262、263、264、265、266、267、268、269、270、271、272、273、274、275、276、277、278、279、280、281、282、283、284、285、286、287、288、289、290、291、292、293、294、295、296、297、298、299、300、301、302、303、304、305、306、307、308、309、310、311、312、313、314、315、316、317、318、319、320、321、322、323、324、325、326、327、328、329、330、331、332、333、334、335、336、337、338、339、340、341、342、343、344、345、346、347、348、349、350、351、352、353、354、355、356、357、358、359、360、361、362、363、364、365、366、367、368、369、370、371、372、373、374、375、376、377、378、379、380、381、382、383、384、385、386、387、388、389、390、391、392、393、394、395、396、397、398、399、400、401、402、403、404、405、406、407、408、409、410、411、412、413、414、415、416、417、418、419、420、421、422、423、424、425、426、427、428、429、430、431、432、433、434、435、436、437、438、439、440、441、442、443、444、445、446、447、448、449、450、451、452、453、454、455、456、457、458、459、460、461、462、463、464、465、466、467、468、469、470、471、472、473、474、475、476、477、478、479、480、481、482、483、484、485、486、487、488、489、490、491、492、493、494、495、496、497、498、499、500、501、502、503、504、505、506、507、508、509、510、511、512、513、514、515、516、517、518、519、520、521、522、523、524、525、526、527、528、529、530、531、532、533、534、535, 536, 537, 538, 539, 540, 541, 542, 543, 544, 545, 546, 547, 548, 549, 550, 551, 552, 553, 554, 555, 556, 557, 558, 559, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569, 570, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 596, 597, 598, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 612, 613, 614, or 615 consecutive amino acids (or any range derivable therefrom), or at most 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, 186187、188、189、190、191、192、193、194、195、196、197、198、199、200、201、202、203、204、205、206、207、208、209、210、211、212、213、214、215、216、217、218、219、220、221、222、223、224、225、226、227、228、229、230、231、232、233、234、235、236、237、238、239、240、241、242、243、244、245、246、247、248、249、250、251、252、253、254、255、256、257、258、259、260、261、262、263、264、265、266、267、268、269、270、271、272、273、274、275、276、277、278、279、280、281、282、283、284、285、286、287、288、289、290、291、292、293、294、295、296、297、298、299、300、301、302、303、304、305、306、307、308、309、310、311、312、313、314、315、316、317、318、319、320、321、322、323、324、325、326、327、328、329、330、331、332、333、334、335、336、337、338、339、340、341、342、343、344、345、346、347、348、349、350、351、352、353、354、355、356、357、358、359、360、361、362、363、364、365、366、367、368、369、370、371、372、373、374、375、376、377、378、379、380、381、382、383、384、385、386、387、388、389、390、391、392、393、394、395、396、397、398、399、400、401、402、403、404、405、406、407、408、409、410、411、412、413、414、415、416、417、418、419、420、421、422、423、424、425、426、427、428、429、430、431、432、433、434、435、436、It may contain 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, 500, 501, 502, 503, 504, 505, 506, 507, 508, 509, 510, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 537, 538, 539, 540, 541, 542, 543, 544, 545, 546, 547, 548, 549, 550, 551, 552, 553, 554, 555, 556, 557, 558, 559, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569, 570, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 596, 597, 598, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 612, 613, 614, or 615 consecutive amino acids (or any range derivable therefrom).

[0066] The polypeptides of the present disclosure may have sequence identity or sequence homology 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 therein), 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 therein), or 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 therein) with one of SEQ ID NOs: 1 to 27.

[0067] This disclosure relates to any one of SEQ ID NO: 1 to 27 at position numbers 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269,270、271、272、273、274、275、276、277、278、279、280、281、282、283、284、285、286、287、288、289、290、291、292、293、294、295、296、297、298、299、300、301、302、303、304、305、306、307、308、309、310、311、312、313、314、315、316、317、318、319、320、321、322、323、324、325、326、327、328、329、330、331、332、333、334、335、336、337、338、339、340、341、342、343、344、345、346、347、348、349、350、351、352、353、354、355、356、357、358、359、360、361、362、363、364、365、366、367、368、369、370、371、372、373、374、375、376、377、378、379、380、381、382、383、384、385、386、387、388、389、390、391、392、393、394、395、396、397、398、399、400、401、402、403、404、405、406、407、408、409、410、411、412、413、414、415、416、417、418、419、420、421、422、423、424、425、426、427、428、429、430、431、432、433、434、435、436、437、438、439、440、441、442、443、444、445、446、447、448、449、450、451、452、453、454、455、456、457、458、459、460、461、462、463、464、465、466、467、468、469、470、471、472、473、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、Starting from 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 537, 538, 539, 540, 541, 542, 543, 544, 545, 546, 547, 548, 549, 550, 551, 552, 553, 554, 555, 556, 557, 558, 559, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569, 570, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 596, 597, 598, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 612, 613, 614, or 615, and any of SEQ ID NO: 1 - 27, with a length 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, 174175、176、177、178、179、180、181、182、183、184、185、186、187、188、189、190、191、192、193、194、195、196、197、198、199、200、201、202、203、204、205、206、207、208、209、210、211、212、213、214、215、216、217、218、219、220、221、222、223、224、225、226、227、228、229、230、231、232、233、234、235、236、237、238、239、240、241、242、243、244、245、246、247、248、249、250、251、252、253、254、255、256、257、258、259、260、261、262、263、264、265、266、267、268、269、270、271、272、273、274、275、276、277、278、279、280、281、282、283、284、285、286、287、288、289、290、291、292、293、294、295、296、297、298、299、300、301、302、303、304、305、306、307、308、309、310、311、312、313、314、315、316、317、318、319、320、321、322、323、324、325、326、327、328、329、330、331、332、333、334、335、336、337、338、339、340、341、342、343、344、345、346、347、348、349、350、351、352、353、354、355、356、357、358、359、360、361、362、363、364、365、366、367、368、369、370、371、372、373、374、375、376、377、378、379、380、381、382、383、384、385、386、387、388、389、390、391、392、393、394、395、396、397、398、399、400、401、402、403、404、405、406、407、408、409、410、411、412、413、414、415、416、417、418、419、420、421、422、423、424、Comprising a nucleic acid molecule or polypeptide comprising 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, 500, 501, 502, 503, 504, 505, 506, 507, 508, 509, 510, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 537, 538, 539, 540, 541, 542, 543, 544, 545, 546, 547, 548, 549, 550, 551, 552, 553, 554, 555, 556, 557, 558, 559, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569, 570, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 596, 597, 598, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 612, 613, 614, or 615 consecutive nucleotides or amino acids.,

