Modified CTLA4 and methods of use thereof

Abstract

In some aspects, polypeptides having high binding affinity to ligands, compositions comprising the same, and methods for using the same are provided. In some embodiments, polypeptides having high binding affinity to CD80, CD86, or both are provided. The immune system is a host defense mechanism that protects organisms from disease. Dysfunction of the immune system can lead to a wide range of diseases (e.g., autoimmune diseases, inflammatory diseases, and cancer). There remains a great need for improved therapeutic compositions and methods that can help regulate the proper function of the immune system, thereby protecting the human body from various diseases and conditions.

Description

[Technical field]

[0001] cross reference This application claims priority to PCT Application No. PCT / CN2018 / 113643, filed November 2, 2018, which is incorporated herein by reference in its entirety for all purposes. Sequence Listing This application contains a Sequence Listing that has been submitted electronically in ASCII format and is incorporated herein by reference in its entirety. This ASCII copy was created on June 4, 2021, is named 46198-702_602_SL.txt, and is 67,015 bytes in size. [Background technology]

[0002] Background of the Disclosure The immune system is a host defense mechanism that protects the organism from disease. Immune system dysfunction can lead to a wide range of diseases, such as autoimmune diseases, inflammatory diseases and cancer. There is still a great need for improved therapeutic compositions and methods that can help regulate the proper function of the immune system, thereby protecting the human body from various diseases and conditions. Summary of the Invention [Means for solving the problem]

[0003] Disclosure Summary In certain embodiments, described herein is a polypeptide comprising a first amino acid sequence having about 70% or more, about 75% or more, about 80% or more, about 85% or more, about 90% or more, about 95% or more, about 96% or more, about 97% or more, about 98% or more, about 99% or more, or about 99.5% or more sequence identity to amino acids 1-124 of SEQ ID NO:2, wherein the polypeptide comprises a mutation at one or more positions selected from positions 18, 40, 68, 77, 86, 92, 107, 117, 118, and 122 relative to SEQ ID NO:2.

[0004] In some embodiments, the polypeptide exhibits enhanced binding affinity for CD80 and / or CD86 compared to abatacept (SEQ ID NO:2) as determined by surface plasmon resonance at 37° C. In some embodiments, the polypeptide comprises a mutation at position 68 relative to SEQ ID NO:2. In some embodiments, the polypeptide comprises a mutation at position 40 relative to SEQ ID NO:2. In some embodiments, the polypeptide further comprises a mutation at one or more positions selected from positions 16, 24, 25, 27, 28, 29, 33, 41, 42, 48, 49, 50, 51, 52, 53, 54, 58, 59, 60, 61, 63, 64, 65, 69, 70, 80, 85, 93, 94, 96, and 105 relative to SEQ ID NO:2. In some embodiments, the mutation comprises an amino acid substitution or deletion.

[0005] In certain embodiments, a polypeptide comprising an amino acid sequence having about 70% or more, about 75% or more, about 80% or more, about 85% or more, about 90% or more, about 95% or more, about 96% or more, about 97% or more, about 98% or more, about 99% or more, or about 99.5% or more sequence identity to amino acids 1-124 of SEQ ID NO:2, wherein the polypeptide has, with respect to SEQ ID NO:2: Described herein are polypeptides that include an amino acid substitution selected from the group consisting of S18R, S18N, A24S, G27DKA, G27DK, G27K, G27R, G27W, G27Y, G27E, G27KK, A29T, A40T, A49P, A50T, G68F, G68K, G68W, G68Y, G68H, G68D, G68E, L77V, D86N, C92S, C92Y, K93V, K93W, K93P, K93C, K93F, K93R, V94L, G105S, G107D, P117S, E118K, D122H, and any combination thereof.

[0006] In some embodiments, the polypeptide exhibits enhanced binding affinity for CD80 and / or CD86 compared to abatacept (SEQ ID NO:2) as determined by surface plasmon resonance at 37° C. In some embodiments, the polypeptide comprises the amino acid substitution G27DKA with respect to SEQ ID NO:2. In some embodiments, the polypeptide comprises the amino acid substitution G68F with respect to SEQ ID NO:2. In some embodiments, the polypeptide comprises the amino acid substitution A40T with respect to SEQ ID NO:2. In some embodiments, the polypeptide further comprises the amino acid substitution K93M with respect to SEQ ID NO:2. In some embodiments, the polypeptide further comprises the amino acid substitution G27DK with respect to SEQ ID NO:2. In some embodiments, the polypeptide further comprises the amino acid substitution G27H with respect to SEQ ID NO:2. In some embodiments, the polypeptide comprises the amino acid substitution P117S with respect to SEQ ID NO:2. In some embodiments, the polypeptide further comprises at least one amino acid substitution selected from the group consisting of A24E, G27H, G27D, A29S, R33W, D41G, T51N, K93M, K93N, and G105D with respect to SEQ ID NO:2.

[0007] In some embodiments, the polypeptide is selected from the group consisting of G27DKA / R33W, G27DKA / G68F, R33W / G68F, G27R / G68Y, G27H / G68F, G27DK / G68F, G27DK / G68D, G27DK / G68E, G27D / G68F, G27E / G68F, G27H / G68D, G27H / G68E, G27DKA / G68F, G27H / G68F, G27DK / G68F, G27DKA / G68F / D122H, G27DKA / G68F / A40T / D122H, G27DKA / G68F / A40T / P117S, G27DKA / G68F / L77V, G27DKA / G68F / C92S / K93M, G27DK / G68F / A49P / A50T, G27DK / G68F / A40T / D86N / G105S, G27KK / G6 8F, G27DKA / G68F / P117S, G27DKA / G68F / A49P / A50T, G27DK / G68F / A40T, G27DK / G68F / L77V / G105S / P117S, G27DK / G68F / L77V, G27DK / G68F / C92S / K93M, G27DK / G68F / P117S, G27DKA / G68F / G105S, G27DKA / G68F / D86N , G27DK / G68F / D122H, G27DK / G68F / A40T / G105S, G27DK / G68F / G105S, G27DK / G68F / D86N, G27DKA / G68F / A40T, G27DKA / G68F / K93M, G27DK / G68F / K93M, and G27DKA / A40T / G68F / K93M. In some embodiments, the polypeptide comprises the amino acid substitution combination G27DKA / A40T / G68F / K93M with respect to SEQ ID NO:2. In some embodiments, the polypeptide comprises the amino acid substitution combination G27DKA / G68F / A40T with respect to SEQ ID NO:2. In some embodiments, the polypeptide comprises the amino acid substitution combination G27DKA / G68F / K93M with respect to SEQ ID NO: 2. In some embodiments, the polypeptide comprises the amino acid substitution combination G27DKA / G68F / A40T / P117S with respect to SEQ ID NO: 2.In some embodiments, the polypeptide comprises the amino acid substitution combination G27DKA / G68F / P117S with respect to SEQ ID NO:2. In some embodiments, the polypeptide comprises the amino acid substitution combination G27H / G68F with respect to SEQ ID NO:2. In some embodiments, the polypeptide comprises the amino acid substitution combination G27DKA / G68F with respect to SEQ ID NO:2. In some embodiments, the polypeptide comprises the amino acid substitution combination G27DK / G68F / K93M with respect to SEQ ID NO:2. In some embodiments, the polypeptide comprises the amino acid substitution combination G27DK / G68F / A40T with respect to SEQ ID NO:2. In some embodiments, the polypeptide comprises the amino acid substitution combination G27DK / G68F / L77V / G105S / P117S with respect to SEQ ID NO:2. In some embodiments, the polypeptide comprises the amino acid substitution combination G27DK / G68F / L77V with respect to SEQ ID NO:2. In some embodiments, the polypeptide comprises the amino acid substitution combination G27DK / G68F / P117S with respect to SEQ ID NO:2. In some embodiments, the polypeptide comprises the amino acid substitution combination G27DK / G68F / D122H with respect to SEQ ID NO:2.

[0008] In some embodiments, the polypeptide comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 amino acid substitutions with respect to amino acids 1-124 of SEQ ID NO: 2. In some embodiments, the polypeptide comprises at most 1, at most 2, at most 3, at most 4, at most 5, at most 6, at most 7, at most 8, at most 9, or at most 10 amino acid substitutions with respect to amino acids 1-124 of SEQ ID NO: 2.

[0009] In certain embodiments, a polypeptide is described herein that comprises an amino acid sequence having about 70% or more, about 75% or more, about 80% or more, about 85% or more, about 90% or more, about 95% or more, about 96% or more, about 97% or more, about 98% or more, about 99% or more, or about 99.5% or more sequence identity to amino acids 1-124 of SEQ ID NO:2, wherein the amino acid substitutions of the polypeptide relative to amino acids 1-124 of SEQ ID NO:2 are selected from the amino acid substitutions described herein.

[0010] In some embodiments, the polypeptide further comprises a second amino acid sequence fused to the first amino acid sequence. In some embodiments, the second amino acid sequence encodes an IgG Fc region. In some embodiments, the IgG Fc region is derived from a human IgG molecule.

[0011] In some embodiments, the second amino acid sequence has about 70% or more, about 75% or more, about 80% or more, about 85% or more, about 90% or more, about 95% or more, about 96% or more, about 97% or more, about 98% or more, about 99% or more, about 99.5% or more, or 100% sequence identity to amino acids 125-357 of SEQ ID NO: 2. In some embodiments, the polypeptide has about 70% or more, about 75% or more, about 80% or more, about 85% or more, about 90% or more, about 95% or more, about 96% or more, about 97% or more, about 98% or more, about 99% or more, about 99.5% or more, or 100% sequence identity to amino acids 1-359 of SEQ ID NO: 6. In some embodiments, the first and second amino acid sequences are fused together via a linker. In some embodiments, the linker comprises 1-10 amino acids. In some embodiments, the linker comprises an amino acid sequence selected from Table 7. In some embodiments, the linker comprises the amino acid sequence Q (SEQ ID NO: 82) or GGGGS (SEQ ID NO: 54).

[0012] In some embodiments, the polypeptide exhibits a binding affinity for CD80 that is at least 1.5 times, at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 10 times, at least 15 times, at least 20 times, at least 25 times, at least 30 times, at least 40 times, at least 50 times, at least 100 times, at least 150 times, at least 200 times, at least 250 times, or at least 300 times greater than the affinity of abatacept (SEQ ID NO: 2) for CD80 as determined by surface plasmon resonance at 37°C. In some embodiments, the polypeptide exhibits a binding affinity for CD86 that is at least 1.5 times, at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 10 times, at least 15 times, at least 20 times, at least 25 times, at least 30 times, at least 40 times, at least 50 times, at least 100 times, at least 150 times, at least 200 times, at least 250 times, or at least 300 times greater than the affinity of abatacept (SEQ ID NO: 2) for CD86 as determined by surface plasmon resonance at 37° C. In some embodiments, the polypeptide exhibits a binding affinity for CD80 that is greater than the affinity of belatacept (SEQ ID NO: 3) for CD80 as determined by surface plasmon resonance at 37° C. In some embodiments, the polypeptide exhibits a binding affinity for CD86 that is greater than the affinity of belatacept (SEQ ID NO: 3) for CD86 as determined by surface plasmon resonance at 37° C. In some embodiments, the polypeptide exhibits a binding affinity for CD80 that is at least 1.5 times, at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 10 times, at least 15 times, at least 20 times, at least 25 times, or at least 30 times greater than the affinity of belatacept (SEQ ID NO: 3) for CD80, as determined by surface plasmon resonance at 37°C.In some embodiments, the polypeptide exhibits a binding affinity for CD86 that is at least 1.5 times, at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 10 times, at least 15 times, at least 20 times, at least 25 times, or at least 30 times greater than the affinity of belatacept (SEQ ID NO: 3) for CD86 as determined by surface plasmon resonance at 37° C. In some embodiments, the polypeptide exhibits a binding affinity for CD80 that is greater than the affinity of SEQ ID NO: 4 for CD80 as determined by surface plasmon resonance at 37° C. In some embodiments, the polypeptide exhibits a binding affinity for CD86 that is greater than the affinity of SEQ ID NO: 4 for CD86 as determined by surface plasmon resonance at 37° C. In some embodiments, the polypeptide exhibits a binding affinity for CD80 that is at least 1.5 times, at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 10 times, at least 15 times, at least 20 times, at least 25 times, or at least 30 times greater than the affinity of SEQ ID NO: 4 for CD80 as determined by surface plasmon resonance at 37° C. In some embodiments, the polypeptide exhibits a binding affinity for CD86 that is at least 1.5 times, at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 10 times, at least 15 times, at least 20 times, at least 25 times, or at least 30 times greater than the affinity of SEQ ID NO: 4 for CD86 as determined by surface plasmon resonance at 37° C. In some embodiments, the polypeptide exhibits a binding affinity for CD80 that is greater than the affinity of SEQ ID NO: 5 for CD80 as determined by surface plasmon resonance at 37° C. In some embodiments, the polypeptide exhibits a binding affinity for CD86 that is greater than the affinity of SEQ ID NO: 5 for CD86 as determined by surface plasmon resonance at 37° C.In some embodiments, the polypeptide exhibits a binding affinity for CD80 that is at least 1.5-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 10-fold, at least 15-fold, at least 20-fold, at least 25-fold, or at least 30-fold greater than the affinity of SEQ ID NO: 4 for CD80 as determined by surface plasmon resonance at 37° C. In some embodiments, the polypeptide exhibits a binding affinity for CD86 that is at least 1.5-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 10-fold, at least 15-fold, at least 20-fold, at least 25-fold, or at least 30-fold greater than the affinity of SEQ ID NO: 4 for CD86 as determined by surface plasmon resonance at 37° C.

[0013] In certain embodiments, described herein are polypeptide-drug conjugates comprising the polypeptides disclosed herein.

[0014] In certain embodiments, described herein are methods of treating a disease or condition comprising administering to a subject in need thereof a polypeptide disclosed herein or a polypeptide-drug conjugate disclosed herein.

[0015] In some embodiments, the disease or condition is an infection, an endotoxic shock associated with an infection, arthritis, rheumatoid arthritis, psoriatic arthritis, systemic juvenile idiopathic arthritis (JIA), inflammatory bowel disease (IBD), systemic lupus erythematosus (SLE), asthma, pelvic peritonitis, Alzheimer's disease, Crohn's disease, ulcerative colitis, irritable bowel syndrome, multiple sclerosis, ankylosing spondylitis, dermatomyositis, uveitis, Peyronie's disease, celiac disease, gallbladder disease, pilonidal disease, psoriasis, inflammatory bowel disease ... disease), peritonitis, psoriasis, vasculitis, surgical adhesions, stroke, type I diabetes, Lyme arthritis, meningoencephalitis, immune-mediated inflammatory disorders of the central and peripheral nervous system, autoimmune disorders, pancreatitis, surgical trauma, graft-versus-host disease, graft rejection, cardiac disease, bone resorption, burn patients, myocardial infarction, Paget's disease, osteoporosis, sepsis, hepatic / pulmonary fibrosis, periodontitis, hypochlorhydria, solid tumors (renal cell carcinoma), liver cancer, multiple myeloma, prostate cancer, bladder cancer, pancreatic cancer, neurological cancers, and B-cell malignancies (e.g., Castleman's disease, certain lymphomas, chronic lymphocytic leukemia, and multiple myeloma).

[0016] In certain embodiments, polypeptides are described herein for use in treating a condition in a subject.

[0017] In some embodiments, the condition is selected from the group consisting of graft rejection, infection, endotoxic shock associated with infection, arthritis, rheumatoid arthritis, psoriatic arthritis, systemic juvenile idiopathic arthritis (JIA), inflammatory bowel disease (IBD), systemic lupus erythematosus (SLE), asthma, pelvic peritonitis, Alzheimer's disease, Crohn's disease, ulcerative colitis, irritable bowel syndrome, multiple sclerosis, ankylosing spondylitis, dermatomyositis, uveitis, Peyronie's disease, celiac disease, gallbladder disease, pilonidal disease, peritonitis, psoriasis, vasculitis, surgical adhesions, stroke, and type I diabetes. disease, Lyme arthritis, meningoencephalitis, immune-mediated inflammatory disorders of the central and peripheral nervous system, autoimmune disorders, pancreatitis, surgical trauma, graft-versus-host disease, cardiac disease, bone resorption, burn patients, myocardial infarction, Paget's disease, osteoporosis, sepsis, hepatic / pulmonary fibrosis, periodontitis, hypochlorhydria, solid tumors (renal cell carcinoma), liver cancer, multiple myeloma, prostate cancer, bladder cancer, pancreatic cancer, neurological cancers, and B-cell malignancies (e.g., Castleman's disease, certain lymphomas, chronic lymphocytic leukemia, and multiple myeloma).

[0018] In certain embodiments, described herein are pharmaceutical compositions comprising a polypeptide disclosed herein or a polypeptide-drug conjugate disclosed herein and a pharma- ceutically acceptable excipient.

[0019] In certain embodiments, described herein are kits that include a polypeptide disclosed herein or a polypeptide-drug conjugate disclosed herein in a container.

[0020] In certain embodiments, described herein is the use of a polypeptide provided herein or a polypeptide-drug conjugate provided herein for the manufacture of a medicament for treating a condition in a subject.

[0021] In certain embodiments, described herein are isolated polynucleotides that encode the polypeptides disclosed herein.

[0022] In certain embodiments, described herein are vectors that comprise the isolated polynucleotides disclosed herein.

[0023] In certain embodiments, cells comprising the vectors disclosed herein are described herein.

[0024] In some embodiments, the cell is a eukaryotic cell. In some embodiments, the cell is a prokaryotic cell. In some embodiments, the cell is a mammalian cell, a bacterial cell, a fungal cell, or an insect cell.

[0025] Incorporation by Reference All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference herein.

