Anti-inflammatory cytokines and methods of use

Abstract

Aspects of the present disclosure provide methods and compositions for treating autoimmune disorders, including, for example, multiple sclerosis and rheumatoid arthritis. Methods for promoting wound healing are also disclosed. Certain aspects are directed to anti-inflammatory cytokines operably linked to albumin proteins. Further aspects relate to methods for delivering anti-inflammatory cytokines to lymph nodes.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority to U.S. Provisional Patent Application No. 63 / 083,722, filed on 25 September 2020, which is incorporated herein by reference in its entirety.

[0002] I. Sequence Listing This application includes a sequence listing submitted in ASCII format, which is incorporated herein by reference in its entirety. The aforementioned ASCII copy, created on 23 September 2021, is named ARCDP0711WO.txt and has a size of 120,558 bytes.

[0003] II. Field of Invention This invention relates at least to the fields of molecular biology, immunology, and medicine. [Background technology]

[0004] III. Background Multiple sclerosis (MS) is a morbid autoimmune disease that can affect millions of people worldwide. Autoreactive immune cells return to the central nervous system (CNS), causing demyelination and resulting in localized damage to the white matter. 1 Lymphocytes and macrophages infiltrating the central nervous system (CNS) cause axonal damage. Recent studies have shown that Th17 cells activated in secondary lymphoid organs (SLOs) migrate to the spinal cord and brain, playing a crucial role in the onset and severity of MS. 2, 3 Therefore, inhibiting lymphocyte migration to the CNS and inducing an immunosuppressive microenvironment in the SLO are considered effective treatments for MS. FTY720 (fingolimod) and the anti-integrin α4 antibody (natalizumab) are used clinically to treat MS by sequestering lymphocytes to the LN and preventing them from reacting with autoantigens in target tissues. 4, 5Experimental autoimmune encephalomyelitis (EAE) is a widely accepted mouse model of MS and reflects many features of disease progression and developmental mechanisms, including lymphocyte migration to the CNS and demyelination.

[0005] Rheumatoid arthritis (RA) is an autoimmune disease that is currently controlled by treatments using inhibitors of inflammatory pathways. The pathological features of RA are synovitis and joint destruction, which cause severe pain and joint dysfunction (48, 49). The causative antigens of RA are not fully understood, but collagen recognition by immune cells plays a crucial role. During the progression of RA, autoantigen-specific T cells, particularly Th17 cells, are activated and produce inflammatory cytokines, including IL-17. In the joints, inflammatory cytokines such as TNF-α and IL-6 induce the activation of macrophages and neutrophils as mediators of the inflammatory response. These inflammatory cells infiltrate the joints and trigger various inflammatory responses, including the activation of osteoclasts that destroy bone within the joints (50). Current RA treatment strategies are symptomatic, and given that many inflammatory cytokines are involved in the progression of RA, various biological therapeutic agents, such as antibodies or soluble receptors against TNF-α, have been developed and approved for clinical use (51).

[0006] Various strategies for treating autoimmune disorders, including MS and RA, have been investigated, including the use of anti-inflammatory cytokines. To date, such strategies have not been transferred to clinical use. Compositions and methods for treating autoimmune disorders, including MS and RA, including the effective delivery of anti-inflammatory cytokines, remain in need. [Overview of the project]

[0007] overview The aspects of this disclosure relate to compositions comprising anti-inflammatory cytokines functionally linked to albumin proteins, and methods of use comprising such compositions, including methods for treating various conditions, including autoimmune or inflammatory conditions.

[0008] The aspects of this disclosure include therapeutic polypeptides, anti-inflammatory polypeptides, anti-inflammatory compositions, pharmaceutical compositions, nucleic acid molecules, vectors, therapeutic cells, methods for treating autoimmune conditions, methods for treating inflammatory conditions, methods for promoting wound healing, methods for treating subjects for multiple sclerosis (MS), methods for treating subjects for rheumatoid arthritis, methods for inhibiting Th17 cell function, methods for reducing inflammation in subjects, methods for targeting anti-inflammatory cytokines to lymph nodes, methods for detecting anti-inflammatory cytokines in lymph nodes, methods for diagnosing subjects having an autoimmune or inflammatory condition, methods for targeting cytokines to the lymph nodes of subjects, and methods for preventing autoimmune or inflammatory conditions.

[0009] The polypeptide of the present disclosure may comprise at least one, two, three, four or more components of: anti-inflammatory cytokines, albumin proteins, albumin-binding proteins, linkers, tags, labels, and anti-inflammatory molecules, the components of which may be in any order starting from the N-terminus. The method of the present disclosure may comprise at least one, two, three, four or more of the following steps: administering a composition to a subject; obtaining a biological sample from the subject; obtaining a lymph sample from the subject; detecting anti-inflammatory cytokines in the lymph sample from the subject; producing a polypeptide comprising anti-inflammatory cytokines; conjugating anti-inflammatory cytokines to albumin proteins via linkers; conjugating anti-inflammatory cytokines to albumin-binding proteins via linkers; diagnosing the subject for an autoimmune or inflammatory condition; treating the subject for an autoimmune or inflammatory condition; promoting wound healing in the subject; and reducing inflammation in the subject.

[0010] Disclosed herein are methods for treating a subject for an autoimmune or inflammatory condition, comprising the step of administering to the subject a viable amount of a composition containing an anti-inflammatory cytokine functionally bound to an albumin protein by subcutaneous, intradermal, or intramuscular administration. Similarly intended are methods for targeting anti-inflammatory cytokines to lymph nodes in a subject by administering a viable amount of a composition containing an anti-inflammatory cytokine functionally bound to an albumin protein. In some cases, the condition is multiple sclerosis (MS). MS may be further defined as primary progressive MS. In some cases, MS includes secondary progressive MS. In some cases, MS is further defined as relapsing-remitting MS. In some cases, MS is further defined as a clinically isolated syndrome. In some cases, the subject is a subject who has experienced or is currently experiencing an acute attack within a period of at least 48 hours prior to administration. In some cases, MS is late MS. Subjects may be defined as having active MS, inactive MS, worsening MS, or non-worsening MS. Subjects with active MS are defined as those who have experienced an episode or MS symptoms and have evidence of disease progression. Subjects with inactive MS are defined as those whose condition is stable and there is no clear evidence of disease progression. Subjects with worsening MS are defined as those who have observed and marked increases in their disability after a relapse. Subjects with non-worsening MS are defined as those who have experienced a relapse but do not show new signs of disability or signs of worsening. In some aspects, MS disease is suppressed by administration of the composition. Suppression may be at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, or 90% suppression. In some aspects, demyelination is inhibited by administration of the composition. Demyelination can be inhibited by at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, or 90%.

[0011] In some aspects, the anti-inflammatory cytokine is IL-4. In some aspects, the anti-inflammatory cytokine functionally linked to albumin protein contains a sequence that has sequence identity with SEQ ID NO:5 of 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.5 or 99.9% (or any range that can be derived therefrom), or contains a sequence that has sequence identity with at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.5 or 99.9% (or any range that can be derived therefrom). In some aspects, the anti-inflammatory cytokine functionally linked to albumin protein contains SEQ ID NO:5. In some aspects, the anti-inflammatory cytokine functionally linked to the albumin protein contains a sequence that has sequence identity with SEQ ID NO:6 of 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.5, or 99.9% (or any range within which it can be derived), or contains a sequence that has sequence identity with at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.5, or 99.9% (or any range within which it can be derived). In some aspects, the anti-inflammatory cytokine functionally linked to the albumin protein contains SEQ ID NO:6. In some aspects, the anti-inflammatory cytokine is IL-33.In some aspects, anti-inflammatory cytokines functionally linked to albumin proteins contain sequences that have sequence identity with SEQ ID NO:9 of 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.5, or 99.9% (or any range that can be derived therefrom), or contain sequences that have sequence identity with at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.5, or 99.9% (or any range that can be derived therefrom). In some aspects, anti-inflammatory cytokines functionally linked to albumin proteins contain SEQ ID NO:9. In some aspects, anti-inflammatory cytokines functionally linked to albumin protein contain sequences that have sequence identity with SEQ ID NO:10 of 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.5, or 99.9% (or any range that can be derived therefrom), or contain sequences that have sequence identity with at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.5, or 99.9% (or any range that can be derived therefrom). In some aspects, anti-inflammatory cytokines functionally linked to albumin protein contain SEQ ID NO:10. In some aspects, the condition is arthritis, multiple sclerosis, or scleroderma. In some aspects, the arthritis is rheumatoid arthritis. In some aspects, the anti-inflammatory cytokine is IL-10.In some aspects, anti-inflammatory cytokines functionally linked to albumin proteins contain sequences that have sequence identity with SEQ ID NO:13 of 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.5, or 99.9% (or any range within which this can be derived), or contain sequences that have sequence identity with at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.5, or 99.9% (or any range within which this can be derived). In some aspects, anti-inflammatory cytokines functionally linked to albumin proteins contain SEQ ID NO:13. In some aspects, anti-inflammatory cytokines functionally linked to albumin protein contain sequences that have sequence identity with SEQ ID NO:14 of 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.5, or 99.9% (or any range that can be derived therefrom), or contain sequences that have sequence identity with at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.5, or 99.9% (or any range that can be derived therefrom). In some aspects, anti-inflammatory cytokines functionally linked to albumin protein contain SEQ ID NO:14. In some aspects, anti-inflammatory cytokines functionally linked to albumin proteins have sequence identity with SEQ ID NO: 52 of 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.5, or 99.9% (or any range within which this can be derived), or contain sequences with sequence identity of at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.5, or 99.9% (or any range within which this can be derived).In some aspects, anti-inflammatory cytokines functionally linked to albumin proteins include SEQ ID NO:52. In some aspects, anti-inflammatory cytokines functionally linked to albumin proteins include sequences with sequence ID NO:53 having sequence identity of 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.5, or 99.9% (or any range derivable within that range), or sequences with sequence identity of at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.5, or 99.9% (or any range derivable within that range). In some aspects, anti-inflammatory cytokines functionally linked to albumin proteins include SEQ ID NO:53. In some aspects, anti-inflammatory cytokines functionally linked to albumin proteins include sequences with sequence ID NO:54 having sequence identity of 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.5, or 99.9% (or any range derivable within that) or sequences with sequence identity of at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.5, or 99.9% (or any range derivable within that) In several contexts, anti-inflammatory cytokines functionally linked to albumin proteins include SEQ ID NO:54.In some aspects, anti-inflammatory cytokines functionally linked to albumin proteins contain sequences that have sequence identity with SEQ ID NO: 55 of 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.5, or 99.9% (or any range within which this can be derived), or contain sequences that have sequence identity with at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.5, or 99.9% (or any range within which this can be derived). In some aspects, anti-inflammatory cytokines functionally linked to albumin proteins contain SEQ ID NO: 55. In some cases, the condition is type 1 diabetes, diabetic peripheral neuropathy, psoriasis, inflammatory bowel disease, or Crohn's disease. In some cases, the condition is acute respiratory distress syndrome (ARDS). It is particularly intended that one or more of these conditions may be excluded from certain cases.

[0012] Disclosed herein are methods for promoting wound healing in a subject, in some aspects, comprising the step of administering to the subject by subcutaneous, intradermal, or intramuscular administration an effective amount of a composition comprising an anti-inflammatory cytokine functionally linked to an albumin protein. In some aspects, the composition increases the rate of wound healing in the subject compared to the rate of wound healing in the subject that has not been administered the composition. In some aspects, the wound is a diabetic ulcer. In some aspects, the anti-inflammatory cytokine is IL-4. In some aspects, anti-inflammatory cytokines functionally linked to albumin proteins contain sequences that have sequence identity with SEQ ID NO:5 of 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.5, or 99.9% (or any range within which this can be derived), or contain sequences that have sequence identity with at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.5, or 99.9% (or any range within which this can be derived). In some aspects, anti-inflammatory cytokines functionally linked to albumin proteins contain SEQ ID NO:5. In some aspects, the anti-inflammatory cytokine functionally linked to the albumin protein contains a sequence that has sequence identity with SEQ ID NO:6 of 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.5, or 99.9% (or any range within which it can be derived), or contains a sequence that has sequence identity with at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.5, or 99.9% (or any range within which it can be derived). In some aspects, the anti-inflammatory cytokine functionally linked to the albumin protein contains SEQ ID NO:6. In some aspects, the anti-inflammatory cytokine is IL-33.In some aspects, anti-inflammatory cytokines functionally linked to albumin proteins contain sequences that have sequence identity with SEQ ID NO:9 of 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.5, or 99.9% (or any range that can be derived therefrom), or contain sequences that have sequence identity with at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.5, or 99.9% (or any range that can be derived therefrom). In some aspects, anti-inflammatory cytokines functionally linked to albumin proteins contain SEQ ID NO:9. In some aspects, anti-inflammatory cytokines functionally linked to albumin proteins have sequence identity with SEQ ID NO:10 of 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.5, or 99.9% (or any range derivable within that range), or contain sequences with sequence identity of at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.5, or 99.9% (or any range derivable within that range). In some aspects, the anti-inflammatory cytokine is IL-10. In some aspects, anti-inflammatory cytokines functionally linked to albumin proteins contain sequences that have sequence identity with SEQ ID NO:13 of 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.5, or 99.9% (or any range within which this can be derived), or contain sequences that have sequence identity with at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.5, or 99.9% (or any range within which this can be derived). In some aspects, anti-inflammatory cytokines functionally linked to albumin proteins contain SEQ ID NO:13.In some aspects, anti-inflammatory cytokines functionally linked to albumin protein contain sequences that have sequence identity with SEQ ID NO:14 of 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.5, or 99.9% (or any range that can be derived therefrom), or contain sequences that have sequence identity with at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.5, or 99.9% (or any range that can be derived therefrom). In some aspects, anti-inflammatory cytokines functionally linked to albumin protein contain SEQ ID NO:14. In the context of this disclosure, the composition may include a hyaluronic acid hydrogel carrier.

[0013] Similarly described are methods for treating or preventing cytokine storm syndrome in a subject, comprising the step of administering to the subject an effective amount of a composition containing IL-27 functionally linked to an albumin-binding polypeptide. In some aspects, the subject has cancer. In some aspects, the subject is being treated with immunotherapy. In some aspects, the immunotherapy includes immune checkpoint blockade (ICB) therapy, adoptive T-cell therapy, cytokine therapy, CAR-T-cell therapy, activation of costimulatory molecules, and combinations thereof. In some aspects, the cancer includes melanoma. In some aspects, the cancer includes renal cancer. In some aspects, the cancer includes stage I, II, III, or IV cancer. In some aspects, the cancer includes metastatic or recurrent cancer. In some aspects, IL-27 includes one of SEQ ID NO: 23-26, as well as combinations and fusions thereof.

[0014] In the context of this disclosure, doses of albumin-cytokine fusion proteins, such as albumin fusions with IL-4, IL-5, IL-10, IL-11, IL-23, IL-27, IL-33, IL-35, IL-36ra, IL-37, or IL-38, are 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.2, 0.21, 0.22, 0.23, 0.24, 0.25, 0.26, 0.27, 0.28, 0.29, 0.3, 0.31, 0.32, 0.33, 0.34, 0.35, 0.36, 0.37, 0.38, 0.39, 0.4, 0.41, 0.42, 0.43, 0.44, 0.45, 0.46, 0.47, 0.48, 0.49, 0.5, 0.51, 0.52, 0.5 3, 0.54, 0.55, 0.56, 0.57, 0.58, 0.59, 0.6, 0.61, 0.62, 0.63, 0.64, 0.65, 0.66, 0.67, 0.68, 0.69, 0.7, 0.71, 0.72, 0.73, 0.74, 0.75, 0.76, 0.77, 0.78, 0.79, 0.8, 0.81, 0.82, 0.83, 0.84, 0.85, 0.86, 0.87, 0.88, 0.89, 0.9, 0.91, 0.92, 0.93, 0.94, 0.95, 0.96, 0.97, 0.98, 0.99, 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, 10.1, 10.2, 10.3, 10.4, 10.5, 10.6, 10.7, 10.8, 10.9, 11, 11.1, 11.2, 11.3, 11.4, 11.5, 11.6, 11.7, 11.8, 11.9, 12, 12.1, 12.2, 12.3, 12.4, 12.5, 12.6, 12.7, 12.8, 12.9, 13, 13.1, 13.2, 13.3, 13.4, 13.5, 13.6, 13.7, 13 0.8, 13.9, 14, 14.1, 14.2, 14.3, 14.4, 14.5, 14.6, 14.7, 14.8, 14.9, 15, 15.1, 15.2, 15.3, 15.4, 15.5, 15.6, 15.7, 15.8, 15.9, 16, 16.1, 16.2, 16.3, 16.4, 16. 5, 16.6, 16.7, 16.8, 16.9, 17, 17.1, 17.2, 17.3, 17.4, 17.5, 17.6, 17.7, 17.8, 17.9, 18, 18.1, 18.2, 18.3, 18.4, 18.5, 18.6, 18.7, 18.8, 18.9, 19, 19.1, 19.2 , 19.3, 19.4, 19.5, 19.6, 19.7, 19.8, 19.9, 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 mg / kg, or any range derivable within that, at least 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.2, 0.21, 0.22, 0.23, 0.24, 0.25, 0.26, 0.27, 0.28, 0.29, 0.3, 0.31, 0.32, 0.33, 0.34, 0.35, 0.36, 0.37, 0.38, 0.39, 0.4, 0.41, 0.42, 0.4 3, 0.44, 0.45, 0.46, 0.47, 0.48, 0.49, 0.5, 0.51, 0.52, 0.53, 0.54, 0.55, 0.56, 0.57, 0.58, 0.59, 0.6, 0.61, 0.62, 0.63, 0.64, 0.65, 0.66, 0.67, 0.68, 0.69, 0.7, 0.71, 0.72, 0.73, 0.74, 0.75, 0.76, 0.77, 0.78, 0.79, 0.8, 0.81, 0.82, 0.83, 0.84, 0.85, 0.86, 0.87, 0.88, 0.89, 0.9, 0.91, 0.92, 0.93, 0.94, 0.95, 0.96, 0.97, 0.98, 0.99, 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, 10.1, 10.2, 10.3, 10.4, 10.5, 10.6, 10.7, 10.8, 10.9, 11, 11.1, 11.2, 11.3, 11.4, 11.5, 11.6, 11.7, 11.8, 11.9, 1 2, 12.1, 12.2, 12.3, 12.4, 12.5, 12.6, 12.7, 12.8, 12.9, 13, 13.1, 13.2, 13.3, 13.4, 13.5, 13.6, 13.7, 13.8, 13.9, 14, 14.1, 14.2, 14.3, 14.4, 14.5, 14.6, 14.7, 14.8, 14.9, 15, 15.1, 15.2, 15.3, 15.4, 15.5, 15.6, 15.7, 15.8, 15.9, 16, 16.1, 16.2, 16.3, 16.4, 16.5, 16.6, 16.7, 16.8, 16.9, 17, 17.1, 17.2, 17.3, 17.4, 17.5, 17.6, 17.7, 17.8, 17.9, 18, 18.1, 18.2, 18.3, 18.4, 18.5, 18.6, 18.7, 18.8, 18.9, 19, 19.1, 19.2, 19.3, 19.4, 19.5, 19.6, 19.7, 19.8, 19.9, 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 mg / kg, or any range deriveable within that, or at most 0.01, 0.02, 0.03、0.04、0.05、0.06、0.07、0.08、0.09、0.1、0.11、0.12、0.13、0.14、0.15、0.16、0.17、0.18、0.19、0.2、0.21、0.22、0.23、0.24、0.25、0.26、0.27、0.28、0.29、0.3、0.31、0.32、0.33、0.34、0.35、0.36、0.37、0.38、0.39、0.4、0.41、0.42、0.43、0.44、0.45、0.46、0.47、0.48、0.49、0.5、0.51、0.52、0.53、0.54、0.55、0.56、0.57、0.58、0.59、0.6、0.61、0.62、0.63、0.64、0.65、0.66、0.67、0.68、0.69、0.7、0.71、0.72、0.73、0.74、0.75、0.76、0.77、0.78、0.79、0.8、0.81、0.82、0.83、0.84、0.85、0.86、0.87、0.88、0.89、0.9、0.91、0.92、0.93、0.94、0.95、0.96、0.97、0.98、0.99、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、10.1、10.2、10.3、10.4、10.5、10.6、10.7、10.8、10.9、11、11.1、11.2、11.3、11.4、11.5、11.6、11.7、11.8、11.9、12、12.1、12.2、12.3、12.4、12.5、12.6、12.7、12.8、12.9、13、13.1、13.2、13.3、13.4、13.5、13.6、13.7、13.8, 13.9, 14, 14.1, 14.2, 14.3, 14.4, 14.5, 14.6, 14.7, 14.8, 14.9, 15, 15.1, 15.2, 15.3, 15.4, 15.5, 15.6, 15.7, 15.8, 15.9, 16, 16.1, 16.2, 16.3, 16.4, 16.5, 16.6, 16.7, 16.8, 16.9, 17, 17.1, 17.2, 17.3, 17.4, 17.5, 17.6, 17.7, 17.8, 1 7.9, 18, 18.1, 18.2, 18.3, 18.4, 18.5, 18.6, 18.7, 18.8, 18.9, 19, 19.1, 19.2, 19.3, 19.4, 19.5, 19.6, 19.7, 19.8, 19.9, 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 mg / kg, or any range derivable within those limits.

[0015] The target dates are the 1st, 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 9th, 10th, 11th, 12th, 13th, 14th, 15th, 16th, 17th, 18th, 19th, 20th, 21st, 22nd, 23rd, 24th, 25th, 26th, 27th, 28th, 29th, 30th, 31st, 32nd, 33rd, 34th, 35th, 36th, 37th, 38th, 39th, 40th, 41st, 42nd, 43rd, 44th, 45th, 46th, 47th, 48th, 49th, 50th, 51st, 52nd, 53rd, 54th, 55th, 56th, 57th, 58th, 59th or 60th, or weeks 1, 2, or 3 weeks. , 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks, 15 weeks, 16 weeks, 17 weeks, 18 weeks, 19 weeks, 20 weeks, 21 weeks, 22 weeks, 23 weeks, 24 weeks, 25 weeks, 26 weeks, 27 weeks, 28 weeks, 29 weeks, 30 weeks, 31 weeks, 32 weeks, 33 weeks, 34 weeks, 35 weeks, 36 weeks, 37 weeks, 38 weeks, 39 weeks, 40 weeks, 41 weeks, 42 weeks, 43 weeks, 44 weeks, 45 weeks, 46 weeks, 47 weeks, 48 ​​weeks, 49 weeks, 50 weeks, 51 weeks, 52 weeks, or 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months or 12 months (also (within any range that can be derived within that range), at least 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, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 31 days, 32 days, 33 days, 34 days, 35 days, 36 days, 37 days, 38 days, 39 days, 40 days, 41 days, 42 days, 43 days, 44 days, 45 days, 46 days, 47 days, 48 ​​days, 49 days, 50 days, 51 days, 52 days, 53 days, 54 days, 55 days, 56 days, 57 days, 58 days, 59 days Days or 60 days, or 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks, 15 weeks, 16 weeks, 17 weeks, 18 weeks, 19 weeks, 20 weeks, 21 weeks, 22 weeks, 23 weeks, 24 weeks, 25 weeks, 26 weeks, 27 weeks, 28 weeks, 29 weeks, 30 weeks, 31 weeks, 32 weeks, 33 weeks, 34 weeks, 35 weeks, 36 weeks, 37 weeks, 38 weeks, 39 weeks, 40 weeks, 41 weeks, 42 weeks, 43 weeks, 44 weeks, 45 weeks, 46 weeks, 47 weeks, 48 ​​weeks, 49 weeks, 50 weeks, 51 weeks, 52 weeks, or 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months,10 months, 11 months, or 12 months (or any range that can be derived within that), or at most 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, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 31 days, 32 days, 33 days, 34 days, 35 days, 36 days, 37 days, 38 days, 39 days, 40 days, 41 days, 42 days , 43 days, 44 days, 45 days, 46 days, 47 days, 48 ​​days, 49 days, 50 days, 51 days, 52 days, 53 days, 54 days, 55 days, 56 days, 57 days, 58 days, 59 days or 60 days, or 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks, 15 weeks, 16 weeks, 17 weeks, 18 weeks, 19 weeks, 20 weeks, 21 weeks, 22 weeks, 23 weeks, 24 weeks, 25 weeks, 26 weeks, 27 weeks, 28 weeks, 29 weeks, 30 weeks, 31 weeks, 32 weeks, 33 weeks, 3 During a specific period such as 4 weeks, 35 weeks, 36 weeks, 37 weeks, 38 weeks, 39 weeks, 40 weeks, 41 weeks, 42 weeks, 43 weeks, 44 weeks, 45 weeks, 46 weeks, 47 weeks, 48 ​​weeks, 49 weeks, 50 weeks, 51 weeks, 52 weeks, or 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, or 12 months (or any range that can be derived within that), 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 doses (or any range within which can be derived), at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 doses (or any range within which can be derived), or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 doses (or any range within which can be derived) may be administered.

[0016] In the context of this disclosure, the doses of albumin-cytokine fusion proteins, such as albumin fusions with IL-4, IL-5, IL-10, IL-11, IL-23, IL-27, IL-33, IL-35, IL-36ra, IL-37, or IL-38, are 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.2, 0.21, 0.22, 0.23, 0.24, 0.25, 0.26, 0.27, 0.28, 0.29, 0.3, 0.31, 0.32, 0.33, 0.34, 0.35, 0.36, 0.37, 0.38, 0.39, 0.4, 0.41, 0.42, 0.43, 0.44, 0.45, 0.46, 0.47, 0.48, 0.49, 0.5, 0.51, 0.52, 0.5 3, 0.54, 0.55, 0.56, 0.57, 0.58, 0.59, 0.6, 0.61, 0.62, 0.63, 0.64, 0.65, 0.66, 0.67, 0.68, 0.69, 0.7, 0.71, 0.72, 0.73, 0.74, 0.75, 0.76, 0.77, 0.78, 0.79, 0.8, 0.81, 0.82, 0.83, 0.84, 0.85, 0.86, 0.87, 0.88, 0.89, 0.9, 0.91, 0.92, 0.93, 0.94, 0.95, 0.96, 0.97, 0.98, 0.99, 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, 10.1, 10.2, 10.3, 10.4, 10.5, 10.6, 10.7, 10.8, 10.9, 11, 11.1,11.2, 11.3, 11.4, 11.5, 11.6, 11.7, 11.8, 11.9, 12, 12.1, 12.2, 12.3, 12.4, 12.5, 12.6, 12.7, 12.8, 12.9, 13, 13.1, 13.2, 13.3, 13.4, 13.5, 13.6, 13.7, 13. 8, 13.9, 14, 14.1, 14.2, 14.3, 14.4, 14.5, 14.6, 14.7, 14.8, 14.9, 15, 15.1, 15.2, 15.3, 15.4, 15.5, 15.6, 15.7, 15.8, 15.9, 16, 16.1, 16.2, 16.3, 16.4, 16.5 , 16.6, 16.7, 16.8, 16.9, 17, 17.1, 17.2, 17.3, 17.4, 17.5, 17.6, 17.7, 17.8, 17.9, 18, 18.1, 18.2, 18.3, 18.4, 18.5, 18.6, 18.7, 18.8, 18.9, 19, 19.1, 19.2, 19.3, 19.4, 19.5, 19.6, 19.7, 19.8, 19.9, 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 mg / kg, or any range derivable within that, at least 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.2, 0.21, 0.22, 0.23, 0.24, 0.25, 0.26, 0.27, 0.28, 0.29, 0.3, 0.31, 0.32, 0.33, 0.34, 0.35, 0.36, 0.37, 0.38, 0.39, 0.4, 0.41, 0.42, 0.43, 0.44, 0.45, 0.46, 0.47, 0.48, 0.49, 0.5, 0.51, 0.52, 0.53, 0.54, 0.55, 0.56, 0.57, 0.58, 0.59, 0.6, 0.61, 0.62, 0.63, 0.64, 0.65, 0.66, 0.67, 0.68, 0.69, 0.7, 0.71, 0.72, 0.73, 0.74, 0.75, 0.76, 0.77, 0.78, 0.79, 0.8, 0.81, 0.82, 0.83, 0.84, 0.85, 0.86, 0.87, 0.88, 0.89, 0.9, 0.91, 0.92,0.93, 0.94, 0.95, 0.96, 0.97, 0.98, 0.99, 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 ,6.4,6.5,6.6,6.7,6.8,6.9,7,7.1,7.2,7.3,7.4,7.5,7.6,7.7,7.8,7.9,8,8.1,8.2,8.3,8.4,8.5,8.6,8.7,8.8,8.9,9,9.1,9.2,9.3,9.4,9.5,9.6,9.7,9.8,9.9,10,10.1,10.2,10.3,10.4,10.5,10.6,10.7,10.8,10.9,11,11.1,11.2,11.3,11.4,11.5,11.6,11.7,11.8,11.9,12, 12.1, 12.2, 12.3, 12.4, 12.5, 12.6, 12.7, 12.8, 12.9, 13, 13.1, 13.2, 13.3, 13.4, 13.5, 13.6, 13.7, 13.8, 13.9, 14, 14.1, 14.2, 14.3, 14.4, 14.5, 14.6, 14.7, 14.8, 14.9, 15, 15.1, 15.2, 15.3, 15.4, 15.5, 15.6, 15.7, 15.8, 15.9, 16, 16.1, 16.2, 16.3, 16.4, 16.5, 16.6, 16.7, 16.8, 16.9, 1 7, 17.1, 17.2, 17.3, 17.4, 17.5, 17.6, 17.7, 17.8, 17.9, 18, 18.1, 18.2, 18.3, 18.4, 18.5, 18.6, 18.7, 18.8, 18.9, 19, 19.1, 19.2, 19.3, 19.4, 19.5, 19.6, 19.7, 19.8, 19.9, 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 mg / kg, or any range deriveable within that, or at most 0.01, 0.02, 0.03,0.04、0.05、0.06、0.07、0.08、0.09、0.1、0.11、0.12、0.13、0.14、0.15、0.16、0.17、0.18、0.19、0.2、0.21、0.22、0.23、0.24、0.25、0.26、0.27、0.28、0.29、0.3、0.31、0.32、0.33、0.34、0.35、0.36、0.37、0.38、0.39、0.4、0.41、0.42、0.43、0.44、0.45、0.46、0.47、0.48、0.49、0.5、0.51、0.52、0.53、0.54、0.55、0.56、0.57、0.58、0.59、0.6、0.61、0.62、0.63、0.64、0.65、0.66、0.67、0.68、0.69、0.7、0.71、0.72、0.73、0.74、0.75、0.76、0.77、0.78、0.79、0.8、0.81、0.82、0.83、0.84、0.85、0.86、0.87、0.88、0.89、0.9、0.91、0.92、0.93、0.94、0.95、0.96、0.97、0.98、0.99、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、10.1、10.2、10.3、10.4、10.5、10.6、10.7、10.8、10.9、11、11.1、11.2、11.3、11.4、11.5、11.6、11.7、11.8、11.9、12、12.1、12.2、12.3、12.4、12.5、12.6、12.7、12.8、12.9、13、13.1、13.2、13.3、13.4、13.5、13.6、13.7、13.8、13.9, 14, 14.1, 14.2, 14.3, 14.4, 14.5, 14.6, 14.7, 14.8, 14.9, 15, 15.1, 15.2, 15.3, 15.4, 15.5, 15.6, 15.7, 15.8, 15.9, 16, 16.1, 16.2, 16.3, 16.4, 16.5, 16.6, 16.7, 16.8, 16.9, 17, 17.1, 17.2, 17.3, 17.4, 17.5, 17.6, 17.7, 17.8, 17 0.9, 18, 18.1, 18.2, 18.3, 18.4, 18.5, 18.6, 18.7, 18.8, 18.9, 19, 19.1, 19.2, 19.3, 19.4, 19.5, 19.6, 19.7, 19.8, 19.9, 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 mg / kg, or any range derivable within that range, and the target days 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 Day, 34th, 35th, 36th, 37th, 38th, 39th, 40th, 41st, 42nd, 43rd, 44th, 45th, 46th, 47th, 48th, 49th, 50th, 51st, 52nd, 53rd, 54th, 55th, 56th, 57th, 58th, 59th or 60th, or 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 1 2 weeks, 13 weeks, 14 weeks, 15 weeks, 16 weeks, 17 weeks, 18 weeks, 19 weeks, 20 weeks, 21 weeks, 22 weeks, 23 weeks, 24 weeks, 25 weeks, 26 weeks, 27 weeks, 28 weeks, 29 weeks, 30 weeks, 3 1 week, 32 weeks, 33 weeks, 34 weeks, 35 weeks, 36 weeks, 37 weeks, 38 weeks, 39 weeks, 40 weeks, 41 weeks, 42 weeks, 43 weeks, 44 weeks, 45 weeks, 46 weeks, 47 weeks, 48 ​​weeks, 49 weeks, 5 Within 0 weeks, 51 weeks, 52 weeks, or 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, or 12 months (or any range within which it can be derived), at least 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,18th, 19th, 20th, 21st, 22nd, 23rd, 24th, 25th, 26th, 27th, 28th, 29th, 30th, 31st, 32nd, 33rd, 34th, 35th, 36th, 37th, 38th, 39th, 40th, 41st, 42nd, 43rd, 44th, 45th, 46th, 47th, 48th, 4 9th, 50th, 51st, 52nd, 53rd, 54th, 55th, 56th, 57th, 58th, 59th or 60th day, or 1st, 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 9th, 10th, 11th, 12th, 13th, 14th, 15th, 16th, 17th, 18th, 19th, 20th week, 2 1 week, 22 weeks, 23 weeks, 24 weeks, 25 weeks, 26 weeks, 27 weeks, 28 weeks, 29 weeks, 30 weeks, 31 weeks, 32 weeks, 33 weeks, 34 weeks, 35 weeks, 36 weeks, 37 weeks, 38 weeks, 39 weeks, 40 weeks, 41 weeks, 42 weeks, 43 weeks, 44 weeks, 45 weeks, 46 weeks, 47 weeks, 48 ​​weeks, 49 weeks, 50 weeks, 51 weeks, 52 weeks, or 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months or 12 months (or any range that can be derived within that), or at most 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13th, 14th, 15th, 16th, 17th, 18th, 19th, 20th, 21st, 22nd, 23rd, 24th, 25th, 26th, 27th, 28th, 29th, 30th, 31st, 32nd, 33rd, 34th, 35th, 36th, 37th, 38th, 39th, 40th, 41st, 42nd, 43rd, 4 4th, 45th, 46th, 47th, 48th, 49th, 50th, 51st, 52nd, 53rd, 54th, 55th, 56th, 57th, 58th, 59th or 60th day, or 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks, 15 weeks, 1 During a specific period such as 6 weeks, 17 weeks, 18 weeks, 19 weeks, 20 weeks, 21 weeks, 22 weeks, 23 weeks, 24 weeks, 25 weeks, 26 weeks, 27 weeks, 28 weeks, 29 weeks, 30 weeks, 31 weeks, 32 weeks, 33 weeks, 34 weeks, 35 weeks, 36 weeks, 37 weeks, 38 weeks, 39 weeks, 40 weeks, 41 weeks, 42 weeks, 43 weeks, 44 weeks, 45 weeks, 46 weeks, 47 weeks, 48 ​​weeks, 49 weeks, 50 weeks, 51 weeks, 52 weeks, or 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 doses (or any range within which can be derived), at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 doses (or any range within which can be derived), or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 doses (or any range within which can be derived) may be administered.

