Anti-TNFR2 Antibodies and Methods of Use Thereof

Abstract

The present disclosure provides antibodies that bind to TNFR2 and the use and related methods of these antibodies.The present disclosure also provides the process for making, preparing and producing the antibodies that bind to TNFR2.The antibodies of the present disclosure are useful in one or more of the diagnosis, prevention or treatment of the disorders or conditions that are mediated by or related to TNFR2 activity. [Figure 1] TIFF2025517490000048.tif114125

Description

[Technical field]

[0001] Reference to sequence listing "This application has been submitted electronically in .xml format and contains a Sequence Listing, which is incorporated by reference herein in its entirety. A copy of said .xml, created on May 5, 2023, is titled PC072865 Sequence Listing.xml and is 42,322 bytes in size."

[0002] background The present invention relates to antibodies that specifically bind to TNFR2, as well as compositions, methods and uses thereof, including the use of the disclosed antibodies to treat autoimmune and autoinflammatory diseases. The present invention also relates to related molecules, such as nucleic acids encoding such antibodies, compositions, and related methods, such as methods for producing and purifying such antibodies and bispecific antibodies, and their use in diagnostic and therapeutic agents. [Background technology]

[0003] TNF is a pleiotropic cytokine expressed on the cell surface of leukocytes and subsequently released via the activity of several proteolytic enzymes. The cellular response to TNF is mediated by two receptors: TNFR1 (TNFRSF1A), which is ubiquitously expressed and mediates proinflammatory responses, and TNFR2 (TNFRSF1B), which is more selectively expressed on certain leukocyte subtypes and is thought to primarily mediate immunomodulatory effects (Salomon 2021). Soluble TNF can activate both receptors, but TNFR2 is more preferentially activated by membrane-associated TNF. Among the activities ascribed to TNFR2 agonism, the most relevant for the treatment of autoimmune and autoinflammatory conditions are the expansion of regulatory T cells, activation-induced cell death and exhaustion of effector T cells, and the enhancement of a regulatory / anti-inflammatory phenotype in B cells, mesenchymal stem cells (MSCs) and myeloid cells, e.g., myeloid-derived suppressor cells (MDSCs) and glia (Faustman and Davis 2010, Salomon 2021). Summary of the Invention [Problem to be solved by the invention]

[0004] [Means for solving the problem]

[0005] The present disclosure provides antibodies that bind to TNFR2 and the use and related methods of these antibodies. The present disclosure also provides a process for making, preparing and producing antibodies that bind to TNFR2. The antibodies of the present disclosure are useful in one or more of the diagnosis, prevention or treatment of disorders or conditions mediated by or related to TNFR2 activity, including but not limited to rheumatoid arthritis (RA), ankylosing spondylitis (AS), Crohn's disease (CD), ulcerative colitis (UC), graft-versus-host disease (GVHD), transplantation, psoriasis (PSO), psoriatic arthritis (PSA), atopic dermatitis (AD), vitiligo, alopecia areata (AA), type 1 diabetes (T1D), multiple sclerosis (MS), irritable bowel disease (IBD), autoimmune hepatitis and systemic lupus erythematosus (SLE). The present disclosure further encompasses the expression of antibodies, and the preparation and manufacture of compositions that include the antibodies of the present disclosure, such as pharmaceuticals for the use of the antibodies.

[0006] Polynucleotides are provided that encode antibodies that bind to TNFR2. Polynucleotides are also provided that encode antibody heavy or light chains, or both. Host cells that express the antibodies are provided. Methods of treatment using the antibodies are provided. Such methods include, but are not limited to, one or more of the following: treating or preventing diseases related to or mediated by TNFR2 expression and / or TNFR2 binding: rheumatoid arthritis (RA), ankylosing spondylitis (AS), Crohn's disease (CD), ulcerative colitis (UC), graft-versus-host disease (GVHD), transplantation, psoriasis (PSO), psoriatic arthritis (PSA), atopic dermatitis (AD), vitiligo, alopecia areata (AA), type 1 diabetes (T1D), multiple sclerosis (MS), irritable bowel disease (IBD), autoimmune hepatitis and systemic lupus erythematosus (SLE).

[0007] The present invention can be more easily understood by referring to the following detailed description of the embodiments of the present invention and the examples contained herein. It should be understood that the present invention is not limited to a specific method of preparation, which may of course vary. It should also be understood that the terminology used herein is only for the purpose of describing specific embodiments, and is not intended to be limiting.

[0008] Exemplary embodiments (E) of the invention provided herein include the following: E1. An isolated antibody that specifically binds to TNFR2, comprising a heavy chain variable region (VH) and a light chain variable region (VL), comprising CDR-H1, CDR-H2 and CDR-H3 sequences of a VH sequence selected from the group consisting of SEQ ID NO:13, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:29 and SEQ ID NO:30; and CDR-L1, CDR-L2 and CDR-L3 sequences of a VL sequence selected from the group consisting of SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8 and SEQ ID NO:9.

[0009] E2. An isolated antibody that specifically binds to TNFR2, comprising a heavy chain variable region (VH) and a light chain variable region (VL), the antibody comprising CDR-H1, CDR-H2 and CDR-H3 sequences according to SEQ ID NO: 30, and CDR-L1, CDR-L2 and CDR-L3 sequences according to SEQ ID NO: 9.