[0026] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings, which are set forth in the following. In certain embodiments, for example, the following items are provided: (Item 1) 1. A polypeptide comprising a first amino acid sequence having about 70% or more, about 75% or more, about 80% or more, about 85% or more, about 90% or more, about 95% or more, about 96% or more, about 97% or more, about 98% or more, about 99% or more, or about 99.5% or more sequence identity to amino acids 1-124 of SEQ ID NO:2, wherein said polypeptide comprises a mutation at one or more positions selected from positions 18, 40, 68, 77, 86, 92, 107, 117, 118, and 122 relative to SEQ ID NO:2. (Item 2) 2. The polypeptide of item 1, wherein the polypeptide exhibits enhanced binding affinity for CD80 and / or CD86 compared to abatacept (SEQ ID NO: 2) as determined by surface plasmon resonance at 37° C. (Item 3) 3. The polypeptide according to item 1 or 2, comprising a mutation at position 68 relative to SEQ ID NO:2. (Item 4) 4. The polypeptide according to any one of items 1 to 3, comprising a mutation at position 40 with respect to SEQ ID NO:2. (Item 5) 5. The polypeptide according to any one of items 1 to 4, wherein the polypeptide further comprises a mutation at one or more positions selected from positions 16, 24, 25, 27, 28, 29, 33, 41, 42, 48, 49, 50, 51, 52, 53, 54, 58, 59, 60, 61, 63, 64, 65, 69, 70, 80, 85, 93, 94, 96, and 105 with respect to SEQ ID NO:2. (Item 6) 6. The polypeptide according to any one of items 1 to 5, wherein the mutation comprises an amino acid substitution or deletion. (Item 7) 2. A polypeptide comprising an amino acid sequence having about 70% or more, about 75% or more, about 80% or more, about 85% or more, about 90% or more, about 95% or more, about 96% or more, about 97% or more, about 98% or more, about 99% or more, or about 99.5% or more sequence identity to amino acids 1 to 124 of SEQ ID NO:2, wherein the polypeptide comprises an amino acid sequence having about 70% or more, about 75% or more, about 80% or more, about 85% or more, about 90% or more, about 95% or more, about 96% or more, about 97% or more, about 98% or more, about 99% or more, or about 99.5% or more sequence identity to amino acids 1 to 124 of SEQ ID NO:2, 18R, S18N, A24S, G27DKA, G27DK, G27K, G27R, G27W, G27Y, G27E, G27KK, A29T, A40T, A49P, A50T, G68F, G68K, G68W, G68Y, G68H, G68D, G68E, L77V, D86N, C92S, C92Y, K93V, K93W, K93P, K93C, K93F, K93R, V94L, G105S, G107D, P117S, E118K, D122H, and any combination thereof. (Item 8) 8. The polypeptide of item 7, wherein the polypeptide exhibits enhanced binding affinity for CD80 and / or CD86 compared to abatacept (SEQ ID NO: 2) as determined by surface plasmon resonance at 37° C. (Item 9) 9. The polypeptide according to any one of items 1 to 8, comprising the amino acid substitution G27DKA with respect to SEQ ID NO: 2. (Item 10) 10. The polypeptide according to any one of items 1 to 9, comprising the amino acid substitution G68F with respect to SEQ ID NO: 2. (Item 11) 11. The polypeptide according to any one of items 1 to 10, comprising the amino acid substitution A40T with respect to SEQ ID NO:2. (Item 12) 12. The polypeptide according to any one of items 1 to 11, further comprising the amino acid substitution K93M with respect to SEQ ID NO:2. (Item 13) 13. The polypeptide according to any one of items 1 to 7 or 10 to 12, further comprising the amino acid substitution G27DK with respect to SEQ ID NO:2. (Item 14) 13. The polypeptide according to any one of items 1 to 7 or 10 to 12, further comprising the amino acid substitution G27H with respect to SEQ ID NO:2. (Item 15) 15. The polypeptide according to any one of items 1 to 14, comprising the amino acid substitution P117S with respect to SEQ ID NO:2. (Item 16) 15. The polypeptide according to any one of items 1 to 14, further comprising at least one amino acid substitution selected from the group consisting of A24E, G27H, G27D, A29S, R33W, D41G, T51N, K93M, K93N, and G105D with respect to SEQ ID NO: 2. (Item 17) With respect to SEQ ID NO: 2, G27DKA / R33W, G27DKA / G68F, R33W / G68F, G27R / G68Y, G27H / G68F, G27DK / G68F, G27DK / G68D, G27DK / G68E, G27D / G68F, G27E / G68F, G27H / G68D, G27H / G68E, G27DKA / G68F, G27H / G68F, G27DK / G68F, G27DK A / G68F / D122H, G27DKA / G68F / A40T / D122H, G27DKA / G68F / A40T / P117S, G27DKA / G68F / L77V, G27DKA / G68F / C92S / K93M, G27DK / G68F / A49P / A50T, G27DK / G68F / A40T / D86N / G105S, G27KK / G68F, G27DKA / G68F / P117S, G 27DKA / G68F / A49P / A50T, G27DK / G68F / A40T, G27DK / G68F / L77V / G105S / P117S, G27DK / G68F / L77V, G27DK / G 68F / C92S / K93M, G27DK / G68F / P117S, G27DKA / G68F / G105S, G27DKA / G68F / D86N, G27DK / G68F / D122H, G27DK 16. The polypeptide according to any one of items 1 to 15, comprising a combination of amino acid substitutions selected from the group consisting of G27DKA / G68F / A40T / G105S, G27DK / G68F / G105S, G27DK / G68F / D86N, G27DKA / G68F / A40T, G27DKA / G68F / K93M, G27DK / G68F / K93M, and G27DKA / A40T / G68F / K93M. (Item 18) 16. The polypeptide according to any one of items 1 to 15, comprising the amino acid substitution combination G27DKA / A40T / G68F / K93M with respect to SEQ ID NO: 2. (Item 19) 16. The polypeptide according to any one of items 1 to 15, comprising the amino acid substitution combination G27DKA / G68F / A40T with respect to SEQ ID NO: 2. (Item 20) 16. The polypeptide according to any one of items 1 to 15, comprising the amino acid substitution combination G27DKA / G68F / K93M with respect to SEQ ID NO: 2. (Item 21) 16. The polypeptide according to any one of items 1 to 15, comprising the amino acid substitution combination G27DKA / G68F / A40T / P117S with respect to SEQ ID NO: 2. (Item 22) 16. The polypeptide according to any one of items 1 to 15, comprising the amino acid substitution combination G27DKA / G68F / P117S with respect to SEQ ID NO: 2. (Item 23) 16. The polypeptide according to any one of items 1 to 15, comprising the amino acid substitution combination G27H / G68F with respect to SEQ ID NO: 2. (Item 24) 16. The polypeptide according to any one of items 1 to 15, comprising the amino acid substitution combination G27DKA / G68F with respect to SEQ ID NO: 2. (Item 25) 16. The polypeptide according to any one of items 1 to 15, comprising the amino acid substitution combination G27DK / G68F / K93M with respect to SEQ ID NO: 2. (Item 26) 16. The polypeptide according to any one of items 1 to 15, comprising the amino acid substitution combination G27DK / G68F / A40T with respect to SEQ ID NO: 2. (Item 27) 16. The polypeptide according to any one of items 1 to 15, comprising the amino acid substitution combination G27DK / G68F / L77V / G105S / P117S with respect to SEQ ID NO: 2. (Item 28) 16. The polypeptide according to any one of items 1 to 15, comprising the amino acid substitution combination G27DK / G68F / L77V with respect to SEQ ID NO: 2. (Item 29) 16. The polypeptide according to any one of items 1 to 15, comprising the amino acid substitution combination G27DK / G68F / P117S with respect to SEQ ID NO: 2. (Item 30) 16. The polypeptide according to any one of items 1 to 15, comprising the amino acid substitution combination G27DK / G68F / D122H with respect to SEQ ID NO:2. (Item 31) 26. The polypeptide according to any one of items 1 to 25, comprising at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 amino acid substitutions with respect to amino acids 1 to 124 of SEQ ID NO: 2. (Item 32) 32. The polypeptide according to any one of items 1 to 31, comprising at most 1, at most 2, at most 3, at most 4, at most 5, at most 6, at most 7, at most 8, at most 9, or at most 10 amino acid substitutions with respect to amino acids 1 to 124 of SEQ ID NO: 2. (Item 33) 32. A polypeptide comprising an amino acid sequence having about 70% or more, about 75% or more, about 80% or more, about 85% or more, about 90% or more, about 95% or more, about 96% or more, about 97% or more, about 98% or more, about 99% or more, or about 99.5% or more sequence identity to amino acids 1 to 124 of SEQ ID NO:2, wherein an amino acid substitution in said polypeptide for amino acids 1 to 124 of SEQ ID NO:2 is selected from the amino acid substitutions according to any one of items 7 to 32. (Item 34) 34. The polypeptide according to any one of items 1 to 33, further comprising a second amino acid sequence fused to the first amino acid sequence. (Item 35) 35. The polypeptide of claim 34, wherein the second amino acid sequence encodes an IgG Fc region. (Item 36) 36. The polypeptide of item 35, wherein the IgG Fc region is derived from a human IgG molecule. (Item 37) 37. The polypeptide according to item 35 or 36, wherein the second amino acid sequence has about 70% or more, about 75% or more, about 80% or more, about 85% or more, about 90% or more, about 95% or more, about 96% or more, about 97% or more, about 98% or more, about 99% or more, about 99.5% or more, or 100% sequence identity to amino acids 125 to 357 of SEQ ID NO: 2. (Item 38) 38. The polypeptide according to any one of Items 34 to 37, wherein the polypeptide has about 70% or more, about 75% or more, about 80% or more, about 85% or more, about 90% or more, about 95% or more, about 96% or more, about 97% or more, about 98% or more, about 99% or more, about 99.5% or more, or 100% sequence identity to amino acids 1 to 359 of SEQ ID NO: 6. (Item 39) 39. The polypeptide according to any one of items 34 to 38, wherein the first amino acid sequence and the second amino acid sequence are fused together via a linker. (Item 40) 40. The polypeptide according to item 39, wherein the linker comprises 1 to 10 amino acids. (Item 41) 41. The polypeptide according to item 39 or 40, wherein the linker comprises an amino acid sequence selected from Table 7. (Item 42) 41. The polypeptide according to item 39 or 40, wherein the linker comprises the amino acid sequence Q (SEQ ID NO: 82) or GGGGS (SEQ ID NO: 54). (Item 43) 43. The polypeptide of any one of items 1 to 42, wherein the polypeptide exhibits a binding affinity for CD80 that is at least 1.5-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 10-fold, at least 15-fold, at least 20-fold, at least 25-fold, at least 30-fold, at least 40-fold, at least 50-fold, at least 100-fold, at least 150-fold, at least 200-fold, at least 250-fold, or at least 300-fold greater than the affinity of abatacept (SEQ ID NO: 2) for CD80 as determined by surface plasmon resonance at 37°C. (Item 44) 44. The polypeptide of any one of items 1 to 43, wherein the polypeptide exhibits a binding affinity for CD86 that is at least 1.5 times, at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 10 times, at least 15 times, at least 20 times, at least 25 times, at least 30 times, at least 40 times, at least 50 times, at least 100 times, at least 150 times, at least 200 times, at least 250 times, or at least 300 times greater than the affinity of abatacept (SEQ ID NO: 2) for CD86 as determined by surface plasmon resonance at 37° C. (Item 45) 45. The polypeptide according to any one of items 1 to 44, wherein the polypeptide exhibits a binding affinity for CD80 that is greater than the affinity of belatacept (SEQ ID NO: 3) for CD80 as determined by surface plasmon resonance at 37°C. (Item 46) 46. ​​The polypeptide according to any one of items 1 to 45, wherein the polypeptide exhibits a binding affinity for CD86 that is higher than the affinity of belatacept (SEQ ID NO: 3) for CD86 as determined by surface plasmon resonance at 37°C. (Item 47) 47. The polypeptide of any one of items 1 to 46, wherein the polypeptide exhibits a binding affinity for CD80 that is at least 1.5-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 10-fold, at least 15-fold, at least 20-fold, at least 25-fold, or at least 30-fold greater than the affinity of belatacept (SEQ ID NO: 3) for CD80 as determined by surface plasmon resonance at 37°C. (Item 48) 48. The polypeptide of any one of items 1 to 47, wherein the polypeptide exhibits a binding affinity for CD86 that is at least 1.5-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 10-fold, at least 15-fold, at least 20-fold, at least 25-fold, or at least 30-fold greater than the affinity of belatacept (SEQ ID NO: 3) for CD86 as determined by surface plasmon resonance at 37°C. (Item 49) 49. The polypeptide according to any one of items 1 to 48, wherein the polypeptide exhibits a binding affinity for CD80 that is greater than the affinity of SEQ ID NO: 4 for CD80 as determined by surface plasmon resonance at 37°C. (Item 50) 50. The polypeptide of any one of items 1 to 49, wherein the polypeptide exhibits a binding affinity for CD86 that is greater than the affinity of SEQ ID NO: 4 for CD86 as determined by surface plasmon resonance at 37° C. (Item 51) 51. The polypeptide of any one of items 1 to 50, wherein the polypeptide exhibits a binding affinity for CD80 that is at least 1.5 times, at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 10 times, at least 15 times, at least 20 times, at least 25 times, or at least 30 times greater than the affinity of SEQ ID NO: 4 for CD80 as determined by surface plasmon resonance at 37° C. (Item 52) 52. The polypeptide of any one of items 1 to 51, wherein the polypeptide exhibits a binding affinity for CD86 that is at least 1.5-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 10-fold, at least 15-fold, at least 20-fold, at least 25-fold, or at least 30-fold greater than the affinity of SEQ ID NO: 4 for CD86 as determined by surface plasmon resonance at 37°C. (Item 53) 53. The polypeptide according to any one of items 1 to 52, wherein the polypeptide exhibits a binding affinity for CD80 that is greater than the affinity of SEQ ID NO: 5 for CD80 as determined by surface plasmon resonance at 37°C. (Item 54) 54. The polypeptide of any one of items 1 to 53, wherein the polypeptide has a binding affinity for CD86 that is greater than the affinity of SEQ ID NO: 5 for CD86 as determined by surface plasmon resonance at 37° C. (Item 55) 55. The polypeptide of any one of items 1 to 54, wherein the polypeptide exhibits a binding affinity for CD80 that is at least 1.5 times, at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 10 times, at least 15 times, at least 20 times, at least 25 times, or at least 30 times greater than the affinity of SEQ ID NO: 4 for CD80 as determined by surface plasmon resonance at 37° C. (Item 56) 56. The polypeptide of any one of items 1 to 55, wherein the polypeptide exhibits a binding affinity for CD86 that is at least 1.5-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 10-fold, at least 15-fold, at least 20-fold, at least 25-fold, or at least 30-fold greater than the affinity of SEQ ID NO: 4 for CD86 as determined by surface plasmon resonance at 37°C. (Item 57) 57. A polypeptide-drug conjugate comprising the polypeptide according to any one of items 1 to 56. (Item 58) A method for treating a disease or condition, comprising administering to a subject in need thereof a polypeptide according to any one of items 1 to 56 or a polypeptide-drug conjugate according to item 57. (Item 59) The disease or condition may be infection, endotoxic shock associated with infection, arthritis, rheumatoid arthritis, psoriatic arthritis, systemic juvenile idiopathic arthritis (JIA), inflammatory bowel disease (IBD), systemic lupus erythematosus (SLE), asthma, pelvic peritonitis, Alzheimer's disease, Crohn's disease, ulcerative colitis, irritable bowel syndrome, multiple sclerosis, ankylosing spondylitis, dermatomyositis, uveitis, Peyronie's disease, celiac disease, gallbladder disease, pilonidal disease, peritonitis, psoriasis, vasculitis, surgical adhesions, stroke, type I diabetes, Lyme arthritis, meningoencephalitis, 59. The method according to item 58, including immune-mediated inflammatory disorders of the central and peripheral nervous system, autoimmune disorders, pancreatitis, surgical trauma, graft versus host disease, graft rejection, cardiac disease, bone resorption, burn patients, myocardial infarction, Paget's disease, osteoporosis, sepsis, hepatic / pulmonary fibrosis, periodontitis, hypochlorhydria, solid tumors (renal cell carcinoma), liver cancer, multiple myeloma, prostate cancer, bladder cancer, pancreatic cancer, neurological cancers, and B-cell malignancies (e.g., Castleman's disease, certain lymphomas, chronic lymphocytic leukemia, and multiple myeloma). (Item 60) 57. The polypeptide according to any one of items 1 to 56 for use in the treatment of a condition in a subject. (Item 61) Such conditions include transplant rejection, infection, endotoxic shock associated with infection, arthritis, rheumatoid arthritis, psoriatic arthritis, systemic juvenile idiopathic arthritis (JIA), inflammatory bowel disease (IBD), systemic lupus erythematosus (SLE), asthma, pelvic peritonitis, Alzheimer's disease, Crohn's disease, ulcerative colitis, irritable bowel syndrome, multiple sclerosis, ankylosing spondylitis, dermatomyositis, uveitis, Peyronie's disease, celiac disease, gallbladder disease, pilonidal disease, peritonitis, psoriasis, vasculitis, surgical adhesions, stroke, type I diabetes, Lyme arthritis, and meningoencephalitis. 61. The polypeptide according to item 60, comprising immune-mediated inflammatory disorders of the central and peripheral nervous system, autoimmune disorders, pancreatitis, surgical trauma, graft-versus-host disease, cardiac disease, bone resorption, burn patients, myocardial infarction, Paget's disease, osteoporosis, sepsis, hepatic / pulmonary fibrosis, periodontitis, hypochlorhydria, solid tumors (renal cell carcinoma), liver cancer, multiple myeloma, prostate cancer, bladder cancer, pancreatic cancer, neurological cancers, and B-cell malignancies (e.g., Castleman's disease, certain lymphomas, chronic lymphocytic leukemia, and multiple myeloma). (Item 62) A pharmaceutical composition comprising the polypeptide according to any one of items 1 to 56 or the polypeptide-drug conjugate according to item 57 and a pharma- ceutically acceptable excipient. (Item 63) A kit comprising the polypeptide according to any one of items 1 to 56 or the polypeptide-drug conjugate according to item 57 in a container. (Item 64) Use of a polypeptide according to any one of items 1 to 56 or a polypeptide-drug conjugate according to item 57 for the manufacture of a medicament for treating a condition of a subject. (Item 65) 57. An isolated polynucleotide encoding the polypeptide according to any one of items 1 to 56. (Item 66) 66. A vector comprising the isolated polynucleotide of item 65. (Item 67) A cell comprising the vector described in item 66. (Item 68) 68. The cell of item 67, which is a eukaryotic cell. (Item 69) 68. The cell according to item 67, which is a prokaryotic cell. (Item 70) 68. The cell of item 67, which is a mammalian cell, a bacterial cell, a fungal cell or an insect cell. [Brief description of the drawings]

[0027] [Figure 1] FIG. 1 is an illustration of a competitive binding assay of an exemplary polypeptide (CTLA4-Fc variant).

[0028] [Diagram 2] 2A and 2B show representative results of competitive binding assays of exemplary polypeptides showing their relative binding affinities to CD80 and CD86, respectively.

[0029] [Diagram 3] 3A and 3B show representative results of another competitive binding assay of an exemplary polypeptide showing its relative binding affinity to CD80 and CD86, respectively.

[0030] [Figure 4] FIG. 4 shows representative results of a T cell proliferation assay of an exemplary polypeptide, illustrating its inhibitory effect on T cell populations.

[0031] [Diagram 5] FIG. 5 illustrates the principle of an assay for testing the inhibitory effect of exemplary polypeptides on IL-2 secretion from T cells upon immune stimulation.

[0032] [Figure 6-1] 6A-6C show representative results of an IL-2 secretion assay of an exemplary polypeptide, illustrating its inhibitory effect on IL-2 secretion from T cells upon immune stimulation. [Figure 6-2] 6A-6C show representative results of an IL-2 secretion assay of an exemplary polypeptide, illustrating its inhibitory effect on IL-2 secretion from T cells upon immune stimulation. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0033] Detailed Description of the Disclosure The disclosed system and method described herein can use conventional techniques and descriptions of molecular biology (including recombinant techniques), cell biology, biochemistry, microarray and sequencing techniques within the skill of a person skilled in the art, unless otherwise stated. Such conventional techniques include polymer array synthesis, hybridization and ligation of oligonucleotides, sequencing of oligonucleotides, and detection of hybridization using labels. Specific illustrations of suitable techniques can be obtained by reference to the examples herein. However, it is of course possible to use equivalent conventional procedures.Such conventional techniques and descriptions can be found in Green et al., eds., Genome Analysis: A Laboratory Manual Series (Vols. I-IV) (1999); Weiner et al., eds., Genetic Variation: A Laboratory Manual (2007); Dieffenbach and Dveksler, eds., PCR Primer: A Laboratory Manual (2003); Bowtell and Sambrook, DNA Microarrays: A Molecular Cloning Manual (2003); Mount, Bioinformatics: Sequence and Genome Analysis (2004); Sambrook and Russell, Condensed Protocols from Molecular Cloning: A Laboratory Manual (2006); and Sambrook and Green, Molecular Cloning: A Laboratory Manual, 4th ed. (2012) (all published by Cold Spring Harbor Laboratory Press); Stryer, L., Biochemistry (4th ed.) WH Freeman, NY (1995); Gait, Oligonucleotide Synthesis: A Practical Methods and techniques for determining the molecular structure of cells can be found in standard laboratory manuals such as, for example, "Analytical Approach," IRL Press, London (1984); Nelson and Cox, Lehninger, Principles of Biochemistry, 6th ed., WH Freeman Pub., New York (2012); RI Freshney, Culture of Animal Cells: A Manual of Basic Technique and Specialized Applications, 6th ed., Wiley-Blackwell (2010); and Berg et al., Biochemistry, 5th ed., WH Freeman Pub., New York (2002), all of which are incorporated by reference in their entirety for all purposes.Before the present composition, research tools and systems and methods are described, but it should be understood that the present disclosure is not limited thereto, since the specific systems and methods, compositions, targets and uses described can of course vary.It should also be understood that the terminology used herein is merely intended to describe specific embodiments, and is not intended to limit the scope of the present disclosure, which will be limited only by the scope of the appended claims.

[0034] As used in this specification and claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. For example, the term "a cell" includes a plurality of cells, including mixtures thereof.

[0035] The term "about" or "approximately" means within an acceptable error range of a particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, "about" can mean within 1 or more than 1 standard deviation, according to the practice in the art. Alternatively, "about" can mean within a range of up to 20%, up to 10%, up to 5%, or up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, preferably within 5-fold, and more preferably within 2-fold of a value. When a particular value is described in the present application and claims, unless otherwise stated, the term "about" should be assumed to mean within an acceptable error range of the particular value.

[0036] The terms "polypeptide", "oligopeptide", "peptide" and "protein" are used interchangeably herein to refer to polymers of amino acids of any length. The polymers may be linear or branched, may contain modified amino acids, and may be interrupted by non-amino acids. The term also encompasses amino acid polymers that are modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component. Also included within this definition are polypeptides that contain, for example, one or more analogs of amino acids (including, for example, unnatural amino acids, etc.) and other modifications known in the art. Because the polypeptides described herein are based on antibodies, it is understood that the polypeptides can occur as single chains or associated chains.

[0037] The term "amino acid" refers to natural, unnatural and synthetic amino acids, including, but not limited to, both D or L optical isomers, and amino acid analogs and peptidomimetics. Standard one-letter or three-letter codes are used to name the amino acids.

[0038] The term "naturally occurring L-amino acid" refers to the L optical isomeric forms of glycine (G), proline (P), alanine (A), valine (V), leucine (L), isoleucine (I), methionine (M), cysteine ​​(C), phenylalanine (F), tyrosine (Y), tryptophan (W), histidine (H), lysine (K), arginine (R), glutamine (Q), asparagine (N), glutamic acid (E), aspartic acid (D), serine (S), and threonine (T).

[0039] As applied to a sequence, the term "non-naturally occurring" as used herein refers to a polypeptide or polynucleotide sequence that has no counterpart, is not complementary to, or does not have a high degree of homology with, a wild-type or naturally occurring sequence found in a mammal, or that contains non-naturally occurring residues (e.g., nucleotide analogs). For example, a non-naturally occurring polypeptide or fragment can share 99%, 98%, 95%, 90%, 80%, 70%, 60%, 50% or less amino acid sequence identity compared to the native sequence when properly aligned.

[0040] The terms "hydrophilic" and "hydrophobic" refer to the degree of affinity that a substance has with water. A hydrophilic substance has a strong affinity for water and tends to dissolve, mix, or get wet with water, while a hydrophobic substance substantially lacks affinity for water and tends to repel and not absorb water, and tends not to dissolve, mix, or get wet with water. Amino acids can be characterized based on their hydrophobicity. Numerous scales have been developed. One example is the scale developed by Levitt, M, et al., J Mol Biol (1976) 104:59, as published in Hopp, TP, et al., Proc Natl Acad Sci USA (1981) 78:3824. Examples of "hydrophilic amino acids" are arginine, lysine, threonine, alanine, asparagine, and glutamine. The hydrophilic amino acids aspartic acid, glutamic acid, and serine, as well as glycine, are of particular interest. Examples of "hydrophobic amino acids" are tryptophan, tyrosine, phenylalanine, methionine, leucine, isoleucine, and valine.

[0041] A "fragment" as applied to a protein is a truncated form of a native biologically active protein that may or may not retain at least a portion of the therapeutic and / or biological activity. A "variant" as applied to a protein is a protein with sequence homology to a native biologically active protein that retains at least a portion of the therapeutic and / or biological activity of the biologically active protein. For example, a variant protein can share at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity compared to the reference biologically active protein. As used herein, the term "biologically active protein portion" includes proteins that have been modified, for example, by site-directed mutagenesis, synthesis of the encoding gene, insertion, or accidentally by mutation.

[0042] In the context of a polypeptide, a "linear sequence" or "sequence" is the order of amino acids in a polypeptide in the amino to carboxyl terminal direction, where residues adjacent to each other in the sequence are adjacent in the primary structure of the polypeptide. A "subsequence" is a linear sequence of a portion of a polypeptide that is known to contain additional residues in one or both directions.

[0043] "Polynucleotide" or "nucleic acid", as used interchangeably herein, refers to a polymer of nucleotides of any length, including DNA and RNA. The nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and / or their analogs, or any substrate that can be incorporated into a polymer by DNA or RNA polymerase. A polynucleotide can contain modified nucleotides, such as methylated nucleotides and their analogs. If present, modifications to the nucleotide structure can be imparted before or after assembly of the polymer. The sequence of nucleotides can be interrupted by non-nucleotide components. A polynucleotide can be further modified after polymerization, such as by conjugation with a labeling component. Other types of modifications include, for example, "caps", those with uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoramidates, carbamates, etc.) and charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.), those containing pendant moieties such as proteins (e.g., nucleases, toxins, antibodies, signal peptides, poly(ply)L-lysine, etc.), those containing intercalating agents (e.g., acridine, psoralens, etc.), those containing chelators (e.g., metals, radioactive metals, boron, metal oxides, etc.), those containing alkylating agents, those with modified linkages (e.g., alpha anomeric nucleic acids, etc.), as well as unmodified forms of the polynucleotide(s), analogs, substitutions of one or more of the naturally occurring nucleotides with internucleotide modifications. Additionally, any of the hydroxyl groups normally present on the sugar can be replaced, for example, by a phosphonate group, a phosphate group, protected by a standard protecting group, or activated to prepare additional linkages to additional nucleotides or conjugated to a solid support. The 5' and 3' terminal OH can be phosphorylated or substituted with an amine or organic capping group moiety of 1-20 carbon atoms. Other hydroxyls can also be derivatized to standard protecting groups.Polynucleotides may also contain analogous forms of ribose or deoxyribose sugars commonly known in the art, including, for example, 2'-O-methyl-, 2'-O-allyl, 2'-fluoro- or 2'-azido-ribose, carbocyclic sugar analogs, α-anomeric sugars, epimeric sugars such as arabinose, xylose or lyxose, pyranose sugars, furanose sugars, sedoheptulose, acyclic analogs, and abasic nucleoside analogs such as methyl riboside. One or more phosphodiester linkages may be replaced by alternative linking groups. These alternative linking groups include, but are not limited to, embodiments in which phosphate is replaced by P(O)S ("thioate"), P(S)S ("dithioate"), (O)NR2 ("amidate"), P(O)R, P(O)OR', CO, or CH2 ("formacetal"), where each R or R' is independently H or a substituted or unsubstituted alkyl (1-20C), aryl, alkenyl, cycloalkyl, cycloalkenyl, or araldyl, optionally containing an ether (-O-) linkage. Not all linkages in a polynucleotide need be identical. The preceding description applies to all polynucleotides referred to herein, including RNA and DNA.

[0044] "Fusion partner" refers to a peptide or polypeptide fused to a CTLA4 variant sequence described herein. The fusion partner may be fused to the CTLA4 variant sequence at the N-terminus and / or C-terminus. Exemplary fusion partners include, but are not limited to, albumin, transferrin, adnectins (e.g., albumin-binding adnectins or pharmacokinetics extending (PKE) adnectins), Fc domains, and unstructured polypeptides (e.g., XTEN and PAS polypeptides (e.g., conformationally disordered polypeptide sequences composed of amino acids Pro, Ala, and / or Ser)), or fragments of any of the foregoing. Fusion partners may be fused to modified CTLA4 variant sequences for any purpose, including, but not limited to, purification, manufacturability, half-life extension, enhanced biophysical properties (e.g., solubility or stability), reduced immunogenicity or toxicity, etc.

[0045] The "variable region" of an antibody refers to the variable region of an antibody light chain or the variable region of an antibody heavy chain, either alone or in combination. The variable regions of the heavy and light chains each consist of four framework regions (FRs) connected by three complementarity determining regions (CDRs), also known as hypervariable regions. The CDRs in each chain are held together in close proximity by the FRs and contribute, together with the CDRs from the other chain, to the formation of the antigen-binding site of the antibody. There are at least two techniques for determining CDRs: (1) an approach based on cross-species sequence variability (i.e., Kabat et al., Sequences of Proteins of Immunological Interest (5th ed., 1991, National Institutes of Health, Bethesda MD)); and (2) an approach based on crystallographic studies of antigen-antibody complexes (Al-lazikani et al. (1997) J. Molec. Biol. 273:927-948). As used herein, CDRs can refer to CDRs defined by either approach or a combination of both approaches.

[0046] A "constant region" of an antibody refers to the constant region of the antibody light chain or the constant region of the antibody heavy chain, either alone or in combination.

[0047] "Linker sequence" refers to an amino acid sequence that is fused at both its N-terminus and C-terminus to another peptide or polypeptide. The linker sequence may be present, for example, in a fusion protein, the termini of which are fused to the CTLA4 variant sequence and the fusion partner sequence (in either order). Exemplary linker sequences may contain between 0 amino acids (i.e., no linker sequence) and 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 60, or 100 amino acids, or more. In some embodiments, the linker sequence may comprise between 1-40, 1-30, 1-20, 1-10, 1-5, 2-40, 2-30, 2-20, 2-10, 5-40, 5-30, 5-20, or 5-10 amino acids. In some embodiments, the linker sequence may comprise between 5-20 amino acids. In some embodiments, the linker sequence may comprise between 10-20 amino acids. Certain exemplary linker sequences described herein may be rich in serine and glycine residues, although it should be understood that the linker sequences are not limited to such sequences.

[0048] "Host cell" includes an individual cell or cell culture that can be or has been the recipient of a vector(s) containing an exogenous polynucleotide. A host cell includes the progeny of a single host cell, and the progeny may not necessarily be completely identical (in morphology or genomic DNA complement) to the original parent cell due to natural, accidental or deliberate mutation. A host cell includes cells transfected in vivo with a polynucleotide(s) of the present disclosure.

[0049] The term "Fc region" is used to define the C-terminal region of an immunoglobulin heavy chain, which may be produced by papain digestion of an intact antibody. The Fc region described herein may be a native sequence Fc region or a variant Fc region. The Fc region described herein, in some cases, comprises two constant domains, a CH2 domain and a CH3 domain. Although the boundaries of the Fc region of an immunoglobulin heavy chain may vary, the human IgG heavy chain Fc region is usually defined as extending from an amino acid residue at position Cys226 or Pro230 to its carboxyl terminus. The numbering of residues in the Fc region is the EU index numbering as in Kabat. Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991. The Fc region of an immunoglobulin generally comprises two constant domains, CH2 and CH3.