[0017] In the context of this disclosure, IL-4-conjugated albumin is administered to subjects in a dose of 0.4–1.5 mg / kg once per week. In the context of this disclosure, subjects are administered one or two doses of IL-4-conjugated albumin per week, with doses of 0.4–1.5 mg / kg. In the context of this disclosure, subjects are administered IL-4-conjugated albumin once a week at a dose of 0.4–1.5 mg / kg.

[0018] In the context of this disclosure, IL-33-conjugated albumin is administered to subjects at a dose of 0.6–12 mg / kg every other day for a total of three doses per week or over a total of three doses per week. IL-33-conjugated albumin may be administered to subjects at a dose of 0.6–12 mg / kg every other day for a period of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 weeks (or any derivable range therein), or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 weeks (or any derivable range therein), for a total of three doses per week.

[0019] In the context of this disclosure, IL-4-linked albumin is administered to subjects three times a week at a dose of 0.5–5 mg / kg. In the context of this disclosure, IL-4-linked albumin is administered to subjects three times a week at a dose of 0.5–5 mg / kg for the treatment of diabetes, such as type 1 diabetes.

[0020] Based on animal studies that present efficacy and toxicity data of specific doses of a compound in mice, such as those described in the examples, those skilled in the art can estimate appropriate doses for humans. This is described in Nair and Jacob, Journal of Basic and Clinical Pharmacy, Vol. 7, Issue 2, March-May 2016, pages 27-31, which is incorporated herein by reference.

[0021] Disclosed herein are methods for treating a subject for multiple sclerosis, comprising the step of administering an effective amount of a composition containing IL-4 functionally linked to an albumin protein to the subject. In some aspects, the IL-4 is human IL-4, and the albumin protein is human serum albumin.

[0022] Disclosed herein are methods for treating a subject for multiple sclerosis, comprising the step of administering to the subject an effective amount of a composition containing IL-33 functionally linked to an albumin protein by subcutaneous, intradermal, or intramuscular administration. In some aspects, the IL-33 is human IL-33, and the albumin protein is human serum albumin.

[0023] Disclosed herein are methods for treating a subject for rheumatoid arthritis, in some aspects, comprising the step of administering to the subject by subcutaneous, intradermal, or intramuscular administration an effective amount of a composition comprising IL-10 functionally linked to an albumin protein. In some aspects, IL-10 is human IL-10, and the albumin protein is human serum albumin.

[0024] Disclosed herein are methods for treating a subject for rheumatoid arthritis, in some aspects, comprising the step of administering to the subject by subcutaneous, intradermal, or intramuscular administration an effective amount of a composition comprising IL-35 functionally linked to an albumin protein. In some aspects, IL-35 is human IL-35, and the albumin protein is human serum albumin.

[0025] Disclosed herein are methods for promoting wound healing in a subject, in some aspects, comprising the step of administering to the subject by subcutaneous, intradermal, or intramuscular administration an effective amount of a composition comprising IL-4 functionally linked to an albumin protein. In some aspects, IL-4 is human IL-4, and the albumin protein is human serum albumin.

[0026] Disclosed herein are methods for promoting wound healing in a subject, in some aspects, comprising the step of administering to the subject by subcutaneous, intradermal, or intramuscular administration an effective amount of a composition comprising IL-33 functionally linked to an albumin protein. In some aspects, the IL-33 is human IL-33, and the albumin protein is human serum albumin.

[0027] Disclosed herein are methods for inhibiting the function of Th17 cells in certain aspects, comprising the step of administering to a subject by subcutaneous, intradermal, or intramuscular administration an effective amount of a composition comprising an anti-inflammatory cytokine functionally linked to an albumin protein.

[0028] Disclosed herein are methods for reducing inflammation in a subject in certain aspects, comprising the step of administering to the subject a viable amount of a composition comprising an anti-inflammatory cytokine functionally linked to an albumin protein, by subcutaneous, intradermal, or intramuscular administration.

[0029] Disclosed herein are methods for targeting anti-inflammatory cytokines to lymph nodes of a subject, comprising the step of administering a composition containing an anti-inflammatory cytokine functionally linked to an albumin protein to the subject by subcutaneous, intradermal, or intramuscular administration. In some cases, the subject has an autoimmune or inflammatory condition. In some cases, the method further comprises the step of identifying the anti-inflammatory cytokine in the lymph nodes of the subject. In some cases, the identification step comprises obtaining a lymph sample from the subject. In some cases, the identification step comprises detecting the presence of the anti-inflammatory cytokine in the lymph sample. In some cases, the anti-inflammatory cytokine remains in the lymph nodes for at least 8 hours after administration of the composition to the subject. In some cases, the anti-inflammatory cytokine remains in the lymph nodes for at least 16 hours after administration of the composition to the subject.

[0030] In some situations, the anti-inflammatory cytokine is IL-4. In some situations, the anti-inflammatory cytokine is IL-5. In some situations, the anti-inflammatory cytokine is IL-10. In some situations, the anti-inflammatory cytokine is IL-11. In some situations, the anti-inflammatory cytokine is IL-23. In some situations, the anti-inflammatory cytokine is IL-27. In some situations, the anti-inflammatory cytokine is IL-33. In some situations, the anti-inflammatory cytokine is IL-35. In some situations, the anti-inflammatory cytokine is IL-36ra. In some situations, the anti-inflammatory cytokine is IL-37. In some situations, the anti-inflammatory cytokine is IL-36ra. In some situations, the anti-inflammatory cytokine is IL-38. In some situations, the anti-inflammatory cytokine is interferon-β. In some situations, the anti-inflammatory cytokine is TGF-β1.

[0031] Disclosed herein are methods for treating a subject for an autoimmune or inflammatory condition, comprising the step of administering to the subject a viable amount of a composition comprising an anti-inflammatory cytokine functionally linked to an albumin-binding polypeptide, by subcutaneous, intradermal, or intramuscular administration. In some aspects, the albumin-binding peptide is an anti-albumin antibody. In some aspects, the albumin-binding protein comprises a sequence having sequence identity with SEQ ID NO: 51 of at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.5, or 99.9% (or any range within which this can be derived). In some aspects, the albumin-binding protein comprises SEQ ID NO: 51.

[0032] In some aspects, albumin protein is human serum albumin. In some aspects, albumin protein is mouse serum albumin.

[0033] In some cases, the composition is administered to the subject by subcutaneous injection. In some cases, the composition is administered to the subject by intradermal injection. In some cases, the composition is administered to the subject by intramuscular injection. In some cases, the composition is administered to the subject by intravenous injection. In some cases, the composition is administered systemically to the subject. In some cases, the albumin protein is functionally linked to the N-terminus of the anti-inflammatory cytokine. In some cases, the anti-inflammatory cytokine is covalently linked to the albumin protein. In some cases, the anti-inflammatory cytokine is covalently linked to the albumin protein via a linker. In some embodiments, albumin is located at the amino-terminal end of the cytokine. In some embodiments, albumin is located at the carboxy-terminal end of the cytokine.

[0034] In some aspects, albumin protein increases the accumulation of anti-inflammatory cytokines in target lymph nodes compared to anti-inflammatory cytokines that are not functionally linked to albumin protein. In some aspects, the composition reduces the number of Th17 cells in the target. In some aspects, the composition inhibits the function of Th17 cells in the target.

[0035] In some aspects, the polypeptides and compositions of this disclosure treat one or more symptoms of MS. Symptoms may include visual alterations, including diplopia, blurred vision or decreased visual acuity; numbness, tingling or weakness (weakness may range from mild to severe); paralysis, spatial disorientation or dizziness; erectile dysfunction (ED, impotence); pregnancy complications; urinary incontinence (or conversely, urinary retention); muscle spasms; impaired muscle coordination; tremors; painful involuntary muscle contractions; slurred speech; and / or fatigue. Symptoms may last for 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 12 hours, 18 hours, 24 hours, or 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, or 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, or 1 month, 2 months, 3 months, 4 months, 5 or 6 months (or any range that can be derived within that), or at least 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 12 hours, 18 hours, 24 hours, or 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, The effects may be reduced by 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 10, 11, 12 weeks, or 1 month, 2 months, 3 months, 4 months, 5 or 6 months (or any range within which can be derived), or by at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100%, or any range within which can be derived.

[0036] In some cases, the composition is administered to the subject via a pre-filled syringe. In some cases, the anti-inflammatory cytokine is administered in doses ranging from 0.1 mg / kg to 50 mg / kg. In some cases, the anti-inflammatory cytokine is administered in doses of at least 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, 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 mg / kg, or any derivable range or value within that range, at most 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, 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 mg / kg, or any derivable range or value within that range, or approximately 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.The drug is administered at doses of 0, 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 mg / kg, or any achievable range or value within that range. In some cases, the subject has been previously treated for the condition. In some cases, the subject has been determined to be resistant to previous treatment. In some cases, the subject has not been previously treated for the condition.

[0037] In some aspects, the method further includes a step of administering an additional anti-inflammatory agent to the target. In some aspects, the method does not include a step of administering an additional anti-inflammatory agent to the target. In some aspects, the composition is administered during the cessation of treatment of the target with the additional anti-inflammatory agent. The additional anti-inflammatory agent is fingolimod, interferon-β, dimethyl fumarate, teriflunomide, integrin α4β1, or an anti-αLβ2 antibody. In some aspects, the additional anti-inflammatory agent is an anti-TNFα agent, an anti-IL-6R agent, an anti-IL-6 agent, or a Janus kinase inhibitor.

[0038] In some aspects, the method includes a step of administering nucleic acids containing sequences encoding anti-inflammatory cytokines and albumin proteins to a target. In some aspects, the nucleic acids are vectors. In some aspects, the method includes a step of administering cells containing the vector to a target. In some aspects, the cells are configured to express the vector.

[0039] In some aspects, the method further includes a step of detecting anti-inflammatory cytokines in the target lymph nodes. In some aspects, the detection step includes obtaining a lymph sample from the subject. In some aspects, the detection step includes detecting the presence of anti-inflammatory cytokines in the lymph sample.

[0040] Throughout this application, the term "approximately" is used to indicate that the value includes inherent error variations with respect to the measurement or quantification method.

[0041] The use of the words "a" or "an" can mean "one" when used with the term "including," but it also coincides with the meanings of "one or more," "at least one," and "one or more."

[0042] The phrase "and / or" means "and" or "or." For example, A, B and / or C includes A alone, B alone, C alone, A and B as a combination, A and C as a combination, B and C as a combination, or A, B and C as a combination. In other words, "and / or" functions as an all-encompassing "or."

[0043] 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”), or “containing” (and any form of “containing,” such as “contains” and “contain”) are inclusive or non-restrictive and do not exclude further, unquoted stages of elements or methods.

[0044] Compositions and methods for use thereof may "contain," "essentially consist of," or "consist of" any of the components or steps disclosed in this application as a whole. Compositions and methods "essentially consisting of" any of the disclosed components or steps shall limit the claims to specific materials or steps that do not substantially affect the basic and novel features of the claimed invention. As used herein and in the claims, 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”), or “containing” (and any form of “containing,” such as “contains” and “contain”) are inclusive or non-restrictive and do not exclude further, unreferenced elements or steps of method. The aspects and aspects described herein in the context of the term “comprising” are also intended to be implemented in the context of the terms “consisting of” or “consisting essentially of.”

[0045] The terms "individual," "subject," and "patient" are used interchangeably and can refer to either a human or a non-human. In some contexts, the subject is human.

[0046] Any method in the context of a therapeutic, diagnostic, or physiological purpose or effect may be described in the use-claim language, such as "use" of any compound, composition, or agent discussed herein, for achieving or performing the described therapeutic, diagnostic, or physiological purpose or effect. For example, this includes the use of albumin conjugated to an anti-inflammatory cytokine for targeting cytokines to lymph nodes of interest. Another example is the use of albumin conjugated to an anti-inflammatory cytokine for treating an autoimmune or inflammatory condition.

[0047] It is particularly intended that any limitations discussed in relation to one aspect or part of the present invention may apply to any other aspect or part of the present invention. Furthermore, any composition of the present invention may be used in any method of the present invention, and any method of the present invention may be used to prepare or utilize any composition of the present invention. Aspects of the aspects described in the examples may also be implemented elsewhere in different examples or elsewhere in this application, for example, in the context of aspects discussed in the summary of the invention, the detailed description of aspects, the claims, and the legend of the figures.

[0048] [Invention 1001] A method for treating multiple sclerosis in a subject, comprising the step of subcutaneously administering a composition to the subject comprising a polypeptide containing IL-33 fused to albumin via a linker, wherein the composition is administered at a dose of 0.6 to 12 mg / kg, the subject receives a total of three doses over one week, and the doses are administered every other day. [Invention 1002] A method for treating a subject for an autoimmune or inflammatory condition, comprising the step of administering an effective amount of a composition comprising an anti-inflammatory cytokine functionally linked to an albumin protein to the subject. [Invention 1003] The method of the present invention 1002, wherein the composition is administered by subcutaneous, intradermal, intramuscular, or intravenous administration. [Invention 1004] The method according to the present invention 1002 or 1003, wherein the composition is administered to a subject by subcutaneous injection. [Invention 1005] The method according to 1002 or 1003 of the present invention, wherein the composition is administered to a subject by intradermal administration. [Invention 1006] The method according to 1002 or 1003 of the present invention, wherein the composition is administered to a subject by intramuscular administration. [Invention 1007] Any method according to invention 1002 to 1006, wherein the subject is a human subject. [Invention 1008] A method according to any one of the present invention 1002 to 1007, wherein an albumin protein is functionally linked to the N-terminus of an anti-inflammatory cytokine. [Invention 1009] A method according to any one of the present invention 1002 to 1008, wherein an anti-inflammatory cytokine is covalently linked to an albumin protein. [Invention 1010] The method of the present invention 1009, wherein an anti-inflammatory cytokine is covalently linked to an albumin protein via a linker. [Invention 1011] A method according to any one of the present invention 1002 to 1010, wherein the aforementioned condition is multiple sclerosis (MS). [Invention 1012] The method of the present invention 1011, wherein MS is further defined as primary progressive MS. [Invention 1013] The method of the present invention 1011, wherein MS is further defined as a secondary progression type MS. [Invention 1014] The method of the present invention 1011, wherein MS is further defined as relapsing-remitting MS. [Invention 1015] The method of the present invention 1011, wherein MS is further defined as a clinically isolated syndrome. [Invention 1016] The method according to any of items 1011 to 1015 of the present invention, wherein the subject is a subject who has experienced or is currently experiencing an acute attack within 48 hours prior to administration. [Invention 1017] Any method 1011 to 1016 of the present invention, wherein MS is a late MS. [Invention 1018] A method according to any one of the present invention 1011 to 1017, wherein the subject is defined as having active MS, inactive MS, worsening MS, or non-worsening MS. [Invention 1019] A method according to any of the present invention 1002 to 1018, wherein the anti-inflammatory cytokine is IL-4. [Invention 1020] The method of the present invention 1019, wherein the anti-inflammatory cytokine functionally linked to the albumin protein includes SEQ ID NO:5. [Invention 1021] The method of the present invention 1019, wherein the anti-inflammatory cytokine functionally linked to the albumin protein includes SEQ ID NO:6. [Invention 1022] A method according to any of the present invention 1002 to 1011, wherein the anti-inflammatory cytokine is IL-33. [Invention 1023] The method of the present invention 1022, wherein the anti-inflammatory cytokine functionally linked to the albumin protein includes SEQ ID NO:9. [Invention 1024] The method of the present invention 1022, wherein the anti-inflammatory cytokine functionally linked to the albumin protein includes SEQ ID NO:10. [Invention 1025] Any method of the present invention 1002 to 1024, further comprising the step of administering an additional anti-inflammatory agent to the target. [Invention 1026] Any method of the present invention 1002 to 1024, which does not include the step of administering further anti-inflammatory agents to the target. [Invention 1027] A method according to any one of the present invention 1002 to 1024, wherein the composition is administered during the cessation of treatment of the target with a further anti-inflammatory agent. [Invention 1028] The method according to any one of the present invention 1025 to 1027, wherein the further anti-inflammatory agent is fingolimod, interferon-β, dimethyl fumarate, teriflunomide, anti-integrin α4β1 antibody, or anti-integrin αLβ2 antibody. [Invention 1029] A method according to any of items 1011 to 1028 of the present invention, wherein MS disease is suppressed by administration of the composition. [Invention 1030] A method according to any of the present invention 1011 to 1029, wherein demyelination is inhibited by administration of the composition. [Invention 1031] A method according to any one of the present invention 1002 to 1010, wherein the condition is arthritis, multiple sclerosis, or scleroderma. [Invention 1032] The method of the present invention 1031, wherein arthritis is rheumatoid arthritis. [Invention 1033] The method of the present invention 1031 or 1032, wherein the anti-inflammatory cytokine is IL-10. [Invention 1034] The method of the present invention 1033, wherein the anti-inflammatory cytokine functionally linked to the albumin protein includes SEQ ID NO:13. [Invention 1035] The method of the present invention 1033, wherein the anti-inflammatory cytokine functionally linked to the albumin protein includes SEQ ID NO:14. [Invention 1036] A method according to any one of the present invention 1031 to 1035, wherein the anti-inflammatory cytokine is IL-35. [Invention 1037] Any method of the present invention 1031 to 1036, further comprising the step of administering an additional anti-inflammatory agent to the target. [Invention 1038] Any method of the present invention 1031 to 1035, which does not include the step of administering further anti-inflammatory agents to the target. [Invention 1039] The method according to any one of the present invention 1031 to 1035, wherein the composition is administered during the cessation of treatment of the target with a further anti-inflammatory agent. [Invention 1040] The method according to any one of the invention 1037 to 1039, wherein the further anti-inflammatory agent is an anti-TNFα agent, an anti-IL-6R agent, an anti-IL-6 agent, or a Janus kinase inhibitor. [Invention 1041] A method according to any one of items 1002 to 1010 of the present invention, wherein the condition is type 1 diabetes, diabetic peripheral neuropathy, psoriasis, inflammatory bowel disease, cytokine storm syndrome, systemic scleroderma, or Crohn's disease. [Invention 1042] A method according to any one of the present invention 1002 to 1041, wherein albumin protein increases the accumulation of anti-inflammatory cytokines in a target lymph node compared to anti-inflammatory cytokines that are not functionally linked to albumin protein. [Invention 1043] Any method 1002 to 1042 of the present invention, wherein the composition reduces the number of Th17 cells in a subject. [Invention 1044] A method according to any of the present invention 1002 to 1043, wherein the composition inhibits the function of Th17 cells in a target. [Invention 1045] A method according to any of items 1002 to 1044 of the present invention, wherein the composition is administered to a subject via a pre-filled syringe. [Invention 1046] A method according to any one of items 1002 to 1045 of the present invention, wherein an anti-inflammatory cytokine is administered at a dose of 0.1 mg / kg to 50 mg / kg. [Invention 1047] A method of any of the present invention 1002 to 1046, wherein the subject has been previously treated for the aforementioned condition. [Invention 1048] The method of the present invention 1047, wherein the subject is determined to be resistant to previous treatment. [Invention 1049] Any method according to item 1002 to 1048 of the present invention, further comprising the step of detecting anti-inflammatory cytokines in the target lymph node. [Invention 1050] The method of the present invention 1049, wherein the detection step includes obtaining a lymph sample from the subject. [Invention 1051] The method of the present invention 1050, wherein the detection step includes detecting the presence of anti-inflammatory cytokines in a lymph sample. [Invention 1052] A method according to any one of the present invention 1002 to 1051, comprising the step of administering nucleic acids containing sequences encoding anti-inflammatory cytokines and albumin proteins. [Invention 1053] The method of the present invention 1052, wherein the nucleic acid is the vector. [Invention 1054] The method of the present invention 1053, comprising the step of administering the vector to cells. [Invention 1055] A method for promoting wound healing in a subject, comprising the step of administering an effective amount of a composition containing an anti-inflammatory cytokine functionally linked to an albumin protein to the subject by subcutaneous, intradermal, or intramuscular administration. [Invention 1056] The method of the present invention 1055, wherein the composition increases the healing rate of a wound in a subject compared to the healing rate of a wound in a subject that has not been administered the composition. [Invention 1057] The method of the present invention 1056, wherein the wound is a diabetic ulcer. [Invention 1058] A method according to any one of the present invention 1055 to 1057, wherein an anti-inflammatory cytokine is covalently linked to an albumin protein. [Invention 1059] The method of the present invention 1058, wherein an anti-inflammatory cytokine is covalently linked to an albumin protein via a linker. [Invention 1060] A method according to any one of the present invention 1055 to 1059, wherein an albumin protein is functionally linked to the N-terminus of an anti-inflammatory cytokine. [Invention 1061] A method according to any of items 1055 to 1060 of the present invention, wherein the anti-inflammatory cytokine is IL-4. [Invention 1062] The method of the present invention 1061, wherein the anti-inflammatory cytokine functionally linked to the albumin protein includes SEQ ID NO:5. [Invention 1063] The method of the present invention 1061, wherein the anti-inflammatory cytokine functionally linked to the albumin protein includes SEQ ID NO:6. [Invention 1064] A method according to any of the present invention 1055 to 1059, wherein the anti-inflammatory cytokine is IL-33. [Invention 1065] The method of the present invention 1064, wherein the anti-inflammatory cytokine functionally linked to the albumin protein includes SEQ ID NO:9. [Invention 1066] The method of the present invention 1064, wherein the anti-inflammatory cytokine functionally linked to the albumin protein includes SEQ ID NO:10. [Invention 1067] A method according to any of the present invention 1055 to 1059, wherein the anti-inflammatory cytokine is IL-10. [Invention 1068] The method of the present invention 1067, wherein the anti-inflammatory cytokine functionally linked to the albumin protein includes SEQ ID NO:13. [Invention 1069] The method of the present invention 1067, wherein the anti-inflammatory cytokine functionally linked to the albumin protein includes SEQ ID NO:14. [Invention 1070] A method according to any of the present invention 1055 to 1069, wherein the composition is not administered to the wound site. [Invention 1071] A method according to any of the present invention 1055 to 1069, wherein the composition comprises a hyaluronic acid hydrogel carrier. [Invention 1072] A method according to any one of the present invention 1055 to 1070, wherein albumin protein increases the accumulation of anti-inflammatory cytokines in a target lymph node compared to anti-inflammatory cytokines that are not functionally linked to albumin protein. [Invention 1073] Any method 1055 to 1072 of the present invention, wherein the composition reduces the number of Th17 cells in a subject. [Invention 1074] A method according to any of the present invention 1055 to 1073, wherein the composition inhibits the function of Th17 cells in a target. [Invention 1075] A method according to any of items 1055 to 1074 of the present invention, wherein the composition is administered to a subject via a pre-filled syringe. [Invention 1076] A method according to any of the present invention 1055 to 1075, wherein the anti-inflammatory cytokine is administered in a dose of 0.1 mg / kg to 50 mg / kg. [Invention 1077] Any method according to item 1055 to 1076 of the present invention, further comprising the step of detecting anti-inflammatory cytokines in the target lymph node. [Invention 1078] The method of the present invention 1077, wherein the detection step includes obtaining a lymph sample from the subject. [Invention 1079] The method of the present invention 1078, wherein the detection step includes detecting the presence of anti-inflammatory cytokines in a lymph sample. [Invention 1080] A method according to any one of the present invention 1055 to 1079, comprising the step of administering nucleic acids containing sequences encoding anti-inflammatory cytokines and albumin proteins. [Invention 1081] The method of the present invention 1080, wherein the nucleic acid is the vector. [Invention 1082] The method of the present invention 1081, comprising the step of administering the vector to cells. [Invention 1083] A method for treating a subject with multiple sclerosis, comprising the step of administering an effective amount of a composition containing IL-4 functionally linked to an albumin protein to the subject. [Invention 1084] A method for treating a subject with multiple sclerosis, comprising the step of administering an effective amount of a composition containing IL-33 functionally linked to an albumin protein to the subject by subcutaneous, intradermal, or intramuscular administration. [Invention 1085] A method for treating a subject with rheumatoid arthritis, comprising the step of administering an effective amount of a composition containing IL-10 functionally linked to an albumin protein to the subject by subcutaneous, intradermal, or intramuscular administration. [Invention 1086] A method for treating a subject with rheumatoid arthritis, comprising the step of administering an effective amount of a composition containing IL-35 functionally linked to an albumin protein to the subject by subcutaneous, intradermal, or intramuscular administration. [Invention 1087] A method for promoting wound healing in a subject, comprising the step of administering an effective amount of a composition containing IL-4 functionally linked to an albumin protein to the subject by subcutaneous, intradermal, or intramuscular administration. [Invention 1088] A method for promoting wound healing in a subject, comprising the step of administering an effective amount of a composition containing IL-33 functionally linked to an albumin protein to the subject by subcutaneous, intradermal, or intramuscular administration. [Invention 1089] A method for inhibiting the function of Th17 cells, comprising the step of administering an effective amount of a composition containing an anti-inflammatory cytokine functionally linked to an albumin protein to a subject by subcutaneous, intradermal, or intramuscular administration. [Invention 1090] A method for reducing inflammation in a subject, comprising the step of administering an effective amount of a composition containing an anti-inflammatory cytokine functionally linked to an albumin protein to the subject by subcutaneous, intradermal, or intramuscular administration. [Invention 1091] A method for targeting an anti-inflammatory cytokine to a target lymph node, comprising the step of administering a composition containing an anti-inflammatory cytokine functionally linked to an albumin protein to the target by subcutaneous, intradermal, or intramuscular administration. [Invention 1092] The method of the present invention 1091, wherein the subject has an autoimmune state or an inflammatory state. [Invention 1093] The method of the present invention 1092, wherein an autoimmune or inflammatory condition is treated by targeting anti-inflammatory cytokines to lymph nodes. [Invention 1094] The method of the present invention 1092 or 1093, wherein the autoimmune or inflammatory condition includes MS, type 1 diabetes, diabetic peripheral neuropathy, psoriasis, inflammatory bowel disease, cytokine storm syndrome, systemic scleroderma, arthritis, rheumatoid arthritis, acute respiratory stress syndrome, or Crohn's disease. [Invention 1095] Any method of the present invention 1091 to 1094, further comprising the step of identifying anti-inflammatory cytokines in the target lymph node. [Invention 1096] The method of the present invention 1095, wherein the identification step includes obtaining a lymph sample from the subject. [Invention 1097] The method of the present invention 1096, wherein the identification step includes detecting the presence of anti-inflammatory cytokines in a lymph sample. [Invention 1098] A method according to any one of the present invention 1091 to 1097, wherein anti-inflammatory cytokines remain in the lymph nodes for at least 8 hours after administration of the composition to a subject. [Invention 1099] A method according to any one of the present invention 1091 to 1097, wherein anti-inflammatory cytokines remain in the lymph nodes for at least 16 hours after administering the composition to a subject. [Invention 1100] A method according to any of the present invention 1091 to 1099, wherein the anti-inflammatory cytokine is IL-4. [Invention 1101] The method of the present invention 1100, wherein IL-4 functionally linked to albumin protein is administered at a dose of 0.4 to 1.5 mg / kg. [Invention 1102] The method of the present invention 1100 or 1101, wherein the subject is administered one or two doses per week. [Invention 1103] The method of the present invention 1100 or 1101, wherein the subject is administered less than three doses per week or over a period of one week. [Invention 1104] A method according to any of the present invention 1091 to 1099, wherein the anti-inflammatory cytokine is IL-5. [Invention 1105] A method according to any of the present invention 1091 to 1099, wherein the anti-inflammatory cytokine is IL-10. [Invention 1106] A method according to any of the present invention 1091 to 1099, wherein the anti-inflammatory cytokine is IL-11. [Invention 1107] A method according to any of the present invention 1091 to 1099, wherein the anti-inflammatory cytokine is IL-23. [Invention 1108] A method according to any of the present invention 1091 to 1099, wherein the anti-inflammatory cytokine is IL-27. [Invention 1109] A method according to any of the present invention 1091 to 1099, wherein the anti-inflammatory cytokine is IL-33. [Invention 1110] The method of the present invention 1109, wherein IL-33 functionally linked to albumin protein is administered at a dose of 0.6 to 12 mg / kg. [Invention 1111] The method according to invention 1109 or 1110, wherein the subject is administered a total of three doses over one week, with each dose administered every other day. [Invention 1112] A method according to any of the present invention 1091 to 1099, wherein the anti-inflammatory cytokine is IL-35. [Invention 1113] A method according to any of the present invention 1091 to 1099, wherein the anti-inflammatory cytokine is IL-36ra. [Invention 1114] A method according to any of the present invention 1091 to 1099, wherein the anti-inflammatory cytokine is IL-37. [Invention 1115] A method according to any of the present invention 1091 to 1099, wherein the anti-inflammatory cytokine is IL-38. [Invention 1116] A method according to any one of the present invention 1091 to 1099, wherein the anti-inflammatory cytokine is interferon-β. [Invention 1117] A method according to any of the present invention 1091 to 1099, wherein the anti-inflammatory cytokine is TGF-β1. [Invention 1118] Any method of the present invention 1091 to 1099, wherein the albumin protein is human serum albumin. [Invention 1119] A method according to any one of the present invention 1091 to 1099, wherein an anti-inflammatory cytokine is covalently linked to an albumin protein. [Invention 1120] A method according to any one of the present invention 1091 to 1099, wherein an anti-inflammatory cytokine is covalently linked to an albumin protein via a linker. [Invention 1121] A method according to any of the present invention 1091 to 1120, wherein an anti-inflammatory cytokine is administered at a dose of 0.1 mg / kg to 50 mg / kg. [Invention 1122] A method for treating a subject with an autoimmune or inflammatory condition, comprising the step of administering to the subject an effective amount of a composition containing an anti-inflammatory cytokine functionally linked to an albumin-binding polypeptide by subcutaneous, intradermal, or intramuscular administration. [Invention 1123] The method of the present invention 1122, comprising albumin-binding polypeptide SEQ ID NO:51. [Invention 1124] A method for treating or preventing cytokine storm syndrome in a subject, comprising the step of administering an effective amount of a composition containing IL-27 functionally linked to an albumin-binding polypeptide to the subject. [Invention 1125] The method of the present invention 1124, wherein the subject has cancer. [Invention 1126] The method of the present invention 1124 or 1125, wherein the subject is treated with immunotherapy. [Invention 1127] Any method of the present invention 1124 to 1126, wherein the immunotherapy includes immune checkpoint block (ICB) therapy, adoptive T-cell therapy, cytokine therapy, CAR-T-cell therapy, activation of costimulatory molecules, and combinations thereof. [Invention 1128] A method according to any one of the present invention 1125 to 1127, wherein the cancer includes melanoma. [Invention 1129] A method according to any one of the present invention 1125 to 1127, wherein the cancer includes kidney cancer. [Invention 1130] A method according to any one of the present invention 1125 to 1129, wherein the cancer is a stage I, II, III, or IV cancer. [Invention 1131] A method according to any one of the present invention 1125 to 1130, wherein the cancer includes metastatic or recurrent cancer. [Invention 1132] The method of the present invention 1022, wherein IL-27 includes one of SEQ ID NO:23~26, as well as combinations and fusions thereof. Other objects, features, and advantages of the present invention will become apparent from the following detailed description. However, while the detailed description and specific examples illustrate certain aspects of the present invention, it should be understood that they are merely illustrative, as various modifications and changes within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description. [Brief explanation of the drawing]

[0049] The following drawings constitute part of this specification and are included to further demonstrate certain aspects of the invention. The invention may be better understood by referring to one or more of these drawings together with the detailed description of the specific aspects presented herein.