[0010] E3. An isolated antibody that specifically binds to TNFR2, comprising a heavy chain variable region (VH) and a light chain variable region (VL), (i) a CDR-L1 sequence according to SEQ ID NO:1; a CDR-L2 sequence according to SEQ ID NO:7 and a CDR-L3 sequence according to SEQ ID NO:3, and a CDR-H1 sequence according to SEQ ID NO:10; a CDR-H2 sequence according to SEQ ID NO:20; a CDR-H3 sequence according to SEQ ID NO:12; or (ii) a CDR-L1 sequence according to SEQ ID NO:1; a CDR-L2 sequence according to SEQ ID NO:2 and a CDR-L3 sequence according to SEQ ID NO:3, and a CDR-H1 sequence according to SEQ ID NO:10; a CDR-H2 sequence according to SEQ ID NO:11; and a CDR-H3 sequence according to SEQ ID NO:12. An antibody comprising:

[0011] E4. An isolated antibody that specifically binds to TNFR2, comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the CDR-L1 sequence is according to SEQ ID NO:1; the CDR-L2 sequence is according to SEQ ID NO:7, the CDR-L3 sequence is according to SEQ ID NO:3, the CDR-H1 sequence is according to SEQ ID NO:10; the CDR-H2 sequence is according to SEQ ID NO:20; and the CDR-H3 sequence is according to SEQ ID NO:12.

[0012] E5. The antibody of any one of E1 to E4, comprising a VH framework sequence derived from a human germline VH sequence selected from the group consisting of IGHV1-46, IGHV4-31, IGHV4-30-4 and IGHV4-4.

[0013] E6. The antibody of any one of E1 to E5, comprising a VH framework sequence derived from a human IGHV1-46 germline sequence.

[0014] E7. The antibody of any one of E1 to E6, comprising a VL framework sequence derived from a human germline VL sequence selected from the group consisting of IGKV1-9, IGKV1-33, IGKV1-27, IGKV1-39, IGKV1-9, IGKV1-1 and IGKV1-11.

[0015] E8. The antibody of any one of E1-E7, comprising a VL framework sequence derived from a human germline IGKV1-9 sequence.

[0016] E9. The antibody of any one of E1-E8, comprising a VL framework sequence and a VH framework sequence, wherein one or both of the VL framework sequence and the VH framework sequence are at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the human germline sequence from which it is derived.

[0017] E10. The antibody of any one of E1-E9, comprising a VL framework sequence and a VH framework sequence, wherein one or both of the VL or VH framework sequences are identical to the human germline sequence from which it is derived.

[0018] E11. An antibody described in any one of E1 to E10, wherein VL comprises an amino acid sequence according to a sequence selected from the group consisting of SEQ ID NO: 4 and SEQ ID NO: 8, and VH comprises an amino acid sequence according to a sequence selected from the group consisting of SEQ ID NO: 13 and SEQ ID NO: 21.

[0019] E12. The antibody according to any one of E1 to E11, comprising a VH sequence of SEQ ID NO: 13 and a VL sequence of SEQ ID NO: 4.

[0020] E13. The antibody according to any one of E1 to E11, comprising a VH sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 21, and a VL sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 8.

[0021] E14. The antibody of any one of E13, comprising a VH sequence of SEQ ID NO:21 and a VL sequence of SEQ ID NO:8.

[0022] E15. The antibody of any one of E14, comprising a VH sequence encoded by the nucleic acid sequence of SEQ ID NO:32.

[0023] E16. The antibody of any one of E14, comprising a VH sequence encoded by the nucleic acid sequence of SEQ ID NO:33.

[0024] E17. The antibody of any one of E1 to E16, comprising a VL sequence encoded by the nucleic acid sequence of SEQ ID NO:31.

[0025] E18. The antibody of any one of E1 to E17, comprising one or both of a VH sequence encoded by a plasmid deposited with the ATCC having ATCC Accession No. PTA-127528 and a VH sequence encoded by a plasmid deposited with the ATCC having ATCC Accession No. PTA-127529.

[0026] E19. The antibody of any one of E1 to E18, comprising a VL sequence encoded by a plasmid deposited with the ATCC having ATCC Accession No. PTA-127531.

[0027] E20. An antibody comprising one or both of a VH sequence encoded by a plasmid deposited with the ATCC having ATCC Accession No. PTA-127528 and a VH sequence encoded by a plasmid deposited with the ATCC having ATCC Accession No. PTA-127529; and a VL sequence encoded by a plasmid deposited with the ATCC having ATCC Accession No. PTA-127531.

[0028] E25. The antibody of E1-E24, further comprising a constant heavy domain (CH1) and a constant light domain (CL).

[0029] E26. The antibody of E25, wherein CH1 is connected to VH and CL is connected to VL to form a Fab domain.

[0030] E26. The antibody of E25, comprising a first and a second Fab domain.

[0031] E27. The antibody of any one of E25 to E26, comprising an antibody Fc domain comprising a first Fc chain and a second Fc chain.

[0032] E28. The antibody of E27, wherein the first Fab domain is covalently fused to a first Fc chain and the second Fab domain is covalently fused to a second Fc chain.

[0033] E29. The antibody described in E27 to E28, wherein the C-terminus of the CH1 domain in the first Fab domain is covalently fused to the N-terminus of the first Fc chain, and the C-terminus of the CH1 domain in the second Fab domain is covalently fused to the N-terminus of the second Fc chain.