[0050] A "native sequence Fc region" comprises an amino acid sequence identical to the amino acid sequence of an Fc region found in nature. A "variant Fc region" comprises an amino acid sequence that differs from the sequence of a native sequence Fc region by at least one amino acid modification. In some cases, the variant Fc region still retains at least one effector function of the native sequence Fc region. In other cases, the variant Fc region may not have the effector function of the native sequence Fc region. The variant Fc region may have at least one amino acid substitution, e.g., about 1 to about 10 amino acid substitutions, or about 1 to about 5 amino acid substitutions, in the native sequence Fc region or the Fc region of the parent polypeptide compared to the native sequence Fc region or the Fc region of the parent polypeptide. The variant Fc region herein may have at least about 80% sequence identity, or at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to a native sequence Fc region and / or the Fc region of a parent polypeptide.

[0051] An "individual" or "subject" is a mammal, more preferably a human. Mammals also include, but are not limited to, farm animals, sport animals, pets, primates, horses, dogs, cats, mice and rats.

[0052] As used herein, "vector" refers to a construct that can deliver one or more gene(s) or sequence(s) of interest to a host cell and preferably express them in the host cell. Examples of vectors include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmids, cosmids or phage vectors, DNA or RNA expression vectors associated with cationic condensing agents, DNA or RNA expression vectors encapsulated in liposomes, and certain eukaryotic cells such as production cells.

[0053] The term "effective amount" or "therapeutically effective amount" refers to an amount of an agent sufficient to produce a beneficial or desired result. The therapeutically effective amount may vary depending on one or more of the following: the subject and disease state being treated, the subject's weight and age, the severity of the disease state, the mode of administration, etc., which can be easily determined by one of ordinary skill in the art. An effective amount of an active agent can be administered in a single dose or multiple doses. A component can be described herein as having at least an effective amount, or at least an amount effective to produce a desired result, such as an amount associated with a particular goal or purpose, such as any described herein.

[0054] The term "effective amount" also applies to a dose that will result in an image for detection by an appropriate imaging method. The specific dose may vary depending on one or more of the following: the particular agent selected, the dosing regimen to be followed, whether it is administered in combination with other compounds, the timing of administration, the tissue to be imaged, and the physical delivery system that carries it.

[0055] As used herein, "pharmaceutical acceptable carrier" or "acceptable pharmaceutical excipient" includes any material that, when combined with an active ingredient, allows the ingredient to retain biological activity and is non-reactive with the subject's immune system. Examples include, but are not limited to, any of the standard pharmaceutical carriers, such as phosphate buffered saline solution, water, emulsions such as oil / water emulsions, and various types of wetting agents. The preferred diluent for aerosol or parenteral administration is phosphate buffered saline or normal (0.9%) saline. Compositions that include such carriers are formulated by well-known conventional methods (see, for example, Remington's Pharmaceutical Sciences, 18th Edition, A. Gennaro, ed., Mack Publishing Co., Easton, PA, 1990; and Remington, The Science and Practice of Pharmacy, 20th Edition, Mack Publishing, 2000).

[0056] Overview

[0057] In one aspect, the disclosure provides polypeptides comprising CTLA4 (cytotoxic T-lymphocyte-associated antigen 4) variant sequences with improved target binding affinity, e.g., modified CTLA4 fusion proteins, compositions and kits comprising the polypeptides, and methods of use thereof.

[0058] T cell lymphocytes play an important role in cell-mediated immunity by providing adaptive responses against specific pathogens. In some cases, T helper cell antigenic responses require the activation of a first signaling pathway by the binding of the T cell receptor to an antigen bound to MHC (major histocompatibility complex) on the surface of an antigen-presenting cell (APC), during which the response also requires the activation of a second signaling pathway (which can produce a costimulatory signal) by the binding of CD28 protein on the T cell surface to CD80 (B7-1) and CD86 (B7-2) on the surface of the APC. The costimulatory pathway mediated by the binding of CD28 to CD80 and CD86 on the surface of the APC can play a role in T cell activation and differentiation, which can also be important in tissue migration and peripheral tolerance induction. Activated T cells can also express CTLA4 (a homolog of CD28 that can bind with higher affinity to CD80 and CD86) on their cell surface. In some cases, CTLA4 expression leads to competitive binding to CD80 and CD86, blocking the CD80 / 86-CD28 interaction and terminating T cell activation. In some cases, these signaling pathways determine the magnitude of the T cell response to an antigen and downstream responses to the antigen, and agents that modulate one or more costimulatory signals, for example, by blocking one or more of the interactions between CD80 / CD86 and CD28 and / or CTLA4, can be effective in treating disorders resulting from a dysregulated immune response.

[0059] In some embodiments, polypeptides comprising CTLA4 variant sequences are provided herein with improved binding affinity to CD80 or CD86. In some embodiments, polypeptides comprising CTLA4 variant sequences provided herein have improved immunosuppressive activity, for example, enhanced inhibitory effect on T cell activation. Without wishing to be bound by a particular theory, in some embodiments, improved binding affinity to CD80 or CD86 is at least partially the result of mutations at one or more positions compared to native human CTLA4 protein having the amino acid sequence of SEQ ID NO: 1. In some embodiments, the subject polypeptide sequence comprises mutations at one or more positions in the binding domain for CD80 or CD86 compared to native CTLA4 protein (SEQ ID NO: 1), thereby potentially inducing a structural change that affects binding affinity to its ligand CD80 or CD86.

[0060] In some embodiments, the present specification provides a polypeptide that is a fusion protein that includes a CTLA4 variant sequence fused to a fusion partner sequence.In some embodiments, without wishing to be bound by a particular theory, the selection of the fusion partner sequence described herein contributes to various advantages (e.g., stability, solubility, deliverability, bioavailability, or productivity) of the subject polypeptide compared to native CTLA4 protein (SEQ ID NO:1) or some other CTLA4 fusion proteins (e.g., abatacept having the amino acid sequence of SEQ ID NO:2 or belatacept having the amino acid sequence of SEQ ID NO:3).

[0061] Sequence identity

[0062] Two polynucleotide or polypeptide sequences are said to be "identical" if the sequence of nucleotides or amino acids in the two sequences are the same when aligned for maximum correspondence as described below. Comparisons between two sequences are typically performed by comparing the sequences over a comparison window to identify and compare local regions of sequence similarity.

[0063] Optimal alignment of sequences for comparison can be performed using the Megalign program in the Lasergene suite of bioinformatics software (DNASTAR, Inc., Madison, Wis.) using default parameters. This program embodies several alignment schemes described in the following references: Dayhoff, MO (1978) A model of evolutionary change in proteins - Matrices for detecting distant relationships. In Dayhoff, MO (ed.) Atlas of Protein Sequence and Structure, National Biomedical Research Foundation, Washington DC Vol. 5, Suppl. 3, pp. 345-358; Hein J., 1990, Unified Approach to Alignment and Phylogenes pp. 626-645 Methods in Enzymology vol. 183, Academic Press, Inc., San Diego, CA; Higgins, DG and Sharp, PM, 1989, CABIOS 5:151-153; Myers, EW and Muller W., 1988, CABIOS 4:11-17; Robinson, ED, 1971, Comb. Theor. 11:105; Santou, N., Nes, M., 1987, Mol. Biol. Evol. 4:406-425; Sneath, PHA and Sokal, RR, 1973, Numerical Taxonomy the Principles and Practice of Numerical Taxonomy, Freeman Press, San Francisco, CA; Wilbur, WJ and Lipman, DJ, 1983, Proc. Natl. Acad. Sci. USA 80:726-730.Alternative alignment programs can be utilized, including but not limited to the BLAST algorithm, which can also be used to assess sequence identity, such as by using default parameters.

[0064] Preferably, the "percentage of sequence identity" is determined by comparing two optimally aligned sequences over a window of comparison (e.g., of at least 20 positions), where the portion of the polynucleotide or polypeptide sequence in the comparison window can contain additions or deletions (i.e., gaps) compared to the reference sequence (which contains no additions or deletions) due to optimal alignment of the two sequences, such as gaps of 20 percent or less (e.g., 5-15 percent or 10-12 percent). The percentage is typically calculated by determining the number of positions where the same nucleic acid base or amino acid residue occurs in both sequences to obtain the number of matched positions, dividing the number of matched positions by the total number of positions in the reference sequence (i.e., the window size), and multiplying this result by 100 to obtain the percentage of sequence identity.

[0065] The sequence identity for the amino acid sequences identified herein is defined as the percentage of amino acid residues in a query sequence that are identical to the amino acid residues of a second reference polypeptide sequence or portion thereof, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, without considering any conservative substitutions as part of the sequence identity. Alignment for the purpose of determining percent amino acid sequence identity can be achieved in various ways within the skill of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine the appropriate parameters for measuring alignment, including any algorithm required to achieve maximum alignment over the entire length of the sequences being compared. Percent identity can be measured over the entire length of the defined polypeptide sequence, or over a shorter length, for example, over the length of a fragment obtained from a larger defined polypeptide sequence, for example, a fragment of at least 15, at least 20, at least 30, at least 40, at least 50, at least 70 or at least 150 contiguous residues. It is understood that such lengths are merely exemplary and any fragment length supported by the sequences shown herein in the tables, figures, or sequence listing may be used to represent lengths for which percentage identity can be measured. In some embodiments, percent identity is determined with respect to the full length of the reference sequence described, such as the sequences provided herein. For example, sequence comparison between two amino acid sequences of the present disclosure (or shorter lengths thereof) can be performed by the computer program Blastp (protein-protein BLAST), provided online by the National Center for Biotechnology Information (NCBI). The percentage amino acid sequence identity of a given amino acid sequence A to a given amino acid sequence B (which can alternatively be expressed as a given amino acid sequence A having a certain % amino acid sequence identity to a given amino acid sequence B) is calculated by the following formula:

number

[0066] CTLA4 variants

[0067] The present disclosure provides compositions comprising polypeptides capable of binding to CD80 or CD86, their uses, and methods of making them. In one aspect, the present disclosure provides polypeptides comprising amino acid sequences that are variants of CTLA4 (CTLA4 variant sequences). The variants of CTLA4 provided herein can be polypeptides comprising sequences related to native CTLA4 sequences. A variant of CTLA4 is a polypeptide comprising a sequence having about 50% or more, about 55% or more, about 60% or more, about 65% or more, about 70% or more, about 75% or more, about 80% or more, about 85% or more, about 90% or more, about 95% or more, about 96% or more, about 97% or more, about 98% or more, about 99% or more, or about 99.5% or more sequence identity to at least a portion of the native CTLA4 sequence (SEQ ID NO:1).

[0068] The native CTLA4 protein can bind to CD80 and CD86 through its extracellular domain or, more specifically, its binding domain that can form a three-dimensional structure that receives and binds CD80 or CD86. The variants of CTLA4 provided herein can have the ability to bind to CD80, CD86, or both through at least a portion of the amino acid sequence for the extracellular domain or binding domain of the native CTLA4 protein. In some cases, a variant of CTLA4 is a polypeptide that includes a sequence having about 50% or more, about 55% or more, about 60% or more, about 65% or more, about 70% or more, about 75% or more, about 80% or more, about 85% or more, about 90% or more, about 95% or more, about 96% or more, about 97% or more, about 98% or more, about 99% or more, or about 99.5% or more sequence identity to part or all of the extracellular domain of a native CTLA4 sequence, e.g., amino acids 36-161 of SEQ ID NO:1, or amino acids 1-124 of abatacept (SEQ ID NO:2). In some cases, a variant of CTLA4 is a polypeptide that includes a sequence having about 50% or more, about 55% or more, about 60% or more, about 65% or more, about 70% or more, about 75% or more, about 80% or more, about 85% or more, about 90% or more, about 95% or more, about 96% or more, about 97% or more, about 98% or more, about 99% or more, or about 99.5% or more sequence identity to the binding domain of a native CTLA4 sequence, e.g., amino acids 58-149 of SEQ ID NO:1, or amino acids 21-112 of SEQ ID NO:2.

[0069] The extracellular domain of the native CTLA4 protein may comprise a sequence as amino acids 36-161 of SEQ ID NO:1 or amino acids 1-124 of SEQ ID NO:2, and the binding domain of the native CTLA4 protein may comprise an amino acid sequence beginning anywhere between amino acids 1-21 and ending anywhere between amino acids 112-124. In some cases, variants of CTLA4 provided herein may include an amino acid sequence having about 50% or more, about 55% or more, about 60% or more, about 65% or more, about 70% or more, about 75% or more, about 80% or more, about 85% or more, about 90% or more, about 95% or more, about 96% or more, about 97% or more, about 98% or more, about 99% or more, or about 99.5% or more sequence identity to an amino acid sequence beginning anywhere between amino acids 1-8, between 8-16, or between 16-21 and ending anywhere between amino acids 112-124 of SEQ ID NO:2. In some cases, a variant of CTLA4 provided herein may comprise an amino acid sequence having about 50% or more, about 55% or more, about 60% or more, about 65% or more, about 70% or more, about 75% or more, about 80% or more, about 85% or more, about 90% or more, about 95% or more, about 96% or more, about 97% or more, about 98% or more, about 99% or more, or about 99.5% or more sequence identity to an amino acid sequence beginning anywhere between amino acids 1-21 and ending anywhere between amino acids 112-115, 115-118, 118-121, or 121-124 of SEQ ID NO:2.In some cases, a variant of CTLA4 provided herein may comprise an amino acid sequence having about 50% or more, about 55% or more, about 60% or more, about 65% or more, about 70% or more, about 75% or more, about 80% or more, about 85% or more, about 90% or more, about 95% or more, about 96% or more, about 97% or more, about 98% or more, about 99% or more, or about 99.5% or more sequence identity to an amino acid sequence beginning anywhere between amino acids 1-8, between 8-16, or between 16-21 and ending anywhere between amino acids 112-115, between 115-118, between 118-121, or between 121-124 of SEQ ID NO:2.

[0070] As provided herein, a variant of CTLA4 can include an amino acid sequence having one or more mutations compared to at least a portion of a native CTLA4 sequence, e.g., SEQ ID NO:1, e.g., the extracellular domain or binding domain of a native CTLA4 sequence, e.g., amino acids 36-161 of SEQ ID NO:1 or amino acids 1-124 of SEQ ID NO:2, or amino acids 56-147 of SEQ ID NO:1 or amino acids 21-112 of SEQ ID NO:2. In some cases, a variant of CTLA4 has at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, or 50 mutations compared to at least a portion of a native CTLA4 sequence, e.g., amino acids 36-161 of SEQ ID NO:1, amino acids 1-124 of SEQ ID NO:2, amino acids 56-147 of SEQ ID NO:1, or amino acids 21-112 of SEQ ID NO:2. In some cases, a CTLA4 variant has up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, up to 9, up to 10, up to 15, up to 20, up to 25, up to 30, up to 35, up to 40, or up to 50 mutations compared to at least a portion of a native CTLA4 sequence, e.g., amino acids 36-161 of SEQ ID NO:1, amino acids 1-124 of SEQ ID NO:2, amino acids 56-147 of SEQ ID NO:1, or amino acids 21-112 of SEQ ID NO:2.

[0071] mutation

[0072] In some embodiments provided herein, the subject polypeptide described herein can have one or more mutations with respect to at least a portion of a reference sequence, e.g., a CTLA4 protein. The mutations can be deletions, insertions or additions, or replacements or substitutions of amino acid residues. "Deletion" refers to a change in an amino acid sequence due to the absence of one or more amino acid residues. "Insertion" or "addition" refers to a change in an amino acid sequence resulting in the addition of one or more amino acid residues compared to a reference sequence. "Replacement" or "substitution" refers to the replacement of one or more amino acids with different amino acids. In the context of the present disclosure, the mutations of a subject polypeptide with respect to at least a portion of a reference sequence, e.g., a native CTLA4 protein, can be determined by comparison of the reference sequence with the subject polypeptide or a fragment thereof. Optimal alignment of sequences for comparison can be performed according to any of the methods known in the art.

[0073] Mutation can be identified by mutation site. Mutation site is the position in the reference sequence where deletion, addition or substitution occurs. The amino acid residues in the reference sequence are numbered from N-terminus to C-terminus, and mutation site is the numbering of the amino acid residue where deletion, addition or substitution occurs. For example, position 26 in the reference sequence is the position where the 26th amino acid residue is located starting from N-terminus.

[0074] In the context of this disclosure, the addition of one or more amino acid residues between a particular position and the position immediately following the particular position (or after the particular position when the amino acid residue at the particular position is the last amino acid residue) on a reference sequence is considered as a substitution of one amino acid residue at that particular position with more than one amino acid residue. For example, a variant amino acid sequence XYYZ is considered to have a substitution of Y at the second position with YY when compared with a reference amino acid sequence XYZ (where X, Y, and Z each represent an individual amino acid residue). Thus, in the context of this disclosure, a mutation at a particular position is intended to mean a deletion of one amino acid residue at that particular position or a substitution of one amino acid residue at that particular position with another amino acid residue or with more than one amino acid residue.

[0075] For the purpose of describing mutations relative to a reference sequence, one-letter amino acid codes can be used. In this regard, for example, when a subject polypeptide is said to include a G to I mutation at position 26, which may be described as "G26I" relative to a reference sequence, it is intended to mean that the 26th amino acid residue, which is a glycine (G) residue according to the reference sequence, is replaced by an alanine residue in the subject polypeptide or portion thereof. In the context of the present disclosure, for example, when a subject polypeptide is said to include a deletion of a glycine (G) residue at position 26, which may be described as "G26del" relative to a reference sequence, it is intended to mean that the 26th amino acid residue, which is a glycine (G) residue according to the reference sequence, is not present in the subject polypeptide or portion thereof. In the context of the present disclosure, for example, when a subject polypeptide is said to include one or more additional amino acid residues after a glycine (G) residue at position 26, which may be set forth as a list of the added amino acid residues followed by "G26_ins," it is intended to mean that the listed one or more amino acid residues are added between the 26th and 27th amino acid residues, which are glycine (G), or after the 26th amino acid residue, which is glycine (G) (if the 26th amino acid residue according to the reference sequence is the last amino acid residue).

[0076] In some embodiments, a subject polypeptide comprises a CTLA4 variant sequence having one or more mutations relative to amino acids 1-124 of SEQ ID NO: 2. In some embodiments, a CTLA4 variant sequence in a subject polypeptide comprises a mutation at any one or more positions relative to amino acids 1-124 of SEQ ID NO: 2. In some embodiments, a CTLA4 variant sequence in a subject polypeptide comprises a mutation at any one or more positions relative to amino acids 1-124 of SEQ ID NO:2, such as positions 16, 18, 24, 25, 27, 28, 29, 30, 32, 33, 40, 41, 42, 48, 49, 50, 51, 52, 53, 54, 56, 58, 59, 60, 61, 63, 64, 65, 68, 69, 70, 77, 80, 85, 86, 92, 93, 94, 96, 105, 106, 107, 122, 117, 118, or any combination thereof. In some embodiments, the CTLA4 variant sequence in a subject polypeptide comprises a mutation at any one or more positions relative to amino acids 1-124 of SEQ ID NO:2, such as positions 18, 24, 27, 29, 33, 40, 41, 49, 50, 51, 68, 77, 86, 92, 93, 94, 105, 107, 122, 117, 118, or any combination thereof. In some embodiments, the CTLA4 variant sequence in a subject polypeptide comprises a mutation at any one or more positions relative to amino acids 1-124 of SEQ ID NO:2, such as positions 18, 40, 68, 77, 86, 92, 107, 117, 118, and 122.

[0077] In some embodiments, a CTLA4 variant sequence in a subject polypeptide comprises any type of mutation at any one or more positions relative to amino acids 1-124 of SEQ ID NO: 2. For example, a CTLA4 variant sequence in a subject polypeptide can have a deletion at any one or more positions relative to amino acids 1-124 of SEQ ID NO: 2. In some embodiments, the CTLA4 variant sequence in the subject polypeptide comprises a deletion at any one or more positions relative to amino acids 1-124 of SEQ ID NO:2, such as positions 16, 18, 24, 25, 27, 28, 29, 30, 32, 33, 40, 41, 42, 48, 49, 50, 51, 52, 53, 54, 56, 58, 59, 60, 61, 63, 64, 65, 68, 69, 70, 77, 80, 85, 86, 92, 93, 94, 96, 105, 106, 107, 122, 117, 118, or any combination thereof. The CTLA4 variant sequence in the subject polypeptide comprises a substitution of any type at any one or more positions relative to amino acids 1-124 of SEQ ID NO:2. In some embodiments, a CTLA4 variant sequence in a subject polypeptide comprises any type of substitution at any one or more positions relative to amino acids 1-124 of SEQ ID NO:2, such as positions 16, 18, 24, 25, 27, 28, 29, 30, 32, 33, 40, 41, 42, 48, 49, 50, 51, 52, 53, 54, 56, 58, 59, 60, 61, 63, 64, 65, 68, 69, 70, 77, 80, 85, 86, 92, 93, 94, 96, 105, 106, 107, 122, 117, 118, or any combination thereof. In some embodiments, the CTLA4 variant sequence in the subject polypeptide comprises any type of substitution at any one or more positions relative to amino acids 1-124 of SEQ ID NO:2, such as positions 18, 24, 27, 29, 33, 40, 41, 49, 50, 51, 68, 77, 86, 92, 93, 94, 105, 107, 122, 117, 118, or any combination thereof.In some embodiments, the CTLA4 variant sequence in the subject polypeptide comprises any type of substitution at any one or more positions relative to amino acids 1-124 of SEQ ID NO:2, such as positions 18, 40, 68, 77, 86, 92, 107, 117, 118, and 122.

[0078] In some embodiments, the CTLA4 variant sequence in a subject polypeptide is selected from the group consisting of S18R, S18N, A24S, A24E, G27D, G27DK, G27DKA, G27E, G27H, G27K, G27KK, G27R, G27W, G27Y, A29S, A29T, A29H, A29Y, T30N, V32I, R33W, A40T, D41G, D41N, A49P, A50T, A50M, T51N, M53Y, M54K, N56D, L57R, L58R, L59R, L60R, L61R, L62R, L63R, L64R, L65R, L66R, L67R, L68R, L69R, L70R, L72R, L74R, L76R, L78R, L79 ... and / or any suitable combination thereof. In some embodiments, a CTLA4 variant sequence in a subject polypeptide comprises one or more amino acid substitutions with respect to amino acids 1-124 of SEQ ID NO:2, such as S18R, S18N, A24S, G27DKA, G27DK, G27K, G27R, G27W, G27Y, G27E, G27KK, A29T, A40T, A49P, A50T, G68F, G68K, G68W, G68Y, G68H, G68D, G68E, L77V, D86N, C92S, C92Y, K93V, K93W, K93P, K93C, K93F, K93R, V94L, G105S, G107D, P117S, E118K, D122H, or any suitable combination thereof. As noted above, a CTLA4 variant sequence in a subject polypeptide may have any type of mutation at any one or more positions. For example, the amino acid G at position 27 relative to amino acids 1-124 of SEQ ID NO:2 may be substituted with any type of amino acid or group of amino acid residues, such as D, DK, DKA, E, H, K, KK, R, W, or Y, or the amino acid K at position 93 relative to amino acids 1-124 of SEQ ID NO:2 may also be substituted with any type of amino acid or group of amino acid residues, such as N, F, M, V, W, P, C, F, or R.

[0079] In some embodiments, the CTLA4 variant sequence in a subject polypeptide can have any combination of mutations for amino acids 1-124 of SEQ ID NO: 2. For example, the CTLA4 variant sequence in a subject polypeptide can have any one of the mutations in Table 5, e.g., G27DKA / R33W, G27DKA / G68F, R33W / G68F, G27R / G68Y, G27H / G68F, G27DK / G68F, G27DK / G68D, G27DK / G68E, G27D / G68F, G27E / G68F, G27H / G68D, G27H / G68E, G27DKA / G68F, G27DK / G68F, G27DK / G68D, G27DK ...F, G27DK / G68D, G27DK / G68E, G27DKA / G68F, G27DK / G68F, G27DK / G68F, G27DK / 68F, G27H / G68F, G27DK / G68F, G27DKA / G68F / D122H, G27DKA / G68F / A40T / D122H, G27DKA / G68F / A40T / P117S, G2 7DKA / G68F / L77V, G27DKA / G68F / C92S / K93M, G27DK / G68F / A49P / A50T, G27DK / G68F / A40T / D86N / G105S, G27KK / G68F, G27DKA / G68F / P117S, G27DKA / G68F / A49P / A50T, G27DK / G68F / A40T, G27DK / G68F / L77V / G105S / P117S, G2 7DK / G68F / L77V, G27DK / G68F / C92S / K93M, G27DK / G68F / P117S, G27DKA / G68F / G105S, G27DKA / G68F / D86N, G27D The CTLA4 variant sequence in the subject polypeptide may have any combination of mutations with respect to amino acids 1-124 of SEQ ID NO:2, such as K / G68F / D122H, G27DK / G68F / A40T / G105S, G27DK / G68F / G105S, G27DK / G68F / D86N, G27DKA / G68F / A40T, G27DKA / G68F / K93M, G27DK / G68F / K93M, or G27DKA / A40T / G68F / K93M. In some embodiments, the CTLA4 variant sequence in the subject polypeptide comprises the amino acid substitution combination G27DKA / A40T / G68F / K93M with respect to SEQ ID NO:2. In some embodiments, the CTLA4 variant sequence in the subject polypeptide comprises the amino acid substitution combination G27DKA / G68F / A40T with respect to SEQ ID NO:2.In some embodiments, the CTLA4 variant sequence in the subject polypeptide comprises the amino acid substitution combination G27DKA / G68F / K93M with respect to SEQ ID NO:2. In some embodiments, the CTLA4 variant sequence in the subject polypeptide comprises the amino acid substitution combination G27DKA / G68F / A40T / P117S with respect to SEQ ID NO:2. In some embodiments, the CTLA4 variant sequence in the subject polypeptide comprises the amino acid substitution combination G27DKA / G68F / P117S with respect to SEQ ID NO:2. In some embodiments, the CTLA4 variant sequence in the subject polypeptide comprises the amino acid substitution combination G27H / G68F with respect to SEQ ID NO:2. In some embodiments, the CTLA4 variant sequence in the subject polypeptide comprises the amino acid substitution combination G27DKA / G68F with respect to SEQ ID NO:2. In some embodiments, the CTLA4 variant sequence in the subject polypeptide comprises the amino acid substitution combination G27DK / G68F / K93M with respect to SEQ ID NO:2. In some embodiments, the CTLA4 variant sequence in the subject polypeptide comprises the amino acid substitution combination G27DK / G68F / A40T with respect to SEQ ID NO:2. In some embodiments, the CTLA4 variant sequence in the subject polypeptide comprises the amino acid substitution combination G27DK / G68F / L77V / G105S / P117S with respect to SEQ ID NO:2. In some embodiments, the CTLA4 variant sequence in the subject polypeptide comprises the amino acid substitution combination G27DK / G68F / L77V with respect to SEQ ID NO:2. In some embodiments, the CTLA4 variant sequence in the subject polypeptide comprises the amino acid substitution combination G27DK / G68F / P117S with respect to SEQ ID NO:2. In some embodiments, the CTLA4 variant sequence in the subject polypeptide comprises the amino acid substitution combination G27DK / G68F / D122H with respect to SEQ ID NO:2.