[0050] [Figure 1A]Figures 1A-1D - IL-4 retains its activity even after fusion with serum albumin (SA). Figure 1A: wt IL-4 and SA-IL-4 were analyzed by SDS-PAGE under non-reducing (N) and reducing (R) conditions using Coomassie blue staining. Figure 1B: Binding of SA-IL-4 to newly isolated immune cells from (left) LN and (right) spleen, measured by flow cytometry (n = 1). Figure 1C: wt IL-4 and SA-IL-4 activity assay. Phosphorylation of STAT6 in T cells was analyzed by flow cytometry after culturing T cells in vitro with the indicated concentrations of wt IL-4 or SA-IL-4 (n = 2). Figure 1D: IL-17 concentrations secreted under Th17 differentiation conditions in the presence of wt IL-4 or SA-IL-4, measured by ELISA (n = 4). Data are mean ± SEM. Two experimental replicas. Statistical analysis was performed using Tukey's test and one-way ANOVA. [Figure 1B] See the explanation in Figure 1A. [Figure 1C] See the explanation in Figure 1A. [Figure 1D] See the explanation in Figure 1A. [Figure 2A]Figures 2A-2J - SA fusion to IL-4 increased the amount of IL-4 in secondary lymphoid organs after intravenous injection. Figures 2A-2B: Amounts of IL-4 in the upper arm and lumbar lining (LN) and (Figure 2B) spleen over time. 40 μg of wt IL-4 or equimolar SA-IL-4 was intravenously (iv) injected into naive mice. LN and spleen were collected and the amount of IL-4 was tested by ELISA (n = 5). Figures 2C-2D: Amounts of IL-4 in the upper arm and lumbar lining (LN) and (Figure 2D) spleen. 40 μg of wt IL-4 or equimolar SA-IL-4 was intraperitoneally (ip) or subcutaneously (sc) injected into naive mice. LN and spleen were collected 4 hours later and the amount of IL-4 was tested by ELISA (n = 4). Figures 2E-2F: Immunofluorescence images of lumbar lining (LN) 1 hour after intravenous injection of DyLight594-labeled IL-4 or SA-IL-4. T cells and high endothelial venules (HEVs) were stained with anti-CD3 antibody or anti-PNAd antibody, respectively (n = 2). Scale bars represent 200 μm (Figure 2E) and 100 μm (Figure 2F). Figure 2G: Binding affinity of SA-IL-4 to FcRn as measured by SPR. Figures 2H-2I: SA(P573K)-IL-4 levels measured in LN (Figure 2H) and spleen (Figure 2I) 1 hour after injection (n = 5). wt IL-4 and SA-IL-4 data from Figures 2A-2B are re-presented. Figure 2J: Transcytoplasmic transport assay. SA-IL-4 or SA(P573K)-IL-4 was added to an insert (apical side) containing cultured human umbilical vein endothelial cells (HUVECs). IL-4 levels in the culture medium from both the insert (apical side) and bottom well (basal side) were measured by ELISA (n = 3). Data are mean ± SEM. Two experimental replicas were used. Statistical analysis was performed using one-way ANOVA with Tukey's test (Figures 2H and 2I). For simple comparisons (Figures 2C, 2D, and 2J), a two-tailed Student's t-test was used. [Figure 2B] See the explanation in Figure 2A. [Figure 2C] See the explanation in Figure 2A. [Figure 2D] See the explanation in Figure 2A. [Figure 2E] See the explanation in Figure 2A. [Figure 2F] See the explanation in Figure 2A. [Figure 2G] See the explanation in Figure 2A. [Figure 2H] See the explanation in Figure 2A. [Figure 2I] See the explanation in Figure 2A. [Figure 2J] See the explanation in Figure 2A. [Figure 3A] Figures 3A-3J - SA fusion to IL-4 increases its concentration in various organs and plasma after injection. Figures 3A-3B: (Figure 3A) Plasma concentrations of wt IL-4 or SA-IL-4 injected IV and (Figure 3B) sc. 10 μg of wt IL-4 or equimolar SA-IL-4 (n = 4 each) was IV injected into naive mice. Blood was collected from 1 minute to 24 hours later, and plasma concentrations of IL-4 were measured by ELISA. Figures 3C-3G: (Figure 3C) Spinal cord, (Figure 3D) Lung, (Figure 3E) Liver, (Figure 3F) LN, and (Figure 3G) Spleen IL-4 levels were measured 4 hours after subcutaneous and intraperitoneal injection of 40 μg of wt IL-4 or equimolar SA-IL-4 (wt IL-4: n = 4, SA-IL-4: n = 6). Figures 3H-3I: IL-4 levels in the spinal cord (n = 4) and lumbar LN (n = 4) of naive and EAE mice were measured by ELISA 1 hour after sc injection of SA-IL-4 (40 μg, IL-4-based) (naive: n = 4, EAE: n = 3). Figure 3J: Binding affinity of mouse plasma-derived SA (IL-4-less) to FcRn as measured by SPR. Data are mean ± SEM. Two-sided Student's t-test was used for Figures 3H and 3J. Experiments were performed once. [Figure 3B] See the explanation in Figure 3A. [Figure 3C] See the explanation in Figure 3A. [Figure 3D] See the explanation in Figure 3A. [Figure 3E] See the explanation in Figure 3A. [Figure 3F] See the explanation in Figure 3A. [Figure 3G] See the explanation in Figure 3A. [Figure 3H] See the explanation in Figure 3A. [Figure 3I] See the explanation in Figure 3A. [Figure 3J] See the explanation in Figure 3A. [Figure 4A] Figures 4A-4C - The SA(P573K) mutation in SA-IL-4 reduced blood concentrations and inactivated FcRn binding. Figure 4A: SA(P573K)-SA-IL-4 was analyzed by SDS-PAGE under non-reducing conditions using Coomassie blue staining. Figure 4B: Binding affinity of SA(P573K)-IL-4 and FcRn as measured by SPR. Binding affinity could not be determined. The first and second trials indicate that the concentration of 62.5 nM was tested twice to verify variability. Figure 4C: Mice were intravenously injected with 40 μg of wt IL-4, SA-IL-4, or SA(P573K)-IL-4. Blood was collected 1 hour later, and plasma IL-4 concentration was determined by ELISA (n = 5). Data are mean ± SEM. Two experimental replicas. [Figure 4B] See the explanation in Figure 4A. [Figure 4C] See the explanation in Figure 4A. [Figure 5A]Figures 5A-5D - SA-IL-4 prevents the progression and onset of EAE disease in the acute phase. Disease progression (Figure 5A) and weight changes (Figure 5B) in C57BL / 6 myelin oligodendrocyte glycoprotein (MOG) 35-55 experimental autoimmune encephalomyelitis (EAE) mice that were injected intraperitoneally with (ip) phosphate-buffered saline (PBS), 10 μg of wt IL-4 into ip, or 10 μg molar equivalent of (sc) SA-IL-4 into ip or subcutaneously every other day from day 8 to 10 after immunization, or administered FTY720 1 mg / kg orally daily. n = 7 / group. The number of mice that developed EAE symptoms is shown in the figure. Figure 5C: Representative spinal cord tissue image. Myelin expression was detected by immunohistochemistry using anti-myelin basic protein antibody (brown). Arrows indicate demyelination. n = 7 / group. The graph shows the percentage of mice that showed demyelination in each treatment group by blinded pathological analysis. Figure 5D: Disease progression in MOG35-55 induced EAE mice injected every other day for 6 days starting day 8 after ip immunization with PBS, SA-IL-4, and SA(P573K)-IL-4 10 μg molar equivalents. n = 6 / group. Two experimental copies. Data are mean ± SEM. Statistical analysis was performed using one-way ANOVA with Tukey's test. [Figure 5B] See the explanation in Figure 5A. [Figure 5C] See the explanation in Figure 5A. [Figure 5D] See the explanation in Figure 5A. [Figure 6] Subcutaneous injection of wt IL-4 did not show a significant therapeutic effect. Disease progression is shown in C57BL / 6 MOG35-55 EAE mice that received sc injections every other day for 16 days starting 8 day after immunization with PBS, 10 μg wt IL-4, or equimolar SA-IL-4. Data are mean ± SEM, n = 7. The experiment was performed once. Statistical analysis was performed using Tukey's test with one-way ANOVA. [Figure 7]Long-term treatment with SA-IL-4 suppresses the onset and progression of EAE disease. Disease progression (n=8) in C57BL / 6 MOG35-55 EAE mice immunized with PBS or SA-IL-4 (10 μg relative to IL-4) via ip injection every other day for 16 days starting 8 days after immunization is shown. The number of EAE-developing mice per total number of mice in each treatment group is shown. Mice were monitored until day 24 to confirm the long-term effect of SA-IL-4 administration. Data are mean ± SEM. Two experimental replicas. Statistical analysis was performed using a two-tailed Student's t-test. **P < 0.01. [Figure 8A] Figures 8A-8B - SA-IL-4 did not affect the number of macrophages and dendritic cells in the spinal cord and influx region LN. Mice were administered wt IL-4, SA-IL-4, or PBS via intravenous injection (ip) or sc-injection (sc) of SA-IL-4 every other day for 10 days starting 8 days post-immunization. FTY720 1 mg / kg body weight was administered orally daily starting 8 days post-immunization. Cells were isolated from the influx region LN (dLN) and spinal cord 17 days after immunization and analyzed by flow cytometry. (Figure 8A) The frequency of F4 / 80+ macrophages in CD11b+ cells and (Figure 8B) the frequency of CD11b+ and CD11c+ DCs in CD45+ cells were analyzed. Data are mean ± SEM (n = 7). The experiment was performed once. Statistical analysis was performed using one-way ANOVA with Tukey's test. [Figure 8B] See the explanation in Figure 8A. [Figure 9A]Figures 9A-9I - SA-IL-4 treatment inhibits leukocyte infiltration into the spinal cord and induces immunosuppressive cells in the influx region (LN). Mice were administered wt IL-4, SA-IL-4, or PBS via ip injection or sc injection of SA-IL-4 every other day for 10 days starting 8 days post-immunization, or FTY720 1 mg / kg body weight orally daily starting 8 days post-immunization. Cells were isolated from the influx region (LN) and spinal cord 17 days after immunization and analyzed by flow cytometry. Figures 9A-9C: Frequency of (Figure 9A) CD45+ leukocytes, (Figure 9B) RoRγt+ Th17 cells, and (Figure 9C) Ly6G+Ly6C+ G-MDSCs in living cells in the spinal cord. IL-4 ip, n = 6; other groups, n = 7. Figures 9D-9I: In the lumbar inflow region (LN) (dLN), the frequencies of (Figure 9D) Ly6G+Ly6C+ G-MDSCs in CD11b+CD45+ cells, (Figure 9E) Ly6G-Ly6C+ M-MDSCs in CD11b+CD45+ cells, (Figure 9F) RoRγt+ Th17 cells in CD4+CD3+ T cells, (Figure 9G) CD86+ M1 macrophages in F4 / 80+CD11b+ macrophages, (Figure 9H) CD206+ M2 macrophages in F4 / 80+CD11b+ macrophages, and (Figure 9I) B220+ B cells in CD11b+CD45+ cells were analyzed. (n = 7 across all groups). Data are mean ± SEM. The experiment was performed once. Statistical analysis was performed using Tukey's test with one-way ANOVA. [Figure 9B] See the explanation in Figure 9A. [Figure 9C] See the explanation in Figure 9A. [Figure 9D] See the explanation in Figure 9A. [Figure 9E] See the explanation in Figure 9A. [Figure 9F] See the explanation in Figure 9A. [Figure 9G] See the explanation in Figure 9A. [Figure 9H] See the explanation in Figure 9A. [Figure 9I] See the explanation in Figure 9A. [Figure 10-1]Figures 10A-10R - SA-IL-4 treatment activates the PD-1 / PD-L1 axis and reduces integrin and cytokine expression in T cells. EAE mice induced with MOG35-55 were injected with PBS, wt IL-4, or SA-IL-4 on days 8, 10, and 12 postimmunization. n = 7 in the PBS-treated group, n = 6 in the other groups. Spinal cord and spleen were isolated on day 13 and immune cells were analyzed (Figures 10A-10I). Frequency of tetramer+ cells (recognizing MOG35-55) in CD4+ T cells in the spinal cord (Figure 10A). In the spleen, (Figure 10B) tetramer + CD4 + αLβ2 integrin + cells in T cells, (Figure 10C) tetramer + CD4 + α4β1 integrin + cells in T cells, (Figure 10D) CD8 + αLβ2 integrin + cells in T cells, and (Figure 10E) CD8 + α4β1 integrin + cells in T cells are shown. (Figure 10F) Mean fluorescence intensity (MFI) of PD-1 in central memory (CM) CD44+CD62L+CD4+ T cells, (Figure 10G) MFI of PD-1 in CM CD44+CD62L+CD8+ T cells, (Figure 10H) MFI of PD-L1 in Ly6C+Ly6G-CD11b+ M-MDSCs, (Figure 10I) Frequency of PD-L1+ in Ly6C+Ly6G-CD11b+ M-MDSCs, (Figure 10J) MFI of PD-L1 in Ly6C+Ly6G+CD11b+ G-MDSCs, (Figure 10K) Frequency of PD-L1+ in Ly6C+Ly6G+CD11b+ G-MDSCs, (Figure 10L) Frequency of IL-23R+ cells in tetramer+ CD4+ T cells, and (Figure 10M) tetramer+ CD4+ FoxP3+CD25+ Treg cells within T cells. Figures 10N-10P: Splenocytes were cultured in vitro for 3 days in the presence of MOG proteins. (Figure 10N) IL-17A, (Figure 10O) IFNγ, and (Figure 10P) GM-CSF concentrations in the culture medium were analyzed by ELISA. Figures 10Q-10R: Splenocytes were cultured ex vivo for 6 hours in the presence of MOG35-55 peptides. Cytokine expression in CD4+ T cells was characterized by flow cytometry. Data are mean ± SEM. The experiment was performed once. Statistical analysis was performed using Tukey's test with one-way ANOVA. [Figure 10-2] See the explanation in Figure 10-1. [Figure 10-3] See the explanation in Figure 10-1. [Figure 10-4] See the explanation in Figure 10-1. [Figure 11-1] Figures 11A-11L: SA-IL-4 treatment in the chronic phase of EAE reduces clinical scores and prevents immune cell infiltration into the spinal cord. EAE was induced in C57BL / 6 mice using MOG35-55. PBS, wt IL-4, or SA-IL-4 were administered via intravenous injection (ip) every other day for 10 days starting 21 postimmunization. Disease progression (Figure 11A) and weight changes (Figure 11B) are shown (n = 6). Figures 11C-11D: PBS, wt IL-4, or SA-IL-4 were administered via sc injection every other day for 12 days starting 21 postimmunization. Disease progression (Figure 11C) and weight changes (Figure 11D) are shown (n = 8 for PBS and SA-IL-4; n = 7 for other treatment groups). Figures 11E-11H: Spinal cord and spleen were collected on day 34, and immune cells were analyzed by flow cytometry. The graphs show the frequency of CD45+ cells in living cells in the spinal cord (Figure 11E), CD4+CD3+CD45+ T cells in living cells in the spinal cord (Figure 11F), tetramer+ (recognizing MOG35-55) RoRγt+CD4+ Th17 cells in living cells in the spinal cord (Figure 11G), and IL-23R+ cells in tetramer+CD4+ cells in the spleen (Figure 11H). Figures 11I-11J: Splenocytes were cultured in vitro for 3 days in the presence of MOG proteins. IL-17A (Figure 11I) and GM-CSF (Figure 11J) concentrations in the culture medium were analyzed by ELISA (n = 8 for PBS and SA-IL-4; n = 7 for other treatment groups). Figures 11K-11L: Splenocytes were cultured in vitro for 6 hours in the presence of MOG35-55 peptides. Cytokine expression in CD4+ T cells was characterized by flow cytometry. PBS and SA-IL-4: n = 8, other treatment groups: n = 7. The experiment was performed once. Data are mean ± SEM. Statistical analysis was performed using one-way ANOVA with Tukey's test. [Figure 11-2]Refer to the description of FIG. 11-1. [Figure 11-3] Refer to the description of FIG. 11-1. [Figure 11-4] Refer to the description of FIG. 11-1. [Figure 11-5] Refer to the description of FIG. 11-1. [Figure 11-6] Refer to the description of FIG. 11-1. [Figure 11-7] Refer to the description of FIG. 11-1. [Figure 11-8] Refer to the description of FIG. 11-1. [Figure 12A] FIGS. 12A-12B - Disease progression (FIG. 12A) and body weight changes (FIG. 12B) associated with disease development in C57BL / 6 myelin oligodendrocyte glycoprotein (MOG) 35-55 experimental autoimmune encephalomyelitis (EAE) mice injected subcutaneously (s.c.) with phosphate-buffered saline (PBS), SA-IL-33 (13-39 μg, based on IL-33), or SA-IL-4 (10 μg, based on IL-4) every other day from day 8 to day 10 after immunization, or dosed daily by oral gavage with FTY720 at 1 mg / kg. n = 6-7 / group. [Figure 12B] Refer to the description of FIG. 12A. [Figure 13A] FIGS. 13A-13D - Albumin fusion to IL-10 provided FcRn binding and resulted in LN accumulation. FIG. 13A: SDS-PAGE analysis of wt IL-10 and SA-IL-10. FIG. 13B: Binding analysis of SA-IL-10 to FcRn. FIG. 13C: Splenocytes (i) or single cells from popliteal LNs (ii) were incubated with SA or SA-IL-10 on ice for 30 minutes. Binding of each protein to immune cells was detected by co-staining with an anti-SA antibody and an antibody to a specific marker of each immune cell population. FIG. 13D: Immunofluorescence images of popliteal LNs after intravenous injection of DyLight594-labeled wt IL-10 or SA-IL-10. T cells and high endothelial venules (HEVs) were stained with an anti-CD3 antibody or an anti-PNAd antibody, respectively. [Figure 13B] Refer to the description of FIG. 13A. [Figure 13C] See the explanation in Figure 13A. [Figure 13D] See the explanation in Figure 13A. [Figure 14A] Figures 14A-14B - Albumin fusion to IL-10 provided prolonged blood circulation. Figure 14A: wt IL-10 or SA-IL-10 (each equivalent to 35 μg of IL-10) was administered to BALB / c mice by tail vein injection. Serum was collected at the indicated time points. Serum concentrations of IL-10 were measured by ELISA (mean ± SEM; n = 5). The plasma half-life of IL-10 was calculated using biphasic exponential decay: MFI(t) = Ae-αt + Be-βt, t1 / 2, α, fast clearance half-life; t1 / 2, β, slow clearance half-life. Area under the curve (AUC) was analyzed by Graphpad Prism. Figure 14B: CAIA (caused arthritis in mice) was selectively induced in the right hind paw by passive immunization with anti-collagen antibodies, followed by subcutaneous injection of LPS into the right hind paw (defined as day 3). The day after LPS injection, DyLight800-labeled wt IL-10 or SA-IL-10 was intravenously injected into CAIA mice. Four hours after injection, the indicated organs were collected and analyzed using the IVIS imaging system (mean ± SEM; n = 4). Statistical analysis was performed using a two-sided Student's t-test. *P < 0.05. [Figure 14B] See the explanation in Figure 14A. [Figure 15A]Figures 15A-15F - Albumin-fused IL-10 accumulated in the LN and suppressed Th17 activation in the LN. CAIA (Chronic Arthritis Induced) was induced by passive immunization with an anti-collagen antibody, followed by intraperitoneal injection of LPS (defined as day 3). On the day of LPS injection, wt IL-10 or SA-IL-10 was intravenously injected into arthritis mice. IL-10 levels and Th17-related cytokines in the LN were measured using ELISA. Figure 15A: Comparison of IL-10 levels 4 hours after injection of each protein. Figure 15B: Pharmacokinetics of wt IL-10 or SA-IL-10 in the LN after intravenous injection (mean ± SEM; n = 4). Figure 15C: AUC of wt IL-10 and SA-IL-10 in various LNs. Figures 15D and 15E: Th17-related cytokine levels in the joint inflow area (popliteal LN) (Figure 15D) and non-inflow area (cervical LN) (Figure 15E). Figure 15F: GM-CSF levels in the popliteal LN. (Mean ± SEM; n = 7) Statistical analysis was performed using analysis of variance (ANOVA) with a two-sided Student's t-test for (Figures 15D and 15E) or Tukey's test for (a and f). *P < 0.05; **P < 0.01; ****P < 0.0001; ns; not significant. [Figure 15B] See the explanation in Figure 15A. [Figure 15C] See the explanation in Figure 15A. [Figure 15D] See the explanation in Figure 15A. [Figure 15E] See the explanation in Figure 15A. [Figure 15F] See the explanation in Figure 15A. [Figure 16A]Figures 16A-16B - Effects of albumin-fused IL-10 on immune cell populations in the spleen (Figure 16A) and LN (Figure 16B). CAIA (caused arthritis in mice) was induced by passive immunization with anti-collagen antibodies, followed by intraperitoneal injection of LPS (defined as day 3). On days 3 and 6, mice were intravenously injected with PBS, wt IL-10, or SA-IL-10. Single cells were extracted from the spleen and popliteal LN the day after the last injection and subsequently analyzed by flow cytometry. The graphs show the frequencies of CD3+ T cells, CD45+ lymphocytes, CD11b+ cells, CD11c+ cells, CD86+ cells, granulocyte MDSCs / neutrophils (Ly6G+ Ly6C+ in CD11b+ cells), monocyte MDSCs (Ly6G- Ly6C+ in CD11b+ cells), macrophages (F4 / 80+ in CD11b+ cells), CD86+ cells within macrophages, and M2 macrophages (CD206+ F4 / 80+ in CD11b+ cells). (Mean ± SEM; n = 6~7) Statistical analysis was performed using Tukey's test for analysis of variance (ANOVA), except for the following graphs: %CD11c+ in CD11b+ cells in Figure 16A and %Ly6G+ in CD11b+ cells in Figure 16B. For the analysis of Ly6C+, the Kruskal-Wallis test followed by Dunn's multiple comparison test was used. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. [Figure 16B] See the explanation in Figure 16A. [Figure 17A]Figures 17A-17C - Albumin-fused IL-10 more effectively suppressed the development of arthritis than wt IL-10. Figure 17A: Arthritis (CAIA) was induced by passive immunization with anti-collagen antibody, followed by intraperitoneal injection of LPS. On the day of LPS injection, PBS, wt IL-10, or SA-IL-10 (equivalent to 43.5 μg of IL-10) was intravenously injected into arthritis mice. Arthritis scores represent the mean + SEM of 7 mice. Figure 17B: Representative H&E images of joints at day 14 in each treatment group. Scale bar, 500 μm. Severity of bone resorption and synovial hyperplasia was scored from 0 to 4 (mean ± SEM; n = 7). Figure 17C: Effect of administration route on the therapeutic effect of SA-IL-10. Arthritis scores represent the mean + SEM of 7 mice. Statistical analysis was performed using Tukey's test for analysis of variance (ANOVA) in cases (a) and (c), and a two-tailed Student's t-test in case (b). **P < 0.01; ***P < 0.001; ****P < 0.0001. [Figure 17B] See the explanation in Figure 17A. [Figure 17C] See the explanation in Figure 17A. [Figure 18A]Figures 18A-18D - Albumin-fused IL-10 showed improved therapeutic effects against established arthritis. Bovine collagen / CFA emulsion was subcutaneously injected into the tail base of DBA / 1J male mice. Three weeks later, bovine collagen / IFA emulsion was further injected as an additional immunization. When the arthritis score reached 2-4 (defined as day 0), mice were intravenously injected with PBS, SA-IL-10 (each equivalent to 43.5 μg of IL-10), or 200 μg of anti-TNF-α antibody. In the studies shown in Figures 18C and 18D, the same treatment was further injected into mice on day 3. In Figures 18A and 18C, arthritis scores represent the mean + SEM of 9-15 mice. Figures 18B and 18D show representative H&E histological images of the joints on day 16. Scale bar, 500 μm. As described in Materials and Methods, the severity of bone resorption and synovial hyperplasia was scored from 0 to 4. Statistical analysis was performed using two-sided Student's t-test. *P < 0.05; **P < 0.01; ***P < 0.001; ns; not significant. [Figure 18B] See the explanation in Figure 18A. [Figure 18C] See the explanation in Figure 18A. [Figure 18D] See the explanation in Figure 18A. [Figure 19A]Figures 19A-19B - Albumin-fused IL-10 suppressed the inflammatory response in the paw. Arthritis (CAIA) was induced by passive immunization with anti-collagen antibodies, followed by intraperitoneal injection of LPS. On the day of LPS injection (defined as day 3), mice with arthritis were intravenously injected with PBS, wt IL-10, or SA-IL-10. Figure 19A: Single cells were extracted from the hind paw on day 11 and subsequently analyzed by flow cytometry. The graph shows the frequencies of CD45+ cells, B cells (B220+ cells within CD45+ lymphocytes), dendritic cells (CD11c+ cells within CD45+ lymphocytes), monocytes (CD11b+ cells within CD45+ lymphocytes), granulocyte MDSCs / neutrophils (Ly6G+ Ly6C+ CD11b+ CD45+), monocyte MDSCs (Ly6G- Ly6C+ CD11b+ CD45+), macrophages (F4 / 80+ CD11b+ CD45+), M2 macrophages (CD206+ F4 / 80+ CD11b+ CD45+), and M1 macrophages (MHC II+ F4 / 80+ CD11b+ CD45+). (Mean ± SEM; n = 7) Figure 19B: Cytokine levels in the hind leg on day 11 (n = 5~7). Statistical analysis was performed using Tukey's test and analysis of variance (ANOVA), except for %CD11c+ in Figure 19A. For the analysis of %CD11c+ in Figure 19A, the Kruskal-Wallis test followed by Dunn's multiple comparison test was used. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. [Figure 19B] See the explanation in Figure 19A. [Figure 20A]Figures 20A-20B - Effects of albumin-fused IL-10 on T cell populations in the paws and blood. CAIA (caused arthritis) was induced by passive immunization with anti-collagen antibodies, followed by intraperitoneal injection of LPS (defined as day 3). On the day of LPS injection, mice were intravenously injected with PBS, wt IL-10, or SA-IL-10. Figure 20A: Single cells were extracted from the hind paws on day 11 and subsequently analyzed by flow cytometry. The graph shows the frequency of NK1.1+ CD3- NK cells, CD3+ T cells, CD3+ CD4+ T cells, Treg (Foxp3+ CD25+) CD3+ CD4+ T cells within CD45+ lymphocytes, CD3+ CD8+ T cells, effector memory T cells (CD62L- CD44+) within CD3+ CD8+ T cells, central memory T cells (CD62L+ CD44+) within CD3+ CD8+ T cells, and PD-1+ cells within CD3+ CD8+ T cells. Figure 20B: Lymphocytes were extracted from blood on day 11, followed by flow cytometry analysis. The graphs depict the frequencies of CD3+ T cells, CD3+CD4+ T cells, Treg (Foxp3+CD25+) CD3+CD4+ T cells, and CD3+CD8+ T cells within CD45+ lymphocytes (mean ± SEM; n = 5-7). Statistical analysis was performed using Tukey's test for analysis of %NK1.1+ in CD45+ cells, %Foxp3+ in CD4+ cells, %CD44+ / CD62L- in CD8+ cells, %CD44+ / CD62L+ in CD8+ cells, and %PD-1+ in CD8+ cells, except for the following graphs: (a) analysis of %NK1.1+ in CD45+ cells, %Foxp3+ in CD4+ cells, %CD44+ / CD62L- in CD8+ cells, and %PD-1+ in CD8+ cells, with the use of the Kruskal-Wallis test followed by Dunn's multiple comparison test. *P < 0.05; **P < 0.01; ***P < 0.001. [Figure 20B] See the explanation in Figure 20A. [Figure 21A]Figures 21A-21B - Safety evaluation of albumin-fused IL-10. Healthy BALB / c mice were intravenously injected with PBS, wt IL-10 or SA-IL-10. Figure 21A: Two days after injection, the white blood cell count, red blood cell count, platelet count, blood hemoglobin concentration and spleen weight were evaluated. Figure 21B: The concentrations of alanine transaminase (ALT), amylase, blood urea nitrogen (BUN), serum calcium, creatine kinase (CK), CO2, total bilirubin (TBli) and total protein in serum were evaluated using a biochemical analyzer. (Mean ± SEM; n = 5) Statistical analysis was performed using analysis of variance (ANOVA) with Tukey's test. *P < 0.05; **P < 0.01. [Figure 21B] See the description of Figure 21A. [Figure 22A] Figures 22A and 22B - PBS and 100 μg of anti-TNF-α were intraperitoneally injected starting on day 0 every two days for 14 days. FTY720 (1 mg / kg body weight) was orally administered daily. SA-IL-10 (equivalent to 43.5 μg of IL-10) was subcutaneously injected on day 0 and day 8. The mouse's hind ankle was subcutaneously exposed to 10 μg of endotoxin-free ovalbumin, 50 μg of alum, and 5 μg of MPLA on day 5. The mice were bled on day 13 (a) and day 19 (b), and the plasma was analyzed for anti-ovalbumin total IgG titer. (Mean ± SEM; n = 5) Statistical analysis was performed using analysis of variance (ANOVA) with Tukey's test. ns; not significant. [Figure 22B] See the description of Figure 22A. [Figure 23] Results from the study described in Example 3 are shown. [Figure 24A] Figures 24A-24B show the results from the study described in Example 4. [Figure 24B] See the description of Figure 24A. [Figure 25] Results from the study described in Example 5 are shown. [Figure 26] The re-epithelialization rate of each wound measured by H&E staining. [Figure 27]Re-epithelialization rate of each wound, measured by H&E staining. [Figure 28] Use of albumin-fusion cytokines for the treatment of scleroderma, as described in Example 7. [Figure 29-1] Figures 29A-D. Optimization of SA-IL-4 dosage. Mice were subcutaneously administered SA-IL-4 once or three times a week. A) Mouse body weight over time. B) CD23 expression level on B cells isolated from peripheral blood mononuclear cells after 4 weeks of treatment. C. Total IgE level in mouse serum isolated from mice after 4 weeks of treatment. D) CD206 expression level in macrophages isolated from peripheral blood mononuclear cells after 1 week of treatment. [Figure 29-2] See the explanation in Figure 29-1. [Figure 30] Figures 30A-B. Wild-type IL-33 causes severe toxicity in EAE-carrying mice. EAE was induced in mice on day 0, and starting on day 8, subcutaneous treatment with 26 μg of wild-type (WT) IL-33, equimolar SA-IL-33, or PBS was performed every other day. A) Survival curves of mice treated with wild-type IL-33, SA-IL-33, or PBS. B) EAE clinical scores of mice. Surviving mice administered WT-IL-33 were removed from the clinical score data after day 11 due to insufficient mice for the study. [Figure 31-1] Figures 31A-D. Three doses of SA-IL-33 are sufficient to prevent EAE while mitigating toxicity. Healthy C57BL / 6 mice were subcutaneously administered various doses of wild-type (WT) or equimolar SA-IL-33 every other day (A-B). Mice were induced to bleed, and serum IgE levels were measured on A) day 5 and B) day 9. Mice were induced to develop EAE on day 0 and subcutaneously treated with 26 μg of SA-IL-33 every other day from day 8, either for three doses or eight doses. C) Clinical EAE scores over time. D) Clinical score on day 20. [Figure 31-2] See the explanation in Figure 31-1. [Figure 32]Representative SA IL-33 size exclusion chromatography plots after affinity and size exclusion chromatography in AKTA pure. [Figure 33] SA IL-33 SDS page (S) and antihistidine Western blot (W) under non-reducing conditions using Coomassie blue staining. [Figure 34] Binding affinity of Fc-ST2 to SA IL-33 on an NTA chip, as measured by SPR. [Figure 35] Figures 35A-B. SA fusion to IL-33 increases its concentration in plasma after subcutaneous injection. (A) Overview of in vivo pharmacokinetic studies of SA IL-33. (B) Comparison of plasma pharmacokinetics of WT vs. SA IL-33. [Figure 36-1] Figures 36A-G. SA IL-33 treatment prevents the development of MOG-induced EAE in the acute phase. (A) SA IL-33 dose escalation study in a prophylactic EAE overview. Mice were treated with 13 ug, 26 ug, or 39 ug (wt IL-33 molar equivalent) by subcutaneous injection on days 8, 10, 12, and 14 post-EAE immunization. (B) Treatment with 26 ug and 39 ug of SA IL-33 per injection prevents the development of EAE. EAE clinical scores from day 7 to day 15 and clinical score on day 15. (C) EAE mice treated with 26 ug of SA IL-33 maintain body weight, while PBS-treated mice lose body weight on day 10 post-immunization. (D) SA IL-33 treatment increases the frequency of ST2+ FoxP3+ CD25+ regulatory T cells in spinal cord inflow lymph nodes and spleen in acute-phase MOG-induced EAE mice. (E) SA IL-33 treatment increases the frequency of Th2 CD4+ T cells in the spinal cord inflow area lymph nodes and spleen, as well as M2 macrophages in the spleen, in acute-phase EAE mice. (F) SA IL-33 treatment increases the frequency of group 2 innate lymphocytes in the spleen of acute-phase MOG-induced EAE mice. (G) SA IL-33 treatment reduces lymphocyte infiltration and cytokine production in the spinal cord of acute-phase MOG-induced EAE mice. [Figure 36-2]See the explanation in Figure 36-1. [Figure 36-3] See the explanation in Figure 36-1. [Figure 37-1] Figures 37A-F. SA IL-33 treatment in the chronic phase of EAE reduces clinical scores, increases body weight, and reduces immune cell infiltration. (A) Overview of SA IL-33 treatment in chronic EAE. Mice were treated with 26 ug (wt IL-33 molar equivalent) by subcutaneous injection on days 20, 22, 24, 26, 28, 30, 32, and 34 after EAE immunization. (B) Treatment with 26 ug of SA IL-33 per injection in the chronic phase reduces EAE clinical scores. EAE clinical scores from day 7 to day 34 and clinical score on day 34. (C) SA IL-33 treatment in the chronic phase of EAE induces body weight gain. (D) SA IL-33 treatment in the chronic phase of EAE increases the frequency of ST2+ FoxP3+ CD25+ regulatory T cells in spinal cord inflow area lymph nodes in MOG-induced EAE mice in the chronic phase. (E) SA IL-33 treatment reduces lymphocyte infiltration and cytokine production in the spinal cord of chronic-phase MOG-induced EAE mice. (F) SA IL-33-treated EAE mouse splenocytes restimulated in vitro with MOG protein or peptide showed reduced TNFα, IL-17A, and IL-17F production compared to PBS-treated mice. [Figure 37-2] See the explanation in Figure 37-1. [Figure 37-3] See the explanation in Figure 37-1. [Figure 37-4] See the explanation in Figure 37-1. [Figure 38-1]Figures 38A-C. SA IL-33 treatment prevents the development of MOG-induced EAE in the acute phase for at least 10 days after discontinuation of treatment. (A) Dose-number study of SA IL-33 in a prophylactic EAE overview. Mice were treated with 26 ug (wt IL-33 molar equivalent) by subcutaneous injection on days 8, 10, and 12 or days 8, 10, 12, 14, 16, 18, 20, and 22 after EAE immunization. (B) Treatment with three doses of 26 ug of SA IL-33 per injection in the acute phase prevents the development of EAE for at least 10 days after discontinuation of treatment. Clinical EAE scores from day 7 to day 20 and clinical score on day 20. (C) EAE mice treated with three doses of 26 ug of SA IL-33 per injection in the acute phase maintain body weight for at least 10 days after discontinuation of treatment. [Figure 38-2] See the explanation in Figure 38-1. [Figure 39] Figures 39A-B. EAE mice experience significantly reduced toxicity compared to naive mice. C57BL / 6 mice were induced with EAE as previously described. On day 21, EAE mice were subcutaneously treated with PBS (EAE) or 10 μg SA-IL-4 (EAE + SA-IL-4) every other day, starting on day 0. Simultaneously, age-matched C57BL / 6 mice were treated with PBS (PBS) or 10 μg SA-IL-4 (SA-IL-4) every other day, starting on day 0. A) Mouse survival curve up to day 15 after the first injection. B) Mouse body weight up to day 15 after the first injection. [Figure 40] Figures 40A-B. Non-obese diabetic (NOD) mice experience reduced toxicity compared to naive mice. NOD mice were subcutaneously treated with 10 μg SA-IL-4 (NOD) three times a week, starting on day 0. Simultaneously, age-matched C57BL / 6 mice were treated with PBS (PBS) or 10 μg SA-IL-4 (SA-IL-4) every other day, starting on day 0. A) Mouse survival curve up to day 45 after the first injection. B) Mouse body weight up to day 45 after the first injection. [Figure 41]Figures 41A-B. Design of SA IL-35 fusion protein. (A) Diagram of SA IL-35 plasmid design. (B) Purified fraction of SA IL-35 after affinity chromatography and size exclusion chromatography. Images of ladder (left) and SDS page SA IL-35 (right). [Figure 42] Albumin-fused IL-35 suppressed the development of arthritis. Arthritis (CAIA) was induced by passive immunization with anti-collagen antibodies, followed by intraperitoneal injection of LPS on day 3. On the day of LPS injection, PBS, SA-IL-10, SA-IL-27, SA-IL-35, or SA-IL-37 were subcutaneously injected into arthritis mice. Mice were euthanized on day 11. The arthritis score represents the mean of 7 mice + SEM. [Modes for carrying out the invention]