[0068] The polypeptides and nucleic acids of the present disclosure are 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273274、275、276、277、278、279、280、281、282、283、284、285、286、287、288、289、290、291、292、293、294、295、296、297、298、299、300、301、302、303、304、305、306、307、308、309、310、311、312、313、314、315、316、317、318、319、320、321、322、323、324、325、326、327、328、329、330、331、332、333、334、335、336、337、338、339、340、341、342、343、344、345、346、347、348、349、350、351、352、353、354、355、356、357、358、359、360、361、362、363、364、365、366、367、368、369、370、371、372、373、374、375、376、377、378、379、380、381、382、383、384、385、386、387、388、389、390、391、392、393、394、395、396、397、398、399、400、401、402、403、404、405、406、407、408、409、410、411、412、413、414、415、416、417、418、419、420、421、422、423、424、425、426、427、428、429、430、431、432、433、434、435、436、437、438、439、440、441、442、443、444、445、446、447、448、449、450、451、452、453、454、455、456、457、458、459、460、461、462、463、464、465、466、467、468、469、470、471、472、473、474、475、476、477、478、479、480、481、482、483、484、485、486、487、488、489、490、491、492、493、494、495、496、497、498、499、500、501、502、503、504、505、506、507、508、509、510、511、512、513、514、515、516、517、518、519、520、521、522、523、524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 537, 538, 539, 540, 541, 542, 543, 544, 545, 546, 547, 548, 549, 550, 551, 552, 553, 554, 555, 556, 557, 558, 559, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569, 570, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 596, 597, 598, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 612, 613, 614, or 615 substitutions (or any range derivable therefrom), at least 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, 177178、179、180、181、182、183、184、185、186、187、188、189、190、191、192、193、194、195、196、197、198、199、200、201、202、203、204、205、206、207、208、209、210、211、212、213、214、215、216、217、218、219、220、221、222、223、224、225、226、227、228、229、230、231、232、233、234、235、236、237、238、239、240、241、242、243、244、245、246、247、248、249、250、251、252、253、254、255、256、257、258、259、260、261、262、263、264、265、266、267、268、269、270、271、272、273、274、275、276、277、278、279、280、281、282、283、284、285、286、287、288、289、290、291、292、293、294、295、296、297、298、299、300、301、302、303、304、305、306、307、308、309、310、311、312、313、314、315、316、317、318、319、320、321、322、323、324、325、326、327、328、329、330、331、332、333、334、335、336、337、338、339、340、341、342、343、344、345、346、347、348、349、350、351、352、353、354、355、356、357、358、359、360、361、362、363、364、365、366、367、368、369、370、371、372、373、374、375、376、377、378、379、380、381、382、383、384、385、386、387、388、389、390、391、392、393、394、395、396、397、398、399、400、401、402、403、404、405、406、407、408、409、410、411、412、413、414、415、416、417、418、419、420、421、422、423、424、425、426、427、428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, 500, 501, 502, 503, 504, 505, 506, 507, 508, 509, 510, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 537, 538, 539, 540, 541, 542, 543, 544, 545, 546, 547, 548, 549, 550, 551, 552, 553, 554, 555, 556, 557, 558, 559, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569, 570, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 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 substitutions (or any range derivable therefrom), or at most 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, 7475、76、77、78、79、80、81、82、83、84、85、86、87、88、89、90、91、92、93、94、95、96、97、98、99、100、101、102、103、104、105、106、107、108、109、110、111、112、113、114、115、116、117、118、119、120、121、122、123、124、125、126、127、128、129、130、131、132、133、134、135、136、137、138、139、140、141、142、143、144、145、146、147、148、149、150、151、152、153、154、155、156、157、158、159、160、161、162、163、164、165、166、167、168、169、170、171、172、173、174、175、176、177、178、179、180、181、182、183、184、185、186、187、188、189、190、191、192、193、194、195、196、197、198、199、200、201、202、203、204、205、206、207、208、209、210、211、212、213、214、215、216、217、218、219、220、221、222、223、224、225、226、227、228、229、230、231、232、233、234、235、236、237、238、239、240、241、242、243、244、245、246、247、248、249、250、251、252、253、254、255、256、257、258、259、260、261、262、263、264、265、266、267、268、269、270、271、272、273、274、275、276、277、278、279、280、281、282、283、284、285、286、287、288、289、290、291、292、293、294、295、296、297、298、299、300、301、302、303、304、305、306、307、308、309、310、311、312、313、314、315、316、317、318、319、320、321、322、323、324、325、326、327、328、329、330、331、332、333、334、335、336、337、338、339、340、341、342、343、344、345、346、347、348、349、350、351、352、353、354、355、356、357、358、359、360、361、362、363、364、365、366、367、368、369、370、371、372、373、374、375、376、377、378、379、380、381、382、383、384、385、386、387、388、389、390、391、392、393、394、395、396、397、398、399、400、401、402、403、404、405、406、407、408、409、410、411、412、413、414、415、416、417、418、419、420、421、422、423、424、425、426、427、428、429、430、431、432、433、434、435、436、437、438、439、440、441、442、443、444、445、446、447、448、449、450、451、452、453、454、455、456、457、458、459、460、461、462、463、464、465、466、467、468、469、470、471、472、473、474、475、476、477、478、479、480、481、482、483、484、485、486、487、488、489、490、491、492、493、494、495、496、497、498、499、500、501、502、503、504、505、506、507、508、509、510、511、512、513、514、515、516、517、518、519、520、521、522、523、524、525、526、527、528、529、530、531、532、533、534、535、536、537、538、539、540、541、542、543、544、545、546、547、548、549、550、551、552、553、554、555、556、557、558、559、560、561、562、563、564、565、566、567、568、569、570、571、572、573、574、575、576、577、578、579、580、It may include 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 substitutions (or any range derivable therefrom).

[0069] The substitution is at one of the amino acid position numbers or nucleic acid position numbers 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266,267、268、269、270、271、272、273、274、275、276、277、278、279、280、281、282、283、284、285、286、287、288、289、290、291、292、293、294、295、296、297、298、299、300、301、302、303、304、305、306、307、308、309、310、311、312、313、314、315、316、317、318、319、320、321、322、323、324、325、326、327、328、329、330、331、332、333、334、335、336、337、338、339、340、341、342、343、344、345、346、347、348、349、350、351、352、353、354、355、356、357、358、359、360、361、362、363、364、365、366、367、368、369、370、371、372、373、374、375、376、377、378、379、380、381、382、383、384、385、386、387、388、389、390、391、392、393、394、395、396、397、398、399、400、401、402、403、404、405、406、407、408、409、410、411、412、413、414、415、416、417、418、419、420、421、422、423、424、425、426、427、428、429、430、431、432、433、434、435、436、437、438、439、440、441、442、443、444、445、446、447、448、449、450、451、452、453、454、455、456、457、458、459、460、461、462、463、464、465、466、467、468、469、470、471、472、473、474、475、476、477、478、479、480、481、482、483、484、485、486、487、488、489、490、491、492、493、494、495、496、497、498、499、500、501、502、503、504、505、506、507、508、509、510、511、512、513、514、515、516、It may be at 517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 537, 538, 539, 540, 541, 542, 543, 544, 545, 546, 547, 548, 549, 550, 551, 552, 553, 554, 555, 556, 557, 558, 559, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569, 570, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 596, 597, 598, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 612, 613, 614, or 615. One or more of these substitutions may be specifically excluded from the aspect.,

[0070] Position numbers 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 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 of one peptide or polypeptide among SEQ ID NO:1 to 27The amino acid at position 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 or 400 can be substituted with alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine or valine.,

[0071] The peptides, polypeptides, and proteins of the present disclosure 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% identity, at least 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity, or at most 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% identity with any one of SEQ ID NO: 1 to 27, and may have amino acid position numbers 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 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,Fragments or segments that may contain, starting from 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, or 200 (or any range derivable therefrom), and ending at amino acid position number 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, or 205 (or any range derivable therefrom).

[0072] A substitution variant typically involves the exchange of one amino acid for another at one or more sites within a protein and may be designed to modulate one or more properties of the polypeptide without losing or sacrificing other functions or properties. The substitution may be conservative, i.e., one amino acid may be substituted for an amino acid of similar shape and charge. Conservative substitutions are well-known in the art and include, for example, the change of alanine to serine, arginine to lysine, asparagine to glutamine or histidine, aspartic acid to glutamic acid, cysteine to serine, glutamine to asparagine, glutamic acid to aspartic acid, glycine to proline, histidine to asparagine or glutamine, isoleucine to leucine or valine, leucine to valine or isoleucine, lysine to arginine, methionine to leucine or isoleucine, phenylalanine to tyrosine, leucine or methionine, serine to threonine, threonine to serine, tryptophan to tyrosine, tyrosine to tryptophan or phenylalanine, and valine to isoleucine or leucine. Alternatively, the substitution may be non-conservative such that the function or activity of the polypeptide is affected. Non-conservative changes typically involve substituting one residue with a chemically different residue, such as a polar or charged amino acid in place of a non-polar or uncharged amino acid, and vice versa. One or more of these substitutions may be specifically excluded from an aspect.

[0073] The protein may be recombinant or synthesized in vitro. Alternatively, a non-recombinant or recombinant protein may be isolated from bacteria. It is also contemplated that bacteria containing such variants can be practiced in compositions and methods. As a result, the protein may not need to be isolated.

[0074] 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.

[0075] Amino acid and nucleic acid sequences may each contain additional residues, such as additional N-terminal or C-terminal amino acids or 5' or 3' sequences, and yet still be essentially as set forth in one of the sequences disclosed herein, so long as the above criteria are met, including maintenance of the biological protein activity when protein expression is involved. The addition of terminal sequences applies particularly to nucleic acid sequences and can include, for example, various non-coding sequences adjacent to either the 5' or 3' portion of the coding region.

[0076] The following considerations are based on changing the amino acids of a protein to create equivalent or even improved second-generation molecules. For example, a particular amino acid can be used in place of another amino acid in a protein structure without significantly losing its interaction binding ability. For example, a structure such as an enzyme catalytic domain or an interaction component can have amino acids substituted so as to maintain such function. Since the biological functional activity of a protein is defined by its interaction ability and properties, specific amino acid substitutions can be made in the protein sequence and in the underlying DNA coding sequence, and yet 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 significantly losing its biological utility or activity.

[0077] In other scenarios, it is intended to modify the function of a polypeptide by introducing one or more substitutions. For example, a specific amino acid can be used in place of another amino acid in a protein structure with the intention of modifying the interaction binding ability of the interacting components. For example, structures such as protein interaction domains, nucleic acid interaction domains, and catalytic sites may have amino acids substituted to modify such functions. Since the biological functional activity of a protein is defined by its interaction ability and properties, specific amino acid substitutions can be made in the protein sequence and in the underlying DNA coding sequence, and yet, proteins with different characteristics can be produced. Thus, the inventors believe that various changes can be made to the DNA sequence of a gene to significantly alter its biological utility or activity.

[0078] When making such changes, the hydrophobic-hydrophilic index of amino acids may be considered. The importance of the hydrophobic-hydrophilic amino acid index in conferring interaction biological functions to proteins is generally understood in the art (Kyte and Doolittle, 1982). It has been recognized that the characteristics of the relative hydrophobic-hydrophilic index of amino acids contribute to the secondary structure of the resulting protein and further define the interaction of the protein with other molecules, such as enzymes, substrates, receptors, DNA, antibodies, antigens, etc.

[0079] It is also understood in the art that effective substitution of similar amino acids can be made based on hydrophilicity. U.S. Patent No. 4,554,101, which is incorporated herein by reference, states that the maximum local average hydrophilicity of a protein, which is governed by the hydrophilicity of its adjacent amino acids, correlates with the biological characteristics of the protein. It is understood that one amino acid can be substituted with another amino acid having a similar hydrophilicity value, and yet, a biologically equivalent and immunologically equivalent protein can be produced.