[0034] E30. The Fc domain is an IgA (e.g., IgA 1 or IgA 2 ), IgD, IgE, IgM or IgG (e.g., IgG 1 , IgG 2 , IgG 3 or IgG4 The antibody described in E31, which is an Fc domain of

[0035] E31. The Fc domain is IgG 1 The antibody of E30, which is an Fc domain of

[0036] E32. The antibody of E31, wherein the CH1 domain in the first Fab domain comprises a sequence according to SEQ ID NO:23.

[0037] E33. The antibody of E31 to E32, wherein the CL in the first Fab domain comprises a sequence according to SEQ ID NO:5.

[0038] E34. The antibody according to E31 to E33, comprising a light chain (LC) comprising a sequence according to SEQ ID NO:9.

[0039] E35. The antibody according to any one of E31 to E34, wherein the first Fab domain and the second Fab domain are identical.

[0040] E36. The antibody described in E31 to E35, wherein the first Fc chain comprises, from the N-terminus to the C-terminus: a first hinge region, a first CH2 region and a first CH3 region, and the second Fc chain comprises, from the N-terminus to the C-terminus: a second hinge region, a second CH2 region and a second CH3 region.

[0041] E37. The antibody of E36, wherein one or both of the first hinge region and second hinge region comprise a sequence according to SEQ ID NO:23.

[0042] E38. The antibody of E36 to E37, wherein one or both of the first CH2 domain and the second CH2 domain comprise a sequence according to SEQ ID NO:25.

[0043] E39. The antibody of E36 to E38, wherein one or both of the first CH3 domain and the second CH3 domain comprise a sequence according to SEQ ID NO:26.

[0044] E40. The antibody of E36 to E39, wherein one or both of the first Fc chain and the second Fc chain comprise a sequence according to SEQ ID NO:37.

[0045] E41. The antibody according to E36 to E40, wherein the first Fc chain and the second Fc chain are identical.

[0046] E42. The antibody according to E32 to E41, comprising a heavy chain (HC) comprising a sequence according to SEQ ID NO:22.

[0047] E43. The antibody of E26 to E42, further comprising a third Fab and a fourth Fab.

[0048] E44. The antibody of E43, wherein the first Fab, the second Fab, the third Fab and the fourth Fab each comprise a CDR-L1 sequence according to SEQ ID NO:1; a CDR-L2 sequence according to SEQ ID NO:7 and a CDR-L3 sequence according to SEQ ID NO:3, and a CDR-H1 sequence according to SEQ ID NO:10; a CDR-H2 sequence according to SEQ ID NO:20; and a CDR-H3 sequence according to SEQ ID NO:12.

[0049] E45. The antibody described in E43 to E44, wherein the first Fab, the second Fab, the third Fab and the fourth Fab each comprise a VH having the sequence according to SEQ ID NO: 21 and a VL having the sequence according to SEQ ID NO: 8.

[0050] E46. The antibody according to E43 to E45, wherein the first Fab, the second Fab, the third Fab and the fourth Fab are identical to each other.

[0051] E47. The antibody according to E43 to E46, wherein the N'-terminus of the first Fab is connected to the C'-terminus of the third Fab.

[0052] E48. The antibody according to any one of E43 to E47, wherein the N'-terminus of the first Fab is connected to the C'-terminus of the third Fab via a first linker.

[0053] E49. The antibody of E48, wherein the first linker comprises a sequence according to SEQ ID NO:27.

[0054] E50. The antibody according to E43 to E49, wherein the N'-terminus of the second Fab is connected to the C'-terminus of the fourth Fab.

[0055] E51. The antibody according to any one of E43 to E50, wherein the N'-terminus of the second Fab is connected to the C'-terminus of the fourth Fab via a second linker.

[0056] E52. The antibody of E51, wherein the second linker comprises a sequence according to SEQ ID NO:27.

[0057] E53. The antibody according to E47 to E48, wherein HC comprises a sequence according to SEQ ID NO: 30.

[0058] E54. An isolated antibody that specifically binds to TNFR2, comprising a heavy chain (HC) comprising a sequence according to SEQ ID NO:30 and a light chain (LC) comprising a sequence according to SEQ ID NO:9.

[0059] E55. The antibody of any one of E1 to E54, comprising a VH sequence encoded by the nucleic acid sequence of SEQ ID NO: 33.

[0060] E56. The antibody of any one of E1 to E55, comprising a VL sequence encoded by the nucleic acid sequence of SEQ ID NO:31.

[0061] E57. An isolated antibody that specifically binds to TNFR2, comprising a heavy chain (HC) sequence encoded by the nucleic acid sequence of SEQ ID NO:33 and a light chain (LC) sequence encoded by the nucleic acid sequence of SEQ ID NO:31.

[0062] E58. The antibody of any one of E1 to E57, comprising an HC sequence encoded by a plasmid deposited with ATCC having ATCC Accession No. PTA-127530.

[0063] E59. The antibody of any one of E1 to E58, comprising an LC sequence encoded by a plasmid deposited with the ATCC and having ATCC Accession No. PTA127532.

[0064] E60. An antibody comprising an HC sequence encoded by a plasmid deposited with the ATCC having ATCC Accession No. PTA-127530, and an LC sequence encoded by a plasmid deposited with the ATCC having ATCC Accession No. PTA-127532.

[0065] E61. The Fc domain is IgG 2 The antibody of E30, which is an Fc domain of

[0066] E62. The antibody of E61, wherein the CH1 domain in the first Fab domain comprises a sequence according to SEQ ID NO:14.

[0067] E63. The antibody of E61 to E62, wherein the CL in the first Fab domain comprises a sequence according to SEQ ID NO:5.