[0051] Detailed explanation Aspects of this disclosure satisfy certain requirements in the art by providing compositions comprising anti-inflammatory cytokines, sometimes linked to albumin proteins, and methods for treating autoimmune or inflammatory conditions and for promoting wound healing. This disclosure is at least in part based on the remarkable discovery that the administration of albumin-linked anti-inflammatory cytokines is effective in treating various autoimmune or inflammatory conditions and in promoting wound healing. Also described herein are methods for targeting anti-inflammatory cytokines to target lymph nodes by linking the cytokines to albumin proteins.

[0052] I. Proteins This disclosure covers a variety of proteins and methods of use. As used herein, “protein” or “polypeptide” means a molecule containing at least five amino acid residues. As used herein, the term “wild-type” means the endogenous version of a molecule that is naturally present in an organism. In some contexts, the wild-type version of a protein or polypeptide is used; however, in many contexts of this disclosure, a modified protein or polypeptide is used to produce an immune response. The above terms may be used interchangeably. “Modified protein” or “modified polypeptide” or “variant” means a protein or polypeptide whose chemical structure, in particular its amino acid sequence, has been altered from that of a wild-type protein or polypeptide. In some contexts, a modified / variant protein or polypeptide has at least one modified activity or function (recognizing that a protein or polypeptide may have multiple activities or functions). A modified / variant protein or polypeptide may be modified with respect to one activity or function, but is particularly intended to retain the wild-type activity or function in other respects, such as immunogenicity.

[0053] Where a protein is specifically referred to herein, it generally refers to a natural (wild-type) or recombinant (modified) protein, or optionally, a protein from which any signal sequence has been removed. Proteins may be isolated directly from organisms in which they are natural, produced by recombinant DNA / exogenous expression methods, or produced by solid-phase peptide synthesis (SPPS) or other in vitro methods. In certain contexts, there are isolated nucleic acid segments and recombinant vectors incorporating nucleic acid sequences encoding polypeptides (e.g., antibodies or fragments thereof). The term “recombinant” may be used in combination with the name of a polypeptide or a particular polypeptide, but generally refers to a polypeptide produced from a nucleic acid molecule that has been manipulated in vitro or is a replica product of such a molecule.

[0054] In certain situations, the size of a protein or polypeptide (wild type or modified type) is 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, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, 700, 725, 750, 775 , 800, 825, 850, 875, 900, 925, 950, 975, 1000, 1100, 1200, 1300, 1400, 1500, 1750, 2000, 2250, 2500 amino acid residues or more, and any range derivable therein, or derivatives of the corresponding amino acid sequences described or referenced herein, may be included, but are not limited to. Polypeptides may be mutated by truncation to be shorter than their corresponding wild-type form, and may be modified by fusing or conjugating heterologous protein or polypeptide sequences with specific functions (e.g., for targeting or localization, for immunogenicity enhancement, for purification purposes, etc.). As used herein, the term “domain” means any distinct functional or structural unit of a protein or polypeptide, and generally means a sequence of amino acids having a structure or function recognizable to those skilled in the art.

[0055] The polypeptides, proteins, or polynucleotides encoding such polypeptides or proteins may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 (or any range derivable therefrom) or more variant amino acids or nucleic acid substitutions, or SEQ ID NO: 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, 6 0, 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, 11 6, 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 or more consecutive amino acids or nucleic acids, or any range that can be derived therefrom, or at most 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 1 4, 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 ,98,99,100,101,102,103,104,105,106,107,108,109,110,111,112,113,114,115,116,117,118,119,120,121,122,123,124,125,126,127,128,1 29, 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, 16 0, 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 or more consecutive amino acids or nucleic acids, or any range that can be derived therefrom and 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 within that range) may be similar, identical, or homologous.

[0056] In some aspects, proteins or polypeptides have amino acid 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 ,78,79,80,81,82,83,84,85,86,87,88,89,90,91,92,93,94,95,96,97,98,99,100,101,102,103,104,105,106,107,108,109,110,111,112,113,114,115,116,117,118,119,120,121,122,123,124,125,126,127,128,129,130,131,132,133,134,135,136,137,138,139,140,141,142,14 3, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203 ,204,205,206,207,208,209,210,211,212,213,214,215,216,217,218,219,220,221,222,223,224,225,226,227,228,229,230,231,232,233,234,235,236,237,238,239,240,241,242,243,244,245,246,247,248,249,250,251,252,253,254,255,256,257,258,259,260,261,262,263,This may include 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 or 304, 305 or 306 (or any range that can be derived from them).

[0057] In some aspects, polypeptides or proteins are found to 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% of one of SEQ ID NO: 1-51. (or any range that can be derived within that range), 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% SEQ IDs that are similar, identical, or homologous by exactly 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% (or any range that can be derived within that) NO: At least one of the numbers 1-51, 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, 1 74, 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, 20 5, 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, 2 99, 300, 301, 302, 303, 304, 305 or 306 (or any range that can be derived from them), 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, 1 38, 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, 16 9, 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, 2 63, 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 or 306 (or any range that can be derived from them), or exactly 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50、51、52、53、54、55、56、57、58、59、60、61、62、63、64、65、66、67、68、69、70、71、72、73、74、75、76、77、78、79、80、81、82、83、84、85、86、87、88、89、90、91、92、93、94、95、96、97、98、99、100、101、102、103、104、105、106、107、108、109、110、111、112、113、114、115、116、117、118、119、120、121、122、123、124、125、126、127、128、129、130、131、132、133、134、135、136、137、138、139、140、141、142、143、144、145、146、147、148、149、150、151、152、153、154、155、156、157、158、159、160、161、162、163、164、165、166、167、168、169、170、171、172、173、174、175、176、177、178、179、180、181、182、183、184、185、186、187、188、189、190、191、192、193、194、195、196、197、198、199、200、201、202、203、204、205、206、207、208、209、210、211、212、213、214、215、216、217、218、219、220、221、222、223、224、225、226、227、228、229、230、231、232、233、234、235、236、237、238、239、240、241、242、243、244、245、246、247、248、249、250、251、252、253、254、255、256、257、258、259、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、It may contain 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, or 306 consecutive amino acids (or any range within which can be derived).

[0058] In some situations, position numbers 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 11 5, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 1 46, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176 ,177,178,179,180,181,182,183,184,185,186,187,188,189,190,191,192,193,194,195,196,197,198,199,200,201,202,203,204,205,206,20 7, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 2 38, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268,Starting with 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, or 305, and with a SEQ ID NO: 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, 6 8, 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, 12 8, 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, 2 64, 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, 29 5, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305 or 306 (or any range that can be derived from them), 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, 1 35, 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, 16 6, 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, 2 29, 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, 26 0, 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 or 306 (or any range that can be derived from them), or exactly 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57 , 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 9 9, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130 ,131,132,133,134,135,136,137,138,139,140,141,142,143,144,145,146,147,148,149,150,151,152,153,154,155,156,157,158,159,160,161,162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, There is a polypeptide containing 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, or 306 consecutive amino acids (or any range within which can be derived).

[0059] The polypeptides of this disclosure have amino acid 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 ,115,116,117,118,119,120,121,122,123,124,125,126,127,128,129,130,131,132,133,134,135,136,137,138,139,140,141,142,143,144,14 5, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206 ,207,208,209,210,211,212,213,214,215,216,217,218,219,220,221,222,223,224,225,226,227,228,229,230,231,232,233,234,235,236,2 37, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 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, 5 67, 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 650 This may include substitutions (or any range derivable therefrom), which may be by alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, or valine.

[0060] Nucleotide sequences of various genes, as well as protein, polypeptide, and peptide sequences, have been previously disclosed and can be found in recognized computerized databases. Two commonly used databases are the Genbank and GenPept databases of the National Center for Biotechnology Information (ncbi.nlm.nih.gov / on the World Wide Web) and the Universal Protein Resource (UniProt; uniprot.org on the World Wide Web). The coding regions of these genes can be amplified and / or expressed using the techniques disclosed herein or as known to those skilled in the art.

[0061] The compositions of this disclosure are intended to contain approximately 0.001 mg to approximately 10 mg of total polypeptides, peptides, and / or proteins per 1 ml. The protein concentrations in the compositions are approximately 0.001, 0.010, 0.050, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, and 10.0. mg / ml or higher (or any derivable range within that), at least approximately 0.001, 0.010, 0.050, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0 It can be mg / ml or more (or any range within which it can be derived) or at most about 0.001, 0.010, 0.050, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0 mg / ml or more (or any range within which it can be derived).

[0062] 1. Variant polypeptide The following is a consideration of altering the amino acid subunits of a protein to produce equivalent or, in some cases, improved second-generation variant polypeptides or peptides. For example, certain amino acids may be used in place of other amino acids in a protein or polypeptide sequence, with or without a significant loss of interaction and binding ability with structures such as the antigen-binding region of an antibody or a binding site on a substrate molecule. Since the interaction ability and properties of a protein determine its functional activity, specific amino acid substitutions can be made in the protein sequence and in its corresponding DNA coding sequence, while still producing a protein with similar or desirable properties. Thus, the inventors intend that various changes can be made in the DNA sequence of a protein-coding gene without significantly losing its biological utility or activity.

[0063] The term "functionally equivalent codon" is used herein to refer to codons that code for the same amino acid, such as the six different codons for arginine. "Neutral substitution" or "neutral mutation," which refers to changes in codons that code for biologically equivalent amino acids, is also considered.

[0064] The amino acid sequence variants of this disclosure may be substitutional, insertional, or deletion variants. Mutations of the polypeptides of this disclosure may affect 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 or more discontinuous or continuous amino acids in the protein or polypeptide compared to the wild type. The variants may include amino acid sequences that are at least 50%, 60%, 70%, 80%, or 90% identical to any sequence provided or referred to herein, including all values ​​and ranges in between. Variants may contain 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more substituted amino acids.

[0065] It is understood that amino acid and nucleic acid sequences may each contain additional residues, such as further N-terminal or C-terminal amino acids, or 5' or 3' sequences, as long as they meet the above criteria, including the maintenance of biological protein activity in which protein expression is involved, and yet may still be essentially identical as described in one of the sequences disclosed herein. Terminal additions are particularly applicable to nucleic acid sequences and may include, for example, various non-coding sequences adjacent to either the 5' or 3' portion of the coding region.

[0066] Deletion variants typically lack one or more residues from the natural or wild-type protein. Individual residues may be deleted, or several adjacent amino acids may be deleted. A cleaved protein can be created by introducing a stop codon (by substitution or insertion) into the encoding nucleic acid sequence.

[0067] Insertion mutants typically involve the addition of amino acid residues at non-terminal points in a polypeptide. This can include the insertion of one or more amino acid residues. Terminal adducts can also be constructed, and these may include fusion proteins that are polymers or chains of one or more peptides or polypeptides described or referenced herein.

[0068] Substitutional variants typically involve the exchange of one amino acid for another at one or more sites within a protein or polypeptide, and may be designed to modify one or more properties of the polypeptide, with or without loss of other functions or properties. Substitutions may be conservative, meaning one amino acid may be replaced with an amino acid having similar chemical properties. “Conservative amino acid substitutions” may involve the exchange of one amino acid class member with another member of the same class. Conservative substitutions are well known in the art and include, for example, the changes 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. Conservative amino acid substitutions may include amino acid residues that do not exist in nature and are usually incorporated by chemical peptide synthesis rather than by synthesis in biological systems. These include peptide mimetic molecules or other inverted or reversed amino acid moieties.

[0069] Alternatively, the substitution may be “non-conservative” in such a way that the function or activity of the polypeptide is affected. Non-conservative changes typically involve substituting one amino acid residue with a chemically different residue, such as using a polar or charged amino acid in place of a non-polar or uncharged amino acid, and vice versa. Non-conservative substitutions may involve the exchange of a member from one amino acid class with a member from another class.

[0070] A. Anti-inflammatory polypeptides This disclosure covers aspects of compositions comprising anti-inflammatory polypeptides and methods of using them. In some aspects, the disclosed anti-inflammatory polypeptides are useful in methods for treating autoimmune or inflammatory conditions. In some aspects, the disclosed anti-inflammatory polypeptides are useful in methods for promoting wound healing. "Anti-inflammatory polypeptide" describes any polypeptide that can reduce, inhibit, prevent or eliminate an inflammatory response in a subject. In some aspects, an anti-inflammatory polypeptide can reduce the number and / or function of Th17 cells in a subject. Examples of anti-inflammatory polypeptides include anti-inflammatory cytokines, polypeptides comprising anti-inflammatory cytokines and albumin proteins, and polypeptides that can bind to inflammatory cytokines and inhibit their activity. In some aspects, the anti-inflammatory polypeptides of this disclosure comprise anti-inflammatory cytokines. In some aspects, the anti-inflammatory polypeptides of this disclosure are anti-inflammatory cytokines. In some aspects, an anti-inflammatory cytokine is functionally linked (e.g., covalently or non-covalently) to one or more further polypeptides. In some aspects, anti-inflammatory cytokines are functionally linked to albumin proteins. In some aspects, the anti-inflammatory polypeptides of this disclosure are anti-inflammatory cytokines covalently linked to albumin proteins. In some aspects, the anti-inflammatory polypeptides of this disclosure are anti-inflammatory cytokines covalently linked to albumin-binding proteins. Anti-inflammatory cytokines may be linked to further polypeptides (e.g., albumin proteins, albumin-binding proteins) via one or more linkers. Further polypeptides (e.g., albumin proteins, albumin-binding proteins) may be linked at the N-terminus of the anti-inflammatory cytokine. Further polypeptides (e.g., albumin proteins, albumin-binding proteins) may be linked at the C-terminus of the anti-inflammatory cytokine. It is also intended that anti-inflammatory cytokines may be conjugated to albumin, for example, through chemical conjugation.In some cases, the linker is a non-amino acid linker, such as a widely available azide or thiol linker.

[0071] Table 1 provides some non-exclusive examples of anti-inflammatory polypeptides intended in this specification.

[0072] [Table 1] TIFF0007896891000002.tif242166TIFF0007896891000003.tif242166TIFF0007896891000004.tif24216 6TIFF0007896891000005.tif242166TIFF0007896891000006.tif242166TIFF0007896891000007.tif96166

[0073] B. Anti-inflammatory cytokines Aspects of this disclosure include anti-inflammatory cytokines. “Anti-inflammatory cytokine” describes cytokines that can control, modulate, or inhibit inflammatory (or “pro-inflammatory”) responses. The anti-inflammatory cytokines of this disclosure may originate from any species. Anti-inflammatory cytokines may be selected for methods disclosed based on desired use and results; for example, mouse anti-inflammatory cytokines may be selected for administration to mouse subjects, while human anti-inflammatory cytokines may be selected for administration to human subjects. In some cases, human anti-inflammatory cytokines may be selected for administration to mouse subjects, for example, if human anti-inflammatory cytokines can have anti-inflammatory effects in mice.

[0074] Table 2 provides some non-exclusive examples of anti-inflammatory cytokines as intended herein.

[0075] [Table 2] TIFF0007896891000009.tif243166TIFF0007896891000010.tif243166TIFF0007896891000011.tif236166TIFF0007896891000012.tif74166

[0076] Certain anti-inflammatory cytokines are described further, for example, in Opal SM, DePalo VA. Chest. 2000 Apr;117(4):1162-72, which is incorporated herein by reference in its entirety.

[0077] C. Albumin protein The aspects of this disclosure relate to albumin proteins, polypeptides linked to albumin proteins, and polypeptides containing albumin proteins. In some aspects, the albumin protein is human albumin (similarly, “human serum albumin” or “HSA”). Human albumin is identified by the NCBI reference sequence NM_000477.7. In some aspects, the albumin protein is mouse albumin (similarly, “mouse serum albumin” or “MSA”). Mouse albumin is identified by the NCBI reference sequence NM_009654.4. In some aspects, the albumin proteins of this disclosure are fully processed albumin proteins that do not contain signal peptides and / or propeptides. Some non-limiting examples of albumin proteins intended herein are provided in Table 3.

[0078] [Table 3]

[0079] D. Albumin-binding protein This disclosure covers albumin-binding proteins, polypeptides linked to albumin-binding proteins, and polypeptides containing albumin proteins. “Albumin-binding protein” describes a protein capable of binding to an albumin protein (e.g., human albumin). In some aspects, the albumin-binding protein is an anti-albumin antibody or antibody-like molecule. In some aspects, the albumin-binding protein is sequence This is a polypeptide containing TIFF0007896891000014.tif4128.

[0080] E. Detected peptide In some respects, the polypeptides described herein may further include detection peptides (similarly, “tags”). A suitable detection peptide is: This includes TIFF0007896891000015.tif19156, etc. In some aspects, the polypeptides described herein include a tag sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% amino acid sequence identity with SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, or SEQ ID NO:42. Other suitable detection peptides are known in the art.

[0081] F. Peptide Linker In some aspects, the polypeptides of this disclosure include peptide linkers (sometimes referred to as linkers). Peptide linkers can be used to isolate any of the peptide domains / regions described herein. For example, linkers may be between albumin proteins and anti-inflammatory cytokines, between anti-inflammatory cytokines and detection peptides or tags, at the N-terminus of a polypeptide, and / or at the C-terminus of a polypeptide. Peptide linkers may have any of a variety of amino acid sequences. Domains and regions may be linked by peptide linkers, which are generally mobile, but do not preclude other chemical bonds. Linkers may be peptides of about 6 to about 40 amino acids in length, or peptides of about 6 to about 25 amino acids in length, or any value or range within which these can be derived. These linkers can be produced by using synthetic oligonucleotides encoding the linkers to link proteins.

[0082] A peptide linker with a certain degree of mobility can be used. The peptide linker can have substantially any amino acid sequence, keeping in mind that a suitable peptide linker generally has a sequence that yields a mobile peptide. The use of small amino acids, such as glycine and alanine, is useful in the creation of mobile peptides. The creation of such sequences is routine for those skilled in the art.

[0083] A suitable linker can be easily selected and can be of any appropriate length, including 1 amino acid (e.g., Gly) to 20 amino acids, 2 amino acids to 15 amino acids, 3 amino acids to 12 amino acids, including 4 to 10 amino acids, 5 to 9 amino acids, 6 to 8 amino acids, or 7 to 8 amino acids, and can be 1, 2, 3, 4, 5, 6, or 7 amino acids.

[0084] A suitable linker can be easily selected and can be any of several suitable lengths, including 1 amino acid (e.g., Gly) to 20 amino acids, 2 amino acids to 15 amino acids, 3 amino acids to 12 amino acids, including 4 to 10 amino acids, 5 to 9 amino acids, 6 to 8 amino acids, or 7 to 8 amino acids, and can be 1, 2, 3, 4, 5, 6, or 7 amino acids.

[0085] Examples of mobile linkers include glycine polymers (G)n, glycine-serine polymers (e.g., (GS)n, (GSGGS)n (SEQ ID NO: 145), (G4S)n, and (GGGS)n, where n is at least an integer of 1). In some cases, n is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 (or any range derivable therein), at most 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 (or any range derivable therein), or exactly 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 (or any range derivable therein). Examples include glycine-alanine polymers, alanine-serine polymers, and other mobile linkers known in the art. Glycine and glycine-serine polymers can be used; both Gly and Ser are relatively unstructured and therefore can function as neutral tethers between components. Glycine polymers can be used; glycine accesses significantly more φ-φ spaces than alanine and is far less restrictive than residues with longer side chains. Exemplary spacers include, but are not limited to, amino acid sequences such as GGGS (SEQ ID NO:43), GGSG (SEQ ID NO:44), GGSGG (SEQ ID NO:45), GSGSG (SEQ ID NO:46), GSGGG (SEQ ID NO:47), GGGSG (SEQ ID NO:48), GSSSG (SEQ ID NO:49), GGGSGGGS (SEQ ID NO:50), etc.

[0086] In further instances, the linker includes (EAAAK)n, where n is an integer of at least 1. In some instances, n is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 (or any range from which it can be derived), at most 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 (or any range from which it can be derived), or exactly 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 (or any range from which it can be derived).

[0087] II. Nucleic acids In certain contexts, nucleic acid sequences can exist in various forms, including: integrated sequences or isolated segments and recombinant vectors of recombinant polynucleotides encoding anti-inflammatory polypeptides; hybridization probes for identifying, analyzing, mutating, or amplifying polynucleotides encoding polypeptides; polynucleotides sufficient for use as PCR primers or sequencing primers; antisense nucleic acids for inhibiting polynucleotide expression; and the aforementioned complementary sequences as described herein. Nucleic acids encoding anti-inflammatory polypeptides are provided in certain contexts. Nucleic acids can be single-stranded or double-stranded and can include RNA and / or DNA nucleotides as well as their synthetic variants (e.g., peptide nucleic acids).

[0088] The term "polynucleotide" refers to a nucleic acid molecule that is either recombinant or isolated from a whole genome. Within the scope of the term "polynucleotide" are oligonucleotides (nucleic acids with a length of 100 residues or less), recombinant vectors, such as plasmids, cosmids, phages, and viruses. A polynucleotide contains a regulatory sequence that, in some respects, is substantially isolated from a native gene or protein-coding sequence. A polynucleotide may be single-stranded (coding or antisense) or double-stranded, and may be RNA, DNA (genomic, cDNA, or synthetic), its analogues, or a combination thereof. Further coding or non-coding sequences may or may not be present within a polynucleotide.

[0089] In this regard, the terms “gene,” “polynucleotide,” or “nucleic acid” are used to mean nucleic acids (including any sequences required for proper transcription, post-translational modification, or localization) that encode proteins, polypeptides, or peptides. As will be understood by those skilled in the art, these terms encompass genome sequences, expression cassettes, cDNA sequences, and smaller genetically engineered nucleic acid segments that express or can be adapted to express proteins, polypeptides, domains, peptides, fusion proteins, and variants. Nucleic acids encoding all or part of a polypeptide may include continuous nucleic acid sequences that encode all or part of such polypeptides. Furthermore, a particular polypeptide may be encoded by nucleic acids, including variants, that have slightly different nucleic acid sequences but still encode the same or substantially similar proteins.

[0090] In certain aspects, there are polynucleotide variants having substantial identity with the sequences disclosed herein; these variants, when compared to the polynucleotide sequences provided herein using the methods described herein (e.g., BLAST analysis with standard parameters), include at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%, or more, including all values ​​and ranges between the following: In certain aspects, the isolated polynucleotide comprises a nucleotide sequence encoding a polypeptide having at least 90%, preferably 95%, and more, identity with the amino acid sequences described herein over the entire length of the sequence; or a nucleotide sequence complementary to the isolated polynucleotide.

[0091] Nucleic acid segments, regardless of the length of the coding sequence itself, may be combined with other nucleic acid sequences such as promoters, polyadenylation signals, additional restriction enzyme sites, multicloning sites, and other coding segments, and therefore their total length can vary considerably. Nucleic acids can be of any length. They can be, for example, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 125, 175, 200, 250, 300, 350, 400, 450, 500, 750, 1000, 1500, 3000, 5000 or more nucleotide lengths, and / or may contain one or more additional sequences, such as regulatory sequences, and / or may be part of a larger nucleic acid, such as a vector. Therefore, nucleic acid fragments of almost any length can be used, and their total length is considered preferable to be limited by the ease of purification and their intended use in the recombinant nucleic acid protocol. In some cases, nucleic acid sequences may encode polypeptide sequences having further heterologous coding sequences, for example, to enable the purification, transport, secretion, or post-translational modification of the polypeptide, or to enable therapeutic utility such as targeting or efficacy. As discussed earlier, tags or other heterologous polypeptides can be appended to the sequence encoding the modified polypeptide, and "heterologous" means a polypeptide that is not the same as the modified polypeptide.