[0080] In some embodiments, a CTLA4 variant sequence in a subject polypeptide comprises an amino acid sequence having about 70% or more, about 75% or more, about 80% or more, about 85% or more, about 90% or more, about 95% or more, about 96% or more, about 97% or more, about 98% or more, about 99% or more, about 99.5% or more, or 100% sequence identity to amino acids 1-126 of SEQ ID NO:6.

[0081] binding affinity

[0082] As described above, the subject polypeptide provided herein comprises a CTLA4 binding domain and can bind to CD80, CD86, or both.The subject polypeptide can typically exhibit high binding affinity to CD80, CD86, or both.In some embodiments, the CD80 is human CD80.In some embodiments, the CD86 is human CD86.

[0083] The binding affinity of molecules to CD80 or CD86 in solution or immobilized on an array can be detected using detection techniques known in the art. Examples of such techniques include immunological techniques such as competitive binding assays and sandwich assays; fluorescence detection using instruments such as confocal scanners, confocal microscopes or CCD-based systems, and techniques such as fluorescence, fluorescence polarization (FP), fluorescence resonance energy transfer (FRET), total internal reflection fluorescence (TIRF), fluorescence correlation spectroscopy (FCS); colorimetric / spectrophotometric techniques; surface plasmon resonance (SPR), where the change in mass of a material adsorbed on a surface is measured; radioisotope-based techniques, including conventional radioisotope binding and scintillation proximity assays (SPA). mass spectrometry such as matrix-assisted laser desorption / ionization mass spectrometry (MALDI) and MALDI-time-of-flight (TOF) mass spectrometry; ellipsometry, an optical method for measuring the thickness of protein films; quartz crystal microbalance (QCM), an ultrasensitive method for measuring the mass of material adsorbed to a surface; scanning probe microscopy such as atomic force microscope (AFM), scanning force microscope (SFM) or scanning electron microscope (SEM); and techniques such as electrochemical, impedance, acoustic, microwave and IR / Raman detection.See, e.g., Mere L, et al., "Miniaturized FRET assays and microfluidics: key components for ultra-high-throughput screening", Drug Discovery Today 4(8):363-369 (1999) and references cited therein; Lakowicz JR, Principles of Fluorescence Spectroscopy, 2nd ed., Plenum Press (1999), or Jain KK: Integrative Omics, Pharmacoproteomics, and Human Body Fluids. In: Thongboonkerd V, ed., ed. Proteomics of Human Body Fluids: Principles, Methods and Applications. Volume 1: Totowa, NJ: Humana Press, 2007, each of which is incorporated by reference in its entirety.

[0084] In some embodiments provided herein, the binding affinity of the subject polypeptide to CD80 or CD86 is measured by surface plasmon resonance. A Biacore® surface plasmon resonance (SPR) system (GE Healthcare, Chicago IL) may be used to measure the binding affinity of the subject polypeptide. Exemplary SPR analysis systems include, but are not limited to, Biacore X100, Biacore T200, Biacore 3000 or Biacore 4000 instruments, and commercial sensor chip series. In a typical application of the Biacore system, the interaction kinetics is analyzed by monitoring the interaction as a function of time over a range of analyte concentrations, and then fitting the entire data set to a mathematical model that describes the interaction. The association phase (during sample injection) contains information about both the association and dissociation processes, whereas only dissociation occurs in the dissociation phase (after sample injection, when buffer flow removes dissociated analyte molecules). A person skilled in the art can select or determine the appropriate parameters and / or conditions for performing a binding affinity assay according to the manufacturer's manual. In some embodiments, the binding affinity of the subject polypeptide is determined by surface plasmon resonance at 37° C. In some embodiments, the binding affinity of the subject polypeptide is determined by surface plasmon resonance at room temperature, e.g., at about 22-25° C. In some embodiments, the binding affinity of the subject polypeptide is determined by surface plasmon resonance at a temperature of 37° C. or less.

[0085] In one aspect, the disclosure provides a polypeptide having a high binding affinity to CD80, CD86, or both. In some embodiments, the polypeptide has a binding affinity to CD80, CD86, or both that is greater than that of abatacept (e.g., SEQ ID NO: 2). For example, the polypeptides provided herein have a binding affinity for CD80, CD86, or both that is at least 1.1 fold, at least 1.2 fold, at least 1.3 fold, at least 1.4 fold, at least 1.5 fold, at least 1.6 fold, at least 1.7 fold, at least 1.8 fold, at least 1.9 fold, at least 2 fold, at least 2.2 fold, at least 2.4 fold, at least 2.6 fold, at least 2.8 fold, at least 3 fold, at least 3.5 fold, at least 4 fold, at least 4.5 fold, at least 5 fold, at least 6 fold, at least 7 fold, at least 8 fold, at least 9 fold, at least 10 fold, at least 12 fold, at least 15 fold, at least 20 fold, at least 25 fold, at least 30 fold, at least 40 fold, at least 50 fold, at least 100 fold, at least 150 fold, at least 200 fold, at least 250 fold, or at least 300 fold greater than that of abatacept (SEQ ID NO:2). In the context of the present disclosure, binding affinities for CD80, CD86, or both, are compared between two different molecules when determined by the same binding assay under the same experimental conditions, e.g., when determined by surface plasmon resonance at 37°C.

[0086] In some embodiments, the polypeptides provided herein have improved binding affinity for CD80 compared to SEQ ID NO: 2, but relatively similar or lower binding affinity for CD86 compared to SEQ ID NO: 2. In some embodiments, the polypeptides provided herein have improved binding affinity for CD86 compared to SEQ ID NO: 2, but relatively similar or lower binding affinity for CD80 compared to SEQ ID NO: 2. In some embodiments, the polypeptides provided herein have improved binding affinity for both CD80 and CD86 compared to SEQ ID NO: 2.

[0087] In some embodiments, the polypeptide has a binding affinity for CD80, CD86, or both that is greater than that of belatacept (e.g., SEQ ID NO: 3). For example, the polypeptides provided herein have a binding affinity for CD80, CD86, or both that is at least 1.1 times, at least 1.2 times, at least 1.3 times, at least 1.4 times, at least 1.5 times, at least 1.6 times, at least 1.7 times, at least 1.8 times, at least 1.9 times, at least 2 times, at least 2.2 times, at least 2.4 times, at least 2.6 times, at least 2.8 times, at least 3 times, at least 3.5 times, at least 4 times, at least 4.5 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times, at least 12 times, at least 15 times, at least 20 times, at least 25 times, at least 30 times, at least 40 times, at least 50 times, or at least 100 times greater than that of belatacept (SEQ ID NO: 3).

[0088] In some embodiments, the polypeptide has a binding affinity for CD80, CD86, or both that is greater than that of SEQ ID NO: 4, 5, or both. For example, the polypeptide has a binding affinity for CD80, CD86, or both that is at least 1.1 times, at least 1.2 times, at least 1.3 times, at least 1.4 times, at least 1.5 times, at least 1.6 times, at least 1.7 times, at least 1.8 times, at least 1.9 times, at least 2 times, at least 2.2 times, at least 2.4 times, at least 2.6 times, at least 2.8 times, at least 3 times, at least 3.5 times, at least 4 times, at least 4.5 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times, at least 12 times, at least 15 times, at least 20 times, at least 25 times, at least 30 times, at least 40 times, at least 50 times, or at least 100 times greater than that of SEQ ID NO: 4, 5, or both.

[0089] In another aspect, the disclosure provides polypeptides having a low binding affinity to CD80, CD86, or both. In some embodiments, the polypeptides have a binding affinity to CD80, CD86, or both that is lower than that of SEQ ID NO:2. For example, the polypeptides provided herein have a binding affinity for CD80, CD86, or both that is at least 1.1 fold, at least 1.2 fold, at least 1.3 fold, at least 1.4 fold, at least 1.5 fold, at least 1.6 fold, at least 1.7 fold, at least 1.8 fold, at least 1.9 fold, at least 2 fold, at least 2.2 fold, at least 2.4 fold, at least 2.6 fold, at least 2.8 fold, at least 3 fold, at least 3.5 fold, at least 4 fold, at least 4.5 fold, at least 5 fold, at least 6 fold, at least 7 fold, at least 8 fold, at least 9 fold, at least 10 fold, at least 12 fold, at least 15 fold, at least 20 fold, at least 25 fold, at least 30 fold, at least 40 fold, at least 50 fold, or at least 100 fold greater than that of SEQ ID NO:2.

[0090] The binding affinity of a subject polypeptide to CD80 or CD86 is k a , k d or K D The term "k a As used herein, the term "k" may refer to the rate constant for the association of a polypeptide to an antigen. d " as used herein may refer to the rate constant for dissociation of a polypeptide from a protein-protein complex. D " as used herein may refer to the equilibrium dissociation constant of a protein-protein interaction. For purposes of this disclosure, K D is the ratio of the two kinetic rate constants k d / k a The smaller the equilibrium dissociation constant, the tighter the subject polypeptide and CD80 or CD86 bind to each other.

[0091] In some embodiments, the polypeptides disclosed herein are at least 102 M -1 s -1 , at least 5 × 10 2 M -1 s -1 , at least 10 3 M -1 s -1 , at least 5 × 10 3 M -1 s -1 , at least 10 4 M -1 s -1 , at least 5 × 10 4 M -1 s -1 , at least 10 5 M -1 s -1 , at least 5 × 10 5 M -1 s -1 , at least 10 6 M -1 s -1 , at least 5 × 10 6 M -1 s -1 , at least 10 7 M -1 s -1 , at least 5 × 10 7 M -1 s -1 , at least 10 8 M -1 s -1 , at least 5 × 10 8 M -1 s -1 , at least 10 9 M -1 s -1 , at least 5 × 10 9 M -1 s -1 k a or any range between any two of these values a In some embodiments, the polypeptides disclosed herein bind to CD80 at 10 5 M -1 s -1 ~10 5 M -1 s -1 Between 5×10 5 M-1 s -1 ~1×10 6 M -1 s -1 Between 7.5×10 5 M -1 s -1 ~2.5×10 6 M -1 s -1 Between 1×10 5 M -1 s -1 ~5×10 6 M -1 s -1 k between a Binds to CD80 at

[0092] In some embodiments, the polypeptides disclosed herein are at least 10 2 M -1 s -1 , at least 5 × 10 2 M -1 s -1 , at least 10 3 M -1 s -1 , at least 5 × 10 3 M -1 s -1 , at least 10 4 M -1 s -1 , at least 5 × 10 4 M -1 s -1 , at least 10 5 M -1 s -1 , at least 5 × 10 5 M -1 s -1 , at least 10 6 M -1 s -1 , at least 5 × 10 6 M -1 s -1 , at least 10 7 M -1 s -1 , at least 5 × 10 7 M -1 s -1 , at least 10 8 M -1 s-1 , at least 5 × 10 8 M -1 s -1 , at least 10 9 M -1 s -1 , at least 5 × 10 9 M -1 s -1 k a or any range between any two of these values a In some embodiments, the polypeptides disclosed herein bind to CD86 at 10 5 M -1 s -1 ~10 5 M -1 s -1 Between 5×10 5 M -1 s -1 ~1×10 6 M -1 s -1 Between 7.5×10 5 M -1 s -1 ~2.5×10 6 M -1 s -1 Between 1×10 5 M -1 s -1 ~5×10 6 M -1 s -1 k between a and binds to CD86.

[0093] In certain embodiments, the polypeptides disclosed herein are -1 or less, 1.5 s -1 or less, 1 s -1 or less, 0.5 s -1 or less, 0.1 s -1 Or smaller, 5 x 10 -2 s -1 or less, 10 -2 s -1 Or smaller, 5 x 10 -3 s -1or less, 10 -3 s -1 Or smaller, 5 x 10 -4 s -1 or less, 10 -4 s -1 Or smaller, 5 x 10 -5 s -1 or less, 10 -5 s -1 Or smaller, 5 x 10 -6 s -1 or less, 10 -6 s -1 Or smaller, 5 x 10 -7 s -1 or less, 10 -7 s -1 Or smaller, 5 x 10 -8 s -1 or less, 10 -8 s -1 Or a smaller k d or any range between any two of these values d In certain embodiments, the polypeptides disclosed herein bind to CD80 at a rate of 2 s -1 or less, 1.5 s -1 or less, 1 s -1 or less, 0.5 s -1 or less, 0.1 s -1 Or smaller, 5 x 10 -2 s -1 or less, 10 -2 s -1 Or smaller, 5 x 10 -3 s -1 or less, 10 -3 s -1 Or smaller, 5 x 10 -4 s -1 or less, 10 -4 s -1 Or smaller, 5 x 10 -5 s-1 or less, 10 -5 s -1 Or smaller, 5 x 10 -6 s -1 or less, 10 -6 s -1 Or smaller, 5 x 10 -7 s -1 or less, 10 -7 s -1 Or smaller, 5 x 10 -8 s -1 or less, 10 -8 s -1 Or a smaller k d or any range between any two of these values d Rapidly binds to CD86.

[0094] In some embodiments, the polypeptides disclosed herein are present in an amount of 5×10 -6 M or smaller, 10 -6 M or smaller, 5×10 -7 M or smaller, 10 -7 M or smaller, 5×10 -8 M or smaller, 10 -8 M or smaller, 5×10 -9 M or smaller, 10 -9 M or smaller, 5×10 -10 M or smaller, 10 10 M or smaller, 5×10 -11 M or smaller, 10 -11 M or smaller, 5×10 -12 M or smaller, 10 -12 M or smaller, 5×10 -13 M or smaller, 10 -13 M or smaller, 5×10 44 M or smaller, 10 -14 M or smaller, 5×10 -15M or smaller, 10 -15 M or smaller K D or any range between any two of these values. D and binds to CD80.

[0095] In some embodiments, the polypeptides disclosed herein are present in an amount of 5×10 -6 M or smaller, 10 -6 M or smaller, 5×10 -7 M or smaller, 10 -7 M or smaller, 5×10 -8 M or smaller, 10 -8 M or smaller, 5×10 -9 M or smaller, 10 -9 M or smaller, 5×10 -10 M or smaller, 10 10 M or smaller, 5×10 -11 M or smaller, 10 -11 M or smaller, 5×10 -12 M or smaller, 10 -12 M or smaller, 5×10 -13 M or smaller, 10 -13 M or smaller, 5×10 44 M or smaller, 10 -14 M or smaller, 5×10 -15 M or smaller, 10 -15 M or smaller K D or any range between any two of these values. D and binds to CD86.

[0096] In certain embodiments, the kinetic properties of the polypeptides disclosed herein are improved compared to abatacept (SEQ ID NO: 2) in comparable assays. For example, in certain embodiments, the polypeptides of the present disclosure have the corresponding kappa activity of abatacept. ak in the range of approximately 1.1 to 1000 times a In some embodiments, the polypeptides of the present disclosure bind to CD80, CD86, or both with a k a Approximately 1.1 times, approximately 1.2 times, approximately 1.5 times, approximately 2 times, approximately 2.5 times, approximately 3 times, approximately 3.5 times, approximately 4 times, approximately 5 times, approximately 7.5 times, approximately 10 times, approximately 20 times, approximately 50 times, approximately 100 times, approximately 200 times, approximately 500 times, approximately 750 times, or approximately 1000 times a k at a rate or in any range between any two of these values a In some embodiments, the polypeptides of the present disclosure bind to CD80, CD86, or both at a rate similar to that of abatacept. d k in the range of approximately 0.0001 to 0.99 times d In some embodiments, the polypeptides of the present disclosure bind to CD80, CD86, or both with a k a Approximately 0.0001 times, approximately 0.0002 times, approximately 0.001 times, approximately 0.002 times, approximately 0.005 times, approximately 0.01 times, approximately 0.02 times, approximately 0.05 times, approximately 0.075 times, approximately 0.1 times, approximately 0.2 times, approximately 0.25 times, approximately 0.3 times, approximately 0.4 times, approximately 0.5 times, approximately 0.075, approximately 0.75 times, approximately 0.8 times, approximately 0.9 times, approximately 0.92 times, approximately 0.94 times, approximately 0.96 times, approximately 0.98 times, or approximately 0.99 times d k at a rate or in any range between any two of these values d In some embodiments, the polypeptides of the present disclosure bind to CD80, CD86, or both at a rate similar to that of abatacept. D K in the range of approximately 0.0001 to 0.99 times D In some embodiments, the polypeptides of the present disclosure bind to CD80, CD86, or both with a K DApproximately 0.0001 times, approximately 0.0002 times, approximately 0.001 times, approximately 0.002 times, approximately 0.005 times, approximately 0.01 times, approximately 0.02 times, approximately 0.05 times, approximately 0.075 times, approximately 0.1 times, approximately 0.2 times, approximately 0.25 times, approximately 0.3 times, approximately 0.4 times, approximately 0.5 times, approximately 0.075, approximately 0.75 times, approximately 0.8 times, approximately 0.9 times, approximately 0.92 times, approximately 0.94 times, approximately 0.96 times, approximately 0.98 times, or approximately 0.99 times d K at a rate or in any range between any two of these values D It binds to CD80, CD86, or both at different rates.

[0097] In some embodiments, the binding of the subject polypeptide has pH dependence. In some embodiments, the pH dependence is defined as the ratio between the binding affinity to both CD80 and CD86 at pH 7.4 and the binding affinity to both CD80 and CD86 at pH 6.0. The pH dependence can be in the form of a fold decrease in binding affinity from pH 7.4 to pH 6.0, or a fold increase in binding affinity from pH 7.4 to pH 6.0. In some embodiments, the pH dependence is defined as the ratio between the K D value and K at pH 7.4 DThe pH dependence is calculated as the ratio between the pH and pH values, i.e., the ratio indicates the fold affinity reduction from pH 7.4 to pH 6.0. When the pH dependence of a subject polypeptide described herein is greater than 1, this means that the polypeptide binds to CD80, CD86, or both in a pH-dependent manner such that its binding to CD80, CD86, or both at pH 7.4 is higher than at pH 6.0. When the pH dependence of a subject polypeptide described herein is less than 1, this means that the polypeptide binds to CD80, CD86, or both in a pH-dependent manner such that its binding to CD80, CD86, or both at pH 6.0 is higher than at pH 7.4. The ability to maintain binding under neutral conditions (e.g., pH is about 7.0, about 7.1, about 7.2, about 7.3, about 7.4, or about 7.5), but is significantly reduced under acidic conditions, allows for dissociation of the subject polypeptide from its binding partner (e.g., CD80 or CD86) in acidic conditions (e.g., inside a lysosome, e.g., pH is less than 7.0, or about 6.5, about 6.0, about 5.5, about, or about 5.0). In some embodiments, the ability to maintain binding under neutral conditions, but is significantly reduced under acidic conditions, allows the subject polypeptide to avoid lysosomal degradation under acidic conditions and return to the plasma where it can again bind to its binding partner (e.g., CD80 and CD86) under neutral conditions. Without wishing to be bound by any theory, it is believed that subject polypeptides having such a pH-dependent binding pattern (e.g., higher binding affinity under neutral conditions than under acidic conditions) have superior properties in terms of antigen neutralization and clearance compared to otherwise identical polypeptides that bind by a pH-independent mechanism.

[0098] In some embodiments, subject polypeptides provided herein have a pH-dependence of binding affinity for CD80, CD86, or both that is greater than 1, e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, or more. In some embodiments, subject polypeptides have a pH-dependence of binding affinity for CD80, CD86, or both that is greater than that of SEQ ID NO: 2. In some embodiments, subject polypeptides have a pH-dependence of binding affinity for CD80, CD86, or both that is at least 1-fold, 2-fold, 5-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 35-fold, 40-fold, 45-fold, 50-fold, 55-fold, 60-fold, 65-fold, 70-fold, 75-fold, or 80-fold greater than that of SEQ ID NO: 2. In some embodiments, the subject polypeptides have a pH-dependence of binding affinity for CD80, CD86, or both that is greater than that of SEQ ID NO: 3. In some embodiments, the subject polypeptides have a pH-dependence of binding affinity for CD80, CD86, or both that is at least 1-fold, 2-fold, 5-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 35-fold, 40-fold, 45-fold, or 50-fold greater than that of SEQ ID NO: 3.

[0099] In some embodiments, the subject polypeptide exhibits a competitive inhibitory effect on other proteins with respect to binding to CD80 or CD86. The binding affinity of the subject polypeptide to CD80 or CD86 can be evaluated by measuring competitive inhibition on other proteins that have binding affinity to CD80 or CD86. For example, a competitive inhibition assay can be performed, in which a reference polypeptide, such as native CTLA4 protein (SEQ ID NO: 1), abatacept (SEQ ID NO: 2), belatacept (SEQ ID NO: 3), SEQ ID NO: 4 or 5, CD28 or its functional equivalent, or a different subject polypeptide provided herein, is expressed in a host cell, such as an immune cell, such as Ramos cell (human Burkitt's lymphoma cell). In some cases, the reference polypeptide is purified and bound to a solid support, such as a microbead. Then, a CD80 or CD86 molecule tagged with a detectable label, such as conjugated with biotin or a fluorescent tag, is incubated with the reference polypeptide expressed by the host cell or bound to a solid support. In some examples, a subject polypeptide is added to the incubation, where the subject polypeptide can compete with the reference polypeptide for binding to detectably labeled CD80 or CD86 molecules. Such competition can be measured by testing detectably labeled CD80 or CD86 molecules that are retained on the surface of host cells or bound to solid supports. The measurement can be performed by any technique available to those skilled in the art, depending on the detectable label used to tag the CD80 or CD86 molecules. For example, flow cytometry, fluorescence imaging, or fluorescence spectroscopy can be used when fluorescent labels are used, while other approaches that measure magnetic force or electrical impedance can be used when magnetic or conductive labels are used, respectively. In these cases, the relative binding affinity of the subject polypeptide to CD80 or CD86 compared to the reference polypeptide is inversely proportional to the CD80 or CD86 molecules that are retained on the surface of cells or bound to solid supports.In other cases, the relative binding affinity of the subject polypeptide to CD80 or CD86 compared to the reference polypeptide is proportional to the CD80 or CD86 molecule carried on the cell surface or bound to a solid support, in which case the subject polypeptide to be tested is expressed by a host cell or bound to a solid support, and the reference polypeptide is used to compete for binding to the CD80 or CD86 molecule. Other formats of competitive inhibition assays available to those skilled in the art can also be used to test the binding affinity of the subject polypeptide. For example, a luminescent oxygen channeling (LOCI) competition assay such as that described in U.S. Pat. No. 6,251,581 or a variation thereof (e.g., AlphaLISA® competition assay) can be applied to measure the binding affinity of the subject polypeptide.

[0100] In some embodiments, the subject polypeptide exhibits immunosuppressive activity. For example, the subject polypeptide can inhibit immune cell activation, such as immune cell proliferation or secretion of cytokines (e.g., IL2).

[0101] Different types of assays are available for evaluating cell proliferation, including but not limited to DNA synthesis cell proliferation assays, metabolic cell proliferation assays, assays that detect proliferation markers, and assays that measure ATP concentration. In DNA synthesis cell proliferation assays, the DNA of proliferating cells is radioactively labeled, the label is washed off, attached to a filter, and then can be measured using a scintillation counter. In metabolic cell proliferation assays, tetrazolium salts can be used, such as MTT, XTT, MTS, and WST, which are reduced in metabolically active cells to form a formazan dye, which then changes the color of the medium. In assays that detect proliferation markers, monoclonal antibodies can be used to target common markers of cell proliferation and / or cell cycle regulation, such as Ki-67, PCNA, topoisomerase IIB, and phospho-histone H3. For the measurement of ATP concentration, bioluminescence-based detection of ATP can be used using the enzyme luciferase and its substrate luciferin.