[0068] E64. The antibody according to E61 to E63, wherein the first Fab domain and the second Fab domain are identical.

[0069] E65. The antibody described in E61 to E64, wherein the first Fc chain comprises, from the N-terminus to the C-terminus: a first hinge region, a first CH2 region and a first CH3 region, and the second Fc chain comprises, from the N-terminus to the C-terminus: a second hinge region, a second CH2 region and a second CH3 region.

[0070] E66. The antibody of E65, wherein one or both of the first hinge region and the second hinge region comprise a sequence according to SEQ ID NO:15.

[0071] E67. The antibody of E65 to E66, wherein one or both of the first CH2 domain and the second CH2 domain comprise a sequence according to SEQ ID NO:16.

[0072] E68. The antibody of E65 to E67, wherein one or both of the first CH3 domain and the second CH3 domain comprise a sequence according to SEQ ID NO:17.

[0073] E69. The antibody of E65 to E68, wherein one or both of the first Fc chain and the second Fc chain comprise a sequence according to SEQ ID NO:18.

[0074] E70. The antibody according to E65 to E69, wherein the first Fc chain and the second Fc chain are identical.

[0075] E71. The antibody according to E75 to E70, comprising a heavy chain (HC) comprising a sequence according to SEQ ID NO:19.

[0076] E72. The antibody according to E65 to E71, comprising a light chain (LC) comprising a sequence according to a sequence selected from the group consisting of SEQ ID NO: 6 and SEQ ID NO: 9.

[0077] E73. The antibody according to E65 to E72, comprising a light chain (LC) comprising a sequence according to SEQ ID NO:9.

[0078] E74. An isolated antibody that specifically binds to TNFR2, comprising a heavy chain (HC) comprising a sequence according to SEQ ID NO: 19 and a light chain (LC) comprising a sequence according to SEQ ID NO: 9.

[0079] E75. EC<5 μM in human TNFR2 potency assay in Jurkat reporter cells 50 The antibody according to any one of E1 to E74, characterized by:

[0080] E76. EC<2μM in human TNFR2 potency assay in Jurkat reporter cells 50 The antibody according to any one of E1 to E75, characterized by:

[0081] E77. EC<10μM in human TNFR2 potency assay on human peripheral blood monocytes 50 The antibody according to any one of E1 to E76, characterized by:

[0082] E78. EC<5μM in human TNFR2 potency assay on human peripheral blood monocytes 50 The antibody according to E1 to E77, characterized by:

[0083] E79. EC<2μM in human TNFR2 potency assay on human peripheral blood monocytes 50 The antibody according to E1 to E78, characterized by:

[0084] E80. EC<20μM in the Cynomolgus TNFR2 potency assay on Cynomolgus peripheral blood monocytes 50 The antibody according to E1 to E79, characterized by:

[0085] E81. EC<15μM in the Cynomolgus TNFR2 potency assay on Cynomolgus peripheral blood monocytes 50 The antibody according to any one of E1 to E80, characterized by:

[0086] E82. EC<5 μM in human TNFR2 potency assay in Jurkat reporter cells 50 The antibody according to E1 to E81, characterized by:

[0087] E83. EC<1 μM in human TNFR2 potency assay in Jurkat reporter cells 50 The antibody according to E1 to E82, characterized by:

[0088] E84. EC<0.5 μM in human TNFR2 potency assay in Jurkat reporter cells 50 The antibody according to any one of E1 to E83, characterized by:

[0089] E85. The antibody of E1-E84, characterized by an EC50 of less than 2 mg / ml for ICAM-1 upregulation in a human TNFR2-expressing primary T cell population derived from human peripheral blood monocytes.

[0090] E86. The antibody of E1-E85, characterized by an EC50 of less than 15 mg / ml for ICAM-1 upregulation in a cynomolgus TNFR2-expressing primary T cell population derived from cynomolgus peripheral blood monocytes.

[0091] E87. The antibody of E1-E86, characterized by an EC50 of less than 1 mg / ml for ICAM-1 upregulation in human TNFR2-expressing primary T cell populations derived from human peripheral blood monocytes.

[0092] E88. The antibody of E1-E87, characterized by an EC50 of less than 0.2 mg / ml for ICAM-1 upregulation in a human TNFR2-expressing primary T cell population derived from human peripheral blood monocytes.

[0093] E89. The antibody of E1-E88, characterized by an EC50 of less than 10 mg / ml for ICAM-1 upregulation in a cynomolgus TNFR2-expressing primary T cell population derived from cynomolgus peripheral blood monocytes.

[0094] E90. The antibody of E1-E89, characterized by an affinity KD for human TNFR2 of less than 1 nM.

[0095] E91. The antibody of E1-E90, characterized by an affinity KD for human TNFR2 of less than 0.5 nM.

[0096] E92. The antibody of E1-E91, characterized by an affinity KD for human TNFR2 of less than 0.1 nM.

[0097] E93. The antibody of E1-E92, characterized by an affinity KD for human TNFR2 of less than 0.07 nM.

[0098] E94. The antibody of E1-E93, characterized by an affinity KD for cynomolgus TNFR2 of less than 1 nM.

[0099] E95. The antibody of E1-E94, characterized by an affinity KD for cynomolgus TNFR2 of less than 0.1 nM.

[0100] E96. An isolated antibody comprising a VH and a VL of an antibody selected from Table 35.

[0101] E97. The antibody according to any one of E1 to E96, for use as a pharmaceutical.