[0092] III. Immunotherapy In some aspects, this method involves the administration of cancer immunotherapy, and / or the subject is one being treated with immunotherapy. Cancer immunotherapy (sometimes called immuno-oncology, abbreviated as IO) is the use of the immune system to treat cancer. Immunotherapy can be classified as active, passive, or hybrid (active and passive). These approaches take advantage of the fact that cancer cells often have molecules on their surface that can be detected by the immune system, known as tumor-associated antigens (TAAs); these are often proteins or other macromolecules (e.g., carbohydrates). Active immunotherapy instructs the immune system to attack tumor cells by targeting TAAs. Passive immunotherapy enhances existing anti-tumor responses and includes the use of monoclonal antibodies, lymphocytes, and cytokines. Immunotherapy is well known in the art, and some are described below.

[0093] A. Immune checkpoint blockade therapy 1. PD-1, PDL1, and PDL2 inhibitors PD-1 can act in the tumor microenvironment where T cells encounter infection or tumors. Activated T cells upregulate PD-1, and continue to express PD-1 in peripheral tissues. Cytokines such as IFN-gamma induce PDL1 expression in epithelial and tumor cells. PDL2 is expressed in macrophages and dendritic cells. The main role of PD-1 is to limit the activity of effector T cells in the periphery and prevent excessive tissue damage during the immune response. The inhibitors of this disclosure may block one or more functions of PD-1 and / or PDL1 activity.

[0094] Alternative names for "PD-1" include CD279 and SLEB2. Alternative names for "PDL1" include B7-H1, B7-4, CD274, and B7-H. Alternative names for "PDL2" include B7-DC, Btdc, and CD273. In some contexts, PD-1, PDL1, and PDL2 are human PD-1, PDL1, and PDL2.

[0095] In some contexts, a PD-1 inhibitor is a molecule that inhibits the binding of PD-1 to its ligand-binding partner. In certain contexts, the PD-1 ligand-binding partner is PDL1 and / or PDL2. In other contexts, a PDL1 inhibitor is a molecule that inhibits the binding of PDL1 to its ligand-binding partner. In certain contexts, the PDL1 binding partner is PD-1 and / or B7-1. In other contexts, a PDL2 inhibitor is a molecule that inhibits the binding of PDL2 to its ligand-binding partner. In certain contexts, the PDL2 binding partner is PD-1. The inhibitor may be an antibody, its antigen-binding fragment, an immunoadhesin, a fusion protein, or an oligopeptide. Exemplary antibodies are described in U.S. Patents 8,735,553, 8,354,509, and 8,008,449, all of which are incorporated herein by reference. Other PD-1 inhibitors for use in the methods and compositions provided herein are known in the art as described in U.S. Patent Publications US2014 / 0294898, US2014 / 022021, and US2011 / 0008369, all of which are incorporated herein by reference.

[0096] In some contexts, PD-1 inhibitors are anti-PD-1 antibodies (e.g., human antibodies, humanized antibodies, or chimeric antibodies). In some contexts, anti-PD-1 antibodies are selected from the group consisting of nivolumab, pembrolizumab, and pidilizumab. In some contexts, PD-1 inhibitors are immunoadhesins (e.g., immunoadhesins containing the extracellular portion or PD-1 binding moiety of PDL1 or PDL2 fused to a constant region (e.g., the Fc region of an immunoglobulin sequence)). In some contexts, PDL1 inhibitors include AMP-224. Nivolumab is also known as MDX-1106-04, MDX-1106, ONO-4538, BMS-936558, and OPDIVO®, and is an anti-PD-1 antibody described in WO2006 / 121168. Pembrolizumab, also known as MK-3475, Merck3475, lambrolizumab, KEYTRUDA®, and SCH-900475, is an anti-PD-1 antibody described in WO2009 / 114335. Pidilizumab, also known as CT-011, hBAT, or hBAT-1, is an anti-PD-1 antibody described in WO2009 / 101611. AMP-224, also known as B7-DCIg, is a PDL2-Fc fusion soluble receptor described in WO2010 / 027827 and WO2011 / 066342. Further PD-1 inhibitors include MEDI0680, also known as AMP-514, and REGN2810.

[0097] In some cases, ICB therapy includes PDL1 inhibitors such as durvalumab, also known as MEDI4736; atezolizumab, also known as MPDL3280A; avelumab, also known as MSB00010118C; MDX-1105; BMS-936559; or combinations thereof. In certain cases, ICB therapy includes PDL2 inhibitors such as rHIgM12B7.

[0098] In some aspects, the inhibitor contains the heavy and light chain CDR or VR of nivolumab, pembrolizumab, or pidilizumab. Thus, in one aspect, the inhibitor contains the CDR1, CDR2, and CDR3 domains of the VH region of nivolumab, pembrolizumab, or pidilizumab, as well as the CDR1, CDR2, and CDR3 domains of the VL region of nivolumab, pembrolizumab, or pidilizumab. In another aspect, the antibody competes for binding to the same epitopes on PD-1, PDL1, or PDL2 as the aforementioned antibody, and / or binds to the same epitopes on PD-1, PDL1, or PDL2 as the aforementioned antibody. In yet another aspect, the antibody has at least about 70, 75, 80, 85, 90, 95, 97, or 99% (or within the derivable range) of variable region amino acid sequence identity with the aforementioned antibody.

[0099] 2. CTLA-4, B7-1, and B7-2 Another immune checkpoint that can be targeted in the methods provided herein is cytotoxic T lymphocyte protein 4 (CTLA-4), also known as CD152. The complete cDNA sequence of human CTLA-4 has Genbank accession number L15006. CTLA-4 is found on the surface of T cells and acts as an "off" switch when it binds to B7-1 (CD80) or B7-2 (CD86) on the surface of antigen-presenting cells. CTLA4 is a member of the immunoglobulin superfamily that is expressed on the surface of helper T cells and transmits inhibitory signals to T cells. CTLA4 is analogous to the T cell co-stimulatory protein CD28, and both molecules bind to B7-1 and B7-2 on antigen-presenting cells. CTLA-4 transmits inhibitory signals to T cells, while CD28 transmits stimulating signals. Intracellular CTLA-4 is also found in regulatory T cells and may be important for their function. When T cells are activated via the T cell receptor and CD28, the expression of CTLA-4, an inhibitory receptor for the B7 molecule, increases. The inhibitors of this disclosure may block one or more functions of CTLA-4, B7-1, and / or B7-2 activity. In some aspects, the inhibitors block the CTLA-4 and B7-1 interaction. In some aspects, the inhibitors block the CTLA-4 and B7-2 interaction.

[0100] In some aspects, ICB therapy includes anti-CTLA-4 antibodies (e.g., human antibodies, humanized antibodies, or chimeric antibodies), their antigen-binding fragments, immunoadhesins, fusion proteins, or oligopeptides.

[0101] Anti-human CTLA-4 antibodies (or VH and / or VL domains derived therefrom) suitable for use in the method of the present invention can be prepared using methods well known in the art. Alternatively, anti-CTLA-4 antibodies recognized in the art can be used. For example, the anti-CTLA-4 antibodies disclosed in U.S. Patent No. 8,119,129, WO01 / 14424, WO98 / 42752; WO00 / 37504 (CP675,206, tremelimumab; formerly also known as ticilimumab), U.S. Patent No. 6,207,156; Hurwitz et al., 1998 can be used in the method disclosed herein. The disclosures of each of the aforementioned publications are incorporated herein by reference. Antibodies that compete with any of these art-recognized antibodies for binding to CTLA-4 can also be used. For example, humanized CTLA-4 antibodies are described in International Patent Application Nos. WO2001 / 014424, WO2000 / 037504, and U.S. Patent No. 8,017,114, all of which are incorporated herein by reference.

[0102] Further anti-CTLA-4 antibodies useful as ICB therapy in the methods and compositions of this disclosure are ipilimumab (also known as 10D1, MDX-010, MDX-101, and Yervoy®) or its antigen-binding fragments and variants (see, for example, WO01 / 14424).

[0103] In some aspects, the inhibitor contains the heavy and light chain CDR or VR of tremelimumab or ipilimumab. Thus, in one aspect, the inhibitor contains the CDR1, CDR2, and CDR3 domains of the VH region of tremelimumab or ipilimumab, as well as the CDR1, CDR2, and CDR3 domains of the VL region of tremelimumab or ipilimumab. In another aspect, the antibody competes for binding to the same epitopes on PD-1, B7-1, or B7-2 as the aforementioned antibody, and / or binds to the same epitopes on PD-1, B7-1, or B7-2 as the aforementioned antibody. In yet another aspect, the antibody has at least about 70, 75, 80, 85, 90, 95, 97, or 99% (or within a derivable range) of variable region amino acid sequence identity with the aforementioned antibody.

[0104] B. Activation of co-stimulatory molecules In some aspects, immunotherapy involves inhibitors of co-stimulatory molecules. In some aspects, inhibitors include inhibitors of B7-1 (CD80), B7-2 (CD86), CD28, ICOS, OX40 (TNFRSF4), 4-1BB (CD137; TNFRSF9), CD40L (CD40LG), GITR (TNFRSF18), and combinations thereof. Inhibitors include inhibitory antibodies, polypeptides, compounds, and nucleic acids.

[0105] C. Dendritic cell therapy Dendritic cell therapy induces an antitumor response by having dendritic cells present tumor antigens to lymphocytes, thereby activating the lymphocytes and stimulating them to kill other cells presenting the antigens. Dendritic cells are antigen-presenting cells (APCs) in the mammalian immune system. In cancer treatment, dendritic cells help target cancer antigens. One example of dendritic cell-based cell carcinoma therapy is cyplücel-T.

[0106] One method to induce dendritic cells to present tumor antigens is by vaccinating them with autologous tumor lysates or short peptides (small portions of proteins corresponding to protein antigens on cancer cells). These peptides are often given in combination with adjuvants (highly immunogenic substances) to enhance the immune and antitumor response. Other adjuvants include proteins or other chemicals that attract and / or activate dendritic cells, such as granulocyte-macrophage colony-stimulating factor (GM-CSF).

[0107] Dendritic cells can also be activated in vivo by inducing GM-CSF expression in tumor cells. This can be achieved by genetically modifying tumor cells to produce GM-CSF, or by infecting tumor cells with an oncolytic virus that expresses GM-CSF.

[0108] Another strategy involves removing dendritic cells from the patient's blood and activating them outside the body. The dendritic cells are activated in the presence of a tumor antigen, which can be a single tumor-specific peptide / protein or tumor cell lysate (a solution of destroyed tumor cells). These cells (with a selective adjuvant) are then injected to trigger an immune response.

[0109] Dendritic cell therapy involves the use of antibodies that bind to receptors on the surface of dendritic cells. Antigens can be added to the antibodies, inducing dendritic cells to mature and providing immunity against tumors. Dendritic cell receptors such as TLR3, TLR7, TLR8, or CD40 are used as antibody targets.

[0110] D. CAR-T cell therapy Chimeric antigen receptors (also known as chimeric immune receptors, chimeric T cell receptors, or artificial T cell receptors, CARs) are engineered receptors that combine the specificity of immune cells to target cancer cells with novel specificity. Typically, these receptors transfer the specificity of monoclonal antibodies to T cells. The receptors are called chimeric because parts from different sources are fused together. CAR-T cell therapy refers to treatments that use such transformed cells for cancer treatment.

[0111] The fundamental principle of CAR-T cell design involves recombinant receptors that combine antigen-binding and T-cell activation functions. The general premise of CAR-T cells is the artificial creation of T cells that target markers found on cancer cells. Scientists can remove T cells from a person, genetically modify them, and return them to the patient to attack cancer cells. Once a T cell is engineered to become a CAR-T cell, it acts as a "living drug." CAR-T cells create a link between an extracellular ligand-recognition domain and an intracellular signaling molecule, which activates the T cell. The extracellular ligand-recognition domain is typically a single-stranded variable fragment (scFv). A critical aspect of the safety of CAR-T cell therapy is ensuring that only cancerous tumor cells, and not normal cells, are targeted. The specificity of CAR-T cells is determined by the selection of the target molecule.

[0112] Exemplary CAR-T therapies include tisagenlecleucel (Kymriah) and axicabtagene ciloleucel (Yescarta). In some aspects, CAR-T therapies target CD19.

[0113] E. Cytokine therapy Cytokines are proteins produced by many types of cells present within tumors. They can modulate the immune response. Tumors often utilize cytokines to promote tumor growth and reduce the immune response. These immunomodulatory effects make it possible to use them as drugs to induce an immune response. Two commonly used cytokines are interferons and interleukins.

[0114] Interferons are produced by the immune system. They are typically involved in antiviral responses, but are also used in cancer treatment. They are classified into three groups: Type I (IFNα and IFNβ), Type II (IFNγ), and Type III (IFNλ).

[0115] Interleukins possess numerous immune system effects. IL-2 is an exemplary interleukin cytokine therapy.

[0116] F. Adoptive T cell therapy Adoptive T-cell therapy is a form of passive immunity involving the transfusion (adoptive cell transfer) of T cells. T cells are found in the blood and tissues and are normally activated when they encounter foreign pathogens. Specifically, T cells are activated when their surface receptors encounter cells that present a portion of a foreign protein on a surface antigen. These can be either infected cells or antigen-presenting cells (APCs). They are found in normal tissues and tumor tissues, in which case they are known as tumor-infiltrating lymphocytes (TILs). They are activated by the presence of APCs such as dendritic cells that present tumor antigens. These cells can attack the tumor, but the intratumoral environment is highly immunosuppressive and prevents immune-mediated tumor death.

[60]

[0117] Several methods have been developed to produce and obtain tumor-targeted T cells. Tumor antigen-specific T cells can be removed from tumor samples (TILs) or filtered from blood. Subsequent activation and culture are performed ex vivo, and the cells are then reinjected. Activation can be achieved through gene therapy or by exposing T cells to tumor antigens.

[0118] Cancer treatment is intended to exclude any of the cancer treatments described herein. Furthermore, aspects of this disclosure include patients who have previously been treated with the treatments described herein, patients who are currently being treated with the treatments described herein, or patients who have not been treated with the treatments described herein. In some aspects, patients are those who have been determined to be resistant to the treatments described herein. In some aspects, patients are those who have been determined to be sensitive to the treatments described herein.

[0119] IV. Treatment method The compositions of this disclosure may be used for in vivo, in vitro, or ex vivo administration. The routes of administration of the compositions may be, for example, intracutaneous, subcutaneous, intravenous, intradermal, intramuscular, topical, local, and / or intraperitoneal. One or more of these routes of administration are particularly intended to be excluded from certain aspects of this disclosure.

[0120] In some aspects, the compositions of the present disclosure are provided via subcutaneous administration (i.e., delivered under the skin). In some aspects, the compositions of the present disclosure are provided via intradermal administration (i.e., delivered under the skin). In some aspects, the compositions of the present disclosure are provided via intramuscular administration (i.e., delivered under the muscle). In some aspects, the compositions of the present disclosure are delivered to the site of a wound. In some aspects, the compositions of the present disclosure are not delivered to the site of a wound (e.g., delivered to a site other than the site of a wound).

[0121] In some aspects, the therapeutic compositions of the present disclosure are administered during the discontinuation of one or more other therapeutic treatments. For example, in some aspects, the disclosed method includes the step of administering an anti-inflammatory polypeptide (e.g., an anti-inflammatory cytokine linked to an albumin protein) to a target during the discontinuation of further anti-inflammatory therapeutic substances (e.g., fingolimod, interferon-β, dimethyl fumarate, teriflunomide, integrin α4β1, anti-αLβ2 antibody, anti-TNFα agent, anti-IL-6R agent, anti-IL-6 agent, or Janus kinase inhibitor).

[0122] A. Autoimmune or inflammatory condition This disclosure relates to methods for treating autoimmune or inflammatory conditions. In some aspects, disclosed herein are methods for treating autoimmune or inflammatory conditions, which include the step of administering a composition containing anti-inflammatory cytokines (e.g., IL-4, IL-10, IL-33, IL-35, etc.) to a subject when the subject has, is at risk of developing, or is suspected of having an autoimmune or inflammatory condition. Such methods may include the step of administering one or more further anti-inflammatory agents. Such methods may exclude the step of administering one or more further anti-inflammatory agents. Further anti-inflammatory agents include, for example, fingolimod, interferon-β, dimethyl fumarate, teriflunomide, integrin α4β1, anti-αLβ2 antibodies, anti-TNFα agents, anti-IL-6R agents, anti-IL-6 agents, and Janus kinase inhibitors (e.g., tofacitinib, baricitinib, upadacitinib).

[0123] Autoimmune or inflammatory conditions (similarly "diseases" or "disorders") suitable for treatment include diabetes (e.g., type 1 diabetes), graft rejection, arthritis (such as acute arthritis, chronic rheumatoid arthritis, gout or gouty arthritis, acute gouty arthritis, acute immune arthritis, chronic inflammatory arthritis, osteoarthritis, type II collagen-induced arthritis, infectious arthritis, Lyme arthritis, proliferative arthritis, psoriatic arthritis, Still's disease, spondyloarthritis, and systemic juvenile-onset rheumatoid arthritis, osteoarthritis, chronic progressive arthritis, osteoarthritis, polyarthritis, reactive arthritis, and Psoriasis (including ankylosing spondylitis), inflammatory hyperproliferative skin diseases, psoriasis such as psoriasis vulgaris, guttate psoriasis, pustular psoriasis, and nail psoriasis, atopic dermatitis including hay fever and Job's syndrome, dermatitis including contact dermatitis, chronic contact dermatitis, exfoliative dermatitis, allergic dermatitis, allergic contact dermatitis, herpetiform dermatitis, nummular eczema, seborrheic dermatitis, nonspecific dermatitis, primary irritant contact dermatitis, and atopic dermatitis, x-linked hyper-IgM syndrome, allergic intraocular inflammatory disease, chronic allergic urticaria and chronic autoimmune urticaria, including chronic idiopathic urticaria. Urticaria such as rash, myositis, polymyositis / dermatomyositis, juvenile dermatomyositis, toxic epidermal necrolysis, scleroderma (including systemic sclerosis), sclerosis such as systemic sclerosis, multiple sclerosis (MS) such as spinal optical MS, primary progressive MS (PPMS), relapsing-remitting MS (RRMS), progressive systemic sclerosis, atherosclerosis, arteriosclerosis, disseminated sclerosis, ataxic sclerosis, neuromyelitis optica (NMO), inflammatory bowel disease (IBD) (e.g., Crohn's disease, autoimmune-mediated gastrointestinal disease, ulcerative colitis, ulcerative colitis, microscopic colitis, collagenous colitis, polycolitis, necrosis) Colitis such as enteritis and autoimmune inflammatory bowel disease, inflammation of the intestines, 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 hematological disorders, rheumatoid arthritis, hereditary angioedema, cranial nerve damage such as meningitis, herpes zoster of pregnancy, bullous pemphigoid of pregnancy, pruritus scrotalis, autoimmune premature ovarian failure, sudden hearing loss due to autoimmune conditions, IgE-mediated diseases such as anaphylaxis and allergic and atopic rhinitis.Encephalitis such as Rasmussen's encephalitis and limbic and / or brainstem encephalitis; uveitis such as anterior uveitis, acute anterior uveitis, granulomatous uveitis, non-granulomatous uveitis, lens antigenic uveitis, posterior uveitis, or autoimmune uveitis; glomerulonephritis (GN) with or without nephrotic syndrome, such as chronic or acute glomerulonephritis including primary GN; immune-mediated GN; membranous GN (membranous nephropathy); idiopathic membranous GN or idiopathic membranous Nephropathy, membranous or membranoproliferative GN (MPGN) including type I and type II, and rapidly progressive GN, proliferative glomerulonephritis, autoimmune polyglandular endocrine deficiency, balanoposthitis, plasmacytic balanoposthitis, balanoposthitis including balanoposthitis, erythema annulare centrifugal, erythema pigmentosum fixed, erythema multiforme, granuloma annulare, lichen sclerosing, lichen simplex, lichen spinal, lichen planus, ichthyosis laminae, exfoliative hyperkeratosis, precancerous keratosis, pyoderma gangrenosum, allergic conditions and This includes, but is not limited to, conditions such as reactions, allergic reactions, allergic or atopic eczema, dyshidrotic eczema, sweaty eczema, and vesicular palmoplantar eczema; asthma such as bronchial asthma, bronchial asthma and autoimmune asthma; conditions with T cell infiltration and chronic inflammatory response; immune reactions to exogenous antigens such as fetal ABO blood group during pregnancy; chronic inflammatory pneumonia; autoimmune myocarditis; leukocytosis; lupus nephritis; lupus encephalitis; lupus in childhood; nonrenal lupus; extrarenal lupus; discoid lupus and discoid lupus erythematosus; alopecia lupus; systemic lupus erythematosus (SLE) such as cutaneous SLE or subacute cutaneous SLE; lupus including neonatal lupus erythematosus (NLE) and disseminated lupus erythematosus; juvenile-onset (Type 1) diabetes mellitus, including pediatric insulin-dependent diabetes mellitus (IDDM); adult-onset diabetes mellitus (Type 2) and autoimmune diabetes mellitus. Cytokine and T lymphocyte-mediated acute and delayed hypersensitivity, sarcoidosis, granulomatous vasculitis including lymphomatous granulomatosis and Wegener's granulomatosis, agranulocytic vasculitis, vasculitis including large vessel vasculitis (including polymyalgia rheumatica and giant cell (Takayasu) arteritis), medium vessel vasculitis (including Kawasaki disease and polyarteritis nodosa), microscopic polyarteritis, immunovasculitis, central nervous system vasculitis, cutaneous vasculitis, hypersensitivity vasculitis, necrotizing vasculitis such as necrotizing vasculitis,and ANCA-associated vasculitis and ANCA-associated small vessel vasculitis such as Churg-Strauss vasculitis or syndrome (CSS), temporal arteritis, aplastic anemia, autoimmune aplastic anemia, Coombs-positive anemia, Diamond-Blackfan anemia, hemolytic anemia or immune hemolytic anemia including autoimmune hemolytic anemia (AIHA), Addison's disease, autoimmune neutropenia, pancytopenia, leukopenia, diseases with extravascular migration of leukocytes, CNS inflammatory diseases, Alzheimer's disease, Parkinson's disease, sepsis, multi-organ injury syndromes such as those secondary to trauma or bleeding, antigenic Body complex-mediated diseases, anti-glomerular basement membrane diseases, antiphospholipid antibody syndromes, allergic neuritis, Behçet's disease / syndrome, Castleman syndrome, Goodpasture syndrome, Raynaud's syndrome, Sjögren's syndrome, Stevens-Johnson syndrome, bullous pemphigoid and cutaneous pemphigoid, pemphigoids (including pemphigus vulgaris, pemphigus phyllodes, mucous membrane pemphigus, and erythematous pemphigus), autoimmune polyglandular autoimmune syndromes, Reiter's disease or syndrome, burns, pre-eclampsia, immune complex disorders such as immune complex nephritis, antibody-mediated nephritis, polyneuropathy, IgM polyneuropathy or chronic neurological disorders such as IgM-mediated neuropathy, autoimmune or immune thrombocytopenia such as idiopathic thrombocytopenic purpura (ITP) including chronic or acute ITP, scleritis such as idiopathic scleritis and episcleritis, autoimmune diseases of the testes and ovaries including autoimmune orchitis and oophoritis, primary hypothyroidism, hypoparathyroidism, autoimmune thyroiditis, thyroiditis such as Hashimoto's disease and chronic thyroiditis (Hashimoto's thyroiditis), or autoimmune endocrine disorders including subacute thyroiditis and autoimmune thyroiditis, idiopathic hypothyroidism, Graves' disease, autoimmune polyglandular syndrome (or Polyglandular syndromes such as polyglandular endocrine disorders, paraneoplastic syndromes including Lambert-Eaton myasthenic syndrome or Eaton-Lambert syndrome, stiff person syndrome or stiff person syndrome, encephalomyelitis such as allergic encephalomyelitis or allergic encephalomyelitis and experimental allergic encephalomyelitis (EAE), experimental autoimmune encephalomyelitis, myasthenia gravis such as thymoma-associated myasthenia gravis, cerebellar degeneration, neuromuscular asthenia, opsoclonus or opsoclonus myoclonus syndrome (OMS), and sensory neuropathy.Multifocal motor neuropathy, Sheehan's syndrome, autoimmune hepatitis, chronic hepatitis, lupoid hepatitis, giant cell hepatitis, chronic active hepatitis or autoimmune chronic active hepatitis, lymphocytic interstitial pneumonia (LIP), bronchiolitis obliterans (non-transplant) vs. NSIP, Guillain-Barré syndrome, Berger's disease (IgA nephropathy), idiopathic IgA nephropathy, linear IgA dermatosis, acute febrile neutrophilic dermatosis, subcorneal pustular dermatosis, transient acanthospermic dermatosis, cirrhosis such as primary biliary cirrhosis or pulmonary cirrhosis, autoimmune enteropathy syndrome, celiac disease or celiac disease, celiac sprue (gluten enteropathy), refractory sprue, idiopathic sprue, cryoglobulinemia, amyotrophic lateral sclerosis (ALS; Lou Gehrig's disease), coronary artery disease, autoimmune inner ear disease (AIED), and other autoimmune conditions. Immunotherapy-related ear diseases, autoimmune hearing loss, polychondritis such as refractory or relapsing or relapsing polychondritis, alveolar proteinosis, Cogan syndrome / non-syphilitic interstitial keratitis, Bell's palsy, Sweet's disease / syndrome, rosacea autoimmune, pain associated with herpes zoster, amyloidosis, noncancerous lymphocytosis, primary lymphocytosis including monoclonal B-cell lymphocytosis (e.g., benign monoclonal immunoglobulinemia and monoclonal immunoglobulinemia of unknown importance, MGUS), peripheral neuropathy, paraneoplastic syndromes , channelopathy such as epilepsy, migraine, arrhythmia, muscle disorders, hearing loss, blindness, periodic paralysis, and central nervous system channelopathy, autism, inflammatory myopathy, focal or segmental or focal segmental glomerulosclerosis (FSGS), endocrine eye disease, uveoretinitis, chorioretinitis, autoimmune liver disease, fibromyalgia, multiple endocrine insufficiency, Schmidt syndrome, adrenal nephritis, gastric atrophy, presenile dementia, demyelinating diseases such as autoimmune demyelinating diseases and chronic inflammatory demyelinating polyneuropathy, Dressler syndrome, alopecia areata, alopecia totalis CREST syndrome (calcification, Raynaud's phenomenon, esophageal motility disorder, finger sclerosis, and telangiectasia), autoimmune infertility in men and women, for example, due to anti-sperm antibodies, mixed connective tissue disease, Chagas disease, rheumatic fever, recurrent miscarriage, farmer's lung, erythema multiforme, postcardiotomy syndrome, Cushing's syndrome, avian lung, allergic granulomatous vasculitis, benign lymphocytic vasculitis, Alport syndrome, alveolitis such as allergic pneumonia and fibrous pneumonia, interstitial lung disease, transfusion reactions, leprosy, malaria, leishmaniasis,Parasitic diseases such as cypanosomiasis, schistosomiasis, and aspergillosis, aspergillosis, Sumpter syndrome, Kaplan syndrome, dengue fever, endocarditis, intramyocardial fibrosis, diffuse interstitial pulmonary fibrosis, interstitial pulmonary fibrosis, pulmonary fibrosis, idiopathic pulmonary fibrosis, cystic fibrosis, endophthalmitis, persistent erythema elevata, fetal erythroblastosis, eosinophilic fasciitis, Schulman syndrome, Felty syndrome, filariasis, chronic circulatory inflammation, metachronous circulatory inflammation, iridocyclitis (acute or chronic) Circulatory inflammation such as Hook's circulatory inflammation, Henoch-Schönlein purpura, human immunodeficiency virus (HIV) infection, SCID, acquired immunodeficiency syndrome (AIDS), echovirus infection, sepsis, endotoxemia, pancreatitis, thyroiditis, parvovirus infection, rubella virus infection, post-vaccination syndrome, congenital rubella infection, Epstein-Barr virus infection, mumps, Evans syndrome, autoimmune gonadal insufficiency, cystitis Denham's chorea, post-streptococcal nephritis, thromboangiitis obliterans, thyroidopathy, tabes dorsalis, choroiditis, giant cell polymyalgia, chronic hypersensitivity pneumonitis, keratoconjunctivitis sicca, epidemic keratoconjunctivitis, idiopathic nephritis syndrome, minimal change nephropathy, benign familial and ischemia-reperfusion injury, transplant organ reperfusion, retinal autoimmunity, arthritis, bronchitis, chronic obstructive airway / lung disease, silicosis, stomatitis, aphthous stomatitis, arthritis, spermatogenesis deficiency, autoimmune hemolysis, bedstool Cryoglobulinemia, Dupuytren's contracture, lens hypersensitive endophthalmitis, allergic enteritis, erythema nodosum, idiopathic facial paralysis, chronic fatigue syndrome, rheumatic fever, Hamann-Ricci disease, sensorineural hearing loss, hemoglobinuria attacks, hypogonadism, focal enteritis, leukopenia, infectious mononucleosis, transverse myelitis, primary idiopathic myxedema, nephrotic syndrome, sympathetic ophthalmitis, granulomatous orchitis, pancreatitis, polyradiculitis of the skin, pyoderma gangrenosum, Quervain's thyroiditis, Acquired splenic atrophy, non-malignant thymoma, vitiligo, toxic shock syndrome, food poisoning, conditions with T cell infiltration, leukocyte adhesion deficiency, immune responses associated with cytokine and T lymphocyte-mediated acute and delayed hypersensitivity, diseases involving extravasation of leukocytes, multiple organ injury syndrome, antigen-antibody complex-mediated diseases, anti-glomerular basement membrane diseases, allergic neuritis, autoimmune polyendocrine syndrome, folliculitis, primary myxedema, autoimmune atrophic gastritis, sympathetic ophthalmitis, rheumatic diseases, mixed connective tissue disease, nephrotic syndrome, pancreatitis, polyglandular autoimmune disease Syndrome, type 1 polyglandular autoimmune syndrome, adult-onset idiopathic hypoparathyroidism (AOIH), cardiomyopathy such as dilated cardiomyopathy, epidermolysis bullosa (EBA), hemochromatosis, myocarditis, nephrotic syndrome, primary sclerosing cholangitis, suppurative or non-suppurative sinusitis, acute or chronic sinusitis, ethmoid, frontal, maxillary, or sphenoid sinusitis, eosinophil-related disorders such as eosinophilia, pulmonary infiltrative eosinophilia, eosinophilia-myalgia syndrome, Loeffler's syndrome, chronic eosinophilic pneumonia, tropical pulmonary eosinophilia, bronchopneumonic aspergillosis, aspergillosis , or granulomas containing eosinophils, anaphylaxis, seronegative spondyloarthritis, autoimmune polyendocrine syndrome, sclerosing cholangitis, sclera, episclera, chronic mucocutaneous candidiasis, Bruton syndrome, transient hypogammaglobulinemia in infancy, Wiscott-Aldrich syndrome, ataxia telangiectasia, collagen disease, rheumatism, autoimmune diseases associated with neurological disorders, lymphadenitis, hypotensive response, vascular dysfunction, tissue damage, cardiovascular ischemia, hyperalgesia, renal ischemia, cerebral ischemia, and diseases with angiogenesis, allergic hypersensitivity, glomerulonephritis, reperfusion Injuries, ischemic reperfusion injury, reperfusion injury of myocardium or other tissues, lymphomatous tracheobronchitis, inflammatory skin diseases, skin diseases with acute inflammatory components, multiple organ failure, bullous diseases, renal cortical necrosis, acute purulent meningitis or other central nervous system inflammatory diseases, inflammatory diseases of the eye and orbit, granulocyte transfusion-associated syndromes, cytokine-induced toxicity, narcolepsy, acute severe inflammation, chronic refractory inflammation, pyelonephritis, intra-arterial hyperplasia, peptic ulcers, valvular heart disease, graft-versus-host disease, contact hypersensitivity, asthmatic airway hypersensitivity, endometriosis, as well as cancer immunotherapy or viral infections (e.g.,Immune responses associated with cytokine storm syndromes (and similarly, "cytokine release syndromes"), such as cytokine storm syndrome caused by SARS-CoV-2 infection, are also considered. It is intended that one or more of these conditions or diseases may be excluded in the context disclosed herein.