[0102] CFSE (carboxyfluorescein succinimidyl ester) cell proliferation assay can be used to measure the effect of subject polypeptides on lymphocyte proliferation and T cell proliferation. CFSE is an effective and common means of monitoring lymphocyte division. CFSE can covalently label long-lived intracellular molecules with the fluorescent dye carboxyfluorescein. Thus, when a CFSE-labeled cell divides, its progeny is endowed with half the number of carboxyfluorescein-tagged molecules, and thus each cell division can be evaluated by measuring the corresponding decrease in cell fluorescence, for example, via flow cytometry or fluorescence imaging. In some cases, primary T cells or immortalized cell lines such as Jurkat cells can be used for this assay upon activation. Other cell proliferation markers such as MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) or BrdU, or measurement techniques such as label-free cell proliferation counts available to those skilled in the art, can also be used to test the inhibitory effect of the subject polypeptide on T cell proliferation, indicating its immunosuppressive activity.

[0103] In some embodiments, a subject polypeptide inhibits T cell proliferation at least as effectively as native CTLA4 protein (SEQ ID NO:1), abatacept (SEQ ID NO:2), belatacept (SEQ ID NO:3), or SEQ ID NO:4 or 5. In further embodiments, a subject polypeptide inhibits T cell proliferation at least as effectively as abatacept (SEQ ID NO:2) or belatacept (SEQ ID NO:3). In some embodiments, a subject polypeptide described herein inhibits T cell proliferation by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more at a concentration of 0.001-100 μg / mL, 0.01-100 μg / mL, 0.1-100 μg / mL, 1-100 μg / mL, 10-100 μg / mL, 0.01-1 μg / mL, 0.01-10 μg / mL, or 0.1-10 μg / mL.

[0104] Another aspect of T cells includes the production and release of cytokines such as IL2. The immunosuppressive activity of the subject polypeptide can be measured by an assay in which IL2 secretion from activated T cells is measured in the presence of the subject polypeptide. IL2 secretion can be measured by any technique available in the art, for example, by ELISA (enzyme-linked immunosorbent assay).

[0105] In some embodiments, the inhibitory effect of a subject polypeptide in either a cell proliferation assay or a cytokine production assay may be examined by testing the inhibitory effect of a series of different concentrations of the subject polypeptide. 50 may be calculated in such assays. In certain embodiments, the subject polypeptide is a native CTLA4 protein (SEQ ID NO: 1), abatacept (SEQ ID NO: 2), belatacept (SEQ ID NO: 3), or IC of SEQ ID NO: 4 or 5. 50 IC ranges from about 0.0001 to 10 times 50 In certain embodiments, the subject polypeptides bind to CD80 and / or CD86 and inhibit cell proliferation, such as T cell proliferation, or cytokine production, such as IL2 production, at IC values ​​of 100-1500 nm, ... 50 IC in the range of about 0.0001 to 0.0005 times, about 0.0005 to 0.001 times, about 0.001 to 0.002 times, about 0.002 to 0.005 times, about 0.005 to 0.0075 times, about 0.0075 to 0.01 times, about 0.01 to 0.02 times, about 0.02 to 0.05 times, about 0.05 to 0.075 times, about 0.075 to 0.1 times, about 0.1 to 0.2 times, about 0.2 to 0.5 times, about 0.5 to 0.75 times, about 0.75 to 1 times, about 1 to 5 times, or about 5 to 10 times 50 value, or an IC within the range of any two of the above values 50 At these levels, it inhibits cell proliferation, such as T cell proliferation, or cytokine production, such as IL2 production.

[0106] In some embodiments, the subject polypeptide is the IC 50 IC in the range of about 0.001 to 0.01 times, about 0.002 to 0.0075 times, about 0.0025 to about 0.005 times, or about 0.003 to 0.004 times 50 In some embodiments, the subject polypeptide inhibits T cell proliferation at an IC value of belatacept (SEQ ID NO: 3). 50 IC in the range of about 0.075 to 0.75 times, about 0.1 to 0.5 times, or about 0.2 to about 0.3 times 50 levels, inhibiting T cell proliferation.

[0107] In some embodiments, the subject polypeptide is the IC 50 IC in the range of about 0.005 to 0.05 times, about 0.0075 to 0.025 times, or about 0.01 to about 0.015 times 50 In some embodiments, the subject polypeptide inhibits IL2 secretion by T cells at an IC value of belatacept (SEQ ID NO: 3). 50 IC of about 0.075 to 0.75 times, about 0.1 to 0.5 times, or about 0.2 to about 0.3 times 50 levels, inhibiting IL2 secretion by T cells.

[0108] Fusion Proteins and Other Modifications

[0109] In some aspects, the disclosure provides a polypeptide that is a fusion protein comprising a CTLA4 variant sequence described herein and a fusion partner sequence.

[0110] The fusion partner sequences provided herein may confer functional properties, including, but not limited to, extended half-life, facilitate protein purification and / or manufacturing, enhanced biophysical properties (e.g., increased solubility or stability), and reduced immunogenicity or toxicity, or any other purpose. For example, the subject fusion proteins may exhibit extended in vivo half-life, thereby facilitating less frequent dosing in a therapeutic regimen (e.g., twice a week, once a week, or once every two weeks, etc.). Exemplary subject polypeptides include the CTLA4 variant sequences described herein fused to a fusion partner sequence (e.g., albumin (e.g., human serum albumin), a PK-extended (PKE) adnectin, XTEN, an Fc domain, or a fragment of any of the foregoing, or a combination of any of the foregoing). Fusion proteins may be produced by expressing a nucleic acid encoding the CTLA4 variant sequence and the fusion partner sequence in the same reading frame, optionally separated by a sequence encoding a linker sequence. The fusion protein may comprise the CTLA4 variant sequence and the fusion partner sequence in any order, for example, one or more fusion partners linked to the N-terminus and / or C-terminus of the CTLA4 variant sequence, or one or more fusion partners linked to both the N-terminus and C-terminus of the CTLA4 variant sequence. The fusion may be formed by attaching a fusion partner to either end (i.e., either the N-terminus or the C-terminus) of the CTLA4 variant sequence (i.e., fusion partner-CTLA4 variant or CTLA4 variant-fusion partner configurations). Additionally, the CTLA4 variant sequence may be fused to one or more fusion partners at both ends, optionally with linker sequences at either or both ends.

[0111] In some embodiments, the CTLA4 variant sequence may be fused to an immunoglobulin Fc domain ("Fc domain"), or a fragment or variant thereof (e.g., a functional Fc region). A functional Fc region may bind to FcRn but does not have effector function. The ability of an Fc region or a fragment thereof to bind to FcRn may be determined by standard binding assays known in the art. Exemplary "effector functions" include C1q binding; complement dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; down-regulation of cell surface receptors (e.g., B cell receptor; BCR), and the like. Such effector functions may be assessed using various assays known in the art for assessing such antibody effector functions.

[0112] In exemplary embodiments, the Fc domain is derived from the IgG1 subclass, although other subclasses (e.g., IgG2, IgG3, and IgG4) may also be used. In some embodiments, an exemplary sequence of a human IgG1 immunoglobulin Fc domain that may be used in a subject polypeptide comprises amino acids 131-357 of SEQ ID NO:7 or SEQ ID NO:2 in Table 6.

[0113] In some embodiments, the Fc region used in the fusion protein may comprise the hinge region of an Fc molecule. An exemplary hinge region comprises the core hinge residues spanning positions 1-16 (i.e., DKTHTCPPCPAPELLG of the exemplary human IgG1 immunoglobulin Fc domain sequence provided above). (SEQ ID NO:83)). In certain embodiments, the fusion protein may adopt a multimeric structure (e.g., a dimer) due in part to cysteine ​​residues at positions 6 and 9 in the hinge region of the exemplary human IgG1 immunoglobulin Fc domain sequence provided above. In some embodiments, the cysteine ​​residues at positions 6 and 9 in the hinge region of the exemplary human IgG1 immunoglobulin Fc domain sequence may be replaced with serine (e.g., amino acids 131-257 of SEQ ID NO:2). In other embodiments, the hinge region, as used herein, may further comprise residues from the CH1 and CH2 regions adjacent to the core hinge sequence of the exemplary human IgG1 immunoglobulin Fc domain sequence provided above. In yet other embodiments, the hinge sequence may be GSTHTCPPCPAPELLG (SEQ ID NO:84) It may comprise or consist of.

[0114] In some embodiments, the hinge sequence may include one or more substitutions that confer desirable pharmacokinetic, biophysical, and / or biological properties. Some exemplary hinge sequences include: EPKSSDKTHTCPPCPAPELLGGPS (SEQ ID NO:85) ,EPKSSDKTHTCPPCPAPELLGGSS (SEQ ID NO:86) ,EPKSSGSTHTCPPCPAPELLGGSS (SEQ ID NO:87) ,DKTHTCPPCPAPELLGGPS (SEQ ID NO:88) , and DKTHTCPPCPAPELLGGSS (SEQ ID NO:89) In one embodiment, residue P at position 18 of the exemplary human IgG1 immunoglobulin Fc domain sequence provided above can be replaced with S to eliminate Fc effector function; (SEQ ID NO:86) ,EPKSSGSTHTCPPCPAPELLGGSS (SEQ ID NO:87) , and DKTHTCPPCPAPELLGGSS (SEQ ID NO:89)In another embodiment, residues DK at positions 1-2 of the exemplary human IgG1 immunoglobulin Fc domain sequence provided above may be replaced with GS to remove a potential clip site; this replacement has the sequence EPKSSGSTHTCPPCPAPELLGGSS (SEQ ID NO:87) In another embodiment, the C at position 103 of the heavy chain constant region of human IgG1 (i.e., domains CH1-CH3) can be replaced with S to prevent inappropriate cysteine ​​bond formation in the absence of light chain; this replacement is represented by the sequence EPKSSDKTHTCPPCPAPELLGGPS (SEQ ID NO:85) ,EPKSSDKTHTCPPCPAPELLGGSS (SEQ ID NO:86) , and EPKSSGSTHTCPPCPAPELLGGSS (SEQ ID NO:87) As exemplified in:

[0115] In some embodiments, the Fc domain comprises an amino acid sequence selected from Table 6. In some embodiments, the Fc domain comprises an amino acid sequence having about 70% or more, about 75% or more, about 80% or more, about 85% or more, about 90% or more, about 95% or more, about 96% or more, about 97% or more, about 98% or more, about 99% or more, or about 99.5% or more sequence identity to amino acids 125-357 of SEQ ID NO:2. It should be understood that the C-terminal lysine of the Fc domain is an optional component of a fusion protein comprising the Fc domain. In some embodiments, the Fc domain comprises an amino acid sequence selected from Table 6, except that the C-terminal lysine is omitted.

[0116] In some embodiments, a subject polypeptide provided herein comprises an amino acid sequence having about 70% or more, about 75% or more, about 80% or more, about 85% or more, about 90% or more, about 95% or more, about 96% or more, about 97% or more, about 98% or more, about 99% or more, about 99.5% or more, or 100% sequence identity to amino acids 1-359 of SEQ ID NO:6.

[0117] In some embodiments, the subject polypeptide is provided comprising a CTLA4 variant sequence fused to an albumin binding sequence.In some embodiments, fusion to serum albumin can increase the half-life of the subject polypeptide or its fragment.Exemplary albumin binding sequences include, but are not limited to, the albumin binding domain from streptococcal protein G (see, for example, Makrides et al., J Pharmacol. Exp. Ther. 277:534-542 (1996) and Sjolander et al., J, Immunol. Methods 201:115-123 (1997)), or albumin binding peptides such as those described in, for example, Dennis, et al., J Biol. Chem. 277:35035-35043 (2002).

[0118] In some embodiments, the CTLA4 variant sequences of the present disclosure are directly fused with serum albumin, including but not limited to human serum albumin. In some embodiments, the CTLA4 variant sequences of the present disclosure are acetylated with fatty acids. In some cases, the fatty acids promote binding to serum albumin. See, for example, Kurtzhals et al., Biochem. J 312:725-731 (1995). The CTLA4 variant sequences can be produced as fusion proteins with human serum albumin (HSA) or a portion thereof. Such fusion constructs can be suitable for enhancing the expression of CTLA4 variants or fragments thereof in eukaryotic host cells (e.g., CHO) or in bacteria (e.g., E. coli). Exemplary HSA moieties include N-terminal polypeptides (amino acids 1-369, 1-419, and intermediate lengths beginning at amino acid 1), as disclosed in U.S. Patent No. 5,766,883 and PCT Publication WO 97 / 24445, which are incorporated herein by reference. In some embodiments, a fusion protein may comprise an HSA protein and a CTLA4 variant or fragment thereof linked to each of the C-terminus and N-terminus of HSA. Exemplary HSA constructs are disclosed in U.S. Patent No. 5,876,969, which is incorporated herein by reference.

[0119] The CTLA4 variant sequence can be fused to an XTEN molecule. XTEN molecules are also referred to as unstructured recombinant polymers, unstructured recombinant polypeptides (URPs), and are generally described in Schellenberger et al., Nat Biotechnol., 2009 December; 27(12): 1186-90, US Publication No. 2012 / 0220011, US Patent No. 7,846,445, and WO / 2012 / 162542, each of which is incorporated herein by reference in its entirety. The half-life of the CTLA4 variant sequence can be altered by changing the composition of the XTEN molecule, for example, by changing its size. For example, the XTEN molecule can be selected to achieve a desired half-life, for example, in the range of 1-50 hours (at least 1 hour, 2 hours, 5 hours, 10 hours, 12 hours, 15 hours, 20 hours, or 25 hours, or longer).

[0120] In some embodiments, the subject polypeptide comprises a CTLA4 variant sequence fused to an adnectin, such as an albumin-binding adnectin or a PKE adnectin. Exemplary adnectins are disclosed in US Publication No. 2011 / 0305663, which is incorporated herein by reference in its entirety. The adnectin can be based on the tenth fibronectin type III domain and can bind to serum albumin. The Adnectin may comprise one or more of a BC loop comprising the amino acid sequence set forth in SEQ ID NO:45, a DE loop comprising the amino acid sequence set forth in SEQ ID NO:46, and an FG loop comprising the amino acid sequence set forth in SEQ ID NO:47, or comprises a polypeptide selected from SEQ ID NOs:48, 49, 50, 51, and 52-72, or comprises a polypeptide at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95% identical to SEQ ID NOs:48, 49, 50, 51, or 52-72, which correspond to SEQ ID NOs:5, 6, 7, 8, 12, 16, 20, and 24-44, respectively, of U.S. Publication No. 2011 / 0305663.

[0121] In certain embodiments, the fusion partner and the CTLA4 variant are fused via a linker sequence. Exemplary linker sequences may include or consist of sequences selected from Table 7, or combinations thereof. Exemplary linker sequences may have a length between 0 (i.e., no linker sequence) and 100 or more amino acids (e.g., between at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 to 60, 50, 40, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, or 11 amino acids). Exemplary non-limiting lengths of the linker sequence include lengths between 1 and 100 amino acids, between 1 and 40 amino acids, between 1 and 20 amino acids, between 1 and 10 amino acids, or between 3 and 5 amino acids.

[0122] In some embodiments, the subject polypeptides described herein are fused to a polymer, such as polyethylene glycol (PEG). A polypeptide or a fragment thereof may be PEGylated, for example, to increase the biological (e.g., serum) half-life of the polypeptide. To PEGylate a polypeptide, the polypeptide is typically reacted with polyethylene glycol (PEG), such as a reactive ester or aldehyde derivative of PEG, under conditions in which one or more PEG groups are attached to the polypeptide. Preferably, the PEGylation is carried out via an acylation reaction or an alkylation reaction with a reactive PEG molecule (or a similar reactive water-soluble polymer). As used herein, the term "polyethylene glycol" is intended to encompass any of the forms of PEG that have been used to derivatize other proteins (e.g., mono (C1-C10) alkoxy- or aryloxy-polyethylene glycol or polyethylene glycol-maleimide). For example, methods for PEGylating proteins such as those disclosed in EP 0 154 316 by Nishimura et al. and EP 0 401 384 by Ishikawa et al. may be used. In some embodiments, polymer, for example, PEG, can be covalently attached to the subject polypeptide described herein at either N- or C-terminus or internal position using conventional chemical methods, for example, chemical conjugation.Without being bound by theory, PEG moiety can contribute to water solubility, high mobility in solution, lack of toxicity and low immunogenicity, extended circulation life, increased stability, easy clearance from body, and altered distribution in body when attached to the polypeptide described herein.

[0123] Other half-life extension technologies that can be used to increase the serum half-life of a subject polypeptide include, but are not limited to, XTEN (Schellenberger et al., Nat. Biotechnol. 27:1186-1192, 2009) and Albu tags (Trussel et al., Bioconjug Chem. 20:2286-2292, 2009).

[0124] In some aspects, the disclosure provides polypeptides comprising CTLA4 variant sequences having chemical modifications (e.g., including but not limited to, conjugation, fusion, and linking chemistries for various functional purposes).

[0125] Agents that alter the immunological reactivity of a subject polypeptide can be used to modify the subject polypeptides provided herein.

[0126] In some embodiments, it is preferable to have low immunogenicity with respect to the target polypeptide. In some embodiments, agents that reduce the immunogenicity of the target polypeptide are used for modification. Agents that reduce immunological reactivity include, but are not limited to, anti-inflammatory agents and immunosuppressants. Anti-inflammatory agents include nonsteroidal anti-inflammatory drugs (NSAIDs) and corticosteroids. NSAIDs include salicylates such as acetylsalicylic acid; diflunisal, salicylic acid, and salsalate; propionic acid derivatives such as ibuprofen; naproxen; dexibuprofen, dexketoprofen, flurbiprofen, oxaprozin, fenoprofen, loxoprofen, and ketoprofen; acetic acid derivatives such as indomethacin, diclofenac, tolmetin, aceclofenac, sulindac, nabumetone, etodolac, and ketorolac; enolic acids such as piroxicam, lornoxicam, meloxicam, isoxicam, tenoxicam, phenylbutazone, and droxicam. These include, but are not limited to, anthranilic acid derivatives, such as mefenamic acid, flufenamic acid, meclofenamic acid, and tolfenamic acid; selective COX-2 inhibitors, such as celecoxib, lumiracoxib, rofecoxib, etoricoxib, valdecoxib, firocoxib, and parecoxib; sulfonanilides, such as nimesulide; and others, such as clonixin and licofelone. Corticosteroids include, but are not limited to, cortisone, dexamethasone, hydrocortisone, methylprednisolone, prednisone, and prednisolone. Immunosuppressants include, but are not limited to, hydroxychloroquine, sulfasalazine, leflunomide, etanercept, infliximab, adalimumab, D-penicillamine, oral gold compounds, injectable gold compounds (intramuscular injection), minocycline, sodium gold thiomalate, auranofin, D-penicillamine, lobenzarit, bucillamine, actarit, cyclophosphamide, azathioprine, methotrexate, mizoribine, cyclosporine, and tacrolimus.

[0127] In some embodiments, it is preferred to have high immunogenicity with respect to the polypeptide of interest. In some embodiments, agents that increase the immunogenicity of the polypeptide of interest are used for modification. Agents that enhance immunological reactivity include, but are not limited to, bacterial superantigens. Agents that facilitate coupling to solid supports include, but are not limited to, biotin or avidin. Immunogen carriers include, but are not limited to, any physiologically acceptable buffer. Biological response modifiers include cytokines, in particular tumor necrosis factor (TNF), interleukin-2, interleukin-4, granulocyte macrophage colony stimulating factor and gamma-interferon.

[0128] Other functional moieties include signal peptides, agents that enhance or reduce immune reactivity, agents that facilitate coupling to solid supports, vaccine carriers, biological response modifiers, paramagnetic labels and drugs.Signal peptides are short amino acid sequences that direct newly synthesized proteins through cell membranes, usually either the endoplasmic reticulum in eukaryotic cells, the inner membrane in bacteria, or both the inner and outer membranes.Signal peptides are typically present in the N-terminal portion of a polypeptide and are typically enzymatically removed during the biosynthesis of the polypeptide and secretion from the cell.Such peptides can be incorporated into the target antibody or a fragment thereof to allow secretion of the synthesized molecule.

[0129] In some embodiments, the subject polypeptide is conjugated to a chemically functional moiety.Typically, the moiety is a label that can produce detectable signals.Such conjugated polypeptides are useful in detection systems, such as, for example, tumor burden quantification, and metastatic imaging and tumor imaging.Such labels are known in the art, and include, but are not limited to, radioisotopes, enzymes, fluorescent compounds, chemiluminescent compounds, bioluminescent compounds, substrate cofactors and inhibitors.For examples of patents that describe the use of such labels, see U.S. Patent Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149; and 4,366,241. The moieties can be covalently linked to the subject polypeptides described herein, can be recombinantly linked, or can be conjugated to the subject polypeptides by means of a secondary reagent, such as an antibody, Protein A, or biotin-avidin complex.

[0130] In some embodiments, the subject polypeptide is conjugated to one or more drug moieties.Suitable drug moieties include immunosuppressants.Non-limiting examples of immunosuppressants that can be conjugated to the subject polypeptide include glucocorticoids, cytostatics, antibodies, immunophilins, statins and other drugs that act against other drugs, such as interferons (e.g., INF-β and INF-γ), opioids, TNF binding agents (e.g., infliximab, etanercept, adalimumab, curcumin and catechin), mycophenolates, IL-1 receptor antagonists, and other small molecule drugs (e.g., fingolimod, myriocin). Exemplary glucocorticoids include, but are not limited to, hydrocortisone, prednisone, prednisolone, methylprednisone, dexamethasone, betamethasone, triamcinolone, beclomethasone, fludrocortisone acetate, deoxycorticosterone acetate, and aldosterone. Exemplary cytostatic agents include, but are not limited to, cyclophosphamide, nitrosoureas, platinum compounds, methotrexate, azathioprine, mercaptopurine, pyrimidine analogs, protein synthesis inhibitors, and antibiotics (e.g., dactinomycin, anthracyclines, mitomycin C, bleomycin, and mithramycin). Exemplary immunosuppressive antibodies include, but are not limited to, anti-CD20 antibodies, anti-IL2 receptor antibodies (daclizumab, basiliximab), Campath-1H, anti-α4β1 integrin antibodies, anti-IL-15 antibodies, anti-IL-6 receptor antibodies, and anti-CD3 antibodies (muromonab). Exemplary agents acting against immunophilins include, but are not limited to, cyclosporine, tacrolimus, and sirolimus.

[0131] In some embodiments, the subject polypeptide is conjugated to an anti-inflammatory agent. Non-limiting examples of anti-inflammatory agents include nonsteroidal anti-inflammatory drugs (e.g., ibuprofen, aspirin, naproxen, diflunisal, ketoprofen, nabumetone, piroxicam, diclofenac, indomethacin, sulindac, tolmetin, etodolac, ketorolac, oxaprozin, and celecoxib), and glucocorticoids that also have immunosuppressant activity.

[0132] In some embodiments, the subject polypeptide is conjugated to an anti-tumor agent, non-limiting examples of which are radioisotopes, vinca alkaloids such as vinblastine, vincristine and vindesine sulfate, adriamycin, bleomycin sulfate, carboplatin, cisplatin, cyclophosphamide, cytarabine, dacarbazine, dactinomycin, duanorubicin hydrochloride, doxorubicin hydrochloride, etoposide, fluorouracil, lomustine, mechlororethamine hydrochloride, melphalan, mercaptopurine, methotrexate, mitomycin, mitotane, pentostatin, pipobroman, procarbaze hydrochloride, streptozotocin, taxol, thioguanine and uracil mustard.

[0133] In some embodiments, the subject polypeptide is conjugated to a toxin, for example, an immunotoxin. A variety of immunotoxins can be used in the subject compositions. Suitable immunotoxins can be found, for example, in Monoclonal Antibody-toxin Conjugates: Aiming the Magic Bullet, Thorpe et al. (1982) Monoclonal Antibodies in Clinical Medicine, Academic Press, pp. 168-190; Vitatta (1987) Science 238:1098-1104; and Winter and Milstein (1991) Nature 349:293-299. Suitable toxins include, but are not limited to, ricin, radionuclides, pokeweed antiviral protein, Pseudomonas exotoxin A, diphtheria toxin, ricin A chain, fungal toxins such as restrictocin, and phospholipase enzymes. See generally, "Chimeric Toxins," Olsnes and Pihl, Pharmac. Ther. 15:355-381 (1981); and "Monoclonal Antibodies for Cancer Detection and Therapy," eds. Baldwin and Byers, pp. 159-179, 224-266, Academic Press (1985).