[0102] E98. The antibody of E97, wherein the use is for the treatment of one or more selected from the group consisting of rheumatoid arthritis (RA), ankylosing spondylitis (AS), Crohn's disease (CD), ulcerative colitis (UC), graft versus host disease (GVHD), transplantation, psoriasis (PSO), psoriatic arthritis (PSA), atopic dermatitis (AD), vitiligo, alopecia areata (AA), type 1 diabetes (T1D), multiple sclerosis (MS), irritable bowel disease (IBD), autoimmune hepatitis, and systemic lupus erythematosus (SLE).

[0103] E99. The antibody according to any one of E96 to E98, wherein the use is for rheumatoid arthritis (RA).

[0104] E100. An isolated polynucleotide comprising one or more nucleotide sequences encoding the antibody of any one of E1-E99.

[0105] E101. The polynucleotide of E100, which is RNA.

[0106] E102. The polynucleotide according to E100 to E101, comprising at least one chemical modification.

[0107] E103. The polynucleotide of E102, wherein the chemical modification is selected from pseudouridine, 1-methylpseudouridine, N1-methylpseudouridine, N1-ethylpseudouridine, 2-thiouridine, 4'-thiouridine, 5-methylcytosine, 2-thio-1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-pseudouridine, 2-thio-5-aza-uridine, 2-thio-dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-pseudouridine, 4-methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-1-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methyluridine, 5-methoxyuridine and 2'-O-methyluridine.

[0108] E104. The polynucleotide according to E100 to E101, which does not include chemical modifications.

[0109] E105. An isolated polynucleotide encoding the HC or LC, or both, of an antibody that binds to TNFR2, said nucleic acid comprising one or more selected from the group consisting of the nucleic acid sequence of SEQ ID NO:31, the nucleic acid sequence of SEQ ID NO:32, the nucleic acid sequence of SEQ ID NO:33.

[0110] E106. An isolated polynucleotide encoding an isolated antibody that specifically binds to TNFR2, the antibody comprising a heavy chain (HC) sequence encoded by the nucleic acid sequence of SEQ ID NO: 33 and a light chain (LC) sequence encoded by the nucleic acid sequence of SEQ ID NO: 31.

[0111] E107. An isolated polynucleotide encoding an isolated antibody comprising an HC sequence encoded by a plasmid deposited with the ATCC having ATCC Accession No. PTA-127530, and an LC sequence encoded by a plasmid deposited with the ATCC having ATCC Accession No. PTA-127532.

[0112] E108. A vector comprising the polynucleotide according to E100 to E107.

[0113] E109. An isolated host cell comprising the polynucleotide according to E100 to E107 or the vector according to E108.

[0114] E110. A method of producing an isolated antibody, comprising culturing a host cell according to E109 under conditions resulting in the production of the antibody, and recovering the antibody.

[0115] E111. A pharmaceutical composition comprising a therapeutically effective amount of the antibody according to E1 to E99 and a pharma- ceutically acceptable carrier.

[0116] E112. A method for treating a medical condition, comprising administering to a subject in need thereof a therapeutically effective amount of an antibody described in any one of E1 to E99, a polynucleotide described in E101, or a pharmaceutical composition described in E111.

[0117] E113. The method of E112, wherein the condition is selected from the group consisting of rheumatoid arthritis (RA), ankylosing spondylitis (AS), Crohn's disease (CD), ulcerative colitis (UC), graft versus host disease (GVHD), transplantation, psoriasis (PSO), psoriatic arthritis (PSA), atopic dermatitis (AD), vitiligo, alopecia areata (AA), type 1 diabetes (T1D), multiple sclerosis (MS), irritable bowel disease (IBD), autoimmune hepatitis and systemic lupus erythematosus (SLE).

[0118] E114. The method according to any one of E112 to E113, comprising a step of subcutaneously administering the antibody or pharmaceutical composition.

[0119] E115. The method according to any one of E112 to E114, wherein said antibody or pharmaceutical composition is administered about twice a week, once a week, once every 2 weeks, once every 3 weeks, once every 4 weeks, once every 5 weeks, once every 6 weeks, once every 7 weeks, once every 8 weeks, once every 9 weeks, once every 10 weeks, twice a month, once a month, once every 2 months, once every 3 months, or once every 4 months. [Brief description of the drawings]