[0124] In some aspects, the disclosed methods are for treating subjects with multiple sclerosis. Methods for treating subjects with multiple sclerosis may include a step of administering albumin-linked IL-4 to the subject. Methods for treating subjects with multiple sclerosis may include a step of administering albumin-linked IL-33 to the subject.

[0125] In some aspects, the disclosed methods are for treating subjects with arthritis. In some aspects, the disclosed methods are for treating subjects with rheumatoid arthritis. The methods for treating subjects with arthritis may include a step of administering IL-10 linked to albumin protein to the subject. The methods for treating subjects with arthritis may include a step of administering IL-35 linked to albumin protein to the subject.

[0126] In some cases, the disclosed methods are for treating subjects with type 1 diabetes. In some cases, the disclosed methods are for treating subjects with diabetic peripheral neuropathy. In some cases, the disclosed methods are for treating subjects with psoriasis. In some cases, the disclosed methods are for treating subjects with inflammatory bowel disease. In some cases, the disclosed methods are for treating subjects with Crohn's disease. In some cases, the disclosed methods are for treating subjects with systemic sclerosis. In some cases, the disclosed methods are for treating subjects with cytokine storm syndrome (including, for example, cytokine storm syndrome caused by cancer immunotherapy and cytokine storm syndrome caused by viral infections such as SARS-CoV-2 infection). In some cases, the disclosed methods are for treating subjects with acute respiratory distress syndrome (ARDS).

[0127] B. Wound healing This disclosure relates to methods for promoting wound healing. In some aspects, disclosed herein are methods for promoting wound healing, comprising the step of administering to a subject a composition comprising an anti-inflammatory cytokine functionally linked to an albumin protein (e.g., IL-4, IL-10, IL-33, IL-35, etc.) when the subject has a wound. In some aspects, the wound is a chronic wound. In some aspects, the wound is a diabetic ulcer. In some aspects, disclosed are methods for promoting wound healing, comprising the step of administering to a subject a composition comprising IL-4 functionally linked to an albumin protein. Such methods may include the step of administering one or more further wound healing agents.

[0128] C. Anti-inflammatory cytokine targeting Aspects of this disclosure relate to methods for targeting anti-inflammatory cytokines to target lymph nodes. In some aspects, the anti-inflammatory cytokines are targeted to target lymph nodes by ligating the cytokines to albumin proteins or albumin-binding proteins. The ligated polypeptides may then be administered to a subject to target the anti-inflammatory cytokines to the target lymph nodes. In some aspects, after administration, the anti-inflammatory cytokines remain in the lymph nodes for at least 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours (or any range within which this can be derived) or longer after the composition has been administered to the subject.

[0129] D. Administration of therapeutic compositions This disclosure relates to compositions and methods, including therapeutic compositions. Different therapeutic methods may be administered in one composition, or in two or more compositions, such as two, three, or four compositions. Various combinations of drugs may be used.

[0130] The therapeutic agents of this disclosure (e.g., anti-inflammatory polypeptides, anti-inflammatory cytokines) may be administered by one or more routes of administration. In some cases, the therapeutic agents may be administered intravenously, intramuscularly, subcutaneously, topically, orally, percutaneously, intraperitoneally, intraorbitally, by implantation, by inhalation, intrathecally, intravenously, or intranasally. One or more of these routes of administration are intended to be excluded from certain aspects of this disclosure. In some cases, the therapeutic agent is administered subcutaneously. In some cases, the therapeutic agent is administered intramuscularly. In some cases, the therapeutic agent is administered intradermally. The appropriate dose may be determined based on the type of disease being treated, the severity and course of the disease, the individual's clinical condition, the individual's medical history and response to treatment, and the discretion of the attending physician.

[0131] Treatment may involve various "unit doses." A unit dose is defined as containing a predetermined amount of the therapeutic composition. The amount administered, as well as the specific route and formulation, is within the scope of the clinical decision-making skills. A unit dose does not necessarily have to be administered as a single injection, but may include a continuous infusion over a set period of time. In some contexts, a unit dose may include a single administerable dose.

[0132] The amount administered depends on the desired therapeutic effect, as well as both the number of treatments and the unit dose. The effective dose (similarly, "effective amount") is understood to be the amount necessary to achieve a particular effect. In certain aspects of practice, doses ranging from 0.1 mg / kg to 50 mg / kg may be intended to influence the protective capacity of these drugs. Therefore, the dosages are intended to include approximately 0.1, 0.5, 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195 and 200, 300, 400, 500, 1000 μg / kg, mg / kg, μg / day or mg / day or any range within which can be derived. Furthermore, such dosages may be administered multiple times, during a single day, and / or over multiple days, weeks, or months.

[0133] In certain situations, the effective dose of a pharmaceutical composition is one that can provide a blood level of approximately 1 μM to 150 μM. In another context, an effective dose provides blood levels of approximately 4 μM to 100 μM; or approximately 1 μM to 100 μM; or approximately 1 μM to 50 μM; or approximately 1 μM to 40 μM; or approximately 1 μM to 30 μM; or approximately 1 μM to 20 μM; or approximately 1 μM to 10 μM; or approximately 10 μM to 150 μM; or approximately 10 μM to 100 μM; or approximately 10 μM to 50 μM; or approximately 25 μM to 150 μM; or approximately 25 μM to 100 μM; or approximately 25 μM to 50 μM; or approximately 50 μM to 150 μM; or approximately 50 μM to 100 μM (or any range within which this can be derived). In another context, a dose can provide the following blood levels of the drug resulting from the therapeutic agent administered to the subject: Approximately 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 or 100 μM or within Any range that can be derived by, at least about 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, 4 8, 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 or 100 μM or any range within that range that can be derived, or at most about 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, or 100 μM or any range derivable within that range. In certain situations, the therapeutic agent administered to a subject is metabolized in the body to a therapeutic agent that has been metabolized, in which case the blood level may refer to the amount of that agent. Alternatively, within the range in which the therapeutic agent is not metabolized by the subject, the blood level discussed herein may refer to the unmetabolized therapeutic agent.

[0134] The appropriate amount of therapeutic composition also depends on the practitioner's judgment and is specific to each individual. Factors influencing the dosage include the patient's physical and clinical condition, route of administration, intended treatment goal (symptom relief vs. cure), and the potency, stability, and toxicity of any specific therapeutic substance or other treatment the subject may be receiving.

[0135] Those skilled in the art will understand and recognize that dosage units of μg / kg or mg / kg body weight can be converted and expressed in equivalent concentration units of μg / ml or mM (blood level), such as 4 μM to 100 μM. It will also be understood that uptake is species and organ / tissue dependent. Applicable conversion factors and physiological assumptions made with respect to uptake and concentration measurements are well known, and those skilled in the art will be able to convert one concentration measurement to another and draw reasonable comparisons and conclusions regarding the dosages, efficacy, and results described herein.

[0136] E. Treatment for patients with cancer In aspects of this disclosure, polypeptides are used to treat subjects having cancer. Cancer may include, but is not limited to, tumors of all types, locations, sizes and characteristics. In some aspects, cancer includes solid tumors. In some aspects, cancer includes, for example, pancreatic cancer, colon cancer, acute myeloid leukemia, adrenocortical carcinoma, AIDS-related cancer, AIDS-related lymphoma, anal cancer, appendiceal cancer, astrocytoma, basal cell carcinoma of the cerebellum or cerebrum in children, cholangiocarcinoma, extrahepatic bladder cancer, bone cancer, osteosarcoma / malignant fibrous histiocytoma, brainstem glioma, brain tumor, astrocytoma brain tumor of the cerebellum, astrocytoma / malignant glioma brain tumor of the cerebrum, ependymal cell tumor, medulloblastoma brain tumor, supratentorial primitive neuroectoderm tumor brain tumor, glioma of the visual pathway and hypothalamus, breast cancer, lymphatic system Cancer, bronchial adenoma / carcinoid tumor, tracheal cancer, lung cancer, Burkitt lymphoma, carcinoid tumor, pediatric carcinoid tumor, gastrointestinal cancer of unknown primary origin, central nervous system lymphoma, primary cerebellar astrocytoma, pediatric cerebral astrocytoma / gliomas, pediatric cervical cancer, pediatric cancer, chronic lymphocytic leukemia, chronic myeloid leukemia, chronic myeloproliferative disorder, cutaneous T-cell lymphoma, fibrinogenic round cell tumor, endometrial cancer, ependymal cell tumor, esophageal cancer, Ewing's tumor, pediatric extragonadal germ cell tumor, extrahepatic cholangiocarcinoma, eye cancer, intraocular melanomaEye cancer, retinoblastoma, gallbladder cancer, gastric / stomach cancer, gastrointestinal carcinoid tumors, gastrointestinal stromal tumors (GIST), germ cell tumors: extracranial, extragonadal or ovarian, gestational trophoblastic tumor, brainstem glioma, glioma, astrocytoma of the cerebrum in children, glioma of the visual pathway and hypothalamus in children, gastric carcinoid, hairy cell leukemia, head and neck cancer, heart cancer, hepatocellular carcinoma (liver cancer), Hodgkin lymphoma, hypopharyngeal cancer, glioma of the hypothalamus and visual pathway, intraocular melanoma in children, islet cell carcinoma (endocrine pancreas), Kaposi's sarcoma, kidney cancer (renal cell carcinoma), laryngeal cancer, leukemia, acute lymphoblastic leukemia (also called acute lymphoblastic leukemia), acute myeloid leukemia (also called acute myelogenous leukemia) Leukemia (also known as chronic lymphocytic leukemia), chronic myelogenous leukemia (also known as chronic myeloid leukemia), hairy cell lip and oral cancer, liposarcoma, liver cancer (primary), non-small cell lung cancer, small cell lung cancer, lymphoma, AIDS-associated lymphoma, Burkitt lymphoma, cutaneous T-cell lymphoma, Hodgkin lymphoma, non-Hodgkin lymphoma (an older classification of all lymphomas other than Hodgkin's), primary central nervous system lymphoma, Waldenström macroglobulinemia, malignant fibrous histiocytoma / osteosarcoma of bone, medulloblastoma in children, intraocular melanoma, Merkel cell carcinoma, malignant mesothelioma in adults, mesothelioma in children, metastatic cervical squamous cell carcinomaNeck cancer, oral cancer, multiple endocrine neoplasia, multiple myeloma / plasmacytic neoplasm, mycosis fungoides, myelodysplastic syndrome, myelodysplastic / myeloproliferative disorder, chronic myeloid leukemia, acute myeloid leukemia in adults, acute myeloid leukemia in children, multiple myeloma, chronic myeloproliferative disorder, cancer of the nasal cavity and paranasal sinuses, nasopharyngeal cancer, neuroblastoma, oral cancer, oropharyngeal cancer, osteosarcoma / malignant fibrous histiocytoma of bone, ovarian cancer, ovarian epithelial carcinoma (surface epithelial-stromal tumor), ovarian germ cell tumor, low-grade ovarian tumor, pancreatic cancer, islet cell cancer of the paranasal sinuses and nasal cavity, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pineal astrocytoma, pineal germ tumor, pineal blastoma and supratentorial primitive neuroectoderm tumor, pituitary adenoma in children, plasmacytoma / multiple myeloma This includes pleuroblastoma, primary central nervous system lymphoma, prostate cancer, rectal cancer, renal cell carcinoma (kidney cancer), transitional cell carcinoma of the renal pelvis and ureter, retinoblastoma, rhabdomyosarcoma, pediatric salivary gland carcinosarcoma, Ewing's sarcoma family tumors, Kaposi's sarcoma, soft tissue sarcoma, Sézary syndrome sarcoma of the uterus, skin cancer (non-melanoma), skin cancer (melanoma), skin cancer, Merkel cell small cell lung cancer, small intestine cancer, soft tissue sarcoma, squamous cell carcinoma, cervical squamous cell carcinoma of unknown primary origin, metastatic gastric cancer, supratentorial primitive neuroectoderm tumor, pediatric T-cell lymphoma, testicular cancer, throat cancer, thymoma, pediatric thymoma, thymic carcinoma, thyroid cancer, urethral cancer, uterine cancer, endometrial sarcoma, vaginal cancer, glioma of the visual pathway and hypothalamus, pediatric vulvar cancer, and Wilms' tumor (kidney cancer). [Examples]

[0137] V. Examples The following embodiments are included to demonstrate certain aspects of the present invention. Those skilled in the art will recognize that the techniques disclosed in the following embodiments are techniques that the inventors have found to function well in the practice of the present invention. However, those skilled in the art will recognize that, in view of this disclosure, many modifications can be made in the specific embodiments disclosed without departing from the spirit and scope of the present invention, but similar or comparable results can still be obtained.

[0138] Example 1 - Long-term retention of albumin-fused IL-4 in secondary lymphoid organs improves experimental autoimmune encephalomyelitis. A. Results 1. SA-IL-4 binds to immune cells and inhibits Th17 differentiation. We recombinantly expressed wild-type (wt) mouse IL-4 and mouse serum albumin (SA) fused mouse IL-4 (Figure 1A). SDS-PAGE revealed that SA fusion to IL-4 increased its molecular size. When added to freshly isolated immune cells from lymph nodes (LNs) and spleens, SA-IL4 selectively bound to antigen-presenting cells (APCs) such as macrophages and dendritic cells (DCs) in vitro compared to other immune cells (Figure 1B).

[0139] The IL-4 receptor is known to be expressed in T cells when stimulated. 13 SA-IL-4 induced downstream phosphorylation of STAT6 in T cells at an EC50 32-fold higher than that of wt IL-4. This suggested that wt IL-4 was more active than SA-IL-4 in vitro (Figure 1C). However, while STAT6 phosphorylation was reduced with engineered IL-4, naive CD4 cells cultured in Th17 cell differentiation medium did not. + It was found that both wt IL-4 and SA-IL-4 are equally effective in inhibiting Th17 differentiation of T cells (Figure 1D). In summary, these results demonstrate that the inventors have successfully created a functionally active SA-IL-4 fusion protein.

[0140] 2. SA-IL-4 increased its blood half-life and persistence in the spleen in both the pulmonary nephrocyte (LN) and spleen. The inventors tested whether intravenous (iv) injection of SA-IL-4 caused accumulation in the spleen and pulmonary nephrocytes (LN) using naive mice. SA fusion to IL-4 significantly increased the amount of IL-4 after iv injection in both the lumbar and brachial LNs and in the spleen (Figures 2A and 2B). Furthermore, SA-IL-4 injected subcutaneously (sc) and intraperitoneally (ip) showed remarkably high levels in both the lumbar and brachial LNs and in the spleen (Figures 2C and 2D). The amount of SA-IL-4 also increased in various organs, such as the liver and lungs, likely due to an extended plasma half-life (Figures 3A-3G). EAE mice showed high accumulation of SA-IL-4 in the lumbar LN and spinal cord (Figures 3J-3I). In vivo distribution analysis based on fluorescence also showed enhanced SA-IL-4 accumulation in the lumbar LN compared to wt IL-4. Surface plasmon resonance (SPR) analysis revealed that SA-IL-4 has a dissociation constant (K) of 385 nM. D SA-IL-4 binds to FcRn (Figure 2E), which is similar to the affinity of SA for FcRn (Figure 3J). The plasma half-life of SA-IL-4 after IV and subcutaneous (sc) injection was significantly prolonged compared to wt IL-4, which was removed from plasma within minutes (Figures 3A and 3B). To investigate the involvement of FcRn in SA-IL-4 accumulation in LN, we created a P573K point mutation in SA that inhibits FcRn binding (Figures 4A and 4B). 14。The SA (P573K) mutation decreased the amount of IL-4 in the LN compared to SA-IL-4 and reduced it to a level similar to that of wt IL-4 (Figure 2E). SA(P573K)-IL-4 had a longer plasma half-life compared to wt IL-4, probably due to an increase in molecular size, but a shorter half-life than SA-IL-4 due to impaired FcRn binding (Figure 4C). It was also confirmed that FcRn was expressed in various cells derived from the LN, liver, and lung, suggesting that the LN localization of SA-IL-4 was not due to specific FcRn expression in the LN. Taken together, these data suggested that FcRn binding was required for SA transport to the LN. The amount of SA(P573K)-IL-4 in the spleen was less than that of SA-IL-4 (Figure 2F).

[0141] The hypothesis was put forward that transcytosis by blood endothelial cells was inhibited by the SA (P573K) mutation, resulting in reduced transport of SA-IL-4 to the LN. To test this, the inventors performed a transcytosis assay using human endothelial cells cultured on cell culture inserts (Figure 2G). The SA (P573K) mutation significantly reduced the transcytosis of SA-IL-4 by endothelial cells. Importantly, mouse SA binds to human FcRn 15 and mouse IL-4 does not bind to human IL-4Rα 16 ; therefore, the inventors were able to test FcRn-mediated transcytosis in this assay. Taken together, it was shown that SA fusion to IL-4 increased the persistence of IL-4 in the LN and spleen, at least in part, through FcRn binding.

[0142] 3. SA-IL-4 treatment strongly suppressed the onset of EAE disease in preventive treatment Next, the inventors used MOG in complete Freund's adjuvant during the acute phase of EAE 35~55Myelin oligodendrocyte glycoprotein (MOG) antigen-induced EAEs, which are disease-induced by the administration of SA-IL-4, were treated with SA-IL-4 (Figures 5A-5D). SA-IL-4 was administered by sc or ip injection. Sc injection was chosen because it is clinically advantageous. IP injection was performed instead of IV injection because the tail becomes flaccid in mice that develop EAEs, making tail vein injection difficult. The inventors further compared the therapeutic effect of SA-IL-4 with that of FTY720. 4 There were no statistically significant differences in clinical scores between the SA-IL-4 (both intraperitoneal and sc injection) group and the FTY720 group, but sc injection of SA-IL-4 completely suppressed disease development in all mice (Figure 5A). Intraperitoneal SA-IL-4 injection and FTY720 prevented the development of EAE in 4 out of 7 mice and suppressed disease severity in the remaining mice. Wt IL-4 treatment did not suppress EAE clinical scores compared to the PBS treatment group, and all of those mice developed the disease. Using body weight change as a clinical indicator of health, mice treated with PBS and wt IL-4 were observed to lose a significant amount of body weight (Figure 5B). Mice that received sc injection of SA-IL-4 gained more body weight than all other groups and indicated better health. The intraperitoneal SA-IL-4 group gained body weight on average, but FTY720-treated mice maintained their body weight. Next, we analyzed spinal demyelination, the main morphological sign of EAE disease (Figure 5C). Mice injected with SA-IL-4 via sc injection showed no detectable demyelination, demonstrating prevention of spinal cord injury. All wt IL-4 treated mice showed demyelination. Next, we monitored the mice long-term up to day 24 and found that intracellular injection of SA-IL-4 inhibited the onset and progression of the disease (Figure 6). It was also confirmed that wt IL-4 did not show a significant therapeutic effect when administered subcutaneously (Figure 7). Furthermore, it was found that introducing the SA(P573K) mutation abolished the therapeutic effect of SA-IL-4, suggesting that FcRn binding of SA-IL-4 plays an important role in EAE treatment. These data suggest that SA fusion to IL-4 strongly improves the therapeutic effect of IL-4 on suppressing EAE disease.

[0143] 4. SA-IL-4 treatment suppressed immune cell infiltration into the spinal cord and induced an immunosuppressive environment in the dLN. Next, immune cells in the spinal cord and spinal cord inflow area lymph nodes (dLNs) after treatment were analyzed. Surprisingly, sc injection of SA-IL-4 significantly suppressed the infiltration of immune cells into the CNS; CD45 detected in the spinal cord. + Immune cells were present in very small numbers (Figure 9A). Consequently, Th17 cells (RORγt+ cells) in the spinal cord were almost undetectable in the sc SA-IL4 treated group (Figure 9B). Intravenous injection of SA-IL-4 suppressed immune cell infiltration in 4 out of 7 mice, which corresponded to the incidence of EAE disease in that group. Administration of FTY720 also suppressed immune cell infiltration into the spinal cord, as expected. wt IL-4 showed no effect on immune cell infiltration, including Th17 cells, into the spinal cord compared to PBS treatment.

[0144] The inventors then analyzed immune cells in lumbar dLN. SA-IL-4 increased granulocyte-like bone marrow-derived suppressor cells (G-MDSCs) but decreased monocyte-like MDSCs (M-MDSCs) (Figures 9C and 9D). CD4 in dLN + The frequency of Th17 cells within T cells was also reduced by SA-IL-4 treatment (both ip and sc) compared to FTY720 treatment (Figure 9E). FTY720 treatment tended to increase the frequency of Th17 cells in dLNs compared to the PBS group, presumably because FTY720 inhibits lymphocyte outflow from LNs. SA-IL-4 treatment reduced the frequency of M1 macrophages and increased M2 macrophages in dLNs (Figure 9F). wt IL-4 did not reduce the frequency of M1 macrophages but increased M2 macrophages. CD11b + Frequency of macrophages within cells (Figure 8A), and CD45 + The frequency of dendritic cells (DCs) within the cell (Figure 8B) was maintained. It has been reported that B cells promote the induction of EAEs by promoting the reactivation of T cells. 17SA-IL-4 (sc) reduced the frequency of B cells compared to both the PBS and FTY720 treatment groups (Figure 9H). In summary, these data demonstrate that SA-IL-4 treatment creates an immunosuppressive environment in the dLN, preventing the infiltration of immune cells into the spinal cord.

[0145] 5. SA-IL-4 treatment reduced IL-17-related cytokine and integrin expression in antigen-reactive CD4+ T cells. The inventors then analyzed the molecular mechanisms of reduced immune cell infiltration in the spinal cord and complete prevention of EAE disease by subcutaneous injection of SA-IL-4. 35~55 The number of reactive T cells was maintained in all treatment groups, suggesting that SA-IL-4 does not alter antigen recognition (Figure 10A). Therefore, it was hypothesized that SA-IL-4 alters T cell functionality. First, T cell migration capacity was tested. Expression levels of αLβ2 (LFA-1) and α4β1 (VLA-4) integrins, adhesion molecules important for lymphocyte migration. 18 It has been reported that it is reduced by IL-4. 19 SA-IL-4 treatment is MOG 35~55 Reactive CD4 + It significantly reduced αLβ2 integrin expression on T cells, but total CD8 + It was found that the expression of α4β1 integrin was not reduced in T cells (Figures 10B-10E). 35~55 Reactive CD4 + In T cells or total CD8 + There was no significant change in T cells. αLβ2 integrin is essential for Th17 cell infiltration into the spinal cord. 18 This suggests that downregulation of integrin expression is one of the mechanisms by which SA-IL-4 reduces lymphocyte migration to the spinal cord.

[0146] The inventors then investigated PD-1 expression in T cells and PD-L1 expression in MDSCs, since the interaction between PD-1 and PD-L1 suppresses T cell activation (Figures 10F-10K). 20Surprisingly, SA-IL-4, not wt IL-4, is the central memory CD4 + T cells and central memory CD8 + PD-1 expression was increased in both T cells (Figures 10F and 10G). Furthermore, SA-IL-4, rather than wt IL-4, increased PD-L1 expression levels and the frequency of PD-L1-expressing cells in both M-MDSCs and G-MDSCs (Figures 10H-10K). These data suggested that T cell suppression could be induced through MDSCs and the PD-1 / PD-L1 axis.

[0147] The inventors then analyzed the expression of Th17-related proteins. IL-23 is an important cytokine for Th17 functionality. IL-4 has been reported to bind to APC and suppress IL-23 and associated Th17 differentiation. 21 SA-IL-4 treatment is MOG 35~55 IL-23R in the reactive T cell repertoire + It was found to reduce the frequency of cells (Figure 10L). SA-IL-4 did not affect the frequency of Treg cells (Figure 10M).

[0148] Next, the inventors re-stimulated splenocytes collected on day 13 with MOG protein (Figures 10N-10P). ELISA of the culture supernatant revealed a decrease in IL-17A expression with SA-IL-4 treatment compared to wt IL-4 treatment, compared to PBS (Figure 10N). The reduction in IL-17 expression was due to MOG in the SA-IL-4 treatment group. 35~55 This indicates a decrease in the number and / or activity levels of reactive Th17 cells. Since IFNγ concentrations were maintained, it was suggested that SA-IL-4 had little effect on Th1 cells (Figure 10O). SA-IL-4 showed a tendency to decrease GM-CSF levels, which has been reported to be a pathogenic cytokine of EAEs. 22 (Figure 10P). Next, the inventors examined MOG in splenic cells. 35~55Cytokine expression in T cells was examined by flow cytometry after peptide restimulation (Figures 10Q and 10R). IL-13 was analyzed to determine whether IL-4 treatment shifted cells toward the Th2 lineage, as well as to identify pathogenic cytokines associated with EAEs, namely GM-CSF, IL-17, IFNγ, and TNFα. SA-IL-4 did not increase IL-13 production upon restimulation, suggesting no shift toward Th2 (Figure 10Q). SA-IL-4 showed CD4 + The frequency of cytokine-expressing cells within the T cell compartment was reduced. These results strongly suggest that Th17 cells in SA-IL-4 treated mice were less pathogenic and less virulent compared to other treatment groups (Figure 10R). In summary, these data indicate that SA-IL-4 modulates multiple immune cell responses in SLO and suppresses the development of EAE disease by preventing immune cell infiltration into the spinal cord, particularly T cell infiltration.

[0149] 6. SA-IL-4 restores paralysis caused by chronic EAE. To determine whether SA-IL-4 had a therapeutic effect in the chronic phase of EAE, the inventors designed an experiment involving the treatment of mice that had already reached the stage of severe paralysis. IL-4 ip injection was initiated on day 21 post-induction (Figures 11A and 11B). Surprisingly, SA-IL-4 demonstrated therapeutic efficacy even when administered at this late point, while wt IL-4 did not. SA-IL-4 treated mice gained weight, while wt IL-4 treated mice did not, indicating disease recovery. The inventors then tested the effect of SA-IL-4 in the chronic phase via subcutaneous injection and compared it to oral FTY720 treatment (Figures 11C and 11D). Mice treated with SA-IL-4 showed a tendency toward lower clinical scores compared to the PBS-treated group. Mice administered with SA-IL-4 gained weight compared to the other groups. FTY720-treated mice and wt IL-4-treated mice did not gain body weight compared to PBS-treated mice.

[0150] The inventors then tested immune cell infiltration into the spinal cord 34 days after induction by flow cytometry (Figures 11E-11G). SA-IL-4 and FTY720 treatments compared with PBS and wt IL-4 treatments in terms of CD4 + T cells and MOG 35~55 SA-IL-4 reduced the number of spinal cord infiltrating immune cells, including reactive Th17 cells, compared to other treatment groups. 35~55 Reactive CD4 + The number of IL-23R-expressing cells in T cells was reduced (Figure 11H). Finally, splenocytes were restimulated with MOG protein. ELISA of the culture supernatant revealed a decrease in IL-17A and GM-CSF concentrations in the SA-IL-4 treated group compared to PBS, but this was not the case after wt IL-4 and FTY720 treatment (Figures 11I and 11J). IL-4 expression was not altered by SA-IL-4 treatment (Figure 11K), and the absence of Th2 bias was confirmed. MOG 35~55 Flow cytometry analysis after peptide restimulation showed that SA-IL-4 was more effective in reducing CD4 compared to other treatments. + It was shown to reduce the frequency of cytokine-expressing cells within the T cell compartment (Figure 11L). In summary, these results indicate that SA-IL-4 treatment has a potent therapeutic effect in the chronic phase of EAE.

[0151] 7. SA-IL-4 did not show significant toxicity after systemic injection. To test whether SA-IL-4 exhibits any adverse effects, the inventors analyzed serum using a biochemical analyzer and blood using a hematologist. SA-IL-4 treatment did not increase organ damage markers or alter blood cell counts. Both SA-IL-4 and wt IL-4 induced splenomegaly. wt IL-4 induced pulmonary edema, indicated by increased pulmonary water content, but SA-IL-4 did not. These data suggest that SA-IL-4 is safe after systemic administration.

[0152] 8. SA-IL-33 and SA-IL-4 prevent the progression and onset of EAE disease. Myelin oligodendrocyte glycoprotein (MOG) 35-55 experimental autoimmune encephalomyelitis (EAE) mice were administered subcutaneously with (sc) phosphate-buffered saline (PBS), subcutaneously with SA-IL-33 (13-39 μg based on IL-33, as shown in Figure 12A), or SA-IL-4 (10 μg, based on IL-4), or orally with FTY720 1 mg / kg daily for 10 days starting 8 day post-immunization. n = 6-7 / group. Figure 12A shows the progression of clinical scores in all groups. SA-IL-33 and SA-IL-4 treatment significantly reduced disease progression and severity compared to the PBS-treated group. Figure 12B shows the progression of body weight in all groups.

[0153] B. Materials and Methods 1. Production and purification of recombinant proteins Sequences encoding mouse SA (amino acid numbers 25-608 of whole serum albumin), mouse IL-4, and the (GGGS)2 linker were synthesized (SEQ ID NO: X6) and subcloned into the mammalian expression vector pcDNA3.1(+) by Genscript. For further purification of the recombinant protein, a sequence encoding 6Hi was added to the C-terminus. The amino acid sequence of the protein is shown in Table 1. HEK-293F cells adapted to the suspension were routinely maintained in serum-free FreeStyle 293 expression medium (Gibco). On the day of transfection, cells were reduced to 1 × 10⁶ 6Fresh medium was inoculated at a density of 10 cells / ml. Plasmid DNA at 2 μg / ml, linear 25 kDa polyethyleneimine at 2 μg / ml (Polysciences), and OptiPRO SFM medium (final concentration 4%, Thermo Fisher) were added sequentially. The culture flask was agitated by orbital shaking at 135 rpm at 37°C in the presence of 5% CO2. Seven days after transfection, the cell medium was collected by centrifugation and filtered through a 0.22 μm filter. The medium was loaded onto a HisTrap HP 5 ml column (GE Healthcare) using AKTA pure 25 (GE Healthcare). After washing the column with wash buffer (20 mM NaH2PO4, 0.5 M NaCl, pH 8.0), the protein was eluted with a gradient of 500 mM imidazole (in 20 mM NaH2PO4, 0.5 M NaCl, pH 8.0). The protein was further purified by size exclusion chromatography using a HiLoad Superdex 200PG column (GE Healthcare) with PBS as the eluent. All purification steps were performed at 4°C. The expressed protein was confirmed to be over 90% pure by SDS-PAGE. The purified protein was tested for endotoxin via the HEK-Blue TLR4 receptor cell line, and the endotoxin level was confirmed to be less than 0.01 EU / mL. The protein concentration was determined from the absorbance at 280 nm using NanoDrop (Thermo Scientific).

[0154] 2. Mouse Eight-week-old C57BL / 6 female mice were obtained from Charles River Laboratories. The mice were housed in the University of Chicago's animal facility for at least one week prior to immunization. All experiments were conducted with the approval of the University of Chicago's Animal Care and Use Committee.

[0155] 3. Binding of proteins to spleen cells or LN-derived cells Single-cell suspensions were obtained by gently disrupting splenic or popliteal lymph nodes through a 70 μm cell strainer. Red blood cells were lysed with ACK lysis buffer for splenocytes (Quality Biological). Cells were counted and resuspended in RPMI-1640 supplemented with 10% FBS and 1% penicillin / streptomycin (all from Life Technologies). 1 × 10⁶ 5 Cells were seeded at 1 / well in a 96-well microplate and incubated on ice for 30 minutes with 2 μg / 100 μl SA and SA-IL4. After four washes with PBS, the cells were further incubated on ice for 20 minutes with rabbit monoclonal anti-mouse serum albumin antibody (clone EPR20195 abcam). After three washes with PBS, the cells were incubated on ice for 20 minutes with 1 μg / ml AlexaFluor 647-labeled anti-rabbit IgG antibody, anti-B220 antibody, anti-CD3 antibody, anti-CD4 antibody, anti-CD8 antibody, anti-CD11c antibody, anti-CD45 antibody, and anti-F4 / 80 antibody. The cells were analyzed by flow cytometry as described below.