[0134] A chemically functional moiety can be produced recombinantly, for example, by creating a fusion gene encoding the polypeptide of interest and the functional moiety, or the polypeptide of interest can be chemically linked to the moiety by any of a variety of well-established chemical procedures. For example, when the moiety is a protein, a variety of coupling agents may be used (e.g., N-succinimidyl-3-(2-pyridyldithiol)propionate (SPDP), succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate, iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCl), active esters (such as disuccinimidyl suberate), aldehydes (such as glutareldehyde), bis-azide compounds (such as bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as tolyene 2,6-diisocyanate), and bis-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene)). The linker may be a "cleavable linker" that facilitates the release of the cytotoxic drug in cells. For example, an acid-labile linker, a peptidase-sensitive linker, a dimethyl linker or a disulfide-containing linker (Chari et al. Cancer Research, 52: 127-131 (1992)) may be used. The moiety may be covalently linked or conjugated with a secondary reagent such as an antibody, protein A or biotin-avidin complex.

[0135] In some embodiments, the subject polypeptides provided herein are bispecific, since they may have two different binding domains, one capable of specifically binding to CD80 or CD86, and the other capable of binding to a different molecule. In other embodiments, the subject polypeptides are specific for more than two different molecules other than CD80 and CD86.

[0136] Polypeptide production

[0137] In one aspect, a polynucleotide encoding a polypeptide of the present disclosure is provided herein. Other aspects of the present disclosure also provide vectors comprising polynucleotide sequences encoding the polypeptides described herein. In some aspects, host cells expressing the polypeptides described herein are also provided.

[0138] The subject polypeptides may be produced as recombinant polypeptides by cloning DNA encoding the subject polypeptides, incorporating the clone into an appropriate vector, and transducing the vector into a host cell. Alternatively, the polynucleotides encoding the polypeptides of the present disclosure may be partially or completely synthesized. In some cases, the subject polypeptides are fusion proteins, and the nucleic acids encoding the fusion partner sequences, e.g., immunoglobulin constant regions, may be obtained by amplifying and modifying germline DNA or cDNA encoding the desired fusion protein, e.g., using polymerase chain reaction (PCR). In some embodiments, the nucleic acid encoding wild-type abatacept may be synthesized and used as a template for mutagenesis, using conventional mutagenesis techniques, to produce the subject polypeptides described herein; alternatively, the nucleic acid encoding the variants may be directly synthesized.

[0139] In some embodiments, the polynucleotides provided herein comprise a nucleic acid sequence encoding a CTLA4 variant operably linked to another nucleic acid sequence encoding another protein, e.g., a fusion partner (e.g., an antibody constant region and / or a flexible linker sequence). The term "operatively linked" as used in this context is intended to mean that the two nucleic acids are linked such that the amino acid sequences encoded by the two nucleic acids are maintained in frame.

[0140] The nucleotide sequence encoding the polypeptide of the present disclosure can also be modified.For example, the polynucleotide encoding the polypeptide of interest can be subjected to codon optimization to achieve the optimized expression of the polypeptide of interest in the desired host cell.For example, in one method of codon optimization, native codons are replaced by the most frequent codons from a reference set of genes, where the rate of codon translation of each amino acid is designed to be high.Further exemplary methods for producing codon-optimized polynucleotides for the expression of desired proteins, which can be applied to the CTLA4 variant sequence and / or fusion partner sequence of the polypeptide of the present disclosure, are described in Kanaya et al., Gene, 238:143-155 (1999), Wang et al., Mol. Biol. Evol., 18(5):792-800 (2001), U.S. Patent No. 5,795,737, U.S. Publication No. 2008 / 0076161 and WO 2008 / 000632.

[0141] The polynucleotides of the disclosure include those that encode functional equivalents of the exemplified polypeptides and fragments thereof. Functional equivalents may be polypeptides having conservative amino acid substitutions, analogs including fusions, and variants.

[0142] If desired, the recombinant polynucleotide may contain heterologous sequences that facilitate detection of expression and purification of the gene product. Examples of such sequences include sequences encoding reporter proteins such as β-galactosidase, β-lactamase, chloramphenicol acetyltransferase (CAT), luciferase, green fluorescent protein (GFP) and derivatives thereof. Other heterologous sequences that facilitate purification include Myc, HA (derived from influenza virus hemagglutinin), His-6, and the like. (SEQ ID NO:90) , FLAG or the Fc portion of an immunoglobulin, glutathione S-transferase (GST), and maltose binding protein (MBP), among others.

[0143] Polynucleotides can be conjugated to various chemically functional moieties as mentioned above.Commonly used moieties include labels that can produce detectable signals, signal peptides, agents that enhance or reduce immune reactivity, agents that facilitate coupling to solid supports, vaccine carriers, biological response modifiers, paramagnetic labels and drugs.Moieties can be covalently linked to polynucleotides by recombinant or other means known in the art.

[0144] The polynucleotide can include additional sequences, such as additional coding sequences within the same transcription unit, control elements such as promoters, ribosome binding sites and polyadenylation sites, additional transcription units under the control of the same or different promoters, sequences allowing for cloning, expression and transformation of host cells, and any such construction as may be desirable according to any of the various embodiments described herein.

[0145] Polynucleotides can be obtained using chemical synthesis, recombinant cloning methods, PCR, or any combination thereof. Those skilled in the art can obtain the desired polynucleotides using the sequence data provided herein by using a DNA synthesizer or ordering from a commercial service.

[0146] The polynucleotide containing the desired sequence can be inserted into a suitable vector, which can then be introduced into a suitable host cell for replication, amplification and expression.Thus, in one aspect, various vectors are provided herein that contain one or more of the polynucleotides of the present disclosure.Also provided is a selectable library of expression vectors that contains at least one vector that encodes a polypeptide of interest.

[0147] The vectors of the present disclosure are generally classified into cloning vectors and expression vectors. Cloning vectors are useful for obtaining duplicate copies of the polynucleotides they contain, or as a means of storing polynucleotides in an archive for future retrieval. Expression vectors (and host cells containing these expression vectors) can be used to obtain polypeptides produced from the polynucleotides they contain. In some embodiments, the subject polypeptide is a fusion protein, and the expression vector may already have a fusion partner sequence, such as an antibody constant region sequence. For example, one approach to convert a protein of the present disclosure that contains only a CTLA4 variant sequence into a subject fusion protein is to insert a nucleic acid encoding the CTLA4 variant sequence into an expression vector that already encodes an immunoglobulin Fc, such that the CTLA4 variant coding sequence is operably linked to an Fc coding sequence in the vector. Additionally or alternatively, the recombinant expression vector may encode a signal peptide that facilitates secretion of the CTLA4 protein from the host cell. The nucleic acid encoding the CTLA4 protein may be cloned into the vector such that the signal peptide is linked in frame to the amino terminus of the CTLA4 protein-encoding nucleic acid. The signal peptide can be a CTLA4 signal peptide or a heterologous signal peptide, such as an immunoglobulin signal peptide. Suitable cloning and expression vectors include any known in the art, such as those used in bacterial, mammalian, yeast, insect and phage display expression systems.

[0148] A suitable cloning vector can be constructed according to standard techniques or can be selected from a large number of cloning vectors available in the art. The cloning vector selected can vary depending on the host cell intended for use, but a useful cloning vector will generally have the ability to self-replicate, can carry a single target of a specific restriction endonuclease, or can carry a marker gene. Suitable examples include plasmids and bacterial viruses, such as pBR322, pMB9, ColE1, pCR1, RP4, pUC18, mp18, mp19, phage DNA (including filamentous and non-filamentous phage DNA), and shuttle vectors such as pSA3 and pAT28. The above and other cloning vectors are available from commercial vendors such as Clontech, BioRad, Stratagene and Invitrogen.

[0149] Expression vectors containing a subject polynucleotide are useful for obtaining a host vector system to produce proteins and polypeptides. Typically, these expression vectors are replicable in the host organism, either as an episome or as an integral part of the chromosomal DNA. Suitable expression vectors for subject polypeptides include plasmids, viral vectors (including phagemids, adenoviruses, adeno-associated viruses, retroviruses, cosmids, etc.). Many expression vectors suitable for expression in eukaryotic cells, including yeast, avian, and mammalian cells, are available. One example of an expression vector is pcDNA3 (Invitrogen, San Diego, Calif.), where transcription is driven by the cytomegalovirus (CMV) early promoter / enhancer. Two types of expression vectors that are particularly useful for expressing the subject polypeptides described herein are phage display vectors and bacterial display vectors.

[0150] It is possible to express the subject polynucleotide of the present disclosure in either prokaryotic or eukaryotic host cells. A host cell line can be selected that regulates the expression of the inserted sequence or modifies and processes the gene product in a desired specific manner. Such modification (e.g., glycosylation) and processing (e.g., cleavage) of protein products can be important for the function of the subject polypeptide. Different host cells have characteristic and specific mechanisms for post-translational processing and modification of proteins and gene products. To ensure the correct modification and processing of expressed foreign proteins, appropriate cell lines or host systems can be selected. For this purpose, eukaryotic host cells can be used that possess the cellular machinery for proper processing of the primary transcript, glycosylation and phosphorylation of the gene product. Such mammalian host cells include, but are not limited to, CHO, VERY, BHK, Hela, COS, MDCK, 293, 3T3, W138, BT483, Hs578T, HTB2, BT2O and T47D, NSO (a mouse myeloma cell line that does not endogenously produce any immunoglobulin chains), CRL7O3O and HsS78Bst cells.

[0151] For long-term, high-yield production of recombinant proteins, stable expression is preferred. For example, cell lines can be engineered that stably express a polypeptide of interest. Rather than using expression vectors that contain a viral origin of replication, host cells can be transformed with DNA controlled by appropriate expression control elements (e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.) and a selectable marker. After introduction of the foreign DNA, engineered cells can be grown in enriched medium for 1-2 days and then switched to selective medium. The selectable marker on the recombinant plasmid confers resistance to selection and allows cells to stably integrate the plasmid into their chromosomes and grow until they form foci, which can then be cloned and expanded into cell lines. This method can be advantageously used to engineer cell lines that express a polypeptide of interest.

[0152] Once a polypeptide molecule of interest has been produced by recombinant expression, it can be purified by any suitable purification method for immunoglobulin molecules, for example, by chromatography (e.g., ion exchange, affinity (particularly by affinity for specific antigens following Protein A) and size classification column chromatography), centrifugation, differential solubility, or any other standard purification technique for proteins. Additionally, a polypeptide of interest can be fused to heterologous polypeptide sequences provided herein or others known in the art to facilitate purification. For example, a polypeptide of interest can be purified by recombinantly adding, inter alia, commercially available poly-histidine tags (His-tags), FLAG-tags, hemagglutinin tags (HA-tags) or myc-tags and utilizing suitable purification methods.

[0153] Treatment

[0154] In another aspect, provided herein are methods of using the subject polypeptides to treat a condition, disease or disorder, such as an inflammatory disorder, an autoimmune disease, cancer, or transplant rejection.

[0155] In some embodiments, the disclosure provides a method of treating an inflammatory disorder in a mammal in need thereof, comprising administering to the mammal a therapeutically effective amount of a subject polypeptide of the disclosure. In some cases, the inflammatory disorder is multiple sclerosis. In other cases, the inflammatory disorder is an autoimmune disease. Examples of autoimmune diseases include acute disseminated encephalomyelitis (ADEM), Addison's disease, antiphospholipid syndrome (APS), aplastic anemia, autoimmune hepatitis, celiac disease, Crohn's disease, diabetes mellitus (type 1), Goodpasture's syndrome, Graves' disease, Guillain-Barré syndrome (GBS), Hashimoto's disease, multiple sclerosis, myasthenia gravis, opsoclonus-myoclonus syndrome (OMS), optic neuritis, Ord's thyroiditis, pemphigus, polyarthritis, primary biliary cirrhosis, psoriasis, These include, but are not limited to, rheumatoid arthritis, inflammatory bowel disease (IBD), juvenile idiopathic arthritis (JIA), psoriatic arthritis, systemic lupus erythematosus (SLE), asthma, Reiter's syndrome, Takayasu's arteritis, temporal arteritis (also known as "giant cell arteritis"), warm autoimmune hemolytic anemia, Wegener's granulomatosis, alopecia universalis, Chagas' disease, chronic fatigue syndrome, autonomic neuropathy, endometriosis, hidradenitis suppurativa, interstitial cystitis, neuromyotonia, sarcoidosis, scleroderma, ulcerative colitis, vitiligo, and vulvodynia. Other disorders include bone resorption disorders and thrombosis.

[0156] In further embodiments, the subject polypeptides of the present disclosure are useful for treating bursitis, lupus, acute disseminated encephalomyelitis (ADEM), Addison's disease, antiphospholipid syndrome (APS), aplastic anemia, autoimmune hepatitis, celiac disease, Crohn's disease, diabetes mellitus (type 1), Goodpasture's syndrome, Graves' disease, Guillain-Barre syndrome (GBS), Hashimoto's disease, inflammatory bowel disease, lupus erythematosus, myasthenia gravis, opsoclonus-myoclonus syndrome (OMS), optic neuritis, Ord's thyroiditis, ostheoarthritis, uveitis retinalis, pemphigus, polyarthritis, primary biliary cirrhosis, Reiter's syndrome, Takayasu's arteritis, temporal arteritis, warm autoimmune hemolytic anemia, Wegener's granulomatosis, alopecia universalis, Chagas disease, chronic fatigue syndrome, autonomic nervous system disorders, and the like. Disorders, endometriosis, hidradenitis suppurativa, interstitial cystitis, neuromyotonia, sarcoidosis, scleroderma, ulcerative colitis, vitiligo, vulvodynia, appendicitis, arteritis, arthritis, blepharitis, bronchiolitis, bronchitis, cervicitis, cholangitis, cholecystitis, chorioamnionitis, colitis, conjunctivitis, cystitis, dacryoadenitis, dermatomyositis, endocarditis, endometritis, enteritis, enterocolitis, epicondylitis, epididymitis, fasciitis, fibromyalgia, gastritis, It is used to treat gastroenteritis, gingivitis, hepatitis, hidradenitis, ileitis, iritis, laryngitis, mastitis, meningitis, myelitis, myocarditis, myositis, nephritis, omphalitis, ovariitis, orchitis, osteitis, otitis, pancreatitis, parotitis, pericarditis, peritonitis, pharyngitis, pleuritis, phlebitis, pneumonitis, proctitis, prostatitis, pyelonephritis, rhinitis, salpingitis, sinusitis, stomatitis, synovitis, tendonitis, tonsillitis, uveitis, vaginitis, vasculitis or vulvitis.

[0157] In yet further embodiments, the present disclosure provides a method of treating cancer in a mammal in need thereof, comprising administering to the mammal a therapeutically effective amount of a subject polypeptide of the present disclosure. In some cases, the cancer is hepatocellular carcinoma. In other cases, the cancer is acute myeloid leukemia, thymus, brain, lung, squamous cell, skin, eye, retinoblastoma, intraocular melanoma, oral and oropharyngeal, bladder, gastric, stomach, pancreas, bladder, breast, cervix, head, neck, renal, kidney, liver, ovary, prostate, colorectal, esophagus, testis, gynecological, thyroid, CNS, PNS, AIDS-related (e.g., lymphoma and Kaposi's sarcoma) or virus-induced cancer.

[0158] In some embodiments, the subject polypeptides of the present disclosure are useful in treating infections, endotoxic shock associated with infection, arthritis, rheumatoid arthritis, psoriatic arthritis, systemic juvenile idiopathic arthritis (JIA), systemic lupus erythematosus (SLE), asthma, pelvic peritonitis, Alzheimer's disease, Crohn's disease, ulcerative colitis, irritable bowel syndrome, Castleman's disease, ankylosing spondylitis, dermatomyositis, uveitis, Peyronie's disease, celiac disease, gallbladder disease, pilonidal disease, peritonitis, psoriasis, vasculitis, surgical adhesions, stroke, type I diabetes, Lyme joints. It is used for the treatment of chronic inflammation, meningoencephalitis, immune-mediated inflammatory disorders of the central and peripheral nervous system, autoimmune disorders, pancreatitis, surgical trauma, graft-versus-host disease, graft rejection, cardiac disease, bone resorption, burn patients, myocardial infarction, Paget's disease, osteoporosis, sepsis, hepatic / pulmonary fibrosis, periodontitis, hypochlorhydria, solid tumors (renal cell carcinoma), prostate and bladder cancer, pancreatic cancer, neurological cancers, and B-cell malignancies (e.g., Castleman's disease, certain lymphomas, chronic lymphocytic leukemia, and multiple myeloma).

[0159] In some embodiments, the subject to be treated is a mammal, such as a human. In other cases, the mammal is a mouse, rat, cat, dog, rabbit, pig, sheep, horse, cow, goat, gerbil, hamster, guinea pig, monkey, or any other mammal. Many such mammals may be subjects known in the art as preclinical models of certain diseases or disorders, including inflammatory diseases, solid tumors, and / or other cancers (e.g., Talmadge et al., 2007, Am. J. Pathol. 170:793; Kerbel, 2003, Canc. Biol. Therap. 2(4 Suppl. 1):S134; Man et al., 2007, Canc. Met. Rev. 26:737; Cespedes et al., 2006, Clin. TransL Oncol. 8:318).

[0160] In another aspect, the present disclosure provides a method of treating a disease, condition, or disorder in a mammal using a subject polypeptide of the present disclosure in conjunction with a second agent, which may be administered together with, prior to, or after the subject polypeptide.

[0161] In some embodiments, the second agent is an immunosuppressant. Immunosuppressants that may be used in combination with the subject polypeptides include, but are not limited to, hydrocortisone, prednisone, prednisolone, methylprednisone, dexamethasone, betamethasone, triamcinolone, beclomethasone, fludrocortisone acetate, deoxycorticosterone acetate, aldosterone, cyclophosphamide, nitrosoureas, platinum compounds, methotrexate, azathioprine, mercaptopurine, pyrimidine analogs, protein synthesis inhibitors, dactinomycin, anthracyclines, mitomycin C, bleomycin, mithramycin, rapamycin, cyclosporine, tacrolimus, sirolimus, mycophenolic acid, mizoribine, 15-deoxyspergualin, mycophenolate mofetil (MMF), anti-thymocyte globulin, anti-CD20 antibodies or derivatives, analogs or antigen-binding fragments thereof, anti-IL2 receptor antibodies (daclizumab, baclofenac ... ciliximab) or derivatives, analogs or antigen binding fragments thereof, Campath-1H, anti-α4β1 integrin antibodies or derivatives, analogs or antigen binding fragments thereof, anti-IL-15 antibodies or derivatives, analogs or antigen binding fragments thereof, anti-IL-6 receptor antibodies or derivatives, analogs or antigen binding fragments thereof, anti-CD3 antibodies (muromonab) or derivatives, analogs or antigen binding fragments thereof, anti-MHC antibodies or derivatives, analogs or antigen binding fragments thereof, anti-CD2 antibodies or derivatives, analogs or antigen binding fragments thereof, anti-CD4 antibodies or derivatives, analogs or antigen binding fragments thereof, anti-CD11a / CD18 antibodies or derivatives, analogs or antigen binding fragments thereof, anti-CD7 antibodies or derivatives, analogs or antigen binding fragments thereof, anti-CD27 antibodies or derivatives, analogs or antigen binding fragments thereof, anti-CD80 and / or anti-CD86 antibodies (e.g., ATCC 611624) HB-253, ATCC CRL-2223, ATCC CRL-2226, ATCC HB-301, ATCC HB-11341, etc.), or derivatives, analogs, or antigen-binding fragments thereof, anti-CD40 antibodies (e.g., ATCCHB-9110), or derivatives, analogs or antigen binding fragments thereof, or derivatives, analogs or antigen binding fragments thereof, anti-CD45 antibodies, or derivatives, analogs or antigen binding fragments thereof, anti-CD58 antibodies, or derivatives, analogs or antigen binding fragments thereof, anti-CD137 antibodies, or derivatives, analogs or antigen binding fragments thereof, anti-ICOS antibodies, or derivatives, analogs or antigen binding fragments thereof, anti-CD150 antibodies, or derivatives, analogs or antigen binding fragments thereof, anti-OX40 antibodies, or derivatives, analogs or antigen binding fragments thereof, anti-4-1BB antibodies, or derivatives, analogs or antigen binding fragments thereof, and low molecular weight adhesion antagonists (e.g., LFA-1 antagonists, selectin antagonists and VLA-4 antagonists).

[0162] In some embodiments, the subject polypeptides are administered in combination with other immunomodulatory compounds (e.g., soluble gp39 (also known as CD40 ligand (CD40L), CD154, T-BAM, TRAP), soluble CD29, soluble CD40, soluble CD80 (e.g., ATCC 68627), soluble CD86, soluble CD28 (e.g., 68628), soluble CD56, soluble Thy-1, soluble CD3, soluble TCR, soluble VLA-4, soluble VCAM-1, soluble LECAM-1, soluble ELAM-1, soluble CD44, antibodies reactive with gp39 (e.g., ATCC HB-10916, ATCC HB-12055, and ATCC HB-12056), antibodies reactive with CD28 (e.g., ATCC HB-11944, or antibodies reactive with CD28 (e.g., ... Clin. Immun. 4(1):18-22, 1980), antibodies reactive with LFA-1 (e.g., ATCC HB-9579 and ATCC TIB-213), antibodies reactive with LFA-2, antibodies reactive with IL-12, antibodies reactive with IFN-γ, antibodies reactive with CD48, antibodies reactive with any ICAM (e.g., ICAM-1 (ATCC CRL-2252), ICAM-2 and ICAM-3), antibodies reactive with CTLA4 (e.g., ATCC HB-304), antibodies reactive with Thy-1, antibodies reactive with CD56, antibodies reactive with CD29, antibodies reactive with TCR, antibodies reactive with VLA-4, antibodies reactive with VCAM-1, antibodies reactive with LECAM-1, antibodies reactive with ELAM-1, antibodies reactive with CD44).

[0163] In some embodiments, the second drug is an antiviral agent. Antiviral agents include, but are not limited to, telaprevir, boceprevir, simeprevir, sofosbuvir, daclastavir, asunaprevir, lamivudine, adefovir, entecavir, tenofovir, telbivudine, interferon alpha, and pegylated interferon alpha. In other embodiments, the second drug is an agent that acts to reduce the symptoms of the inflammatory conditions described herein. Antiinflammatory agents include nonsteroidal anti-inflammatory drugs (NSAIDs) and corticosteroids. NSAIDs include salicylates such as acetylsalicylic acid; diflunisal, salicylic acid and salsalate; propionic acid derivatives such as ibuprofen; naproxen; dexibuprofen, dexketoprofen, flurbiprofen, oxaprozin, fenoprofen, loxoprofen and ketoprofen; acetic acid derivatives such as indomethacin, diclofenac, tolmetin, aceclofenac, sulindac, nabumetone, etodolac and ketorolac; piroxicam, lornoxicam, These include, but are not limited to, enolic acid derivatives such as meloxicam, isoxicam, tenoxicam, phenylbutazone and droxicam; anthranilic acid derivatives such as mefenamic acid, flufenamic acid, meclofenamic acid and tolfenamic acid; selective COX-2 inhibitors such as celecoxib, lumiracoxib, rofecoxib, etoricoxib, valdecoxib, firocoxib and parecoxib; sulfonanilides such as nimesulide; and others such as clonixin and licofelone. Corticosteroids include, but are not limited to, cortisone, dexamethasone, hydrocortisone, methylprednisolone, prednisone and prednisolone.