[0080] As outlined above, amino acid substitutions are generally based on the relative similarity of the amino acid side chain substituents, such as hydrophobicity, hydrophilicity, charge, size, etc. Exemplary substitutions that take into account the various features described 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.

[0081] In certain aspects, all or part of the proteins described herein can also be synthesized in solution or on a solid support according to the prior art. Various automated synthesizers are commercially available and can be used according to known protocols. See, for example, Stewart and Young, (1984); Tarn et al., (1983); Merrifield, (1986); and Barany and Merrifield (1979), each of which is hereby incorporated by reference. Alternatively, recombinant DNA techniques can be used in which a nucleotide sequence encoding a peptide or polypeptide is inserted into an expression vector, which is then transformed or transfected into a suitable host cell and cultured under conditions suitable for expression.

[0082] One aspect involves the use of gene transfer into cells, including microorganisms, for protein production and / or presentation. A gene for a protein of interest can be introduced into a suitable host cell, followed by culturing the cells under appropriate conditions. Nucleic acids encoding substantially any polypeptide can be used. The construction of recombinant expression vectors and the elements contained therein are discussed herein. Alternatively, the protein produced may be an endogenous protein normally synthesized by the cells used for protein production.

[0083] III. Nucleic Acids In certain aspects, the present disclosure relates to recombinant polynucleotides encoding the polypeptides of the present disclosure. Accordingly, certain aspects relate to nucleotides encoding the polypeptides, chimeric polypeptides, or multimeric polypeptides of the present disclosure.

[0084] As used herein, the term "polynucleotide" refers to a nucleic acid molecule that is either recombinant or isolated free of total genomic nucleic acid. Included within the scope of the term "polynucleotide" are oligonucleotides (nucleic acids that are 100 residues or less in length), for example, recombinant vectors including plasmids, cosmids, phages, viruses, and the like. A polynucleotide may, in certain aspects, include regulatory sequences that are substantially isolated from natural gene or protein coding sequences. A polynucleotide may be single-stranded (coding or antisense) or double-stranded, and may be RNA, DNA (genomic, cDNA or synthetic), an analog thereof, or a combination thereof. Additional coding or non-coding sequences may or may not be present within the polynucleotide.

[0085] In this regard, the terms "gene", "polynucleotide", or "nucleic acid" are used to refer to nucleic acids that encode proteins, polypeptides or peptides (including any sequences required for proper transcription, post-translational modification or localization). As will be understood by those skilled in the art, this term encompasses genomic sequences, expression cassettes, cDNA sequences, as well as genetically engineered smaller nucleic acid segments that can express or be adapted to express proteins, polypeptides, domains, peptides, fusion proteins, and variants. Nucleic acids encoding all or part of a polypeptide can be 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 (or any range derivable therefrom) of nucleotides, nucleosides or base pairs (including any values and ranges therebetween) of a continuous nucleic acid sequence.In addition, it is also conceivable that certain polypeptides can be encoded by nucleic acids that contain variants encoding the same or substantially similar proteins but have slightly different nucleic acid sequences.

[0086] In certain aspects, the invention relates to isolated nucleic acid segments and recombinant vectors incorporating the polypeptides of the present disclosure or nucleic acid sequences encoding the peptides. The term "recombinant" can be used in combination with polynucleotides or polypeptides and generally refers to polypeptides or polynucleotides that are produced and / or manipulated in vitro or polypeptides or polynucleotides that are replication products of such molecules.

[0087] In other aspects, the invention relates to isolated nucleic acid segments and recombinant vectors incorporating the polypeptides of the present disclosure or nucleic acid sequences encoding the peptides.

[0088] The nucleic acid segments used in the present disclosure may be combined with other nucleic acid sequences, such as promoters, polyadenylation signals, additional restriction enzyme sites, multicloning sites, other coding segments, etc., and thus their full lengths can vary considerably. Therefore, nucleic acid fragments of almost all lengths can be used, although preferably their full lengths are intended to be limited by ease of preparation and use in the intended recombinant nucleic acid protocol. In some cases, the nucleic acid sequence can encode a polypeptide sequence having additional heterologous coding sequences, for example, to enable purification, transport, secretion, post-translational modification of the polypeptide, or to enable therapeutic utility such as targeting or efficacy. As discussed above, tags or other heterologous polypeptides can be added to the sequence encoding the modified polypeptide, and "heterologous" refers to a polypeptide that is not the same as the modified polypeptide.

[0089] In certain aspects, the present disclosure provides polynucleotide variants having substantial identity to the sequences disclosed herein, which have at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%, or more sequence identity (including any value and range therebetween) compared to the polynucleotide sequences of the present disclosure using methods described herein (e.g., BLAST analysis using standard parameters).

[0090] The present disclosure also contemplates the use of polynucleotides that are complementary to all of the above polynucleotides.

[0091] A. Vector The polypeptide of the present disclosure may be encoded by a nucleic acid molecule contained in a vector. The term "vector" is used to refer to a carrier nucleic acid molecule into which a heterologous nucleic acid sequence can be inserted for introduction into a cell in which it can be replicated and expressed. The nucleic acid sequence may be "heterologous", which in context means that the nucleic acid sequence is foreign to the cell into which the vector is introduced or to the nucleic acid into which the nucleic acid sequence is incorporated, and this includes sequences that are homologous to sequences in the cell or nucleic acid but are normally not found at a particular location within the host cell or nucleic acid. Vectors include DNA, RNA, plasmids, cosmids, viruses (bacteriophages, animal viruses and plant viruses), and artificial chromosomes (e.g., YAC). Those skilled in the art will be well equipped to construct vectors through standard recombinant techniques (e.g., Sambrook et al., 2001; Ausubel et al., 1996, both incorporated herein by reference). In addition to encoding the polypeptide of the present disclosure, the vector can encode one or more other polypeptide sequences such as one or more other bacterial peptides, tags, or immunogenic enhancing peptides. Useful vectors for encoding such fusion proteins include pIN vectors (Inouye et al., 1985), vectors encoding a string of histidines, and pGEX vectors for use in the production of glutathione S-transferase (GST) soluble fusion proteins for subsequent purification and separation or cleavage.

[0092] The term "expression vector" refers to a vector that contains a nucleic acid sequence encoding at least a portion of a gene product that can be transcribed. In some cases, the RNA molecule is then translated into a protein, polypeptide, or peptide. Expression vectors can contain various "control sequences" that refer to nucleic acid sequences necessary for the transcription and possibly translation of a coding sequence operably linked in a particular host organism. In addition to control sequences that govern transcription and translation, vectors and expression vectors may also perform other functions and contain nucleic acid sequences described herein.

[0093] B. Promoters and Enhancers A "promoter" is a control sequence. A promoter is typically a region of a nucleic acid sequence where the initiation and rate of transcription are controlled. This may include genetic elements to which regulatory proteins and molecules, such as RNA polymerase and other transcription factors, can bind. The phrases "functionally disposed", "functionally linked", "under control", and "under transcriptional control" mean that the promoter is in the correct functional position and / or orientation in relation to a nucleic acid sequence to control the initiation and expression of that sequence. A promoter may or may not be used together with an "enhancer", which refers to a cis-acting regulatory sequence involved in the transcriptional activation of a nucleic acid sequence.

[0094] Of course, it may be important to use promoters and / or enhancers that effectively induce the expression of a DNA segment in the cell type or organism selected for expression. Those skilled in the art of molecular biology generally understand the use of promoters, enhancers, and combinations of cell types for protein expression (see Sambrook et al., 2001, which is incorporated herein by reference). The promoter used may be constitutive, tissue-specific, or inducible, and in certain aspects, can induce high-level expression of the introduced DNA segment under specific conditions, such as the large-scale production of a recombinant protein or peptide.

[0095] The specific promoter used to control the expression of a polynucleotide encoding a peptide or protein of the present invention is not considered to be critically important as long as the polynucleotide can be expressed in the target cell, preferably a bacterial cell. When human cells are targeted, it is preferred to position the polynucleotide coding region adjacent to and under the control of a promoter that can be expressed in human cells. Generally speaking, such a promoter can include any of a bacterial promoter, a human promoter, or a viral promoter.

[0096] C. Initiation Signals and Internal Ribosome Entry Sites (IRESs) Specific initiation signals may also be required for efficient translation of the coding sequence. These signals include the ATG start codon or adjacent sequences. It may be necessary to provide an exogenous translation control signal that includes the ATG start codon. One of ordinary skill in the art will be able to readily determine this and provide the necessary signals.

[0097] In certain aspects of the invention, internal ribosome entry site (IRES) elements are used to create multi-gene or polycistronic messages. IRES elements can bypass the ribosome scanning model of 5'-methylated Cap-dependent translation and initiate translation at internal sites (Pelletier and Sonenberg, 1988; Macejak and Sarnow, 1991). IRES elements can be linked to different reading frames. Multiple reading frames, each separated by an IRES, can be transcribed together to create a polycistronic message. Multiple genes can be efficiently expressed using a single promoter / enhancer and a single message can be transcribed (see U.S. Patent Nos. 5,925,565 and 5,935,819, which are incorporated herein by reference).