[0156] 4. Analysis of STAT6 phosphorylation by flow cytometry EasySep Mouse CD4 + Using a T cell isolation kit (Stem Cell), mouse CD4 cells were extracted from the spleen of C57BL / 6 mice. + T cells were purified. Purified CD4 + T cells (10 6 Cells (100 cells / ml) were pre-coated with 5 μg / ml anti-CD3 antibody (clone 17A2, Bioxcell) and activated for 2 days in 6-well plates supplemented with soluble 2 μg / ml anti-CD28 antibody (clone 37.51, BioLegend). The culture medium was IMDM (Gibco) containing 10% heat-inactivated FBS, 1% penicillin / streptomycin, and 50 μM 2-mercaptoethanol (Sigma Aldrich). After 2 days of culture, activated CD4 +T cells were stimulated with 50 ng / ml recombinant mouse IL-2 (Peprotech) for 3 hours to induce IL-4Rα expression. After IL-2 stimulation, the cells were washed and allowed to stand in fresh medium for 3 hours. The cells were then transferred to a 96-well plate (50,000 cells / well). The indicated amount of wt IL-4 or SA-IL-4 was added to CD4. + T cells were exposed to 37°C for 15 minutes to induce STAT6 phosphorylation. The cells were immediately fixed with BD Phosflow Lyse / Fix buffer at 37°C for 10 minutes, and then permeabilized on ice with BD Phosflow Perm Buffer III for 30 minutes. Cells were stained with AlexaFluor 647 anti-pSTAT6 antibody (clone J71-773.58.11, BD), which recognizes Tyr641 phosphorylation. Staining was performed in the dark at room temperature (RT) for 1 hour. Cells were acquired using BD LSR, and data were analyzed using FlowJo (Treestar). + The mean fluorescence intensity (MFI) of the population was plotted against cytokine concentration. Dose-response curves were fitted using Prism (v8, GraphPad).

[0157] 5. Surface Plasmon Resonance (SPR) SPR measurements were performed using a Biacore X100 SPR system (GE Healthcare). Mouse FcRn recombinant protein (Acro Biosystems) was immobilized on a C1 chip (GE Healthcare) via amine coupling at approximately 200 resonance units (RUs) according to the manufacturer's instructions. SA-IL4 or SA (mouse SA, Sigma-Aldrich) was flowed at 30 μL / min in running buffer (0.01 M monobasic anhydrous sodium phosphate, pH 5.8, 0.15 M NaCl) at decreasing concentrations. The sensor chip was regenerated with PBS, pH 7.4 after each cycle. Specific binding of the SA fusion protein to FcRn was calculated by comparing it to a defunctionalized channel used as a reference. The experimental results were fitted to Langmuir binding kinetics using BIAevaluation software (GE Healthcare).

[0158] 6. Differentiation of Th17 cells in vitro under culture conditions Follow the manufacturer's instructions for the EasySep® Mouse Naive CD4. + Using the T Cell Isolation Kit (STEMCELL Technologies), naive CD4 cells were extracted from spleen cells. + T cells were isolated. 10 5 Cells were plated into 96-well plates and cultured for 3 days. As the Th17 induction medium, IMDM containing 5% FBS and 20 ng / mL rmIL-6 (peprotech), 10 ng / mL rmTGF-β (peprotech), 10 ng / mL rmIL-23 (peprotech), and 5 μg / mL anti-IFNγ (BioXcell) was used. The 96-well plates were coated with 2 μg / mL anti-CD3 (clone 2C11, BioXcell), and 2 μg / mL anti-CD28 (clone 37.51, BioXcell) was added to the medium. IL-17A concentration in the medium was measured using the IL-17 Ready-Set-Go! Mouse Uncoated ELISA kit (Invitrogen) according to the manufacturer's protocol. Data were analyzed using Prism software (v6, GraphPad).

[0159] 7. Plasma pharmacokinetics of proteins wt IL-4 or SA-IL-4 (equivalent to 10 μg of IL-4) was intravenously injected into female C57BL / 6 mice. Blood samples were collected in low-protein-binding tubes at 1 minute, 10 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, and 24 hours after injection. Plasma IL-4 concentrations were measured using the IL-4 Ready-Set-Go! Mouse Uncoated ELISA kit (Invitrogen) according to the manufacturer's protocol.

[0160] 8. Pharmacokinetics of proteins in lymph nodes, spleen, lungs, liver, and spinal cord wt IL-4, SA-IL-4, or SA(P573K)-IL-4 (equivalent to 40 μg of IL-4) was intravenously injected into healthy or EAE-induced (16 days post-induction; see induction protocol below) C57BL / 6 mice. Post-injection samples were collected from the lumbar and upper arm lymph nodes, spleen, liver, lungs, and spinal cord, and then homogenized using Lysing Matrix D and FastPrep-24 5G (MP Biomedical) at 5000 beats / min for 40 seconds in T-PER tissue protein extraction reagent (Thermo Scientific) containing cOmplete® proteinase inhibitor cocktail (Roche). After homogenization, samples were incubated overnight at 4°C. Samples were centrifuged (5000 g, 5 min), and total protein and IL-4 concentrations were analyzed using a BCA assay kit (Thermo Fisher) and an IL-4 Mouse Uncoated ELISA kit (Invitrogen), respectively. Simultaneously, cytokine levels in LN extracts were measured using either the Mouse Uncoated ELISA kit (Invitrogen) or the Ready-SET-Go! ELISA kit (eBioscience) according to the manufacturer's protocol.

[0161] 9. Fluorescence-based detection of IL-4 in LN To prepare fluorescently labeled wt IL-4 and SA-IL-4, the proteins were incubated in an 8-fold molar excess of DyLight 800 NHS ester (Thermo Fisher) at RT for 1 hour, and unreacted dye was removed using a Zebaspin spin column (Thermo Fisher) according to the manufacturer's instructions. 10 μg of DyLight 800-labeled wt IL-4 and SA-IL-4 with equivalent fluorescence were intravenously injected into naive C57BL / 6 mice. Four hours later, the iliac lining (LN) was imaged using a Xenogen IVIS Imaging System 100 (Xenogen) under the following conditions: f / stop: 2; optical filter excitation 745 nm; excitation 800 nm; exposure time: 5 seconds; small binning.

[0162] 10. Analysis of FcRn expression in cells from various organs C57BL / 6 mice were euthanized, and the lymph nodes in the arms, axillae, and inguinal regions were isolated along with the liver and lung lobes and digested. Briefly, the lung lobes were cut into small pieces with scissors and then digested in 5 ml of DMEM (Gibco) containing 5% FBS, 1 mg / ml collagenase IV (LS004188, Worthington Biochemical), 3.3 mg / ml collagenase D (11088866001, Sigma), 20 μg / ml DNAse I (LS006333, Worthington Biochemical) and 1.2 mM CaCl2 at 37°C for 1 hour on a shaker. Lymph nodes were punctured with a needle and digested in 750 μl of DMEM (Gibco) containing 5% FBS, 1 mg / ml collagenase IV, 40 μg / ml DNAse I and 1.2 mM CaCl2 at 37°C for 30 minutes with magnetic agitation. Subsequently, 750 μl of 3.3 mg / ml collagenase D, 40 μg / ml DNase I, and 1.2 mM CaCl2 were added to DMEM containing 5% FBS, and each sample was further digested for 15 minutes. The liver was cut into pieces and digested in 5 ml of DMEM containing 5% FBS, 1 mg / ml collagenase IV, 1 mg / ml collagenase D, 40 μg / ml DNase I, and 1.2 mM CaCl2 on a shaker at 37°C for 1 hour. After quenching the medium with 5 mM EDTA, a single-cell suspension was prepared using a 70 μm cell strainer (22-363-548, Fisher). The liver samples were centrifuged at 50 × g for 5 minutes to pellet the hepatocytes, which were then discarded. Finally, the erythrocytes were lysed in 1 ml of ACK buffer for 90 seconds and neutralized in 10 ml of DMEM medium containing 5% FBS. After digestion, the single-cell suspension was counted, and 1 to 2 million cells were stained. For antibodies against surface targets, staining was performed in PBS containing 2% FBS; for intracellular targets, the cells were stained in PBS containing 2% FBS with 0.5% saponin for 2 hours.The following anti-mouse antibodies were used for flow cytometry: CD45 APC-Cy7 (clone 30-F11, BioLegend), CD31 BUV395 (clone 390, BD Biosciences), gp38 PE-Cy7 (clone 8.1.1, BioLegend), FcRn (R and D systems, 1:50 dilution), F4 / 80 PE (clone BM8, BioLegend), CD11c BV421 (clone N418, BioLegend), CD11b BV786 (clone M1 / 70, BD Biosciences), and CD146 BV605 (clone ME-9F1, BD Biosciences). For FcRn staining, cells were stained with Alexa Fluor 647 donkey anti-goat IgG (Jackson ImmunoResearch, 1:400 dilution). Cell viability was determined using the immobilizable viability dye eFluor 455UV dye (65-0868-14, eBioscience).

[0163] 11. Transendothelial transport assay Human umbilical vein endothelial cells (HUVEC; Lonza) were maintained in EGM-2 medium (Lonza) and used up to passage number 9. 10 5Cells were seeded in 6.5 mm diameter, 0.4 μm pore inserts (Corning) pre-coated with 50 μg / ml rat tail collagen type I (Corning) in PBS, and incubated for 3 days to obtain a dense monolayer. The medium in the insert and bottom well was changed to EGM-2 without growth factors, and incubated for 2 hours. SA-IL-4 or SA(P573K)-IL-4 (10 μg / ml) was added to the insert (apical side), followed by incubation for 3 hours. Medium was collected from both the insert (apical side) and the bottom well (basal side), and IL-4 was measured by mouse IL-4 ELISA (R&D systems). Transendothelial transport was calculated as the ratio of IL-4 transported to the basal side of the insert to the total amount of IL-4 applied to the apical side. To confirm the integrity of the monolayer membrane, cells on the insert membrane were fixed in PBS 2% PFA for 15 minutes, permeabilized with TBS 1% Triton for 10 minutes, and stained with goat anti-human VE-Cadherin (R&D systems) in TBS 0.5% casein for 1 hour at RT, followed by staining with donkey anti-goat 594 (Invitrogen) secondary antibody in TBS 0.5% casein for 1 hour at RT. The membrane was mounted in DAPI-containing Prolong Gold Antifade Reagent (Invitrogen) and imaged with an Olympus IX2-DSU fluorescence microscope and a 60x objective lens. Z projections were generated from the fluorescence image stack using ImageJ and the Stack Focuser plugin.

[0164] 12. Immunofluorescence As described above, wt IL-4 and SA-IL-4 were fluorescently labeled with DyLight 594 NHS ester (Thermo Fisher). Mice were euthanized 1 hour after intravenous injection of fluorescently labeled IL-4 (40 μg for wt IL-4 and the same fluorescence dose for SA-IL-4). Mouse linolones (LNs) were collected, fixed overnight in 2% PFA in PBS, and washed with PBS. After overnight incubation in 30% sucrose solution, the LNs were embedded in an Optimum Cutting Temperature compound. Next, 5 μm frozen sections were cut using a cryostat. Next, sections were blocked in RT with 2% BSA in PBS and incubated in RT for 2 hours with the following primary antibodies: 10 μg / ml hamster anti-mouse CD3ε antibody (clone: ​​145-2C11, BioLegend) and 2.5 μg / ml rat anti-mouse PNAd (clone: ​​MECA-79, BioLegend). After washing with PBS-T, the tissue was stained in RT for 1 hour with the following fluorescently labeled secondary antibodies: Alexa Fluor 647 goat anti-hamster (1:400, Jackson ImmunoResearch) and Alexa Fluor 488 donkey anti-rat (1:400, Jackson ImmunoResearch). After washing the tissue three times, it was covered with Prolong gold antifade mountant containing 4',6-diamidino-2-phenylindole (DAPI; Thermo Fisher Scientific). For CD3 staining, an IX83 microscope (Olympus) was used for imaging at 10x magnification, and for PNAd staining, a Leica SP8 3D laser scanning confocal microscope was used at 20x magnification. Images were processed using ImageJ software (NIH).

[0165] 13. EAE Model MOG was administered to the dorsal flank of 9-12 week old C57BL / 6 juvenile female mice. 35~55Subcutaneous immunization with a complete Freund's adjuvant (CFA) emulsion was followed by intraperitoneal administration of pertussis toxin (PTX) in PBS, first on the day of immunization and then again on the following day. MOG 35~55 CFA emulsion and PTX were purchased from Hooke Laboratories. Following the initial immunization, the severity of EAE was monitored, and clinical scores were measured daily from day 8 post-immunization. Clinical scores were determined by AI, MN, or AS based on the Hooke Laboratories criteria (available on the World Wide Web at hookelabs.com / services / cro / eae / MouseEAEscoring.html) under blinded treatment group divisions. IL-4, SA-IL-4, and PBS were administered intraperitoneally or subcutaneously (into the left dorsal flank of mice; approximately 2 cm away from the emulsion injection site) in 100 μl of PBS every other day. FTY720 (1 mg / kg body weight) was administered orally daily.

[0166] 14. Histological features of the spinal cord Thoracic and lumbar vertebrae were harvested from EAE mice and dissected at the thoracolumbar junction. The tissue was fixed overnight in 2% PFA. After washing with PBS, the tissue was decalcified overnight using Decalcifier II (Leica Biosystem). Next, the tissue was embedded in paraffin. After paraffin embedding, the block was cut into 5 mm sections. After deparaffinization and rehydration, the tissue sections were treated with Target Recovery Solution (S1699, DAKO) and heated in a steamer at a temperature of over 95°C for 20 minutes. The tissue sections were incubated with anti-mouse aMBP (abcam ab40390) in a humidity chamber at RT for 1 hour. After washing with TBS, the tissue sections were incubated with biotinylated anti-rat IgG (10 mg / mL, Vector Laboratories) at RT for 30 minutes. Antigen-antibody binding was detected by the Elite Kit (PK-6100, Vector Laboratories) and the DAB (DAKO, K3468) system. The slides were imaged using EVOS FL Auto (Life Technologies).

[0167] 15. Flow cytometry EAE mice were treated with PBS, wt IL-4, or SA-IL-4 (equivalent to 10 μg of IL-4) every other day, starting 8 days after immunization. Spinal cord, spleen, and lumbar spinal cord tissue were collected 13, 17, or 34 days after immunization. Spinal cord tissue was digested at 37°C for 30 minutes in Dulbecco's modified Eagle medium (DMEM) supplemented with 2% FBS, 2 mg / mL collagenase D (Sigma-Aldrich), and 40 μg / mL DNase I (Roche). Single-cell suspensions were obtained by gently disrupting cells through a 70 μm cell strainer. For the spleen, erythrocytes were lysed with ACK lysis buffer (Quality Biological) and then stained with antibodies for flow cytometry.Antibodies were used against the following molecules: anti-mouse CD3ε (145-2C11, BD Biosciences), CD4 (RM4-5, BD Biosciences), anti-mouse CD8α (53-6.7, BD Biosciences), anti-mouse CD45 (30-F11, BD Biosciences), CD44 (IM7, BD Biosciences), CD62L (MEL-14, BD Biosciences), F4 / 80 (T45-2342, BD Biosciences), CD86 (GL1, BD Biosciences), CD206 (C068C2, BioLegend), Ly6G (1A8, BioLegend), Ly6C (HK1.4, BioLegend), CD11b (M1 / 70, BioLegend), CD11c (HL3, BD Biosciences), B220 (RA3-6B2, BioLegend), PD-1 (29F.1A12, BD Biosciences), PD-L1 (MIH7, BioLegend), IL-23R (O78-1208, BD Biosciences), integrin αL (HI111, BD Biosciences), integrin β2 (M18 / 2, BD Biosciences), integrin β1 (HMb1-1, BD Biosciences), integrin α4 (R1-2, BD Biosciences), GM-CSF (MP1-22E9, BD Biosciences), IL-13 (17-7222-80, eBioscience), IL-17 (TC11-18H10.1, BD Biosciences), IFNγ (XMG1.2, BD Biosciences), TNFα (eBioscience, MP6-XT22), and FoxP3 (MF23, BD Biosciences), RoRγt antibody (Q31-378, BD Biosciences). For the detection of MOG-recognizing T cells, T-Select I-Ab MOG. 35~55 Tetramer-PE (MBL International Corporation) or MOG 38~49Tetramer-PE (NIH Tetramer Core Facility) was used. MOG 38~49 Nonspecific binding of Tetramer was not considered. Identification of fixable live / dead cells was performed using Fixable Viability Dye eFluor 455 (eBioscience), Live / Dead Fixable Violet (eBioscience), or Live / Dead Fixable Aqua (eBioscience) according to the manufacturer's instructions. Staining was performed on ice for 20 minutes. For intracellular staining, cells were fixed at 4°C for 20 minutes using Cytofix / Cytoperm (BD Bioscience). For permeabilization, cells were stained in perm / wash buffer (BD Bioscience) at 4°C for 30 minutes. After the washing step, cells were stained with specific antibodies on ice for 20 minutes before fixation. All flow cytometry analyses were performed using a Fortessa (BD Biosciences) flow cytometer and analyzed using FlowJo software (Tree Star).

[0168] 16. Restimulation of splenic cells Single-cell suspensions were prepared from dLN and spleen. For cytokine production analysis, 5 × 10⁶ cells were used. 5 Individual lymphocytes and 2 × 10⁶ 6 Individual splenocytes were plated into a 96-well round-bottom plate. Cells were treated with 10 μM MOG. 35~55 Cells were stimulated with a peptide (Genscript). After 2 hours, GolgiPlug (brefelzin A) and GolgiStop (monensin) were added according to the manufacturer's protocol to block intracellular cytokine secretion. Four hours after the addition of GolgiPlug and GolgiStop, cells were stained for flow cytometry. For fixation, Cytofix / Cytoperm (BD Bioscience) was used at 4°C for 20 minutes. For permeabilization, Perma / wash buffer (BD Bioscience) was used, and cells were stained in Perma / wash buffer at 4°C for 30 minutes. For 3 days of restimulation, 2.5 × 10⁶ cells were used.5 Individual lymphocytes or 1 × 10⁶ 6 Individual splenocytes were plated into a 96-well round-bottom plate. Cells were treated with 10 μM MOG. 35~55 The mice were stimulated with either 100 μg / ml MOG protein (Anaspec) (for 6 hours of culture followed by flow cytometry) or 100 μg / ml MOG protein (for 72 hours of culture). After 72 hours, the supernatant was collected for ELISA analysis using the Ready-Set-Go! Kit (Invitrogen) or the LEGEND MAX mouse GM-CSF ELISA kit (BioLegend).

[0169] 17. Safety assessment of SA-IL-4 C57BL / 6 mice were intravenously injected with PBS, wt IL-4, or SA-IL-4 (equivalent to 10 μg of IL-4). Two days later, blood samples collected from the mice were analyzed using a COULTER Ac·T 5diff CP blood analyzer (Beckman Coulter) according to the manufacturer's instructions. Lungs and spleens were collected and weighed. Lung water content was determined by weighing before and after overnight freeze-drying using a FreeZone 6 Benchtop Freeze Dryer (Labconco). Serum samples collected from mice injected with PBS, wt IL-4, and SA-IL-4 were analyzed using a Biochemistry Analyzer (Alfa Wassermann Diagnostic Technologies) according to the manufacturer's instructions.

[0170] 18. Statistical analysis Statistical significance between experimental groups was determined using Prism software (v6 GraphPad). Using one-way ANOVA followed by Tukey's HSD post-hoc test, the variances between groups were found to be similar by the Brown-Forsyth test. For single comparisons, a two-tailed Student's t-test was used. A p-value less than 0.05 is considered statistically significant.

[0171] Example 2 - Enhanced lymph node transport of manipulated IL-10 suppresses rheumatoid arthritis in a mouse model. A. Results 1. Albumin-fused IL-10 binds to neonatal Fc receptors (FcRn) and APCs and accumulates in the LN. Wild-type (wt) mouse IL-10 and SA-fusion mouse IL-10 were expressed by recombination, and the molecular weight of the fusion protein was correspondingly higher than that of wt IL-10 as determined by SDS-PAGE; furthermore, the majority of SA-IL-10 existed as monomers under non-reducing conditions (Figure 13A). Surface plasmon resonance (SPR) analysis revealed that SA-IL-10 had micromolar K levels. d It was revealed that it binds to FcRn (Figure 13B). The binding ability of these proteins to splenocytes and unicellular cells isolated from popliteal LN was further evaluated by flow cytometry (Figure 13C). SA-fused IL-10 showed high binding to macrophages and dendritic cells in both splenocytes and LN-derived cells. After intravenous administration of fluorescently labeled SA-IL-10, a significantly higher fluorescence signal was observed in popliteal LN compared to wt IL-10 (Figure 13D). Interestingly, the higher fluorescence signal was located around high endothelial venules (HEVs) where antigen-presenting cells (APCs) were present (43).

[0172] 2. Albumin-fused IL-10 exhibits long-term blood circulation. SA is known to exhibit long-lasting circulation in vascular endothelial cells via FcRn-mediated regeneration (44,45). SA-IL-10 showed significantly longer-lasting blood circulation compared to wt IL-10 (Figure 14A). Figure 14B shows fluorescence signals from major organs of mice intravenously injected with DyLight800-labeled protein. Reflecting its long-lasting circulatory characteristics, SA-IL-10 showed higher signals in the heart, lungs, and spleen than wt IL-10.

[0173] 3. Albumin-fused IL-10 reduces immune activity after accumulating in the linden. SA-fused IL-10 showed micromolar affinity for FcRn (Figure 13B) and accumulated in the LN after intravenous injection (Figure 13D). Next, the amount of IL-10 in the LN and its pharmacokinetics were quantitatively evaluated (Figures 15A-15C). After intravenous injection of wt IL-10 or SA-IL-10 into CAIA mice, IL-10 concentrations in the LN at various time points were detected using ELISA. Four hours after injection, SA-IL-10 showed significantly higher IL-10 signals in the joint inflow area (popliteal fossa) LN and mesenteric LN, and relatively high signals in the non-inflow area (cervical) LN compared to wt IL-10 (Figure 15A). Mice injected with SA-IL-10 also showed a peak in IL-10 concentration around one hour after injection (Figure 15B) and a 5-10 times higher AUC in the LN than wt IL-10 (Figure 15C). These data suggested that SA-IL-10 accumulated in the LN immediately after intravenous injection and showed higher retention in the LN compared to wt IL-10.

[0174] High concentrations and AUC of SA-IL-10 in the LN may affect the phenotypes of various immune cells in the LN and other secondary lymphoid organs. Therefore, immune cell populations in the spleen and popliteal LN were analyzed by flow cytometry (Figure 16A-B). Intravenous injection of SA-IL-10 was observed in CD3 in the spleen. + T cells and CD45 + It induced a significant decrease in lymphocyte frequency (Figure 16A). Furthermore, CD86 + Dendritic cells, granulocyte-derived bone marrow suppressor cells (G-MDSCs), and CD86 + The frequency of M1 macrophages decreased, and CD206 +The frequency of M2 macrophages increased after injection of SA-IL-10 compared to PBS or wt IL-10. A similar trend was observed in popliteal LNs (Figure 16B). These data suggested that SA-IL-10 suppressed APC activity while simultaneously activating immunosuppressive M2 macrophages. Inactivation of APCs and high accumulation of IL-10 in LNs may suppress the activity of Th17 cells, which play a crucial role in the development of RA (46, 47). Th17-related cytokines (IL-17, IL-6, and TGF-β) were measured in LNs from the joint inflow area (popliteal) and non-inflow area (cervical) and compared with treatment with wt IL-10. IL-17 levels were statistically reduced in popliteal LNs after treatment with SA-IL-10, while levels in cervical LNs were not statistically reduced by any IL-10 variant (Figures 15D and 15E). Treatment with SA-IL-10 reduced the concentration of GM-CSF in the popliteal LN, but did not reduce wt IL-10 (Figure 15F).

[0175] 4. Albumin-fused IL-10 suppresses the onset of rheumatoid arthritis. The therapeutic effects of engineered IL-10 in a passive collagen antibody-induced arthritis (CAIA) model were evaluated (Figures 17A-17C). Intravenous injection of SA-IL-10 significantly suppressed the development of arthritis, while mice injected with PBS or wt IL-10 showed severe inflammation in the paws (Figure 17A). Histological analysis showed that intravenous administration of SA-IL-10 significantly suppressed the inflammatory response in the paws and mitigated joint pathology compared to PBS-treated mice (Figure 17B). The effects of administration routes on therapeutic efficacy were also investigated by comparing intravenous, topical (sole), and subcutaneous (distal and mid-dorsal) administration (Figure 17C). Surprisingly, SA-IL-10 showed very high inhibitory effects against CAIA via all administration routes tested (Figure 17C).

[0176] As a second arthritis model, an active collagen-induced arthritis (CIA) model was used to evaluate SA-IL-10 against RA treatment. A single injection of SA-IL-10 into CIA mice induced a significant suppression of arthritis establishment compared to PBS (Figure 18A). Most mice treated with PBS showed severe inflammation in the paws, as indicated by histological features and scores (Figure 18B). In contrast, mice treated with SA-IL-10 showed paw conditions nearly identical to naive mice, and most mice had histological scores of 1 or less (Figure 18B). Treatment with anti-TNF-α antibody (αTNF-α), a mouse model of an antibody drug clinically used for RA treatment, also suppressed the increase in clinical scores compared to PBS-treated CIA mice (Figure 18C), but joint histological features and scores did not recover even after two injections of αTNF-α (Figure 18D). In summary, these results indicated that SA-IL-10, administered topically, intravenously, and even subcutaneously, had a high anti-inflammatory effect, and that its therapeutic effect was equivalent to or greater than that of αTNF-α treatment.

[0177] 5. Albumin-fused IL-10 suppresses inflammatory responses in the feet. Next, the immune cell population in the hind paw was analyzed using flow cytometry (Figure 19A). After intravenous injection of SA-IL-10, CD45 + The frequency of immune cells was significantly reduced compared to the group treated with PBS or wt IL-10. CD45 + Within the cell, the frequencies of B cells and dendritic cells became comparable to those in healthy mice, and CD11b + The number of cells also decreased significantly to the level of healthy mice. CD11b + Within the cells, the G-MDSC population decreased, and the frequency of macrophages recovered to the level of healthy mice. Furthermore, CD206 + The frequency of M2 macrophages was significantly increased by SA-IL-10 injection compared to PBS or wt IL-10 treatment, even exceeding the frequency in healthy mice. Analysis of T cell populations in the paw revealed that SA-IL-10 increased CD4 in CAIA mice. +Cells and Foxp3 + It was revealed that SA-IL-10 suppressed changes in Tregs (Figure 20A). Furthermore, SA-IL-10 suppressed the decrease in Treg frequency in the blood (Figure 20B). Reflecting these changes in the immune cell population, various inflammatory cytokines in the paws were significantly reduced by intravenous injection of SA-IL-10, and their levels were comparable to those in healthy mice (Figure 19B).

[0178] 6. Albumin-fused IL-10 does not show toxicity after injection. Finally, a safety assessment was conducted to determine if engineered IL-10 exhibited any adverse effects. Representative blood parameters and spleen weight, measured by a blood analyzer, did not show significant changes between treatment groups (Figure 21A). Various biochemical markers in serum were also examined using a biochemical analyzer (Figure 21B). In the engineered IL-10 treatment group, most markers, with the exception of amylase (which did not increase but rather decreased slightly), showed similar levels compared to the PBS treatment group, indicating that engineered IL-10 possesses high safety after systemic administration. Furthermore, while FTY720, a clinically approved drug for treating multiple sclerosis (fingolimod), showed some immunosuppressive effects under experimental conditions (though not significant), SA-IL-10 injection did not affect anti-OVA IgG titers, similar to the results observed with αTNF-α (Figures 22A and 22B). These results indicate that engineered IL-10 possesses high safety after systemic administration.

[0179] B. Materials and Methods 1. Production and purification of recombinant proteins Mouse serum albumin without propeptides (amino acid numbers 25-608 of whole serum albumin), mouse IL-10, and sequences encoding the (GGGS)2 linker were synthesized (SEQ ID NO: X15) and subcloned into the mammalian expression vector pcDNA3.1(+) by Genscript. For further purification of the recombinant protein, a sequence encoding 6Hi was added to the C-terminus. HEK-293F cells adapted to the suspension were routinely maintained in serum-free FreeStyle 293 expression medium (Gibco). On the day of transfection, cells were 1 × 10⁶ 6 Fresh medium was inoculated at a density of 100 cells / mL. Plasmid DNA at 2 μg / mL, linear 25 kDa polyethyleneimine at 2 μg / mL (Polysciences), and OptiPRO SFM medium (final concentration 4%, Thermo Fisher) were added sequentially. The culture flask was agitated by orbital shaking at 135 rpm at 37°C in the presence of 5% CO2. Seven days after transfection, the cell medium was collected by centrifugation and filtered through a 0.22 μm filter. The medium was loaded onto a HisTrap HP 5 ml column (GE Healthcare) using AKTA pure 25 (GE Healthcare). After washing the column with wash buffer (20 mM NaH2PO4, 0.5 M NaCl, pH 8.0), the protein was eluted with a gradient of 500 mM imidazole (in 20 mM NaH2PO4, 0.5 M NaCl, pH 8.0). The protein was further purified by size exclusion chromatography using a HiLoad Superdex 200PG column (GE Healthcare) with PBS as the eluent. All purification steps were performed at 4°C. Protein expression was confirmed to be over 90% pure by SDS-PAGE. The purified protein was tested for endotoxin via the HEK-Blue TLR4 receptor cell line, and the endotoxin level was confirmed to be less than 0.01 EU / mL. Protein concentration was determined from absorbance at 280 nm using NanoDrop (Thermo Scientific).

[0180] 2. Detection of SA-IL-10 binding to FcRn SPR measurements were performed using a Biacore X100 instrument. Recombinant mouse FcRn (Acro Biosystems) were immobilized on a C1 chip (GE Healthcare) via amine coupling for approximately 200 RU, according to the manufacturer's instructions. SA-IL-10 was flowed at room temperature at 30 μL / min in running buffer (0.01 M monobasic anhydrous sodium phosphate, pH 5.8, 0.15 M NaCl) while decreasing the concentration. The sensor chip was regenerated with PBS, pH 7.4 after each cycle. Specific binding of the SA fusion protein to FcRn was calculated by comparing it with a defunctionalized channel used as a reference. The K of SA-IL-10 was determined by fitting a 1:1 Langmuir binding model to the data using BIAevaluation software (GE Healthcare). d The value was determined.

[0181] 3. Mouse Seven-week-old BALB / c female mice and eight-week-old DBA / 1J male mice were obtained from the Jackson Laboratory. The experiment was conducted with the approval of the University of Chicago's Institutional Animal Care and Use Committee.

[0182] 4. Binding of proteins to spleen cells or LN-derived cells Single-cell suspensions were obtained by gently disrupting the spleen and popliteal LN through a 70 μm cell strainer. Red blood cells were lysed with ACK lysis buffer for splenocytes (Quality Biological). Cells were counted and resuspended in RPMI-1640 supplemented with 10% FBS and 1% penicillin / streptomycin (all from Life Technologies). 1 × 10⁶ 5Cells were seeded at 1 / well in a 96-well microplate and incubated on ice for 30 minutes with 2 μg / 100 μL of SA or SA-IL-10. After four washes with PBS, the cells were further incubated on ice for 20 minutes with anti-mouse albumin antibody (abcam). After three washes with PBS, cells were incubated on ice for 20 minutes with 1 μg / mL AlexaFluor 647-labeled anti-rabbit IgG (Jackson ImmunoResearch), anti-B220 (RA3-6B2, BioLegend), anti-CD3e (145-2C11, BD Biosciences), anti-CD4 (RM4-5, BD Biosciences), anti-CD8 (53-6.7, BD Biosciences), anti-CD11c (HL3, BD Biosciences), anti-CD45 (30-F11, BD Biosciences), and anti-F4 / 80 (T45-2342, BD Biosciences) antibodies. Cells were analyzed by flow cytometry as described below.