[0120] [Figure 1] FIG. 1 shows the analytical SEC profiles of the commercial MR2-1 clone and the one-step purified clone 162. [Diagram 2] 2 is a representation of the structure of TetraFab, showing an extra Fab domain (designated outer VH-outer CH1) appended onto the inner VH (designated inner VH-inner CH1). The outer and inner VHs are linked by a G4S domain. [Diagram 3] Figure 3 shows survival curves for TetraFab-2053 vs. control Ig. Statistics: Log-rank (Mantel-Cox) test. Data also shown in Table 21. [Figure 4] Figure 4 illustrates percentage change in body weight versus baseline. Data also shown in Table 22. [Diagram 5] Figure 5 shows mean plasma human IFNγ (pg / ml). ***p<0.001 TF2053 vs. control Ig. Data also shown in Table 23. [Figure 6] Figure 6 shows mean plasma human IL10 (pg / ml). ***p<0.001 TF2053 vs. control Ig. Data also shown in Table 24. [Figure 7] Figure 7 shows mean plasma human IL17A (pg / ml). **p<0.01 TF2053 vs. control Ig. ***p<0.001 TF2053 vs. control Ig. Data also shown in Table 25. [Figure 8]Figure 8 shows mean plasma human TNFα (pg / ml). ***p<0.001 TF2053 vs. control Ig. Statistics: unpaired two-tailed Student's t-test. Data also shown in Table 26. [Figure 9] Figure 9 shows mean liver weight per body weight (mg / kg) at sacrifice. ***p<0.001 TF2053 vs. control Ig. Data also shown in Table 27. [Figure 10] Figure 10 shows the mean spleen weight per body weight (mg / kg) at sacrifice. ***p<0.001 TF2053 vs. control Ig. Statistics: unpaired two-tailed Student's t-test. Data also shown in Table 28. [Figure 11] Figure 11 shows the mean percentage of human CD45+ cells in whole blood leukocytes. **p<0.01. ***p<0.001. ****p<0.0001. Data also shown in Table 29. [Figure 12] Figure 12 shows the mean percentage of human CD4+ among human CD45+ / CD3+ whole blood lymphocytes. ****p<0.0001. Data also shown in Table 30. [Figure 13] Figure 13 shows the mean percentage of human CD8+ among human CD45+ / CD3+ whole blood lymphocytes. ****p<0.0001. Data also shown in Table 31. [Figure 14] Figure 14 shows the mean percentage of anergic (PD1+KLRG1-CD57-) cells among human CD8+ / CD3+ / CD45+ blood lymphocytes. ****p<0.0001. Data also shown in Table 32. [Figure 15] Figure 15 shows the mean percentage of exhausted (PD1+KLRG1+CD57-) cells among human CD8+ / CD3+ / CD45+ blood lymphocytes. **p<0.01. Data also shown in Table 33. [Figure 16]Figure 16 shows the mean percentage of senescent (PD1-KLRG1+CD57+) cells among human CD8+ / CD3+ / CD45+ blood lymphocytes. *p<0.05. **p<0.01. ****p<0.0001. Statistics: Unpaired two-tailed T-test between groups at each time point. Data also shown in Table 34. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0121] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0122] All references cited herein, including patent applications, patent publications, and UniProtKB accession numbers, are hereby incorporated by reference as if each individual reference was specifically and individually indicated to be incorporated by reference in its entirety.

[0123] The techniques and procedures described and referred to herein are generally well understood and commonly employed by those of skill in the art using conventional methodologies, such as, for example, the widely used methodologies described in: Sambrook et al., Molecular Cloning: A Laboratory Manual 3rd Edition (2001) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (FMA Usubel et al., eds., (2003)); Series METHODS IN ENZYMOLOGY (Academic Press, Inc.): PCR 2: A PRACTICAL APPROACH (MJ MacPherson, BD Hames, and GR Taylor, eds. (1995)), Harlow and Lane, eds. (1988) ANTIBODIES, A LABORATORY MANUAL, and ANIMAL CELL CULTURE (RI Freshney, ed. (1987)); Oligonucleotide Synthesis (MJ Gait, ed., 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (ed. J. E. Elis, 1998) Academic Press; Animal Cell Culture (ed. R. I. Freshney, 1987); Introduction to Cell and Tissue Culture (J. P. Mather and P. E. Oberts, 1998) Plenum Press; Cell and Tissue Culture Laboratory Procedures (eds. A. Doyle, J. B. Griffiths and D. G. Newell, 1993-8) J. Wiley and Sons; Handbook of Experimental Immunology (eds. D. M. Weir and C. C. Blackwell); Gene Transfer Vectors for Mammalian Cells (J. M. Miller and M. P.Calos, 1987); PCR: The Polymerase Chain Reaction, (Mullis et al., 1994); Current Protocols in Immunology (JE Coligan et al., 1991); Short Protocols in Molecular Biology (Wiley and Sons, 1999); Immunobiology (C.A. Janeway and P. Travers, 1997); Antibodies (P. Finch, 1997); Antibodies: A Practical Approach (D. Catty, ed., IRL Press, 1988-1989); Monoclonal Antibodies: A Practical Approach (P. Shepherd and C. Dean, eds., Oxford University Press, 2000); Using Antibodies: A Laboratory Manual (E. Harlow and D. Lane (Cold Spring Harbor Laboratory Press, 1999)); The Antibodies (M. Zanetti and JD Capra, eds., Harwood Academic Press, 1999) Publishers, 1995); and their latest editions.

[0124] definition Unless otherwise defined herein, scientific and technical terms used in connection with the present invention shall have the meanings that are commonly understood by those of ordinary skill in the art.

[0125] As used herein, the singular forms "a," "an," and "the" include plural references unless specifically stated otherwise. For example, "an" antibody includes one or more antibodies.

[0126] When aspects or embodiments of the invention are described in terms of a Markush group or other grouping of alternatives, the invention includes the entire recited group as a whole, but also each member of the group individually and all possible subgroups of the main group, as well as the main group in which one or more of the group members are absent. The invention also envisions the explicit exclusion of one or more of any of the group members in the claimed invention.

[0127] Any examples following the term "eg" or "for example" are not meant to be exhaustive or limiting.

[0128] As used herein, the term "about" when used to modify a numerically defined parameter (e.g., *** A dose of about 5 mg means that the parameter may vary by 10% below or above the stated value for that parameter. For example, a dose of about 5 mg means 5% ± 10%, i.e., it may vary between 4.5 mg and 5.5 mg.