[0098] D. Selectable and Screenable Markers In certain aspects of the invention, cells containing the nucleic acid constructs of the disclosure can be identified in vitro or in vivo by encoding a screenable or selectable marker in an expression vector. When transcribed and translated, the marker confers an identifiable change on the cell, enabling easy identification of cells containing the expression vector. Generally, a selectable marker confers a property that enables selection. A positively selectable marker enables selection in the presence of the marker, whereas a negatively selectable marker prevents selection in the presence of the marker. An example of a positively selectable marker is a drug resistance marker.

[0099] E. Host cell As used herein, the terms "cell", "cell line", and "cell culture" can be used interchangeably. All of these terms include any and all progeny of their next generation. It is understood that all progeny may not be identical due to deliberate or accidental mutations. In the context of expressing a heterologous nucleic acid sequence, "host cell" refers to a prokaryotic or eukaryotic cell and includes any transformable organism capable of replicating a vector or expressing a heterologous gene encoded by a vector. A host cell can be and is used as a recipient for a vector or virus. A host cell may be "transfected" or "transformed", which refers to the process by which an exogenous nucleic acid, such as a sequence encoding a recombinant protein, is introduced or transferred into the host cell. Transformed cells include the primary subject cells and their progeny.

[0100] Host cells can be derived from prokaryotes or eukaryotes, including bacteria, yeast cells, insect cells, and mammalian cells, for the replication of vectors or the expression of some or all of the nucleic acid sequences. A number of cell lines and cultures are available for use as host cells and can be obtained from the American Type Culture Collection (ATCC), which functions as a living culture and an archive of genetic material (www.atcc.org).

[0101] F. Expression Systems There are a number of expression systems that contain at least a portion or all of the compositions discussed above. Prokaryotic and / or eukaryotic-based systems can be used in conjunction with the present invention to produce nucleic acid sequences, or their homologous polypeptides, proteins, and peptides. Many such systems are commercially available and widely accessible.

[0102] The insect cell / baculovirus system can result in high-level protein expression of heterologous nucleic acid segments, as described in U.S. Patent Nos. 5,871,986 and 4,879,236, which are hereby incorporated by reference in their entirety, and can be purchased, for example, from INVITROGEN® under the name MAXBAC® 2.0 and from CLONTECH® under the name BACPACK™ BACULOVIRUS EXPRESSION SYSTEM.

[0103] In addition to the expression system of the present invention disclosed, other examples of expression systems include the COMPLETE CONTROL□ Inducible Mammalian Expression System of STRATAGENE (registered trademark), which includes a synthetic ecdyson receptor or its pET expression system, an Escherichia coli expression system. Another example of an inducible expression system is available from INVITROGEN (registered trademark) and has the T-REX (trademark) (tetracycline-regulated expression) system, an inducible mammalian expression system using a full-length CMV promoter. INVITROGEN (registered trademark) also provides a yeast expression system called the Pichia methanolica Expression System, which is designed for high-level production of recombinant proteins in the methylotrophic yeast Pichia methanolica. How to express vectors such as expression constructs to produce nucleic acid sequences, or their homologous polypeptides, proteins, or peptides will be known to those skilled in the art.

[0104] IV. Combination Therapy The administration of the compositions and related methods of the present disclosure, particularly the polypeptides of the present disclosure, can also be used in combination with the administration of additional therapies such as additional therapeutic substances described herein, or in combination with other conventional therapeutic substances known in the art for the treatment of autoimmune or inflammatory conditions.

[0105] The therapeutic compositions and treatments disclosed herein can precede, proceed concurrently with, and / or follow another treatment or agent at intervals ranging from several minutes to several weeks. In instances where agents are applied separately to a cell, tissue, or organism, generally, it will be ensured that no significant period elapses between the times of each delivery such that the therapeutic agents can still exert a favorable combined effect on the cell, tissue, or organism. For example, in such cases, it is contemplated that the cell, tissue, or organism can be contacted with two, three, four or more agents or treatments substantially simultaneously (i.e., within less than about 1 minute). In other instances, one or more therapeutic agents or treatments can be administered or provided 1 minute, 5 minutes, 10 minutes, 20 minutes, 30 minutes, 45 minutes, 60 minutes, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 22 hours, 23 hours, 24 hours, 25 hours, 26 hours, 27 hours, 28 hours, 29 hours, 30 hours, 31 hours, 32 hours, 33 hours, 34 hours, 35 hours, 36 hours, 37 hours, 38 hours, 39 hours, 40 hours, 41 hours, 42 hours, 43 hours, 44 hours, 45 hours, 46 hours, 47 hours, 48 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks or 8 weeks or within or within any range derivable therein before and / or after the administration or performance of another therapeutic agent or treatment.

[0106] In some instances, two or more treatment courses can be utilized. It is contemplated that multiple courses can be implemented.

[0107] V. Treatment Methods 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, intracutaneous, subcutaneous, intravenous, intradermal, intramuscular, topical, local and / or intraperitoneal administration. It is particularly contemplated that one or more of these routes of administration are excluded from certain aspects of the present disclosure.

[0108] In some aspects, the compositions of the present disclosure are provided via subcutaneous administration (i.e., provided subcutaneously). In some aspects, the compositions of the present disclosure are provided via intradermal administration (i.e., provided intradermally). In some aspects, the compositions of the present disclosure are provided via intramuscular administration (i.e., provided intramuscularly).

[0109] In some aspects, the therapeutic compositions of the present disclosure are administered during the discontinuation of one or more other therapies. For example, in some aspects, what is disclosed is a method comprising the step of administering the composition of the present disclosure to a subject during the discontinuation of one or more additional anti-inflammatory therapeutic substances (such as 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).

[0110] 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, local and intraperitoneal administration.

[0111] Aspects of the present disclosure relate to methods for treating autoimmune or inflammatory conditions. In some aspects, disclosed herein is a method for treating an autoimmune or inflammatory condition comprising administering a composition of the present disclosure to a subject having an autoimmune or inflammatory condition, a subject at risk of developing an autoimmune or inflammatory condition, or a subject suspected of having an autoimmune or inflammatory condition. Such methods may include administering one or more additional anti-inflammatory agents. Such methods may exclude administering one or more additional anti-inflammatory agents. Additional anti-inflammatory agents include, for example, fingolimod, interferon-β, dimethyl fumarate, teriflunomide, integrin α4β1, anti-αLβ2 antibody, anti-TNFα agents, anti-IL-6R agents, anti-IL-6 agents, and Janus kinase inhibitors (e.g., tofacitinib, baricitinib, upadacitinib).