[0183] 5. Plasma pharmacokinetics of proteins IL-10 or SA-IL-10 (equivalent to 35 μg of IL-10) was intravenously injected into female BALB / c mice. Blood samples were collected in low-protein-binding tubes at 1, 5, 10, and 30 minutes post-injection, as well as at 1, 4, 8, and 24 hours, and then incubated overnight at 4°C. Serum IL-10 concentrations were measured using the IL-10 Mouse Uncoated ELISA kit (Invitrogen) according to the manufacturer's protocol. Exponential biphasic decay (Y = Ae -αt + Be -βt The half-life was calculated using fitting. Fast clearance half-life, t 1 / 2,α ; Slow clearance half-life, t 1 / 2,β The data was analyzed using Prism software (v8, GraphPad).

[0184] 6. CAIA Model Arthritis was induced in female BALB / c mice by intraperitoneal injection of an anti-collagen antibody cocktail (1.0 mg / mouse, Chondrex) on day 0, followed by intraperitoneal injection of LPS (25 μg / mouse, Chondrex) on day 3. On day 3, prior to LPS injection, mice were injected intravenously, subcutaneously (mid-dorsal), or via the sole of the foot with PBS, wt IL-10, SA-IL-10 (each equivalent to 43.5 μg of IL-10), or 200 μg of rat anti-mouse TNF-α antibody (clone XT3.11, Bio X Cell). Joint swelling was scored daily according to the manufacturer's protocol (Chondrex). On the final day of scoring, the hind feet were fixed in 10% neutral formalin (Sigma-Aldrich), decalcified in Decalcifer II (Leica), and then subjected to histological analysis. Paraffin-embedded feet were sliced ​​to a thickness of 5 μm and stained with H&E. Images were scanned with a Pannoramic digital slide scanner and analyzed using Pannoramic Viewer software. The severity of bone resorption and synovial hyperplasia in the arthritis model was scored on a three-point scale (0-2) following previously reported criteria with slight modifications: 0, normal to minimal infiltration of the pannus in the subchondral bone of the cartilage and marginal zone; 1, mild to moderate infiltration of the marginal zone with mild cortical and medullary bone destruction; 2, severe infiltration associated with complete or near-complete destruction of the joint structure. The scores for both hind feet were totaled for each mouse (total score per mouse, 0-4). Histopathological analysis was performed blinded.

[0185] 7. CIA Model Male DBA / 1J mice (8 weeks old) were immunized by subcutaneous injection of bovine collagen / complete Freund's adjuvant (CFA) emulsion (Hooke Kit, Hooke Laboratories) at the base of the tail. Three weeks later, a replenishment antigen injection of bovine collagen / incomplete Freund's adjuvant (IFA) emulsion (Hooke Kit, Hooke Laboratories) was administered. After the replenishment antigen injection, mice were examined daily, and joint swelling was scored according to the manufacturer's protocol (Hooke Laboratories). When a total score of 2-4 was achieved (defined as day 0), mice were intravenously injected with PBS, SA-IL-10 (each equivalent to 43.5 μg of IL-10), or 200 μg of rat anti-mouse TNF-α antibody (clone XT3.11, Bio X Cell). On the final day of scoring, the hind legs were collected and histological analysis was performed as described above.

[0186] 8. In vivo biodistribution studies To prepare the fluorescently labeled protein, wt IL-10 and SA-IL-10 were incubated in an 8-fold molar excess of DyLight 800 NHS ester (Thermo Fisher) at room temperature for 1 hour, and unreacted dyes were removed using a Zebaspin spin column (Thermo Fisher) according to the manufacturer's instructions. BALB / c mice were intraperitoneally injected with an anti-collagen antibody cocktail (1.0 mg / mouse) on day 0, followed by an injection of 10 μg of LPS into the right hind leg on day 3. The following day, 20 μg of DyLight 800-labeled protein was intravenously injected. Four hours later, organs taken from the disease model were imaged using a Xenogen IVIS Imaging System 100 (Xenogen) under the following conditions: f / stop: 2; optical filter excitation 745 nm; excitation 800 nm; exposure time: 5 seconds; small binning. To normalize the fluorescence signal from each organ, the weight of each organ was measured.

[0187] 9. LN microscopy BALB / c mice were intravenously injected with equimolar amounts of DyLight594-labeled wt IL-10 (43.5 μg) or SA-IL-10 labeled with dye. 24 hours after injection, popliteal lymph nodes (LNs) were harvested and frozen in dry ice with an optimal cutting temperature (OCT) compound. Tissue slices (10 μm) were obtained from the frozen sections. The tissue was fixed with 2% paraformaldehyde in PBS at room temperature for 15 minutes. After washing with PBS-T, the tissue was blocked with 2% BSA in PBS-T at room temperature for 1 hour. The tissue was stained with anti-mouse CD3 antibody (1:100, 145-2C11, BioLegend) or anti-mouse peripheral lymph node addressin (PNAd) antibody (1:200, MECA79, BioLegend) and Alexa Fluor 488 donkey anti-rat antibody (1:400, Jackson ImmunoResearch). After washing the tissue three times, it was covered with Prolong gold antifade mountant containing 4',6-diamidino-2-phenylindole (DAPI; Thermo Fisher Scientific). For CD3 staining, an IX83 microscope (Olympus) was used for imaging at 10x magnification, and for PNAd staining, a Leica SP8 3D laser scanning confocal microscope was used at 20x magnification. Images were processed using ImageJ software (NIH).

[0188] 10. LN Pharmacokinetics CAIA mice were intravenously injected with wt IL-10 or SA-IL-10 (each equivalent to 35 μg of IL-10). LNs from the popliteal fossa, mesentery, and neck were collected 30 minutes after injection, and at 1, 4, 8, and 24 hours. These were then homogenized using Lysing Matrix D and FastPrep-24 5G (MP Biomedical) at 5,000 beats / min for 40 seconds in T-PER tissue protein extraction reagent (Thermo Scientific) containing cOmplete® proteinase inhibitor cocktail (Roche). After homogenization, the samples were incubated overnight at 4°C. The samples were centrifuged (5,000 g, 5 min), and total protein and IL-10 concentrations were analyzed using a BCA assay kit (Thermo Fisher) and an IL-10 Mouse Uncoated ELISA kit (Invitrogen), respectively. Simultaneously, cytokine levels in LN extracts were measured using either the Mouse Uncoated ELISA kit (Invitrogen) or the Ready-SET-Go! ELISA kit (eBioscience) according to the manufacturer's protocol. To detect GM-CSF, CAIA mice were intravenously injected twice at 3-day intervals with either wt IL-10 or SA-IL-10 (equivalent to 35 μg of IL-10 each). Popliteal LN was collected the day after the last injection for GM-CSF detection.

[0189] 11. Flow cytometry CAIA mice were intravenously injected with PBS, wt IL-10, or SA-IL-10 (each equivalent to 43.5 μg of IL-10). After 8 days, blood and hind feet were collected. Red blood cells in the blood were lysed with ACK lysis buffer (Quality Biological) and then stained with antibodies for flow cytometry. The feet were digested at 37°C for 60 minutes in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 2% FBS, 2 mg / mL collagenase D, and 40 μg / mL DNase I (Roche). Single-cell suspensions were obtained by gently disrupting the cells through a 70 μm cell strainer. Antibodies against the following molecules were used: anti-mouse CD3 (145-2C11, BD Biosciences), CD4 (RM4-5, BD Biosciences), anti-mouse CD8α (53-6.7, BD Biosciences), anti-mouse CD25 (PC61, BD Biosciences), anti-mouse CD45 (30-F11, BD Biosciences), CD44 (IM7, BD Biosciences), CD62L (MEL-14, BD Biosciences), PD-1 (29F.1A12, BD Biosciences), NK1.1 (PK136, BD Biosciences), Foxp3 (MF23, BD Biosciences), F4 / 80 (T45-2342, BD Biosciences), MHC II (M5 / 114.15.2, BioLegend), CD206 (C068C2, The following stains were used: BioLegend, Ly6G (1A8, BioLegend), Ly6C (HK1.4, BioLegend), CD11b (M1 / 70, BioLegend), CD11c (HL3, BD Biosciences), and B220 (RA3-6B2, BioLegend). Identification of fixable live / dead cells was performed using Fixable Viability Dye eFluor 455 (eBioscience) according to the manufacturer's instructions. Unless otherwise instructed, staining was performed on ice for 20 minutes, and intracellular staining was performed using the Foxp3 staining kit according to the manufacturer's instructions (BioLegend).After the washing stage, cells were stained with specific antibodies on ice for 20 minutes before fixation. All flow cytometry analyses were performed using a Fortessa (BD Biosciences) flow cytometer and analyzed using FlowJo software (Tree Star).

[0190] 12. Safety Evaluation BALB / c mice were intravenously injected with PBS, wt IL-10, or SA-IL-10 (each equivalent to 43.5 μg of IL-10). Two days after injection, blood samples collected from the mice were analyzed using a COULTER Ac·T 5diff CP blood analyzer (Beckman Coulter) according to the manufacturer's instructions. Spleen weight was also measured. Serum samples collected from protein-injected mice were analyzed using a Biochemistry Analyzer (Alfa Wassermann Diagnostic Technologies) according to the manufacturer's instructions. For general immunosuppression assessment, PBS and 100 μg of anti-TNF-α were intraperitoneally injected every two days for 14 days, starting from day 0. FTY720 (1 mg / kg body weight) was administered orally daily. SA-IL-10 (equivalent to 43.5 μg of IL-10) was subcutaneously injected on days 0 and 8. On day 5, the fore hocks of C57BL / 6 mice were subcutaneously exposed to 10 μg of endotoxin-free ovalbumin, 50 μg of alum, and 5 μg of monophosphoryl lipid A (MPLA). On days 13 and 19, the mice were induced to bleed, and their plasma was analyzed for total anti-ovalbumin IgG titers.

[0191] 13. Statistical analysis Statistical significance between experimental groups was determined using Prism software (v8, GraphPad). Using one-way ANOVA followed by Tukey's HSD post-hoc test, the variances between groups were found to be similar by the Brown-Forsyth test. For nonparametric data, the Kruskal-Wallis test followed by Dunn's multiple comparison test was used. For single comparisons, the two-tailed Student's t-test was used. (Symbol) * , ** ,*** and **** showed P-values of less than 0.05, 0.01, 0.001, and 0.0001, respectively; ns, not significant.

[0192] Example 3 - Albumin-fused IL-35 suppressed the onset of arthritis Collagen-induced arthritis (CAIA) was induced by passive immunization with anti-collagen antibody, followed by intraperitoneal injection of LPS. On the day of LPS injection, PBS or human IL-35-mouse SA fusion protein (equivalent to 54 μg of wt IL-35) was intravenously injected into arthritic mice. The arthritis scores of each group are shown in Fig. 23. SA-IL-35 significantly reduced the disease progression. The arthritis scores represent the mean + SEM from 7 mice. Statistical analysis was performed using a two-sided Student's t-test, and the p-value was less than 0.01 between PBS and IL-35-MSA.

[0193] Example 4 - SA-IL-4 promotes wound healing through angiogenesis Full-thickness back skin wounds were created in db / db type 2 diabetic mice. Seven days later, the wound closure and the number of blood vessels in the granulation tissue area were evaluated by histomorphometry. Wounds in db / db diabetic mice were injected every other day from day 0 to day 6. The treatment groups were subcutaneous (s.c.) phosphate-buffered saline (PBS), subcutaneous 10 μg of wild-type (WT) IL-4, or SA-IL-4 (equimolar: 10 μg, based on IL-4). The graph data are mean ± standard error of the mean (SEM), and statistical comparisons were performed using one-way analysis of variance. * P <.05; ** P <.01.

[0194] Figure 24A shows the results of wound closure for all treatment groups. SA-IL-4 treatment significantly improved wound closure. Figure 24B shows the results for the number of blood vessels in the granulation tissue. SA-IL-4 treatment significantly improved the number of blood vessels, indicating an increase in angiogenesis.

[0195] Example 5 - SA-IL-27 reduces plasma IFNγ concentration after cancer immunotherapy in tumor-bearing mice Plasma IFNγ concentration indicates serum cytokine release syndrome after cancer immunotherapy. 7 days before cytokine treatment, 5 × 10 5 Mice were inoculated with B16F10 melanoma cells and treated once intravenously with CBD-IL-12 (25 μg, IL-12-based) and SA-IL-27 (10 μg, IL-27-based) on day 0. Plasma IFNγ levels on day 2 are shown in Figure 25. SA-IL-27 significantly reduced IFNγ levels compared to mice treated with CBD-IL-12 alone. * =P<0.05 CBD-IL-12: Collagen-binding domain fused IL-12 recombinant protein.

[0196] Example 6 - Use of albumin-fusion cytokines for wound healing As of 2020, 34.2 million people were diagnosed with diabetes mellitus, and it is one of the leading causes of death in the United States. Beyond mortality, there are many complications often associated with diabetes, one of which is impaired healing. This most commonly manifests as lower extremity wounds (i.e., foot ulcers). These ulcers can lead to amputation, a procedure in which diabetic patients account for 85% of the patient population. Wound healing is a problem that has been extensively researched, particularly in the context of diabetes, but there is currently no satisfactory solution. Current clinical treatments are severely limited to management strategies such as wound unloading and surgical debridement, as opposed to long-term solutions. The few available treatments have shown little improvement in patient outcomes. For example, current research focuses on the use of growth factors such as vascular endothelial growth factor A (VEGF-A), a factor associated with angiogenesis. While VEGF-A treatment has shown some improvement in re-epithelialization of chronic wounds, VEGF-A has been associated with undesirable side effects, such as persistent vascular leakage leading to hypotension. Genentech has developed terbermine, a topical recombinant VEGF-A treatment, but it has not yet received clinical approval.

[0197] Our approach to improving chronic wound healing focuses on immunomodulation in the diabetic wound environment. Many immune cell populations are involved in regulating wound closure, and in diabetic wounds, many of these immune cells are dysregulated. Monocytes and macrophages play crucial roles in wound healing in normal skin, but are impaired in several ways in the diabetic wound environment. Diabetic wounds have demonstrated an increase in the numbers of both monocytes and macrophages, as well as a failure of pro-inflammatory macrophages to transition to anti-inflammatory macrophages. This failure of pro-inflammatory to anti-inflammatory macrophage transition is a target for treatment with engineered cytokines. Interleukin-4 (IL-4) and interleukin-10 (IL-10) are both widely considered anti-inflammatory cytokines. Both IL-4 and IL-10 are central to the pathway that induces macrophage polarization from pro-inflammatory to anti-inflammatory phenotypes. The inventors modified both IL-4 and IL-10 through engineering changes to increase their circulating half-life and local wound retention, respectively. These novel engineered cytokine constructs demonstrated the ability to improve wound closure through re-epithelialization in a diabetic mouse model.

[0198] A. Method Male db / db mice aged 8-10 weeks from Jackson Laboratories were used. The backs of each mouse were broadly shaved and washed with 70% ethanol and iodine. Four symmetrical full-thickness wounds were made in the shaved area using a 6 mm diameter biopsy punch. Immediately afterward, the wounds were bandaged with non-adhesive Adaptic® dressings and secured with Tegaderm® seals and tissue adhesive. On day 4, cytokine therapy was administered, with subcutaneous administration of 40 μg of IL-10 molar equivalents of MSA-IL-10 and topical application of 200 μg of IL-4 molar equivalents of A3-MSA-IL-4 in a hyaluronic acid hydrogel carrier. The mouse wounds in Figure 26 were kept open with splints to prevent contraction. On day 11, the mice were euthanized and the wounds were excised for histological analysis.

[0199] B. Results Following wound excision and histological analysis, the inventors were able to confirm that the manipulated cytokine therapy resulted in a significant increase in wound closure, defined by re-epithelialization, compared to the PBS control and, in the case of A3-MSA-IL-4, the hyaluronic acid-only control. These results are shown in Figures 26-27.

[0200] Example 7: Use of albumin-fusion cytokines for the treatment of scleroderma Scleroderma is a chronic autoimmune disease in which skin tissue is replaced by thickened tissue containing excess collagen. Cytokine therapy for scleroderma has not been previously tested. The inventors attempted to evaluate whether SA-mouse IL-10 and SA-human IL-35 fusion proteins would have any effect. First, bleomycin was dissolved in PBS at a concentration of 1 mg / ml. Then, mice were anesthetized with isoflurane. Hair removal lotion was applied to the backs of the mice, and after waiting 30-60 seconds, the lotion and hair were removed with tissue paper. Next, bleomycin (100 μg = 100 μl) was subcutaneously injected into one site on the back of the mice five days a week for two weeks using a 27-gauge needle. On days 7 and 10, the inventors sc-treated the mice with SA-IL-10 (40 μg / injection) or 20 μg / injection. As a control, 100 μl of PBS was sc-injected using a 27-gauge needle. Finally, a circle was drawn with a marking pen to indicate the injection site. The graph in Figure 28 shows the clinical scores for scleroderma, measured histologically and blindly. Mean + SD.

[0201] These studies demonstrate that SA IL-33 significantly reduces toxicity compared to wild-type IL-33. Mice were induced to develop an EAE on day 0, and subcutaneous administration of wild-type or equimolar SA IL-33 began on day 8, every other day. On day 11, after two treatments, mice treated with wild-type IL-33 experienced severe toxicity and death (Figure 30A). No deaths were observed in mice treated with SA-IL-33, and the mice continued to receive SA-IL-33, being protected from developing EAE (Figure 30B). In healthy mice, a total of three doses administered every other day had little effect on serum IgE production compared to wild-type IL-33 (Figure 31A). After five doses, mice treated with SA-IL-33 had serum IgE levels comparable to those treated with wild-type IL-33 (Figure 31B). To determine whether three doses of medication were sufficient to protect against EAE, mice were induced to develop EAE and treated with SA-IL-33 every other day, starting on day 8 after induction. Compared to eight doses, three doses were sufficient to protect against EAE, suggesting that a three-dose regimen is sufficient to protect against the disease (Figure 31C-D).

[0202] Example 8: Management of IL-4 and IL-33 toxicity by SA fusion and dose selection IL-4 is a multifaceted cytokine and has been used as a potential therapeutic agent in cancer patients with malignant melanoma and metastatic renal cell carcinoma. These studies have shown a significant number of patients with grade 3 or 4 toxicity requiring discontinuation of treatment. Dosage strategies in these studies included daily or three-times-weekly administration of low-dose subcutaneous wild-type IL-4. Similarly, mice constitutively overexpressing IL-4 exhibit toxicity associated with B-cell hyperactivation and excessive IgE response. We demonstrate that more frequent dosing, i.e., three times-weekly dosing, leads to higher levels of side effects, including weight loss, B-cell activation, and serum IgE levels, compared to less frequent (once-weekly) dosing (Figures 29A-C). Importantly, we observe that a single low dose of SA-IL-4 is sufficient to drive upregulation of the CD206 mannose receptor, an indicator of efficacy at this dosing level (Figure 29D).

[0203] These studies similarly demonstrate that SA IL-33 significantly reduces toxicity compared to wild-type IL-33. Mice were induced to develop an EAE on day 0 and began receiving subcutaneous administration of wild-type or equimolar SA IL-33 every other day starting on day 8. On day 11, after two treatments, mice treated with wild-type IL-33 experienced severe toxicity and death (Figure 30A). No deaths were observed in mice treated with SA-IL-33, and the mice continued to receive SA-IL-33 and were protected from developing EAE (Figure 30B). In healthy mice, a total of three doses administered every other day had little effect on serum IgE production compared to wild-type IL-33 (Figure 31A). After five doses, mice treated with SA-IL-33 had serum IgE levels comparable to those treated with wild-type IL-33 (Figure 31B). To determine whether three doses of medication were sufficient to protect against EAE, mice were induced to develop EAE and treated with SA-IL-33 every other day, starting on day 8 after induction. Compared to eight doses, three doses were sufficient to protect against EAE, suggesting that a three-dose regimen is sufficient to protect against the disease (Figure 31C-D).

[0204] Example 9: SA-IL-33 for treating multiple sclerosis (EAE) Figures 32-42 further illustrate aspects of the SA-IL-33 experiment in mice.

[0205] method Production and purification of recombinant SA IL-33 Sequences encoding mouse SA IL-33 (amino acid numbers 25-608 of whole serum albumin) without propeptides, mouse IL-33, and the (GGGS)2 linker were synthesized and subcloned into the mammalian expression vector pcDNA3.1(+) by Genscript. For further purification of the recombinant protein, a sequence encoding 6-His was added to the carboxyl terminus. HEK-293F cells adapted to suspension were routinely maintained in serum-free FreeStyle 293 expression medium (Gibco). On the day of transfection, cells were inoculated into fresh medium at a density of 1 × 10⁶ cells per ml; 2 μg ml of plasmid DNA, 2 μg ml of linear 25 kDa polyethyleneimine (Polysciences), and OptiPRO SFM medium (final concentration 4%; Thermo Fisher) were added sequentially. The culture flask was stirred by orbital shaking at 135 rpm at 37°C in the presence of 5% CO₂. Seven days after transfection, the cell medium was collected by centrifugation and filtered through a 0.22 μm filter. The medium was loaded onto a HisTrap HP 5 ml column (GE Healthcare) using AKTA pure 25 (GE Healthcare). After washing the column with wash buffer (20 mM NaH2PO4 and 0.5 M NaCl, pH 8.0), the protein was eluted using a gradient of 500 mM imidazole (in wash buffer). The protein was further purified by size exclusion chromatography using a HiLoad Superdex 200PG column (GE Healthcare) with PBS as the eluent. All purification steps were performed at 4°C. The expressed protein was confirmed to be over 90% pure by SDS-PAGE. The purified protein was tested for endotoxin via the HEK-Blue TLR4 receptor cell line, and the endotoxin level was confirmed to be less than 0.01 EU mL-1. Protein concentration was determined from absorbance at 280 nm using a NanoDrop spectrophotometer (Thermo Scientific).

[0206] SPR SPR measurements were performed using the Biacore X100 SPR system (GE Healthcare). Biacore Sensor Chips were immobilized with his-tagged 10 ug / mL of SA IL-33 or his-tagged unmodified IL-33 until the resonance unit (RU) reached 1000. As a control, a reference surface was coated with 10 mg / mL of his-tagged SA (without cytokines) at 1000 RU. The concentration of the analyte Fc-ST2 was reduced from 120 nM to 1.875 nM over a 60-second contact time. Specific binding of SA IL-33 to Fc-ST2 was calculated by subtracting the value of the reference channel coated with SA alone from the value of SA IL-33. The experimental results were then fitted to Langmuir binding kinetics using BIAevaluation software (GE Healthcare).

[0207] mouse Female C57BL / 6 mice (8 weeks old) were obtained from Charles River Laboratories. The mice were housed in the University of Chicago's animal facility for at least one week prior to immunization. All experiments were conducted with the approval of the University of Chicago's Animal Care and Use Committee.

[0208] Protein plasma pharmacokinetics Female C57BL / 6 mice were subcutaneously injected with either WT IL-33 or SA-IL-33 (equivalent to 26 μg IL-33). Blood samples were collected in low-protein-binding tubes at 2, 4, 8, 12, 24, 36, 48, 72, and 96 hours post-injection. Plasma IL-33 concentrations were measured using the IL-33 mouse uncoated ELISA kit (R&D Systems) according to the manufacturer's protocol.

[0209] EAE Model Young female C57BL / 6 mice (9-12 weeks old) were subcutaneously immunized in the dorsal flank with a complete Freund's adjuvant (MOG35-55 / CFA Emulsion, Hooke Laboratories) emulsion, followed by intravenous administration of pertussis toxin in PBS, first on the day of immunization and then again the following day. After the initial immunization, the severity of EAE was monitored, and clinical scores were measured daily from day 8 post-immunization. Clinical scores were determined based on Hooke Laboratories criteria under blinded treatment group classification. WT IL-33, SA-IL-33, and PBS were administered every other day in 200 μl of PBS (into the left dorsal flank of the mouse; approximately 2 cm away from the emulsion injection site). FTY720 (1 mg kg-1 body weight) was administered orally daily.

[0210] Flow cytometry EAE mice were treated with PBS, WT IL-33 (Biolegend), or SA IL-33 (equivalent to 26 μg of IL-33) every other day, starting 8 days after immunization. Spinal cord, spleen, and cervical and iliac lymph node tissues were harvested 15, 23, or 35 days after immunization. Lymph node and spinal cord tissues were digested at 37°C for 45 minutes in DMEM medium supplemented with 2% FBS and 2 mg ml-1 collagenase D (Sigma-Aldrich). Single-cell suspensions were obtained by gently disrupting cells through a 70 μm cell strainer. For spleen, erythrocytes in the blood were lysed with ACK lysis buffer (Quality Biological) and then stained with antibodies for flow cytometry. For detection of MOG-recognized T cells, T-Select I-Ab MOG35~55 tetramer-PE (MBL International Corporation) or MOG 38~49 Tetramer-PE (NIH Tetramer Core Facility) was used. MOG 38~49Non-specific binding of the tetramers was not considered. Discrimination of fixable live / dead cells was performed using Fixable viability dye eFluor 455 (eBioscience), Live / dead fixable violet (eBioscience), or Live / dead fixable aqua (eBioscience) according to the manufacturer's instructions. Staining was performed for 20 minutes on ice. For intracellular staining, cells were fixed with Cytofix / Cytoperm (BD Bioscience) for 20 minutes at 4°C. For permeabilization, Perm / Wash buffer (BD Bioscience) was used and cells were stained in Perm / Wash buffer for 30 minutes at 4°C. After the washing step, cells were stained with specific antibodies for 20 minutes on ice before fixation. All flow cytometry analyses were performed using a Fortessa (BD Biosciences) flow cytometer and analyzed using FlowJo software (Tree Star).

[0211] Restimulation of splenocytes Single cell suspensions were prepared from dLNs and spleen. For analysis of cytokine production, 5×10 5 lymphocytes and 2×10 6 splenocytes were plated in 96-well round-bottom plates. Cells were stimulated with 10 μM MOG 35~55 peptide (Genscript). After 2 hours, GolgiPlug (brefeldin A) and GolgiStop (monensin) were added according to the manufacturer's protocol to block secretion of intracellular cytokines. Cells were stained for flow cytometry after 4 hours. For fixation, Cytofix / Cytoperm (BD Bioscience) was used for 20 minutes at 4°C. For permeabilization, Perm / Wash buffer (BD Bioscience) was used and cells were stained in Perm / Wash buffer for 30 minutes at 4°C. For restimulation over 3 days, 2.5×10 5 splenocytes were plated in 96-well round-bottom plates. Cells were stimulated with 10 μM MOG 35~55 (Genscript) or 100 μg ml -1The cells were stimulated with MOG protein (Anaspec). After 72 hours, the supernatant was collected for analysis using a LegendPlex Multiplex Cytokine Array (Biolegend).

[0212] statistical analysis Statistical significance between experimental groups was determined using Prism software (v9 GraphPad). Using one-way analysis of variance (ANOVA) followed by Tukey's HSD post-hoc test, the variances between groups were found to be similar using the Brown-Forsyth test. For single comparisons, a two-tailed Student's t-test was used. A p-value less than 0.05 was considered statistically significant.

[0213] Example 10: Albumin-fused human IL-35 protein has a significant therapeutic effect against arthritis. Serum albumin-fused interleukin 35 (SA IL-35) was manipulated by recombinant fusion of mouse SA with human IL-35. For this purpose, as shown in Figure 1a, a single-stranded SA-IL-35 plasmid DNA construct was synthesized consisting of the IL-27β subunit of IL-35 (also known as Ebi3), a mobile (GGGS)4 linker, the IL-12α subunit of IL-35 (also known as IL-12p35), a mobile (GGGS)5 linker, mouse SA without propeptides, and a 6-histidine tag sequence along the C-terminus, starting from the N-terminus. This construct was then subcloned into the mammalian expression vector pcDNA3.1(+). This protein was produced in suspension-adapted HEK-293F cells and purified by affinity and size exclusion chromatography. The molecular weight and purity of this protein were qualitatively evaluated by SDS-PAGE. SDS-PAGE samples were stained with Commissar Blue. The SDS-PAGE for SA-IL-35 is shown in Figure 41B.

[0214] The inventors then evaluated SA IL-35 as a prophylactic measure 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 BALB / c mice, as depicted in Figure 2a. On day 3 after collagen-II antibody immunization, CAIA mice were treated with intravenous injection of 43.5 ug of SA IL-35 (wild-type IL-35 molar equivalent), followed by intraperitoneal injection of 25 mg of LPS. Clinical scores of the forelimbs and hindlimbs of the mice were recorded daily from day 3 to day 11. The severity of arthritis was on a scale of 0 to 4, where 0 represented a healthy paw, 1 represented swelling and / or redness of one joint, 2 represented swelling and / or redness of two or more joints, 3 represented swelling and / or redness of the entire paw, and 4 represented maximum swelling. The experimental results are described below. Prophylactic treatment with a single dose of SA IL-35 prevented the development of severe disease in a CAIA mouse model of arthritis. This data is shown in Figure 42.

[0215] All methods disclosed and asserted herein can be prepared and carried out without excessive experimentation, given the present invention. While the compositions and methods of the present invention have been described in terms of certain aspects, it will be apparent to those skilled in the art that the methods and the steps or order of steps described herein can be modified without departing from the concept, spirit, and scope of the present invention. More specifically, certain chemically and physiologically related agents can be used instead of the agents described herein, and it will be apparent that the same or similar results can be obtained at the same time. All such similar substitutions and modifications, which will be apparent to those skilled in the art, are considered to be within the spirit, scope, and concept of the present invention as defined by the appended claims.

[0216] References The following references are incorporated herein by reference to the extent that they provide exemplary procedures or other details that supplement what is described herein. TIFF0007896891000016.tif158160TIFF0007896891000017.tif238161TIFF0007896891000018.tif238161TIFF0007896891000019.tif238160TIFF0007896891000020.tif121160

Claims

1. A composition comprising an anti-inflammatory cytokine functionally linked to an albumin protein for use in a method for targeting an anti-inflammatory cytokine to a target lymph node, wherein the method comprises the step of administering the composition to a subject by subcutaneous, intradermal or intramuscular administration, wherein the anti-inflammatory cytokine is IL-33, the subject has multiple sclerosis, and the anti-inflammatory cytokine is covalently linked to an albumin protein.

2. The composition according to claim 1, wherein multiple sclerosis is treated by targeting anti-inflammatory cytokines to lymph nodes.

3. The composition according to claim 1 or 2, wherein the anti-inflammatory cytokine is administered in a dose of 0.1 mg / kg to 50 mg / kg.

4. The composition according to claim 3, wherein the anti-inflammatory cytokine is administered in a dose of 0.6 to 12 mg / kg.

5. The composition according to claim 3 or 4, wherein the subject is administered a total of three doses over one week, and the doses are administered every other day.

6. The composition according to any one of claims 1 to 5, wherein the method further comprises the step of identifying anti-inflammatory cytokines in the target lymph node.

7. The composition according to any one of claims 1 to 6, wherein the method further comprises the step of identifying anti-inflammatory cytokines in a target lymph node, the identification step comprising obtaining a lymph sample from the target.

8. The composition according to any one of claims 1 to 7, wherein the method further comprises the step of identifying anti-inflammatory cytokines in a target lymph node, the identification step comprising obtaining a lymph sample from the target, and the identification step comprising detecting the presence of anti-inflammatory cytokines in the lymph sample.

9. The composition according to any one of claims 1 to 8, wherein the anti-inflammatory cytokine remains in the lymph nodes for at least 8 hours or at least 16 hours after administration of the composition to a subject.

10. The composition according to any one of claims 1 to 9, wherein the albumin protein is human serum albumin.

11. The composition according to any one of claims 1 to 10, wherein an anti-inflammatory cytokine is covalently linked to an albumin protein via a linker.

12. A composition comprising a polypeptide containing IL-33 fused to albumin via a linker, for use in a method for treating multiple sclerosis in a subject, The method comprises the step of subcutaneously administering the composition to a subject, wherein the composition is administered at a dose of 0.6 to 12 mg / kg, the subject receives a total of three doses over one week, and the doses are administered every other day.

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