[0129] antibody "Antibody" refers to an immunoglobulin molecule capable of specifically binding to a target, e.g., a polypeptide, carbohydrate, polynucleotide, lipid, etc., via at least one antigen binding site located in the variable region of the immunoglobulin molecule. As used herein, the term "antibody" can encompass any type of antibody (e.g., monospecific, bispecific), including portions of intact antibodies that retain the ability to bind to a given antigen (e.g., "antigen-binding fragments"), and any other modified configuration of an immunoglobulin molecule that contains an antigen-binding site.

[0130] Antibodies include antibodies of any class, e.g., IgG, IgA, or IgM (or subclasses thereof), and antibodies need not be of any particular class. Depending on the antibody amino acid sequence of the constant region of its heavy chain (HC), immunoglobulins can be assigned to different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these are further subdivided into subclasses (isotypes), e.g., IgG 1 , IgG 2 , IgG 3 , IgG 4 , IgA 1 and IgA 2 The heavy chain constant regions that correspond to the different classes of immunoglobulins are called alpha, delta, epsilon, gamma, and mu, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known.

[0131] Examples of antigen-binding fragments and modified configurations of antibodies include: (i) Fab fragments (monovalent fragments consisting of the VL, VH, CL and CH1 domains); (ii) F(ab')2 fragments (bivalent fragments containing two Fab fragments linked by disulfide bridges at the hinge region); and (iii) Fv fragments consisting of the VL and VH domains of a single arm of an antibody. Furthermore, although the two domains of the Fv fragment: VL and VH, are encoded by separate genes, they can be joined using recombinant methods by a synthetic linker that allows them to be produced as a single protein chain in which the VL and VH regions pair to form a monovalent molecule (also known as single-chain Fv (scFv)); see, for example, Bird et al., Science 1988;242:423-426 and Huston et al., Proc. Natl. Acad. Sci. 1988 USA 85:5879-5883. Other forms of single chain antibodies, such as diabodies, are also encompassed.

[0132] Additionally, antibodies lacking a C-terminal lysine (K) amino acid residue on the heavy chain polypeptide are further encompassed (e.g., human IgG1 heavy chains contain a terminal lysine). As is known in the art, the C-terminal lysine may be omitted during antibody production, resulting in an antibody having a heavy chain lacking a C-terminal lysine. Alternatively, the antibody heavy chain may be produced using a nucleic acid that does not contain a C-terminal lysine.

[0133] Variable region 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. As known in the art, 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, which contribute to the formation of the antigen-binding site of the antibody. In particular, when a variant of a subject variable region is desired that has a substitution at an amino acid residue outside the CDR region (i.e., in the framework region), a suitable amino acid substitution, preferably a conservative amino acid substitution, can be identified by comparing the subject variable region with the variable regions of other antibodies that contain the CDR1 and CDR2 sequences in the same canonical class as the subject variable region (Chothia and Lesk, J Mol Biol 196(4):901-917, 1987).

[0134] In certain embodiments, the delineation of CDRs and the identification of the residues that constitute the binding site of an antibody are achieved by solving the structure of an antibody or the structure of an antibody-ligand complex. In certain embodiments, it can be achieved by any of a variety of techniques known to those skilled in the art, such as X-ray crystallography. In certain embodiments, various methods of analysis can be used to identify or approximate CDR regions. In certain embodiments, various methods of analysis can be used to identify or approximate CDR regions. Examples of such methods include, but are not limited to, Kabat definition, Chothia definition, AbM definition, contact definition, extended definition, and conformation definition.

[0135] The Kabat definition is a standard for numbering residues in an antibody and is typically used to identify CDR regions. See, for example, Johnson and Wu, 2000, Nucleic Acids Res., 28:214-8. The Chothia definition is similar to the Kabat definition, but the Chothia definition takes into account the location of certain structural loop regions. See, for example, Chothia et al., 1986, J. Mol. Biol., 196:901-17; Chothia et al., 1989, Nature, 342:877-83. The extended definition is a combination of the Kabat definition and the Chothia definition. The AbM definition uses an integrated suite of computer programs created by the Oxford Molecular Group to model antibody structures. See, e.g., Martin et al., 1989, Proc Natl Acad Sci (USA), 86:9268-9272; "AbM™ A Computer Program for Modeling Variable Regions of Antibodies", Oxford, UK; Oxford Molecular, Ltd. The AbM definition models the tertiary structure of antibodies from the primary sequence using a combination of knowledge databases and ab initio methods, such as those described by Samudrala et al., 1999, "Ab Initio Protein Structure Prediction Using a Combined Hierarchical Approach", in PROTEINS, Structure, Function and Genetics Suppl., 3:194-198. The contact definition is based on an analysis of available complex crystal structures. See, e.g., MacCallum et al., 1996, J. Mol. Biol., 5:732-45. In another approach, referred to herein as "conformational definition" of CDRs, the positions of CDRs can be identified as residues that contribute enthalpic-wise to antigen binding. See, e.g., Makabe et al., 2008, Journal of Biological Chemistry, 283:1156-1166.Still other CDR boundary definitions may not strictly follow one of the above approaches, but nevertheless overlap with at least a portion of Kabat CDRs, but may be shortened or extended in light of predictions or experimental findings that certain residues or groups of residues do not significantly affect antigen binding.As used herein, CDRs may refer to CDRs defined by any approach known in the art, including combinations of approaches.The methods used herein may utilize CDRs defined according to any of these approaches.For any given embodiment that contains more than one CDR, CDRs may be defined according to any one or more of Kabat, Chothia, extended, AbM, contact or conformation definitions.