[0112] Autoimmune or inflammatory conditions suitable for treatment include diabetes (e.g., type 1 diabetes), graft rejection, arthritis (rheumatoid arthritis, e.g., acute arthritis, chronic rheumatoid arthritis, gout or gouty arthritis, acute gouty arthritis, acute immune arthritis, chronic inflammatory arthritis, osteoarthritis, type II collagen-induced arthritis, infectious arthritis, Lyme arthritis, proliferative arthritis, psoriatic arthritis, Still's disease, spondyloarthritis, and systemic juvenile idiopathic arthritis), osteoarthrosis, chronic progressive arthritis, degenerative arthritis, primary chronic polyarthritis, reactive arthritis, and ankylosing spondylitis), inflammatory hyperproliferative skin diseases, psoriasis, e.g., plaque psoriasis, guttate psoriasis, pustular psoriasis, and nail psoriasis, atopy including atopic diseases such as hay fever and Job syndrome, dermatitis, e.g., contact dermatitis, chronic contact dermatitis, exfoliative dermatitis, allergic dermatitis, allergic contact dermatitis, dermatitis herpetiformis, nummular eczema, seborrheic dermatitis, nonspecific dermatitis, primary irritant contact dermatitis, and atopic dermatitis, X-linked hyper IgM syndrome, allergic intraocular inflammatory diseases, urticaria including chronic idiopathic urticaria such as chronic allergic urticaria and chronic autoimmune urticaria, myositis, polymyositis / dermatomyositis, juvenile dermatomyositis, toxic epidermal necrolysis, scleroderma (including systemic scleroderma), sclerosis such as systemic sclerosis, multiple sclerosis (MS), e.g., spino-optical MS, primary progressive MS (PPMS), and relapsing-remitting MS (RRMS), progressive systemic sclerosis, atherosclerosis, arteriosclerosis, disseminated sclerosis, ataxic sclerosis, neuromyelitis optica (NMO), inflammatory bowel disease (IBD) (e.g., Crohn's disease, autoimmune-mediated gastrointestinal diseases, ulcerative colitis, colitis ulcerosa, microscopic colitis, collagenous colitis, polypous colitis, necrotizing enteritis, transmural colitis, etc. of the colon, and autoimmune inflammatory bowel diseases), intestinal inflammation, pyoderma gangrenosum, erythema nodosum, primary sclerosing cholangitis, respiratory distress syndrome including adult or acute respiratory distress syndrome (ARDS), meningitis, inflammation of all or part of the uvea, iritis, choroiditis, autoimmune blood disorders, rheumatoid spondylitis, rheumatoid synovitis, hereditary angioedema, cranial nerve damage as in meningitis, herpes gestationis, pemphigoid gestationis, scrotal pruritus,Autoimmune premature ovarian insufficiency, sudden deafness due to autoimmune status, IgE-mediated diseases such as anaphylaxis and allergic and atopic rhinitis, encephalitis such as Rasmussen encephalitis and limbic system and / or brainstem encephalitis, anterior uveitis, acute anterior uveitis, granulomatous uveitis, non-granulomatous uveitis, lens antigenic uveitis, posterior uveitis, or uveitis such as 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 insufficiency, balanitis, for example, plasmacytic balanoposthitis, balanoposthitis, centrifugal annular erythema, dyschromic fixed erythema (erythema dyschromicum perstans), erythema multiforme, granuloma annulare, lichen nitidus, lichen sclerosus et atrophicus, lichen simplex chronicus, lichen spinulosus, lichen planus, ichthyosis hystrix, exfoliative keratosis, premalignant keratosis, pyoderma gangrenosum, allergic states and responses, allergic reactions, allergic or atopic eczema, asteatotic eczema, dyshidrotic eczema, and eczema including vesicular palmar and plantar eczema, asthma, for example, bronchial asthma (asthma bronchiale), bronchial asthma, and autoimmune asthma, conditions with T cell infiltration and chronic inflammatory reaction, immune responses to foreign antigens such as fetal A-B-O blood type during pregnancy, chronic pulmonary inflammatory diseases, autoimmune myocarditis, leukocyte adhesion deficiency, lupus, for example, lupus nephritis, lupus encephalitis, pediatric lupus, non-renal lupus, extrarenal lupus, discoid lupus and discoid erythematosus, alopecia areata lupus, systemic lupus erythematosus (SLE), for example, cutaneous SLE or subacute cutaneous SLE, neonatal lupus syndrome (NLE), and disseminated erythematosus, juvenile-onset (type I) diabetes including pediatric insulin-dependent diabetes (IDDM), and adult-onset diabetes (type II diabetes) as well as conditions such as autoimmune diabetes, but not limited thereto. Immune responses related to acute and delayed hypersensitivity mediated by cytokines and T lymphocytes, sarcoidosis, lymphomatoid granulomatosis,Granulomatosis including Wegener's granulomatosis, agranulocytosis, vasculitides, e.g., vasculitis, large vessel vasculitis (including rheumatoid polymyalgia and giant cell (Takayasu) arteritis), medium vessel vasculitis (including Kawasaki disease and polyarteritis nodosa / peripheral arteritis nodosa), microscopic polyarteritis, immune vasculitis, CNS vasculitis, cutaneous vasculitis, allergic vasculitis, necrotizing vasculitis, e.g., systemic necrotizing vasculitis, and ANCA-associated vasculitis, e.g., Churg-Strauss vasculitis or syndrome (CSS), and ANCA-associated small vessel vasculitis, temporal arteritis, aplastic anemia, autoimmune aplastic anemia, Coombs positive anemia, Diamond-Blackfan anemia, hemolytic anemia or immune hemolytic anemia including autoimmune hemolytic anemia (AIHA), Addison's disease, autoimmune neutropenia, pancytopenia, leukopenia, diseases with extravascular migration of leukocytes, CNS inflammatory disorders, dementia, mental health disorders, depression, Alzheimer's disease, Parkinson's disease, sepsis, multiple organ injury syndromes such as those secondary to trauma or hemorrhage, antigen-antibody complex-mediated diseases, anti-glomerular basement membrane diseases, antiphospholipid antibody syndrome, allergic neuritis, Behcet's disease / syndrome, Castleman syndrome, Goodpasture syndrome, Raynaud's syndrome, Sjogren syndrome, Stevens-Johnson syndrome, pemphigoid such as bullous pemphigoid and dermatitis herpetiformis, pemphigus (including pemphigus vulgaris, pemphigus foliaceus, mucous membrane pemphigoid pemphigus, pemphigus erythematosus), autoimmune polyglandular endocrinopathies, Reiter's disease or syndrome, burns, preeclampsia, immune complex disorders such as immune complex nephritis, antibody-mediated nephritis, polyneuropathy, chronic neuropathies such as IgM polyneuropathy or IgM-mediated neuropathy, autoimmune or immune-mediated thrombocytopenias such as idiopathic thrombocytopenic purpura (ITP) including chronic or acute ITP, idiopathic cerato-scleritis, scleritis such as episcleritis, autoimmune orchitis and ovaritis including autoimmune diseases of the testis and ovary, primary hypothyroidism, hypoparathyroidism, autoimmune endocrine diseases, e.g., thyroiditis, e.g., autoimmune thyroiditis, Hashimoto's disease, chronic thyroiditis (Hashimoto's thyroiditis), or subacute thyroiditis, autoimmune thyroid diseases, idiopathic hypothyroidism, Graves' disease, polyendocrine syndromes such as autoimmune polyendocrine syndrome (or polyendocrine endocrine disorder syndrome),Paraneoplastic syndromes including Lambert-Eaton myasthenic syndrome or Eaton-Lambert syndrome, neurological paraneoplastic syndromes such as stiff-man syndrome or stiff-person syndrome, encephalomyelitis such as allergic encephalomyelitis or encephalomyelitis allergica and experimental allergic encephalomyelitis (EAE), experimental autoimmune encephalomyelitis, myasthenia gravis such as thymoma-related myasthenia gravis, cerebellar degeneration, neuromyotonia, opsoclonus or opsoclonus-myoclonus syndrome (OMS), and sensory neuropathy, multifocal motor neuropathy, Sheehan syndrome, autoimmune hepatitis, chronic hepatitis, lupoid hepatitis, giant cell hepatitis, chronic active hepatitis or autoimmune chronic active hepatitis, lymphocytic interstitial pneumonia (LIP), bronchiolitis obliterans (non-transplant) vs NSIP, Guillain-Barré syndrome, Berger disease (IgA nephropathy), idiopathic IgA nephropathy, linear IgA dermatosis, acute febrile neutrophilic dermatosis, subcorneal pustular dermatosis, transient acantholytic dermatosis, cirrhosis such as primary biliary cirrhosis and pneumonocirrhosis, autoimmune enteropathy syndrome, Celiac disease or Coeliac disease, celiac sprue (gluten enteropathy), refractory sprue, idiopathic sprue, cryoglobulinemia, amyotrophic lateral sclerosis (ALS; Lou Gehrig's disease), coronary artery disease, autoimmune ear diseases such as autoimmune inner ear disease (AIED), autoimmune deafness, relapsing or recurrent or refractory relapsing polychondritis such as polychondritis, alveolar proteinosis, Cogan syndrome / non-syphilitic interstitial keratitis, Bell palsy, Sweet disease / syndrome, autoimmune uveitis, pain associated with herpes zoster, amyloidosis, non-cancerous lymphocytosis, primary lymphocytosis including monoclonal B-cell lymphocytosis (e.g., benign monoclonal gammopathy and monoclonal gammopathy of undetermined significance, MGUS), peripheral neuropathy, paraneoplastic syndrome, channelopathies such as epilepsy, migraine, arrhythmia, myopathy, hearing loss, blindness, periodic paralysis, and CNS channelopathies, autism, inflammatory myopathy, focal or segmental or focal segmental glomerulosclerosis (FSGS), endocrine ophthalmopathy, uveitis, choroiditis, autoimmune liver disorder,Fibromyalgia, multiple endocrine deficiencies, Schmidt syndrome, adrenalitis, gastric atrophy, senile dementia, demyelinating diseases such as autoimmune demyelinating diseases and chronic inflammatory demyelinating polyneuropathy, Dressler syndrome, alopecia greata, alopecia totalis, CREST syndrome (calcinosis, Raynaud's phenomenon, esophageal motility disorder, acrosclerosis), and telangiectasia), autoimmune infertility in men and women, for example, due to anti-sperm antibodies, mixed connective tissue disease, Chagas disease, rheumatic fever, recurrent miscarriage, farmer's lung, erythema multiforme, postcardiotomy syndrome, Cushing syndrome, bird fancier's lung, allergic granulomatous vasculitis, benign lymphocytic vasculitis, Alport syndrome, pneumonitis such as allergic pneumonitis and fibrosing alveolitis, interstitial lung disease, transfusion reaction, leprosy, malaria, leishmaniasis, kypanosomiasis, schistosomiasis, helminthiasis such as ascariasis, aspergillosis, Samter 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, erythema elevatum et diutinum, erythroblastosis fetalis, eosinophilic fasciitis, Schurman syndrome, Felty syndrome, filariasis, chronic cyclitis, heterochronic cyclitis, iridocyclitis (acute or chronic), or Fuch's cyclitis, Henoch-Schönlein purpura, human immunodeficiency virus (HIV) infection, SCID, acquired immunodeficiency syndrome (AIDS), echovirus infection, sepsis, endotoxemia, pancreatitis, thyrotoxicosis, parvovirus infection, rubella virus infection, post-vaccination syndrome, congenital rubella infection, Epstein-Barr virus infection, mumps, Evans syndrome, autoimmune gonadal insufficiency, Sydenham chorea, post-streptococcal glomerulonephritis, obliterative thrombangiitis, thyrotoxicosis, tabes dorsalis, choroiditis, giant cell polymyalgia, chronic hypersensitivity pneumonia, dry keratoconjunctivitis, epidemic keratoconjunctivitis, idiopathic nephrotic syndrome, minimal change nephropathy, benign familial and ischemia-reperfusion injury, transplanted organ reperfusion, retinal autoimmunity, joint inflammation, bronchitis, chronic obstructive airway / lung disease, silicosis, stomatitis, aphthous stomatitis, atherosclerotic disorders, asperniogenese, autoimmune hemolysis,Boeck'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, hemoglobinuric attack, hypogonadism, regional enteritis, leukopenia, infectious mononucleosis, transverse myelitis, primary idiopathic myxedema, nephrosis, sympathetic ophthalmia, granulomatous orchitis, pancreatitis, acute polyneuritis, pyoderma gangrenosum, de Quervain's thyroiditis, acquired spenic atrophy, non-malignant thymoma, vitiligo, toxic shock syndrome, food poisoning, conditions accompanied by infiltration of T cells, leukocyte adhesion deficiency, immune responses associated with acute and delayed hypersensitivity mediated by cytokines and T lymphocytes, diseases accompanied by extravascular migration of leukocytes, multiple organ injury syndrome, antigen-antibody complex-mediated diseases, anti-glomerular basement membrane diseases, allergic neuritis, autoimmune polyendocrine insufficiency, oophoritis, primary myxedema, autoimmune atrophic gastritis Sympathetic ophthalmia, rheumatic diseases, mixed connective tissue disease, nephrotic syndrome, pancreatitis, polyendocrinopathy, polyglandular autoimmune syndrome type I, adult-onset idiopathic hypoparathyroidism (AOIH), cardiomyopathies such as dilated cardiomyopathy, epidermolysis bullosa acquisita (EBA), hemochromatosis, myocarditis, nephrotic syndrome, primary sclerosing cholangitis, purulent or non-purulent rhinitis, acute or chronic rhinitis, ethmoiditis, frontal sinusitis, maxillary sinusitis, or sphenoid sinusitis, eosinophil-related disorders such as eosinophilia, pulmonary infiltrative eosinophilia, eosinophilia-myalgia syndrome, Reiter's syndrome, chronic eosinophilic pneumonia, tropical pulmonary eosinophilia, bronchopulmonary aspergillosis, aspergilloma, or granuloma containing eosinophils, anaphylaxis, seronegative spondyloarthritis, autoimmune polyendocrine syndrome, sclerosing cholangitis, sclera, episclera, chronic mucocutaneous candidiasis, Bruton's syndrome, transient hypogammaglobulinemia of infancy, Wiskott-Aldrich syndrome, ataxia telangiectasia syndrome, telangiectasis, autoimmune disorders associated with collagen diseases, rheumatism, nerve diseases, lymphadenitis, decreased blood pressure response, vascular dysfunction, tissue damage, cardiovascular ischemia, hyperalgesia, renal ischemia, cerebral ischemia, and diseases associated with angiogenesis, allergic hypersensitivity, glomerulonephritis, reperfusion injury, ischemic reperfusion injury, reperfusion injury of the myocardium or other tissues, lymphomatoid tracheobronchitis, inflammatory skin diseases, skin diseases with acute inflammatory components, multiple organ failure, vesicular diseases, renal cortical necrosis, acute purulent meningitis or other central nervous system inflammatory disorders, inflammatory disorders of the eye and orbit, granulocyte transfusion-related syndrome, cytokine-induced toxicity, narcolepsy, acute severe inflammation, chronic refractory inflammation, pyelonephritis, intravascular hyperplasia, peptic ulcer, valvulitis, graft-versus-host disease, contact hypersensitivity, asthmatic airway hypersensitivity, ischemic stroke, pulmonary hypertension, adverse events of immunotherapy, atherosclerosis, wound healing, peripheral arterial disease, cerebral ischemia-reperfusion (I / R) injury, myocardial infarction and immune responses associated with endometriosis are also contemplated. The compositions and polypeptides may be used to treat inflammation associated with stroke, cerebrovascular accident, heart attack and / or acute respiratory distress syndrome. The compositions and polypeptides may be used to treat inflammation and post-infection symptoms associated with viral infections such as COVID, SARS and MERS and / or inflammation and post-infection symptoms associated with the administration of vaccines.