[0164] In further embodiments, the second therapeutic agent is an anti-cancer agent (e.g., a chemotherapeutic agent). The chemotherapeutic agent can be selected from the group consisting of mitotic inhibitors, alkylating agents, antimetabolites, intercalating antibiotics, growth factor inhibitors, cell cycle inhibitors, enzymes, topoisomerase inhibitors, biological response modifiers, antihormones, angiogenesis inhibitors and antiandrogens. Non-limiting examples of chemotherapeutic agents, cytotoxic agents and non-peptide small molecules include Gleevec® (imatinib mesylate), Velcade® (bortezomib), Casodex (bicalutamide), Iressa® (gefitinib) and adriamycin, as well as a number of chemotherapeutic agents. Non-limiting examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclosphosphamide (CYTOXAN™); alkylsulfonates such as busulfan, improsulfan, and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines, including altretamine, triethylenemelamine, trietylenephosphoramide, triethylenethiophosphoramide, and trimethylolmelamine; chlorambucil, chlornaphazine, and the like. azine, collophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, nitrogen mustards such as uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimustine; aclacinomycin, actinomycin, autramycin, azaserine, bleomycin, cactinomycin, calicheamicin, carabicin, carminomycin,Carzinophilin, Casodex™, chromomycin, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, esorubicin, idarubicin, marcellomycin, mitomycin, mycophenolic acid, nogalamycin, olivomycin, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin, and the like. Antibiotics; metabolic antagonists such as methotrexate and 5-fluorouracil (5-FU); folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogues such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogues such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens such as calusterone, dromostanol propionate, epithiostanol, mepitiostane, testolactone; aminoglutethimide, mitotane,Anti-adrenal drugs such as trilostane; folic acid supplements such as folic acid; aceglatone; aldophosphamide glycosides; aminolevulinic acid; amsacrine; bestrabucil; bisantrene; edatrexate; defofamine; demecolcine; diaziquone; elfomithine; elliptinium acetate; etoglucide; gallium nitrate; hydroxyurea; lentinan; lonidamine; mitoguazone; mitoxantrone; mopidamol; nitracrine; pentostatin; phenamet; pirarubicin; podophyllinic acid acid); 2-ethylhydrazide; procarbazine; PSK.R™; razoxane; schizofiran; spirogermanium; tenuazonic acid; triazicon; 2,2',2''-trichlorotriethylamine; urethane; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxanes such as paclitaxel (TAXOL™, Bristol-Myers Squibb Oncology, Princeton, NJ) and docetaxel (TAXOTERE™, Rhone-Poulenc Rorer, Antony, France); retinoic acid; esperamicin; capecitabine; and pharma- ceutically acceptable salts, acids, or derivatives of any of the above. Suitable chemotherapeutic cell conditioners include, for example, antiestrogens, including tamoxifen (Nolvadex™), raloxifene, aromatase-inhibiting 4(5)-imidazole, 4-hydroxytamoxifen, trioxifene, keoxifene, LY 117018, onapristone, and toremifene (Fareston); and flutamide, nilutamide, bicalutamide,Also included are anti-hormonal agents that act to regulate or inhibit hormone action in tumors, such as antiandrogens such as leuprolide and goserelin; chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; navelbine; novantrone; teniposide; daunomycin; aminopterin; xeloda; ibandronate; camptothecin-11 (CPT-11); the topoisomerase inhibitor RFS 2000; and difluoromethylornithine (DMFO). If desired, the subject polypeptide may be any of the following: Herceptin®, Avastin®, Erbitux®, Rituxan®, Taxol®, Arimidex®, Taxotere®, ABVD, AVICINE, Abagovomab, Acridinecarboxamide, Adecatumumab, 17-N-allylamino-17-demethoxygeldanamycin, Alpharadin, Alvocidib, 3-aminopyridine-2-carboxaldehyde thiosemicarbazone, Amonafide, Anthracenedione, Anti-CD22 Immunotoxin, Anti-tumor drugs, Anti-tumor herbs, Apaziquone, Atiplimod, Azathioprine, Belotecan, Bendamustine, BIBW 2992, Biricodar, Brostallicin, Bryostatin, Buthionine sulfoximine, CBV (chemotherapy), Calyculin, Cell cycle non-specific antitumor agent, Dichloroacetic acid, Discodermolide, Elsamitrucin, Enocitabine, Epothilone, Eribulin, Everolimus, Exatecan, Exisulind, Ferruginol, Forodesin, Fosfestrol, ICE chemotherapy regimen, IT-101, Imexon, Imiquimod, Indolocarbazole, Irofulven,Laniquidar, Larotaxel, Lenalidomide, Lucantone, Lurtotecan, Mahosfamide, Mitozolomide, Nafoxidine, Nedaplatin, Olaparib, Ortataxel, PAC-1, Pawpaw, Pixantrone, Proteasome Inhibitors, Rebeccamycin, Resiquimod, Rubitecan, SN-38, Salinosporamide A, Sapacitabi It can be used in combination with commonly prescribed anti-cancer drugs such as Sapacitabine, Stanford V, Swainsonine, Talaporfin, Tariquidar, Tegafur-uracil, Temodar, Tesetaxel, Triplatin Tetranitrate, Tris(2-chloroethyl)amine, Troxacitabine, Uramustine, Vadimezan, Vinflunine, ZD6126 and Zosuquidar.

[0165] The specific dose will vary depending on the particular polypeptide selected, the dosing regimen to be followed, whether it is administered in combination with other agents, the timing of administration, the tissue to which it is administered, and the physical delivery system by which it is delivered. In some embodiments, a subject polypeptide is administered to a subject within the range of 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, 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, or 70 mg per week on average over the course of a treatment cycle. For example, the subject polypeptide is administered to the subject within about 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, or 55 mg per week. In some embodiments, the subject polypeptide is administered to the subject within about 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, or 55 mg per week.

[0166] In some embodiments, a subject polypeptide is administered to a subject in an amount that is greater than 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10 mg per day, on average, over the course of a treatment cycle. For example, a subject polypeptide is administered to a subject in an amount that is greater than about 6-10 mg, about 6.5-9.5 mg, about 6.5-8.5 mg, about 6.5-8 mg, or about 7-9 mg per day, on average, over the course of a treatment cycle.

[0167] In some embodiments, the subject polypeptide is administered at a dose of about 0.01 mg / kg, 0.02 mg / kg, 0.03 mg / kg, 0.04 mg / kg, 0.05 mg / kg, 0.06 mg / kg, 0.07 mg / kg, 0.08 mg / kg, 0.09 mg / kg, 0.1 mg / kg, 0.2 mg / kg, 0.3 mg / kg, 0.4 mg / kg, 5 mg / kg, 6 mg / kg, 7 mg / kg, 8 mg / kg, 9 mg / kg, 10 mg / kg, 15 mg / kg, 16 mg / kg, 17 mg / kg, 18 mg / kg, 19 mg / kg, 20 mg / kg, 21 mg / kg, 22 mg / kg, 23 mg / kg, 24 mg / kg, 25 mg / kg, 26 mg / kg, 27 mg / kg, 28 mg / kg, 29 mg / kg, 30 mg / kg, 31 mg / kg, 32 mg / kg, 33 mg / kg, 34 mg / kg, 35 mg / kg, 36 mg / kg, 37 mg / kg, 38 mg / kg, 39 mg / kg, 40 mg / kg, 41 mg / kg, 42 mg / kg, 43 mg / kg, 44 mg / kg, 45 mg / kg, 46 mg / kg, 47 mg / kg, 48 mg / kg, 49 mg / kg, 50 mg / kg, 51 mg / kg, 52 mg / kg, 53 mg / kg, 54 mg / kg, 55 mg / kg, 56 mg / kg, 57 mg / kg, 58 mg / kg, 59 mg / kg, 60 mg / kg, 61 mg / kg, 62 mg / kg, 63 mg / kg, 64 mg The dose is administered to the subject within the range of about 0.01 mg / kg to 50 mg / kg per day, such as 11 mg / kg, 12 mg / kg, 13 mg / kg, 14 mg / kg, 15 mg / kg, 16 mg / kg, 17 mg / kg, 18 mg / kg, 19 mg / kg, 20 mg / kg, 25 mg / kg, 30 mg / kg, 35 mg / kg, 40 mg / kg, 45 mg / kg, or 50 mg / kg, or values ​​less than or greater than these values. In some embodiments, a subject polypeptide is administered to a subject within the range of about 0.1 mg / kg to 400 mg / kg per week, such as about 0.1 mg / kg, 0.2 mg / kg, 0.3 mg / kg, 0.4 mg / kg, 0.5 mg / kg, 0.6 mg / kg, 0.7 mg / kg, 0.8 mg / kg, 0.9 mg / kg, 1 mg / kg, 5 mg / kg, 10 mg / kg, 15 mg / kg, 20 mg / kg, 25 mg / kg, 30 mg / kg, 35 mg / kg, 40 mg / kg, 45 mg / kg, 50 mg / kg, 100 mg / kg, 150 mg / kg, 200 mg / kg, 250 mg / kg, 300 mg / kg, 350 mg / kg, or 400 mg / kg per week, or less or more than these.In some embodiments, the subject polypeptides are administered at about 0.4 mg / kg, 0.5 mg / kg, 1 mg / kg, 5 mg / kg, 10 mg / kg, 15 mg / kg, 20 mg / kg, 25 mg / kg, 30 mg / kg, 35 mg / kg, 40 mg / kg, 45 mg / kg, 50 mg / kg, 100 mg / kg, 150 mg / kg, 200 mg / kg, 250 mg / kg, 300 mg / kg, 350 mg / kg, 400 mg / kg, 450 ...50 mg / kg, 600 mg / kg, 650 mg / kg, 700 mg / kg, 750 mg / kg, 800 mg / kg, 850 mg / kg, 900 mg / kg, 900 mg / kg, 1000 mg / kg, 1000 mg / kg, 1000 mg / kg, 1000 mg / kg, 1000 mg / kg, 1000 mg / kg, 1000 mg / kg, 1000 mg / kg, 1000 mg / kg, 1000 mg / kg, 1000 mg / kg, 1000 mg / kg, 1000 mg / kg, 1000 mg / The subject is administered about 0.4 mg / kg to 1500 mg / kg per month, such as, less than, or greater than, 400 mg / kg, 450 mg / kg, 500 mg / kg, 550 mg / kg, 600 mg / kg, 650 mg / kg, 700 mg / kg, 750 mg / kg, 800 mg / kg, 850 mg / kg, 900 mg / kg, 950 mg / kg, or 1000 mg / kg. In some embodiments, the subject polypeptide is administered about 1 mg / m per week. 2 , 5 mg / m 2 , 10 mg / m 2 , 15 mg / m 2 , 20 mg / m 2 , 25 mg / m 2 , 30 mg / m 2 , 35 mg / m 2 , 40 mg / m 2 , 45 mg / m 2 , 50 mg / m 2 , 55 mg / m 2 , 60 mg / m 2 , 65 mg / m 2 , 70 mg / m 2 , 75 mg / m 2 , 100 mg / m 2 , 125 mg / m 2 , 150 mg / m 2 , 175 mg / m 2 or 200 mg / m 2 , values ​​below or above these limits, etc., approximately 0.1 mg / m per week 2 ~200mg / m 2The target dose is administered to the subject within the range of about 1 or more, about 2 or more, about 3 or more, about 4 or more, about 5 or more, about 6 or more, about 7 or more, about 8 or more, about 9 or more, about 10 or more, about 11 or more, about 12 or more, about 13 or more, about 14 or more, about 15 or more, about 16 or more, about 17 or more, about 18 or more, about 19 or more, about 20 or more, about 25 or more, about 30 or more or more doses. For example, a dose of about 1 mg / kg per week can be delivered weekly over the course of a week at a dose of about 1 mg / kg per week, about 2 mg / kg can be administered every two weeks, or about 4 mg / kg can be administered every four weeks. The dosing schedule can be repeated according to any regimen described herein, including any of the dosing schedules described herein. In some embodiments, the subject polypeptide is administered at a dose of about 1 mg / m 2 , 5 mg / m 2 , 10 mg / m 2 , 15 mg / m 2 , 20 mg / m 2 , 25 mg / m 2 , 30 mg / m 2 , 35 mg / m 2 , 40 mg / m 2 , 45 mg / m 2 , 50 mg / m 2 , 55 mg / m 2 , 60 mg / m 2 , 65 mg / m 2 , 70 mg / m 2 , 75 mg / m 2 , 100 mg / m 2 , 130 mg / m 2 , 135 mg / m 2 , 155 mg / m 2 , 175 mg / m 2, 200 mg / m 2 , 225 mg / m 2 , 250 mg / m 2 , 300 mg / m 2 , 350 mg / m 2 , 400 mg / m 2 , 420 mg / m 2 , 450 mg / m 2 or 500 mg / m 2 , values ​​below or above these values, such as approximately 0.1 mg / m 2 ~500mg / m 2 is administered to the subject in the range of

[0168] The dose of the subject polypeptide may be about, at least about, or at most about 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 225, 250, 275, 300, 35 ... It can be 25, 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, 1000mg or mg / kg, or any range derivable therefrom. It is considered that mg / kg dosage refers to the mg amount of subject polypeptide per kg of total subject body weight. When multiple doses are given to a patient, it is considered that each dose can vary in amount or be the same.

[0169] Pharmaceutical Compositions

[0170] In another aspect, provided herein are pharmaceutical compositions comprising a subject polypeptide and a pharma- ceutically acceptable carrier, excipient or stabilizer, including, but not limited to, inert solid diluents and fillers, diluents, sterile aqueous solutions and various organic solvents, permeation enhancers, solubilizers and adjuvants (Remington's Pharmaceutical Sciences, 16th ed., Osol, A. ed. (1980)).

[0171] The subject pharmaceutical compositions may be in a form suitable for oral administration, for example, as tablets, capsules, pills, powders, sustained release formulations, solutions, suspensions, parenteral injection, as sterile solutions, suspensions or emulsions, topical administration, as ointments or creams, or rectal administration, as suppositories. Suitable examples of sustained release preparations include semipermeable matrices of solid hydrophobic polymers containing the subject polypeptide, which matrices are in the form of shaped articles, for example, films or microcapsules. Examples of sustained release matrices include polyesters, hydrogels (e.g., poly(2-hydroxyethyl-methacrylate) or poly(vinyl alcohol)), polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and gamma-ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as LUPRON DEPOT™ (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D-(−)-3-hydroxybutyric acid. Some sustained release formulations allow for release of molecules over a period of weeks to months or even up to several years. In some embodiments, a subject pharmaceutical composition releases a subject polypeptide as described herein for at least several weeks, such as at least 1 week, 2 weeks, 3 weeks, or 4 weeks. In further embodiments, a subject pharmaceutical composition releases a subject polypeptide as described herein for several months, such as at least 1 month, 2 months, 3 months, 4 months, 5 months, or 6 months.

[0172] The pharmaceutical composition may be in a unit dosage form suitable for single administration of a precise dosage. The pharmaceutical composition may further comprise a subject polypeptide as an active ingredient, and may comprise a conventional pharmaceutical carrier or excipient. In addition, the pharmaceutical composition may comprise other medicinal or pharmaceutical agents, carriers, adjuvants, etc.

[0173] Exemplary parenteral administration forms include solutions or suspensions of the active polypeptide and / or the PEG-modified polypeptide in sterile aqueous solutions, for example, aqueous propylene glycol or dextrose solutions. Such dosage forms can be suitably buffered, if desired, with salts such as histidine and / or phosphate salts.

[0174] In some embodiments, the present disclosure provides an injectable pharmaceutical composition comprising a subject polypeptide and a pharmaceutical excipient suitable for injection. Exemplary components and amounts of agents in such compositions are as described herein.

[0175] Forms in which the compositions of the present disclosure can be incorporated for administration by injection include elixirs, mannitol, dextrose or sterile aqueous solutions and similar pharmaceutical vehicles, as well as aqueous or oily suspensions or emulsions including sesame oil, corn oil, cottonseed oil or peanut oil.

[0176] Aqueous solutions in saline can be used for injection. Ethanol, glycerol, propylene glycol, liquid polyethylene glycol, and the like (and suitable mixtures thereof), cyclodextrin derivatives, and vegetable oils can also be used. For example, in the case of dispersions, proper fluidity can be maintained by the use of a coating such as lecithin to maintain the required particle size, and by the use of surfactants. Prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like.

[0177] Sterile injectable solution can be prepared by incorporating the subject polypeptide of the present disclosure or its functional fragment in a desired amount in a suitable solvent with various other components as listed above, followed by sterilization by filtration.Generally, dispersion is prepared by incorporating various sterilized active components into a sterile vehicle that contains a basic dispersion medium and other components.For the preparation of sterile injectable solution, a particular desired preparation method is vacuum drying and freeze-drying technology, which produces a powder of active components and any additional desired components from the solution that has been previously sterilized and filtered.

[0178] In some embodiments, the present disclosure provides a pharmaceutical composition for oral administration comprising a subject polypeptide of the present disclosure or a functional fragment thereof and a pharmaceutical excipient suitable for oral administration.

[0179] In some embodiments, provided herein is a solid pharmaceutical composition for oral administration, comprising: (i) an effective amount of a subject polypeptide of the present disclosure or a functional fragment thereof; optionally (ii) an effective amount of a second agent; and (iii) a pharmaceutical excipient suitable for oral administration. In some embodiments, the composition further comprises (iv) an effective amount of a third agent.

[0180] In some embodiments, the pharmaceutical composition is a liquid pharmaceutical composition suitable for oral intake.The pharmaceutical composition suitable for oral administration can be presented as a powder or in granules, in a solution or suspension in aqueous or non-aqueous liquid, in oil-in-water emulsion or in water-in-oil liquid emulsion, in separate dosage forms such as capsules, cachets or tablets, or liquids or aerosol sprays, each containing a predetermined amount of active ingredient.Such dosage forms can be prepared by any of the methods of pharmacy, which typically includes the step of bringing into association the active ingredient with a carrier, which constitutes one or more necessary ingredients.In general, the composition is prepared by uniformly and intimately mixing the active ingredient with a liquid carrier or a finely divided solid carrier or both, and then, if necessary, shaping the product into the desired presentation.

[0181] The present disclosure further encompasses anhydrous pharmaceutical compositions and dosage forms comprising active ingredients, since water can facilitate the degradation of some polypeptides. For example, in the pharmaceutical arts, water can be added (e.g., 5%) as a means of simulating long-term storage to determine characteristics such as the shelf life or stability of a formulation over time. Anhydrous pharmaceutical compositions and dosage forms can be prepared using anhydrous or low moisture containing ingredients and low moisture or low humidity conditions. Pharmaceutical compositions and dosage forms containing lactose can be made anhydrous if substantial contact with moisture and / or humidity is expected during manufacturing, packaging and / or storage. Anhydrous pharmaceutical compositions can be prepared and stored such that their anhydrous nature is maintained. Thus, anhydrous compositions can be packaged using materials known to prevent exposure to water such that they can be included in suitable prescribed kits. Examples of suitable packaging include, but are not limited to, hermetically sealed foils, plastics, etc., unit dose containers, blister packs and strip packs.

[0182] The subject polypeptide of the present disclosure can be combined in intimate admixture with a pharmaceutical carrier according to conventional pharmaceutical compounding techniques. The carrier can take a wide variety of forms depending on the form of preparation desired for administration. In some embodiments, in preparing compositions for oral dosage forms, without using lactose, any of the usual pharmaceutical media can be used as carriers, such as water, glycols, oils, alcohols, flavorings, preservatives, colorings, etc., for oral liquid preparations (such as suspensions, solutions and elixirs) or aerosols; or carriers such as starch, sugar, microcrystalline cellulose, diluents, granulating agents, lubricants, binders and disintegrants for oral solid preparations. For example, suitable carriers include powders, capsules and tablets, as well as solid oral preparations. If desired, tablets can be coated by standard aqueous or non-aqueous techniques.

[0183] Binders suitable for use in pharmaceutical compositions and dosage forms include, but are not limited to, corn starch, potato starch or other starches, gelatin, natural and synthetic gums such as acacia, sodium alginate, alginic acid, other alginates, powdered tragacanth, guar gum, cellulose and its derivatives (e.g., ethyl cellulose, cellulose acetate, calcium carboxymethylcellulose, sodium carboxymethylcellulose), polyvinylpyrrolidone, methylcellulose, pregelatinized starch, hydroxypropyl methylcellulose, microcrystalline cellulose, and mixtures thereof.

[0184] Examples of fillers suitable for use in pharmaceutical compositions and dosage forms include, but are not limited to, talc, calcium carbonate (e.g., granules or powder), microcrystalline cellulose, powdered cellulose, dextrates, kaolin, mannitol, silicic acid, sorbitol, starch, pregelatinized starch, and mixtures thereof.

[0185] Disintegrants can be used in the composition to provide tablets that disintegrate when exposed to an aqueous environment. Too much disintegrant can produce tablets that can disintegrate in the bottle. Too little can be insufficient for disintegration to occur, thus altering the rate and extent of release of the active ingredient(s) from the dosage form. Thus, a dosage form can be formed using a sufficient amount of disintegrant that is neither too little nor too much to adversely alter the release of the active ingredient(s). The amount of disintegrant used can vary based on the type of formulation and the mode of administration, and can be readily discerned by one of ordinary skill in the art. About 0.5 to about 15 weight percent of disintegrant or about 1 to about 5 weight percent of disintegrant can be used in the pharmaceutical composition. Disintegrants that can be used in forming pharmaceutical compositions and dosage forms include, but are not limited to, agar-agar, alginic acid, calcium carbonate, microcrystalline cellulose, croscarmellose sodium, crospovidone, polacrilin potassium, sodium starch glycolate, potato or tapioca starch, other starches, pregelatinized starch, other starches, clays, other algins, other celluloses, gums, or mixtures thereof.

[0186] Lubricants that can be used in forming pharmaceutical compositions and dosage forms include, but are not limited to, calcium stearate, magnesium stearate, mineral oil, light mineral oil, glycerin, sorbitol, mannitol, polyethylene glycol, other glycols, stearic acid, sodium lauryl sulfate, talc, hydrogenated vegetable oils (e.g., peanut oil, cottonseed oil, sunflower oil, sesame oil, olive oil, corn oil, and soybean oil), zinc stearate, ethyl oleate, ethyl laureate, agar, or mixtures thereof. Additional lubricants include, for example, syloid silica gel, coagulated aerosol of synthetic silica, or mixtures thereof. Lubricants can be optionally added in an amount of less than about 1 weight percent of the pharmaceutical composition.

[0187] When aqueous suspensions and / or elixirs are desired for oral administration, the active ingredient therein may be combined with diluents such as water, ethanol, propylene glycol, glycerin, and various combinations thereof, as well as various sweetening or flavoring agents, coloring substances or dyes, and, if desired, emulsifying and / or suspending agents.

[0188] Tablets may be uncoated or may be coated by known techniques to delay disintegration and absorption in the gastrointestinal tract, thereby providing a sustained effect over a longer period of time.For example, time-delay materials such as glyceryl monostearate or glyceryl distearate can be used.The preparation for oral use can also be presented as a hard gelatin capsule in which active ingredient is mixed with an inert solid diluent, such as calcium carbonate, calcium phosphate or kaolin, or as a soft gelatin capsule in which active ingredient is mixed with water or an oil medium, such as peanut oil, liquid paraffin or olive oil.

[0189] Surfactants that can be used to form pharmaceutical compositions and dosage forms include, but are not limited to, hydrophilic surfactants, lipophilic surfactants, and mixtures thereof. That is, a mixture of hydrophilic surfactants can be used, a mixture of lipophilic surfactants can be used, or a mixture of at least one hydrophilic surfactant and at least one lipophilic surfactant can be used.

[0190] Surfactants with lower HLB values ​​are more lipophilic or hydrophobic and have greater solubility in oils, while surfactants with higher HLB values ​​are more hydrophilic and have greater solubility in aqueous solutions. Hydrophilic surfactants are generally considered to be compounds with HLB values ​​greater than about 10, as are anionic, cationic or zwitterionic compounds for which the HLB scale is generally not applicable. Similarly, lipophilic (i.e., hydrophobic) surfactants are compounds with HLB values ​​equal to or less than about 10. However, the HLB values ​​of surfactants are merely a rough guide that is generally used to enable the formulation of industrial, pharmaceutical and cosmetic emulsions.

[0191] Hydrophilic surfactants can be either ionic or nonionic.Suitable ionic surfactants include but are not limited to alkyl ammonium salts; fusidate salts; fatty acid derivatives of amino acids, oligopeptides and polypeptides; glyceride derivatives of amino acids, oligopeptides and polypeptides; lecithin and hydrogenated lecithin; lysolecithin and hydrogenated lysolecithin; phospholipids and their derivatives; lysophospholipids and their derivatives; carnitine fatty acid ester salts; salts of alkyl sulfates; fatty acid salts; sodium docusate; acyl lactylates; mono- and diacetylated tartaric acid esters of mono- and diglycerides; succinylated mono- and di-glycerides; citrate esters of mono- and diglycerides; and mixtures thereof.

[0192] Of the above mentioned groups, ionic surfactants include, by way of example: lecithin, lysolecithin, phospholipids, lysophospholipids and derivatives thereof; carnitine fatty acid ester salts; salts of alkyl sulfates; fatty acid salts; sodium docusate; acylactylates; mono- and diacetylated tartaric acid esters of mono- and diglycerides; succinylated mono- and diglycerides; citric acid esters of mono- and diglycerides; and mixtures thereof.

[0193] Ionic surfactants include ionized forms of lecithin, lysolecithin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidic acid, phosphatidylserine, lysophosphatidylcholine, lysophosphatidylethanolamine, lysophosphatidylglycerol, lysophosphatidic acid, lysophosphatidylserine, PEG-phosphatidylethanolamine, PVP-phosphatidylethanolamine, lactylic esters of fatty acids, and stearoyl-2-lactylate. , stearoyl lactylate, succinylated monoglycerides, mono / diacetylated tartaric acid esters of mono / diglycerides, citrate esters of mono / diglycerides, cholyl sarcosine, caproate, caprylate, caprate, laurate, myristate, palmitate, oleate, ricinoleate, linoleate, linolenate, stearate, lauryl sulfate, tetracecyl sulfate, docusate, lauroyl carnitine, palmitoyl carnitine, myristoyl carnitine, and salts and mixtures thereof.

[0194] Hydrophilic nonionic surfactants may include, but are not limited to, alkyl glucosides; alkyl maltosides; alkyl thioglucosides; lauryl macrogol glycerides; polyoxyalkylene alkyl ethers such as polyethylene glycol alkyl ethers; polyoxyalkylene alkylphenols such as polyethylene glycol alkylphenols; polyoxyalkylene alkylphenol fatty acid esters such as polyethylene glycol fatty acid monoesters and polyethylene glycol fatty acid diesters; polyethylene glycol glycerol fatty acid esters; polyglycerol fatty acid esters; polyoxyalkylene sorbitan fatty acid esters such as polyethylene glycol sorbitan fatty acid esters; hydrophilic transesterification products of polyols with at least one member of the group consisting of glycerides, vegetable oils, hydrogenated vegetable oils, fatty acids, and sterols; polyoxyethylene sterols, derivatives and analogs thereof; polyoxyethylated vitamins and derivatives thereof; polyoxyethylene-polyoxypropylene block copolymers; and mixtures thereof; polyethylene glycol sorbitan fatty acid esters and hydrophilic transesterification products of polyols with at least one member of the group consisting of triglycerides, vegetable oils, and hydrogenated vegetable oils. The polyol can be glycerol, ethylene glycol, polyethylene glycol, sorbitol, propylene glycol, pentaerythritol or a saccharide.