[0113] VI. Pharmaceutical Compositions and Methods In some aspects, the pharmaceutical composition is administered to a subject. Different aspects involve 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. Further, such compounds can be administered in combination with additional treatments.

[0114] The composition can be formulated for parenteral administration, for example, formulated for injection via intravenous, catheterized injection, intraarterial injection, intramuscular, subcutaneous, or even intraperitoneal routes. Typically, such compositions can be prepared as an injectable, either as a liquid solution or a suspension; solid forms suitable for use in preparing solutions or suspensions by the addition of a liquid prior to injection can also be prepared; and the preparation can be emulsified. The preparation of such formulations will be understood by those skilled in the art in light of the present disclosure. In some aspects, the administration is systemic.

[0115] Other routes of administration are contemplated. For example, the constructs and agents can be administered in association with a carrier. In some aspects, the carrier is a nanoparticle or microparticle.

[0116] Particles can have variable-sized structures and are variously known as microspheres, microparticles, nanoparticles, nanospheres, or liposomes. Such particulate formulations can be formed by covalent or non-covalent attachment of constructs to the particles. As used herein, "particle", "microparticle", "bead", "microsphere", and grammatical equivalents mean small individual particles that can be administered to a subject. In certain aspects, the particles are substantially spherical in shape. As used herein, the term "substantially spherical" means that the shape of the particle deviates from a sphere by no more than about 10%. Particles typically consist of a substantially spherical core and optionally one or more layers. The core can vary in size and composition. In addition to the core, the particles can have one or more layers to provide functions appropriate for the use of interest. When present, the thickness of the layer can vary depending on the needs of a particular application. For example, the layer can impart useful optical properties.

[0117] Pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions; formulations 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 form must be sterile and must be fluid to the extent that it can be easily injected. It should also be stable under the conditions of manufacture and storage and must be protected against the contaminating action of microorganisms such as bacteria and fungi.

[0118] In addition, the carrier can be a solvent or dispersion medium including, for example, water, ethanol, polyols (such as glycerol, propylene glycol and liquid polyethylene glycol, etc.), suitable mixtures thereof and vegetable oils. Appropriate fluidity can be maintained, for example, by the use of coatings, such as lecithin, by maintaining the required particle size in the case of dispersants, and by the use of surfactants. The suppression of the action of microorganisms can be brought about by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, thimerosal, etc. In many cases, it is preferable to include isotonic agents, such as saccharides and sodium chloride. The long-term absorption of the injectable composition can be brought about by the use of absorption retardants in the composition, such as aluminum monostearate and gelatin.

[0119] The sterile injectable solution is prepared by incorporating the required amount of the active compound into a suitable solvent having the various other ingredients listed above as required, and then filtering and sterilizing. Generally, the dispersant is prepared by incorporating various sterilized active ingredients into a sterilized medium containing a basic dispersion medium and the other required ingredients among those listed 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, by which powders of the active ingredient plus further desired ingredients are obtained from its previously sterilized filtered solution.

[0120] As used herein, the term "pharmaceutically acceptable" means that a compound, substance, composition and / or dosage form is suitable for contact with human and animal tissues within the scope of sound medical judgment, and is without excessive toxicity, irritation, allergic response or other complications that pose problems, and is in balance with a reasonable benefit / risk ratio. The term "pharmaceutically acceptable carrier" means a pharmaceutically acceptable substance, composition or medium involved in the support or transport of a chemical substance, such as a liquid or solid bulking agent, diluent, excipient, solvent or encapsulating material.

[0121] As used herein, "pharmaceutically acceptable salts" refers to derivatives of the compounds of the present disclosure in which the parent compound is modified by converting any existing acidic or basic moieties into their salt forms. Examples of pharmaceutically acceptable salts include, but are not limited to, salts of basic residues such as amines with mineral or organic acids; alkali or organic salts of acidic residues such as carboxylic acids, and the like. Pharmaceutically acceptable salts include, for example, conventional non-toxic salts or quaternary ammonium salts of the parent compound formed with non-toxic inorganic or organic acids. Pharmaceutically acceptable salts can be synthesized from the parent compound containing a basic or acidic moiety by conventional chemical methods.

[0122] Some variation in dosage will necessarily occur depending on the condition of the subject. The person responsible for administration will determine the appropriate dosage for the individual subject in any given case. The effective amount of a therapeutic or prophylactic composition is determined based on the intended purpose. The terms "unit dose" or "dosage" refer to physically discrete units suitable for use in a subject, each unit containing a predetermined quantity of the composition calculated to produce the desired response discussed above, accompanied by its administration, i.e., the appropriate route and regimen. The amount administered depends on the desired effect and is according to both the number of treatments and the unit dose. Also, the exact amount of the composition depends on the judgment of the practitioner and is specific to each individual. Factors affecting dosage include the physical and clinical condition of the subject, the route of administration, the intended purpose of treatment (alleviation or cure of symptoms), and the potency, stability and toxicity of the specific composition.

[0123] Once formulated, the solutions are administered in a manner compatible with the dosage formulation and in an amount effective therapeutically or prophylactically. The formulations are readily administered in various dosage forms, such as, for example, solutions for injection of the type described above.

[0124] Typically, for a human adult (weighing approximately 70 kilograms), a compound of about 0.1 mg to about 3000 mg (including any value and range therebetween), or about 5 mg to about 1000 mg (including any value and range therebetween), or about 10 mg to about 100 mg (including any value and range therebetween) is administered. It will be understood that such dosage ranges are merely exemplary and that administration can be adjusted according to factors known to those of ordinary skill in the art.

[0125] In certain scenarios, the object is approximately 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, 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, at least 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, 10000 milligrams (mg) or micrograms (mcg) or μg / kg or micrograms / kg / min or mg / kg / min or micrograms / kg / hour or mg / kg / hour, or μM or mM, 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, 1213、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, 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 agents discussed herein are administered. Any range derivable therein is contemplated.,

[0126] The dosage may be administered as needed, or every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 18 or 24 hours (or any range derivable therein) or 1, 2, 3, 4, 5, 6, 7, 8, 9 times or more per day (or any range derivable therein). The dosage may be administered initially before or after the symptoms of the medical condition. In some aspects, the patient may receive the first dose of the regimen 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 hours (or any range derivable therein) or 1, 2, 3, 4 or 5 days (or any range derivable therein) after the patient develops or exhibits symptoms of the medical condition. The patient may be treated for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 days or more (or any range derivable therein), or until the symptoms of the medical condition disappear or are reduced, or until 6, 12, 18 or 24 hours or 1, 2, 3, 4 or 5 days after the symptoms of the infection disappear or are reduced.

[0127] VII. Sequence TIFF2025521602000012.tif176163TIFF2025521602000013.tif237163TIFF2025521602000014.tif242163TIFF2025521602000015.tif243163TIFF2025521602000016.tif244163TIFF2025521602000017.tif243163TIFF2025521602000018.tif243163TIFF2025521602000019.tif243163TIFF2025521602000020.tif246163TIFF2025521602000021.tif241163TIFF2025521602000022.tif241163TIFF2025521602000023.tif243163TIFF2025521602000024.tif246163TIFF2025521602000025.tif243163TIFF2025521602000026.tif246163TIFF2025521602000027.tif244163TIFF2025521602000028.tif243163TIFF2025521602000029.tif243163TIFF2025521602000030.tif243163TIFF2025521602000031.tif246163TIFF2025521602000032.tif192163

Example

[0128] VIII. Example The following examples are included to demonstrate preferred embodiments of the present disclosure. Those skilled in the art will recognize that the techniques disclosed in the following examples are those that the inventors have found to function well in the practice of the present disclosure and, therefore, may be considered to constitute preferred forms for their implementation. However, those skilled in the art should recognize that, in light of the present disclosure, many modifications can be made to the specific embodiments disclosed without departing from the spirit and scope of the present disclosure and still obtain the same or similar results.

[0129] In the examples, figures, and figure descriptions, amino acids K22, R196, R73, and R194 refer to the amino acids at position numbers 23, 197, 74, and 194 of the IL-35 amino acid sequence shown in SEQ ID NO:1 and the EBI3 subunit sequence shown in SEQ ID NO:2, respectively.

[0130] Example 1: Secretion Assay of IL-35-HSA Mutants An expression plasmid was constructed based on pcDNA 3.1 (Thermo Fisher Scientific, US). The cDNA fragment encoding the full length (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 cloned into pcDNA 3.1 digested with the same restriction enzymes after digestion with NheI and EcoRV. Site-directed mutagenesis was performed using the standard method of inverse PCR with pcDNA-IL35-HSA / WT as the template to construct each expression plasmid of the IL-35-HSA mutant. Sequencing confirmed that the mutations were correctly introduced. The constructed mutants are shown in Table 1.

[0131] The expression plasmid was linearized with the restriction enzyme ScaI and transfected into CHO cells according to the manual of Lipofectamine 2000 (Thermo Fisher Scientific, US). Twenty-four hours after transfection, antibiotic selection was performed with 500 ug / ml of G418 (Thermo Fisher Scientific, US) to construct a stable expression pool.

[0132] The constructed stable expression pool was seeded into a 125 mL flask at a seeding density of 2×10 6 cells / mL and incubated for 72 hours. The medium was centrifuged at 10,000g for 10 minutes, and the supernatant was collected.

[0133] Next, the secretion levels of IL-35-HSA WT and IL-35-HSA mutants were analyzed by SDS-PAGE, and the image analysis of the obtained signal intensities was performed using ImageJ software (available online at / / imagej.nih.gov / ij / ) to evaluate the amount of secreted protein. As a result, increased secretion was shown in the K22A and R196A mutants (Table 1, Figures 1-3).

[0134] (Table 1) TIFF2025521602000033.tif153160

[0135] Example 2: Comprehensive evaluation of IL-35-HSA, IL-35-Fc and IL-35 mutants in secretion The inventors performed amino acid substitutions to all 19 different amino acids to compare the expression of IL-35 proteins.

[0136] Expression plasmids in which each of the 4 sites (K22, R196, R73, R194) of EBI3 was substituted with 19 different amino acids were constructed using the same method as described in Example 1 (Table 2). The constructed mutants are shown in Table 2. The expression plasmids were transfected into CHO cells according to the Lipofectamine 2000 manual (5×10 4cells / well, 96-well plate, DNA 1 μg / well). The transfection method followed the Lipofectamine 2000 manual. In the case of IL-35, the p35 / WT plasmid was also transfected simultaneously. The transfectants were incubated for 72 hours. The medium was centrifuged at 10,000 g for 10 minutes, and the supernatant was collected. The amount of protein secreted into the culture supernatant was quantified by Human EBI3 DuoSet ELISA (R&D SYSTEMS, P#: DY6456-05) (n = 3). From the concentration of the secreted protein, the relative amount of the secreted protein was calculated with each control set to 1 (Tables 3 - 5). Among the four site mutations, the introduction of mutations to K22 and R196 significantly increased the secretion amounts of IL-35-HSA, IL-35-Fc, and IL-35. Regarding the introduction of mutations to R73 and P194, the effect on the secretion amounts of IL-35-HSA, IL-35-Fc, and IL-35 increased slightly. Among the 19 types of amino acids, the most secreted amino acids were A, G>V, I>L>H, and S>T. It was found that non-polar amino acids were preferred. The inventors also prepared and included different versions of Fc (SEQ ID NO:70 - 74) because different blood half-lives were expected.

[0137] (Table 2) TIFF2025521602000034.tif187159

[0138] (Table 3) IL-35-HSA Secretion (Relative Ratio, After Sorting) TIFF2025521602000035.tif214159

[0139] (Table 4) IL-35-Fc Secretion (Relative Ratio, After Sorting) TIFF2025521602000036.tif213158

[0140] (Table 5) IL-35 Secretion (Relative Ratio, After Sorting) TIFF2025521602000037.tif214159

[0141] Example 3: Verification of the linker connecting IL-35 and HSA To compare the expression of the IL-35 protein, the inventors created different linker amino acid sequences between IL-35 and HSA.

[0142] IL-35 and HSA were connected using the linkers shown in Table 6, and the secretion amount of IL-35-HSA was evaluated. Expression vectors connecting IL-35 and HSA with each linker were constructed using the PCR method. The expression plasmid was transfected into CHO cells according to the Lipofectamine 2000 manual (5×10 4 cells / well, 96-well plate, DNA 1 μg / well). The transfection method followed the Lipofectamine 2000 manual. The transfectants were incubated for 24 hours. The medium was centrifuged at 10,000 g for 10 minutes, and the supernatant was collected. Next, each secretion level of IL-35-HSA was analyzed by Western blotting, and the image analysis of the obtained signal intensity was performed using ImageJ software (available online at imagej.nih.gov / ij / ), and the amount of secreted protein was evaluated. The relative amount of secreted IL-35-HSA was calculated with linker 12 as 1 (Table 6). The secretion of IL-35-HSA was significantly increased in linkers 1, 10, and 11 compared to linker 12.

[0143] (Table 6) TIFF2025521602000038.tif95158Table 6 shows SEQ ID NO:75 - 85 and 3, respectively.

[0144] Example 4: Evaluation of mutants in which four cysteines in EBI3 were replaced with serine To compare the expression of the IL-35 protein, the inventors performed the substitution of cysteine with serine in the EBI3 portion.

[0145] An expression vector of a mutant in which four cysteines in EBI3 were replaced with serine was prepared in the same manner as described in Example 1. The expression plasmid was transfected into CHO cells according to the manual of Lipofectamine 2000 (5×10 4 cells / well, 96-well plate, 1 μg / well of DNA). The transfection method followed the manual 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, each secretion level of IL-35-HSA was analyzed by Western blotting, and image analysis of the obtained signal intensity was performed using ImageJ software (available online at imagej.nih.gov / ij / ), and the amount of the secreted protein was evaluated. The relative amount of the secreted protein was calculated with IL-35-HSA / WT as 1. When cysteines were replaced with serine at four positions in EBI3, the secretion of IL-35-HSA increased slightly (Figure 5).

[0146] Example 5: Monomerization of IL-35-HSA by Additives The present inventors tested additives for preventing dimerization of IL35.