[0195] Other hydrophilic non-ionic surfactants are PEG-10 laurate, PEG-12 laurate, PEG-20 laurate, PEG-32 laurate, PEG-32 dilaurate, PEG-12 oleate, PEG-15 oleate, PEG-20 oleate, PEG-20 dioleate, PEG-32 oleate, PEG-200 oleate, PEG-400 oleate, PEG-15 stearate, PEG-32 distearate. Salt, PEG-40 Stearate, PEG-100 Stearate, PEG-20 Dilaurate, PEG-25 Glyceryl Trioleate, PEG-32 Dioleate, PEG-20 Glyceryl Laurate, PEG-30 Glyceryl Laurate, PEG-20 Glyceryl Stearate, PEG-20 Glyceryl Oleate, PEG-30 Glyceryl Oleate, PEG-30 Glyceryl Laurate, PEG-40 Glyceryl Laurate Polyglyceryl-10 Laurate, PEG-40 Palm Kernel Oil, PEG-50 Hydrogenated Castor Oil, PEG-40 Castor Oil, PEG-35 Castor Oil, PEG-60 Castor Oil, PEG-40 Hydrogenated Castor Oil, PEG-60 Hydrogenated Castor Oil, PEG-60 Corn Oil, PEG-6 Capric / Caprylic Glycerides, PEG-8 Capric / Caprylic Glycerides, Polyglyceryl-10 Laurate, PEG-30 Cholesterol, PEG-25 Phytosterols, PEG-30 Soy Sterols Examples of suitable glyceryl ethers include, but are not limited to, glyceryl ether, PEG-20 trioleate, PEG-40 sorbitan oleate, PEG-80 sorbitan laurate, polysorbate 20, polysorbate 80, POE-9 lauryl ether, POE-23 lauryl ether, POE-10 oleyl ether, POE-20 oleyl ether, POE-20 stearyl ether, tocopheryl PEG-100 succinate, PEG-24 cholesterol, polyglyceryl-10 oleate, Tween 40, Tween 60, sucrose monostearate, sucrose monolaurate, sucrose monopalmitate, PEG 10-100 nonylphenol series, PEG 15-100 octylphenol series, and poloxamers.

[0196] Suitable lipophilic surface-active substances include, by way of example only: fatty alcohols, glycerol fatty acid esters, acetylated glycerol fatty acid esters, lower alcohol fatty acid esters, propylene glycol fatty acid esters, sorbitan fatty acid esters, polyethylene glycol sorbitan fatty acid esters, sterols and sterol derivatives, polyoxyethylated sterols and sterol derivatives, polyethylene glycol alkyl ethers, sugar esters, sugar ethers, lactic acid derivatives of mono- and diglycerides, hydrophobic transesterification products of at least one member of the group consisting of glycerides, vegetable oils, hydrogenated vegetable oils, fatty acids and sterols with polyols, oil-soluble vitamins / vitamin derivatives, and mixtures thereof. Of this group, preferred lipophilic surface-active substances include glycerol fatty acid esters, propylene glycol fatty acid esters and mixtures thereof, or are hydrophobic transesterification products of at least one member of the group consisting of vegetable oils, hydrogenated vegetable oils and triglycerides with polyols.

[0197] In one embodiment, the composition comprises a solubilizer to ensure good solubilization and / or dissolution of the compound and minimize precipitation of the compound.This may be particularly advantageous for compositions for parenteral use, such as compositions for injection.Solubilizers can also be added to increase the solubility of hydrophilic drugs and / or other components, such as surfactants, or to maintain the composition as a stable or homogeneous solution or dispersion.

[0198] Examples of suitable solubilizing agents include, but are not limited to, alcohols and polyols such as ethanol, isopropanol, butanol, benzyl alcohol, ethylene glycol, propylene glycol, butanediol and its isomers, glycerol, pentaerythritol, sorbitol, mannitol, transcutol, dimethyl isosorbide, polyethylene glycol, polypropylene glycol, polyvinyl alcohol, hydroxypropyl methylcellulose and other cellulose derivatives, cyclodextrin and cyclodextrin derivatives; ethers of polyethylene glycol having an average molecular weight of about 200 to about 6000, such as tetrahydrofurfuryl alcohol PEG ether (glycofurol) or methoxy PEG; 2-pyrrolidone, 2-piperidone, ε-caprolid ... amides and other nitrogen-containing compounds such as tetrahydrofuran, N-alkylpyrrolidone, N-hydroxyalkylpyrrolidone, N-alkylpiperidone, N-alkylcaprolactam, dimethylacetamide and polyvinylpyrrolidone; esters such as ethyl propionate, tributyl citrate, acetyl triethyl citrate, acetyl tributyl citrate, triethyl citrate, ethyl oleate, ethyl caprylate, ethyl butyrate, triacetin, propylene glycol monoacetate, propylene glycol diacetate, ε-caprolactone and its isomers, δ-valerolactone and its isomers, β-butyrolactone and its isomers; and other solubilizing agents known in the art such as dimethylacetamide, dimethylisosorbide, N-methylpyrrolidone, monooctanoin, diethylene glycol monoethyl ether and water.

[0199] Mixtures of solubilizers can also be used. Examples include, but are not limited to, triacetin, triethyl citrate, ethyl oleate, ethyl caprylate, dimethylacetamide, N-methylpyrrolidone, N-hydroxyethylpyrrolidone, polyvinylpyrrolidone, hydroxypropylmethylcellulose, hydroxypropylcyclodextrin, ethanol, polyethylene glycol 200-100, glycofurol, transcutol, propylene glycol and dimethylisosorbide. Particularly preferred solubilizers include sorbitol, glycerol, triacetin, ethyl alcohol, PEG-400, glycofurol and propylene glycol.

[0200] The amount of solubilizer that can be included is not particularly limited. The amount of a given solubilizer can be limited to the amount tolerated by the organism, which can be easily determined by those skilled in the art. In some situations, for example, to maximize the concentration of the drug, it may be advantageous to include an amount of solubilizer that far exceeds the amount tolerated by the organism, and the excess solubilizer is removed using conventional techniques such as distillation or evaporation before administering the composition to the subject. Thus, when present, the solubilizer can be in a weight ratio of 10%, 25%, 50%, 100% or up to about 200% by weight based on the combined weight of the drug and other excipients. If desired, very small amounts of solubilizer can be used, such as 5%, 2%, 1% or even less. Typically, the solubilizer can be present in an amount of about 1% to about 100% by weight, more typically about 5% to about 25% by weight.

[0201] The composition may further comprise one or more pharma- ceutically acceptable additives and excipients, including, but not limited to, detackifiers, antifoaming agents, buffers, polymers, antioxidants, preservatives, chelating agents, viscomodulators, tonicifiers, flavorants, colorants, odorants, opacifiers, suspending agents, binders, fillers, plasticizers, lubricants, and mixtures thereof.

[0202] In addition, acids or bases can be incorporated into the composition to facilitate processing, enhance stability, or for other reasons. Examples of pharma-ceutically acceptable bases include amino acids, amino acid esters, ammonium hydroxide, potassium hydroxide, sodium hydroxide, sodium bicarbonate, aluminum hydroxide, calcium carbonate, magnesium hydroxide, magnesium aluminum silicate, synthetic aluminum silicate, synthetic hydrocalcite, magnesium aluminum hydroxide, diisopropylethylamine, ethanolamine, ethylenediamine, triethanolamine, triethylamine, triisopropanolamine, trimethylamine, tris(hydroxymethyl)aminomethane (TRIS), and others. Also suitable are bases that are salts of pharma- ceutically acceptable acids such as acetic acid, acrylic acid, adipic acid, alginic acid, alkanesulfonic acid, amino acids, ascorbic acid, benzoic acid, boric acid, butyric acid, carbonic acid, citric acid, fatty acids, formic acid, fumaric acid, gluconic acid, hydroquinosulfonic acid, isoascorbic acid, lactic acid, maleic acid, oxalic acid, para-bromophenylsulfonic acid, propionic acid, p-toluenesulfonic acid, salicylic acid, stearic acid, succinic acid, tannic acid, tartaric acid, thioglycolic acid, toluenesulfonic acid, uric acid, etc. Salts of polyprotic acids such as sodium phosphate, disodium hydrogen phosphate, and sodium dihydrogen phosphate can also be used. When the base is a salt, the cation can be any convenient and pharma-ceutically acceptable cation such as ammonium, alkali metals, alkaline earth metals, etc. Examples include, but are not limited to, sodium, potassium, lithium, magnesium, calcium, and ammonium.

[0203] Suitable acids are pharma- ceutically acceptable organic or inorganic acids. Examples of suitable inorganic acids include hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, nitric acid, boric acid, phosphoric acid, etc. Examples of suitable organic acids include acetic acid, acrylic acid, adipic acid, alginic acid, alkanesulfonic acid, amino acids, ascorbic acid, benzoic acid, boric acid, butyric acid, carbonic acid, citric acid, fatty acids, formic acid, fumaric acid, gluconic acid, hydroquinosulfonic acid, isoascorbic acid, lactic acid, maleic acid, methanesulfonic acid, oxalic acid, para-bromophenylsulfonic acid, propionic acid, p-toluenesulfonic acid, salicylic acid, stearic acid, succinic acid, tannic acid, tartaric acid, thioglycolic acid, toluenesulfonic acid, uric acid, etc.

[0204] In another aspect of the present disclosure, a kit is provided that includes a unit dose containing a subject polypeptide composition of the present disclosure and instructions for use. The kit may further include one or more unit doses containing one or more additional reagents, such as the immunosuppressant reagents, cytotoxic or radiotoxic agents described above, or one or more additional therapeutic proteins (e.g., antibodies) described herein. The kit typically includes a label indicating the intended use of the contents of the kit. The term label includes any written or recorded material supplied on or with the kit or that otherwise accompanies the kit.

[0205] The kits of the present disclosure may also include diagnostic agents and / or other therapeutic agents. In one embodiment, the kits include a subject polypeptide of the present disclosure and a diagnostic agent that can be used in a diagnostic method for diagnosing the status or presence of a disease, condition or disorder in a subject as described herein. EXAMPLES

[0206] The following examples are presented for the purpose of illustrating various embodiments of the present disclosure, and are not intended to limit the present disclosure in any manner. These examples, together with the methods described herein, are representative of currently preferred embodiments, are exemplary, and are not intended as limitations on the scope of the present invention. Those skilled in the art will recognize variations thereof and other uses that are encompassed within the spirit of the present disclosure as defined by the scope of the claims.

[0207] Example 1: Binding affinity assay All SPR measurements were performed on a BIAcore 3000 instrument (GE Biosciences, Piscataway, NJ). For operation and control of the BIAcore 3000 instrument, BIAcore software - BIAcore 3000 Control software V3.2 was used. For analysis of SPR data from the BIAcore 3000 instrument, BiaEvaluation software V4.1 was used and data were plotted using Graph Pad Prism software version 5. Binding affinity of CTLA4 variants to CD80 or CD86 was measured at 25°C in HBS-EP buffer (10 mM HEPES, 150 mM NaCl, 3.4 mM EDTA, 0.005% P20). The flow rate for affinity studies was 30 μL / min. As ligands for the construction of the reference channel of the chip, the indicated CTLA4 variants were used. Analyte binding to the immobilized ligand (CD80 / CD86) was measured, with sIL-6R concentrations ranging from 1.2 to 100 nM (3x dilution). Each sample was injected at a flow rate of 30 μL / min for 3 min to bind to the peptide bound to the chip. The bound analyte was then dissociated by pumping analyte-free binding buffer over the chip at the same flow rate. After 500 s, remaining bound analyte was removed by injecting regeneration solution (1 M formic acid). Data were analyzed using the Kinetics Wizard and manual fit programs, both included within the BiaEvaluation software V4.1. a is the on-rate; k d is the off-rate; KD is the equilibrium dissociation constant; the relative affinity is K D (Abatacept) / K D (variant) k a , k d , K D and affinity relative to abatacept (samples 70# or 103#) are shown in Tables 1-1, 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, and 1-8.

[0208] All SPR measurements were performed on a BIAcore 3000 instrument (GE Biosciences, Piscataway, NJ). [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 1-6] [Table 1-7] [Table 1-8]

[0209] Example 2: Determination of pH Dependence of Binding Affinity for CD80 and CD86 SPR measurements at pH 7.4 and pH 6.0 were carried out in parallel according to the protocol detailed in Example 1. D The pH dependence was calculated as K at pH 6.0. D value and K at pH 7.4 DThe KD values ​​were calculated as the ratio between the values. This indicates the affinity reduction fold from pH 7.4 to pH 6.0. If the pH dependence of the subject variants described herein is greater than 1, this means that the variant binds to CD80 or CD86 in a pH-dependent manner such that its binding to CD80 or CD86 at pH 7.4 is higher than that at pH 6.0. If the pH dependence of the subject variants described herein is less than 1, this means that the variant binds to CD80 or CD86 in a pH-dependent manner such that its binding to CD80 or CD86 at pH 6.0 is higher than that at pH 7.4. SPR measurements were performed on exemplary variants 133# and 142#, and abatacept and belatacept were used as references. The resulting KD values ​​are shown in Tables 2-1 and 3-1 below, respectively. The pH dependences thus determined by comparing the binding affinities at pH 7.4 and pH 6.0 are shown in Tables 2-2 and 3-2, respectively. As shown in Tables 2-2 and 3-2, both exemplary variants 133# and 142# exhibited a higher pH dependence for CD80 and CD86 affinity binding than abatacept, indicating a more significant decrease in affinity binding from pH 7.4 to pH 6.0; exemplary variant 133# exhibited a higher pH dependence for CD80 and CD86 affinity binding than belatacept; and exemplary variant 142# exhibited a much higher pH dependence for CD86 affinity binding than both abatacept and belatacept. These data indicate superior properties of these exemplary CTLA4-Ig variants in terms of antigen neutralization and clearance. [Table 2-1] [Table 2-2] [Table 3-1] [Table 3-2]

[0210] Example 3: Evaluation of relative binding affinities Exemplary CTLA4-Ig variants were examined by competitive inhibition assay for their binding affinity to CD80 and CD86.In this competitive inhibition assay, abatacept (WT CTLA4-Ig) construct was transfected into Ramos cells (human Burkitt's lymphoma cells), and abatacept was expressed and displayed on the cell surface of Ramos cells.Then, as shown in FIG. 1, the Ramos cells expressing abatacept were incubated with biotin-conjugated CD80 or CD86, which can be bound by abatacept expressed on the cell surface.Then, exemplary CTLA4-Ig variants or reference variants (e.g., abatacept or belatacept) were added to the incubated cells. Competition between the subsequently added exemplary CTLA4-Ig variant and surface-displayed abatacept for binding to CD80-biotin or CD86-biotin will result in a portion of the surface-bound CD80-biotin or CD86-biotin being shed from the cell surface. The amount of CD80-biotin or CD86-biotin shed from the cell surface may be proportional to the binding affinity of the exemplary variant to CD80 or CD86 compared to abatacept. As a result, the amount of CD80-biotin or CD86-biotin remaining on the cell surface is inversely proportional to the relative binding affinity of the exemplary variant. The amount of CD80 or CD86 bound to the cell surface was examined by flow cytometry after incubating the cells with streptavidin-APC and indicated by the level of fluorescent signal provided by the APC dye bound to the cell surface. As shown in Figures 2A and 2B, the lower the fluorescent signal, the higher the binding affinity of the exemplary variants to CD80 or CD86 examined. For example, exemplary variants, mutants 142# (as shown by curves #1 and #6 in Figures 2A and 2B, respectively) and 133# (curves #2 and #7) showed higher affinity to CD80 and CD86 compared to abatacept (curves #4 and #9) and belatacept (curves #3 and #8).

[0211] Example 4: Evaluation of relative binding affinities Exemplary CTLA4-Ig variants were examined by different competitive inhibition assays for their binding affinity to CD80 and CD86.In this competitive inhibition assay, CD28-Ig constructs were transfected into Ramos cells (human Burkitt's lymphoma cells), and CD28-Ig was expressed and presented on the cell surface of Ramos cells.CD28 may be an important co-stimulatory receptor in T cell activation, so CD28 may have natural binding affinity to CD80 and CD86.Then, CD28-Ig-expressing Ramos cells were incubated with biotin-conjugated CD80 or CD86, which can be bound by CD28-Ig expressed on the cell surface.Then, exemplary CTLA4-Ig variants or reference variants (e.g., abatacept or belatacept) were added to the incubated cells. Competition between the subsequently added exemplary CTLA4-Ig variants and surface-displayed CD28 for binding to CD80-biotin or CD86-biotin will result in some of the surface-bound CD80-biotin or CD86-biotin being shed from the cell surface. The amount of CD80-biotin or CD86-biotin shed from the cell surface may be proportional to the binding affinity of the exemplary variant to CD80 or CD86 compared to CD28. As a result, the amount of CD80-biotin or CD86-biotin remaining on the cell surface is inversely proportional to the relative binding affinity of the exemplary variant. The amount of CD80 or CD86 bound to the cell surface was examined by flow cytometry after incubating the cells with streptavidin-APC, and was indicated by the fluorescent signal level provided by the APC dye bound to the cell surface. As shown in Figures 3A and 3B, the lower the fluorescent signal, the higher the binding affinity of the exemplary variant to the examined CD80 or CD86.For example, exemplary variants, mutants 142# (as shown by curves #11 and #16, respectively, in Figures 3A and 3B) and 133# (curves #12 and #17) showed higher affinity for CD80 and CD86 compared to abatacept (curves #14 and #19) and belatacept (curves #13 and #8).

[0212] Example 5: Evaluation of immunosuppressive effects on T cell proliferation The immunosuppressive effect of the exemplary CTLA4-Ig variants was investigated in an assay in which the inhibitory effect of the exemplary CTLA4-Ig on primary T cell proliferation was tested. In the experiment, T cells were isolated from healthy human peripheral blood mononuclear cells (PBMCs) and labeled with the fluorescent dye carboxyfluorescein succinimidyl ester (CFSE), which is used to track cell proliferation. The isolated primary T cells were stimulated with anti-CD3 antibody OKT3 and CD86-Fc coated on the surface of cell culture dishes. Different concentrations of abatacept or the exemplary CTLA4-Ig variants were added to the T cell cultures, which could inhibit the proliferation of T cells. After 4 days of culture at 37° C., the T cells were subjected to flow cytometry analysis for their proliferation. The half-maximal inhibitory concentration (IC 50 ) was calculated for each tested exemplary variant and abatacept, as shown in FIG. 4.

[0213] Example 6: Evaluation of immunosuppressive effects on IL2 secretion The immunosuppressive effect of the exemplary CTLA4-Ig variants was examined in an assay testing the inhibitory effect of the exemplary CTLA4-Ig on T cell IL12 secretion. Raji cells (human B-lymphocytes) can express CD80 and CD86 on the surface. Jurkat cells (human T-lymphocytes) can activate their surface receptor CD28 when stimulated by PHA (phytohemagglutinin P). Thus, as shown in FIG. 5, in the presence of PHA, co-cultured Raji cells and Jurkat cells can bind their surface CD28 and CD80 / CD86 together, which can lead to the activation of Jurkat cells and the secretion of IL2 from the activated Jurkat cells. In the experiment, Jurkat and Raji cells were co-cultured in the presence of PHA, and different concentrations of abatacept or exemplary CTLA4-Ig variants were added to the co-culture, which could inhibit Jurkat cell activation and thus IL2 secretion, as illustrated in FIG. 5. The higher the immunosuppressive activity of the exemplary variant, the lower the level of IL2 detected. IL2 secretion was examined by ELISA after 24 hours of stimulation with 5 μg / mL PHA. IC 50 was calculated for each tested exemplary variant and abatacept, as shown in Figures 6A-6C. More testing results for exemplary polypeptides are shown in Table 4. [Table 4]

[0214] Exemplary polypeptides tested in Examples 1-6 are polypeptides containing the mutations listed in Table 5 with respect to SEQ ID NO:2. [Table 5-1] [Table 5-2] [Table 5-3]

[0215] While preferred embodiments of the present disclosure have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will occur to those skilled in the art without departing from the present invention. It is understood that various alternatives to the disclosed embodiments described herein may be employed in practicing the present disclosure. The following claims define the scope of the present disclosure, and it is intended that methods and structures within the scope of these claims and their equivalents be covered thereby. [Table 6-1] [Table 6-2] [Table 7-1] [Table 7-2] [Table 7-3] [Table 8-1] [Table 8-2]

Claims

1. A polypeptide comprising a first amino acid sequence having 95% or greater sequence identity to amino acids 1-124 of SEQ ID NO:2, With respect to SEQ ID NO:2: (1) G68F, (2) G27DKA, G27DK, or G27H, and (3) 0 to 3 additional mutations selected from the group consisting of A40T, K93M, P117S, G105S, L77V, and D122H. and having up to five mutations, including The polypeptide exhibits at least a 10-fold greater binding affinity for CD80 as compared to the affinity of abatacept (SEQ ID NO: 2) for CD80 as determined by surface plasmon resonance at 25°C.

2. With respect to SEQ ID NO: 2, G27DKA / G68F, G27H / G68F, G27DK / G68F, G27DKA / G68F / A40T / P117S, G27DKA / G68F / P117S, G27DK / G68F / A40T, G27DK / G68F / L77V / G105S / P117S, G27DK / G68F / L77V, G27 2. The polypeptide of claim 1, comprising a combination of amino acid substitutions selected from the group consisting of G27DK / G68F / P117S, G27DK / G68F / D122H, G27DKA / G68F / A40T, G27DKA / G68F / K93M, G27DK / G68F / K93M, and G27DKA / A40T / G68F / K93M.

3. 2. The polypeptide of claim 1, comprising the amino acid substitution combination G27DKA / A40T / G68F / K93M relative to SEQ ID NO:

2.

4. 2. The polypeptide of claim 1, comprising the amino acid substitution combination G27DKA / G68F / A40T with respect to SEQ ID NO:

2.

5. 2. The polypeptide of claim 1, comprising the amino acid substitution combination G27DKA / G68F / K93M relative to SEQ ID NO:

2.

6. 2. The polypeptide of claim 1, comprising the amino acid substitution combination G27DKA / G68F with respect to SEQ ID NO:

2.

7. 2. The polypeptide of claim 1, comprising the amino acid substitution combination G27DK / G68F / K93M relative to SEQ ID NO:

2.

8. The polypeptide of any one of claims 1 to 7, further comprising a second amino acid sequence fused to the first amino acid sequence.

9. 9. The polypeptide of claim 8, wherein the second amino acid sequence encodes an IgG Fc region, and the second amino acid sequence has at least 90% sequence identity to SEQ ID NO:7 or has at least 90% sequence identity to amino acids 131-357 of SEQ ID NO:

2.

10. The polypeptide of claim 9 , wherein the IgG Fc region is derived from a human IgG molecule.

11. The polypeptide of any one of claims 8 to 10, wherein the polypeptide has 90%, 95%, 96%, 97%, 98%, 99%, 99.5% or 100% sequence identity to amino acids 1 to 359 of SEQ ID NO:

6.

12. A polypeptide-drug conjugate comprising the polypeptide according to any one of claims 1 to 11.

13. A pharmaceutical composition comprising a polypeptide according to any one of claims 1 to 11 or a polypeptide-drug conjugate according to claim 12 and a pharma- ceutically acceptable excipient.

14. A kit comprising the polypeptide according to any one of claims 1 to 11 or the polypeptide-drug conjugate according to claim 12 in a container.

15. 14. The pharmaceutical composition of claim 13 for use in treating a condition in a subject.

16. The condition may be selected from the group consisting of graft rejection, infection, endotoxic shock associated with infection, arthritis, rheumatoid arthritis, psoriatic arthritis, systemic onset juvenile idiopathic arthritis (JIA), inflammatory bowel disease (IBD), systemic lupus erythematosus (SLE), asthma, pelvic peritonitis, Alzheimer's disease, Crohn's disease, ulcerative colitis, irritable bowel syndrome, multiple sclerosis, ankylosing spondylitis, dermatomyositis, uveitis, Peyronie's disease, celiac disease, gallbladder disease, pilonidal disease, peritonitis, psoriasis, vasculitis, surgical adhesions, stroke, type I diabetes, Lyme arthritis, myelopathy, pulmonary tuberculosis ...

16. The pharmaceutical composition for use according to claim 15, comprising meningoencephalitis, immune-mediated inflammatory disorders of the central and peripheral nervous system, autoimmune disorders, pancreatitis, surgical trauma, graft versus host disease, cardiac disease, bone resorption, burn patients, myocardial infarction, Paget's disease, osteoporosis, sepsis, hepatic / pulmonary fibrosis, periodontitis, hypochlorhydria, solid tumors, renal cell carcinoma, liver cancer, multiple myeloma, prostate cancer, bladder cancer, pancreatic cancer, neurological cancers, B-cell malignancies, Castleman's disease, certain lymphomas, chronic lymphocytic leukemia or multiple myeloma.

17. An isolated polynucleotide encoding the polypeptide of any one of claims 1 to 11.

18. A vector comprising the isolated polynucleotide of claim 17.

19. A cell comprising the vector of claim 18.

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