[0147] IL-35-HSA was observed to multimerize during culture in medium and during the purification process. Additives were investigated to monomerize the multimerized IL-35-HSA. 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 dispensing an equal amount into each tube, each additive was added. The additives used were ascorbic acid (final 1 mM), methionine (final 1 mM), glutathione (final 1 mM), and cysteine (final 1, 10, 50 mM), respectively. Then, the samples were incubated at 37 °C for 15 minutes. Thereafter, each secretion profile of IL-35-HSA was analyzed by Western blotting (Figure 6). As a result, under the conditions of pH 7 and pH 9, the multimer consisting of IL-35-HSA completely disappeared by the addition of methionine, glutathione, and cysteine, and all became monomers. The inventors also tested anionic cyclodextrin (0.001 - 1000 mM), reducing agents (mercaptoethanol and DTT) (0.001 - 1000 mM), and cationic agents (arginine 0.001 - 1000 mM), PEG polymers (0.001 - 1000 mM).

[0148] Example 6 The reducing conditions can prevent 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 in PBS using a size exclusion column. Under the reducing conditions of SDS-PAGE after a 50 mM DTT reaction, the multimer consisting of IL-35-HSA completely disappeared and all became monomers. (Figure 7)

[0149] Example 7 Deletion of the first 20 amino acids at the N-terminus of Ebi3 The N-terminus of the Ebi3 protein analyzed by AlphaFold2 does not contribute to the protein structure. Therefore, the inventors created N-terminal deletion variants (10 amino acid deletion: Δ10 and 20 amino acid deletion: Δ20). 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 in PBS using a size exclusion column. Non-reducing conditions of SDS-PAGE showed that a 20 amino acid deletion, rather than a 10 amino acid deletion, resolved super-large aggregation. (Figure 8)

[0150] Example 8 IL-35-HSA was detected in the lymph nodes 8 days after subcutaneous injection of IL-35-HSA. Forty micrograms of IL-35-HSA was subcutaneously injected into the back of the mouse. Four hours and 8 days after injection, the inventors harvested the lymph nodes (Figure 9). After homogenization of the lymph nodes, the presence of human IL-35 was tested using an ELISA kit (R and D systems DY6456-05) that detects human EBI3 protein. This kit does not detect the mouse endogenous EBI3 protein. The signal at 450 nm indicates that IL-35-HSA is detectable and persists in the lymph nodes for a long time.

[0151] Example 9 IL-35-HSA injection reduces cytokine release syndrome Ten micrograms of IL-35-HSA was subcutaneously injected twice on days 0 and 7. On day 9, 2.5 μg of TNFα protein was intraperitoneally injected. Two hours after TNFα injection, blood was collected and serum IL-6 was analyzed by ELISA (Figure 10A). The TNFα-induced IL-6 concentration in the serum was decreased by IL-35-HSA injection. Flow cytometry analysis showed an increase in dendritic cells (DCs) in the lymph nodes, which exhibited an immunotolerogenic CD206+ phenotype rather than a pro-inflammatory phenotype (Figures 10B - D).

[0152] Example 10: Fusion of Collagen-Binding Domain to IL-35 for Localization The inventors have previously found that a collagen-binding domain derived from the von Willebrand factor A3 domain extends tissue retention after injection due to its collagen affinity in tissues. Prolonged retention of IL-35 at the disease site may be beneficial for extension. The inventors produced a collagen-binding domain-fused IL-35 protein (SEQ ID NO: 53-55). Collagen-binding IL35 can be injected via various injection routes including intravenous, intradermal, and subcutaneous routes.

[0153] Example 11: Evaluation of the Efficacy of SA IL-35 in a Collagen Antibody-Induced Arthritis Model A. Results: The supernatant of mutant SA IL-35 derived from CHO cells 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 and is depicted in Figure 1. Next, mutant SA IL-35 was evaluated as a prophylactic treatment to prevent the development of collagen antibody-induced arthritis (CAIA). The CAIA model was induced on day 0 by intraperitoneal injection of a collagen-II antibody cocktail into 8-week-old female BALB / c mice (Jackson laboratories), as depicted in Figure 11A. The antibody cocktail consisted of four monoclonal antibodies that induce arthritis and are specific for epitopes located on type II collagen [1]. Antibodies that bind to the epitope induce the formation of immune complexes, which subsequently deposit in cartilage and synovium, causing a complement-dependent inflammatory reaction that results in joint swelling [1]. On day 3 after immunization with collagen II antibody, CAIA mice were treated with 40 μg of mutant SA IL-35 (wild-type IL-35 molar equivalent) by subcutaneous injection in the dorsal flank, followed by an intraperitoneal injection of 25 μg of LPS. From day 3 to day 12, the clinical scores of the forelimbs and hindlimbs of the mice were recorded daily. The severity of arthritis was scored on a scale of 0 to 4, where score 0 indicates a healthy limb, score 1 indicates swelling and / or redness of one joint, score 2 indicates swelling and / or redness of two or more joints, score 3 indicates swelling and / or redness of the entire limb, and score 4 indicates maximal swelling. The clinical score results of the experiment are depicted in Figures 11B-C. Prophylactic treatment with a single dose of mutant SA IL-35 prevented the development of severe disease in the CAIA mouse model of arthritis.

[0154] To evaluate the effect of mutant SA IL-35 on immune cell populations in secondary lymphoid organs, single cell suspensions from the draining lymph nodes (popliteal) and spleen were collected at the endpoint and stained for various markers of T cell and myeloid phenotypes and activation as previously described [2]. In the T cell compartment, treatment with mutant SA IL-35 increased the expression of PD-1 on CD4+ and CD8+ T cells in the lymph nodes (Figure 12A) and also increased the expression of PD-1 on CD4+ T cells in the spleen (Figure 12B). In the myeloid compartment, treatment with mutant SA IL-35 reduced the expression of CD40 and CD86 on CD11b+ myeloid cells in the lymph nodes (Figure 12C). Treatment also reduced the percentage of CD11b+ F4 / 80+ macrophages in the lymph nodes and the expression of CD86 on macrophages, but increased the ratio of M2 macrophages to M1 macrophages (Figure 12C). In the spleen, treatment with mutant SA IL-35 reduced the percentage of CD11b+ myeloid cells and the expression of CD86 on CD11b+ cells (Figure 12D). Treatment also reduced the percentage of CD11b+ F4 / 80+ macrophages, but increased the percentage of Arg1+ M2 macrophages (Figure 12D). In the dendritic cell compartment, treatment with mutant SA IL-35 reduced the percentage of CD11c+ dendritic cells in the lymph nodes and the expression of CD86 and CD40 on CD11c+ dendritic cells (Figure 12E). Treatment also reduced the percentage of CD11c+ CD11b+ dendritic cells and the expression of CD86, CD40 and MHC class II on these cells (Figure 12E). In the myeloid-derived suppressor cell compartment, treatment with mutant SA IL-35 reduced the expression of CD86 and CD40 on CD11b+ Ly6Chi Ly6G- MDSC (Figure 12F).

[0155] B. Methods: 1. Collagen-induced arthritis model: (Source Yuba et al 2021) Arthritis was induced as previously described [2] in 8-week-old female BALB / c mice (Jackson Labs). On day 0, mice were injected intraperitoneally with 1 mg / mouse of anti-collagen antibody cocktail (Chondrex), followed by 25 μg of lipopolysaccharide (Chondrex). Three days after immunization with collagen II antibody, CAIA mice were treated with 40 μg of mutant SA IL-35 (wild-type IL-35 molar equivalent) by subcutaneous injection in the dorsal flank, followed by an intraperitoneal injection of 25 μg of LPS. From day 3 to day 12, the clinical scores of the forelimbs and hindlimbs of the mice were recorded daily according to the manufacturer's protocol (Chondrex).

[0156] 2. Flow cytometry analysis: Popliteal lymph node and spleen tissues were digested in DMEM medium (Sigma-Aldrich) supplemented with 2% FBS, 2 mg / ml -1 of collagenase D at 37 °C for 45 minutes. Single cell suspensions were obtained by gently crushing through a 70 μm cell strainer. In the case of the spleen, red blood cells in the blood were lysed with ACK lysis buffer (Quality Biological), followed by antibody staining for flow cytometry. Cells were blocked with CD16 / 32 antibody and stained on ice for 20 minutes with Live / dead fixable aqua (eBioscience) according to the manufacturer's instructions. After the washing step, cells were stained on ice for 20 minutes with surface antibodies. Intracellular staining was performed using the FoxP3 staining kit according to the manufacturer's instructions (BioLegend). After one wash, cells were stained overnight at room temperature with intracellular antibodies. Flow cytometry analysis was performed using a Fortessa flow cytometer (BD Biosciences) and analyzed using FlowJo software (Tree Star).

[0157] Although specific embodiments have been described in some detail or with reference to one or more individual embodiments, those skilled in the art will be able to make numerous changes to the disclosed embodiments without departing from the scope of the invention. Further, where appropriate, any aspect of any of the above-described embodiments can be combined with any aspect of any of the other described embodiments to form additional embodiments having equivalent or different characteristics and addressing the same or different problems. Similarly, it will be understood that the above-described advantages and benefits may relate to one embodiment or to several embodiments. Any reference to a patent publication or other publication is, in this specification, a specific incorporation by reference of the disclosure of that publication. The claims should not be construed to include such limitations, unless a means-plus-function or step-plus-function limitation is expressly recited in a given claim using the phrases "means for" or "step for", respectively.

[0158] References: TIFF2025521602000039.tif48160

Claims

[Claim 1] A polypeptide comprising 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 include K23, R74, R195, R197, or a combination thereof.

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