[0136] Constant region The "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. The IgG heavy chain constant region contains three sequential immunoglobulin domains (CH1, CH2 and CH3) along with a hinge region between the CH1 and CH2 domains. The IgG light chain constant region contains a single immunoglobulin domain (CL).

[0137] Fc Domains and Fc Chains "Fc domain" refers to the portion of an immunoglobulin (Ig) molecule that correlates with the crystallizable fragment obtained by papain digestion of an Ig molecule. As used herein, the term refers to the two-chain constant region of an antibody, each chain excluding the first constant region immunoglobulin domain. Within the Fc domain, there are two "Fc chains" (e.g., "first Fc chain" and "second Fc chain"). "Fc chain" generally refers to the C-terminal portion of an antibody heavy chain. Thus, Fc chain refers to the last two constant region immunoglobulin domains (CH2 and CH3) of IgA, IgD and IgG heavy chains, and the last three constant region immunoglobulin domains of IgE and IgM heavy chains, and optionally the flexible hinge N-terminal to these domains.

[0138] Although the boundaries of an Fc chain may vary, a human IgG heavy chain Fc chain is usually defined to include residues C226 or P230 at its carboxyl terminus, numbering according to the EU index in Edelman et al., Proc. Natl. Acad. Sci. USA 1969;63(1):78-85, and as described in Kabat et al., 1991. Typically, an Fc chain includes from about amino acid residues 236 to about 447 of the human IgG1 heavy chain constant region. "Fc chain" can refer to this polypeptide in isolation or in the context of a larger molecule (e.g., in an antibody heavy chain or an Fc fusion protein).

[0139] A "functional" Fc domain refers to an Fc domain that retains at least one effector function of a native sequence Fc domain. 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 receptors); and B cell activation. Such effector functions generally require that the Fc domain be combined with a binding domain (e.g., an antibody variable region) and can be assessed using a variety of assays known in the art for assessing such antibody effector functions.

[0140] A "native sequence" Fc chain refers to an Fc chain that comprises an amino acid sequence identical to the amino acid sequence of an Fc chain found in nature. A "variant" Fc chain comprises an amino acid sequence that differs from that of a native sequence Fc chain by virtue of at least one amino acid modification.

[0141] Monoclonal antibodies "Monoclonal antibody" (mAb) refers to an antibody derived from a single copy or clone, including, for example, any eukaryotic, prokaryotic, or phage clone. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Moreover, in contrast to polyclonal antibody preparations which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies used in accordance with the present invention may be made by the hybridoma method first described by Kohler and Milstein, 1975, Nature 256:495, or may be made by recombinant DNA methods such as those described in U.S. Pat. No. 4,816,567. In another example, the monoclonal antibodies can be isolated from phage libraries, such as those generated using the techniques described in McCafferty et al., 1990, Nature 348:552-554.

[0142] Human antibodies "Human antibody" refers to an antibody that has an amino acid sequence corresponding to that of an antibody produced by a human or that is produced using any technique for producing a fully human antibody. For example, a fully human antibody can be obtained by using commercially available mice that have been engineered to express specific human immunoglobulin proteins, or by library (e.g., phage, yeast, or ribosome) display techniques to prepare a fully human antibody. This definition of a human antibody specifically excludes humanized antibodies that contain non-human antigen-binding residues.

[0143] Chimeric antibodies "Chimeric antibody" refers to an antibody in which the variable region sequences are derived from one species and the constant region sequences are derived from another species, e.g., the variable region sequences are derived from a murine antibody and the constant region sequences are derived from a human antibody.

[0144] Humanized antibodies "Humanized" antibody refers to a non-human (e.g., murine) antibody that is a chimeric antibody containing minimal sequence derived from non-human immunoglobulin. Preferably, a humanized antibody is a human immunoglobulin (recipient antibody) in which residues from the recipient's CDRs are replaced by residues from the CDRs of a non-human species (donor antibody), e.g., mouse, rat, or rabbit, that have the desired specificity, affinity, and capacity. Humanized antibodies may contain residues that are found neither in the recipient antibody nor in the imported CDR or framework sequences, but are included to further refine and optimize antibody performance.

[0145] antigen "Antigen" refers to a molecular entity used to immunize an immunocompetent vertebrate to produce an antibody that recognizes the antigen, or to screen an expression library (e.g., phage, yeast or ribosome display library, among others) for antibody selection. As used herein, antigen is more broadly named and is generally intended to include the target molecule that is specifically recognized by an antibody, and thus includes fragments or mimetics of the molecule used in the immunization process to raise antibodies or in library screening to select antibodies.

[0146] Epitope "Epitope" refers to the area or region of an antigen to which an antibody specifically binds, as determined by any method known in the art, for example, the area or region that contains the residue that interacts with the antibody.There are many methods known in the art for mapping and characterizing the location of epitopes on proteins, including elucidation of the crystal structure of antibody-antigen complexes, competitive assays, gene fragment expression assays, epitope mapping and synthetic peptide-based assays, as described, for example, in Chapter 11 of Harlow and Lane, Using Antibodies, a Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1999.In addition or alternatively, during the discovery process, the generation and characterization of antibodies can reveal information about desired epitopes.From this information, it is then possible to competitively screen antibodies for binding to the same epitope.

[0147] Furthermore, the epitope to which the antibody binds can be determined in a systematic screening by using overlapping peptides derived from the antigen and determining the binding by the antibody. According to gene fragment expression assay, the open reading frame encoding the antigen can be fragmented randomly or by specific genetic structure, and the reactivity of the expressed fragment of the antigen with the antibody to be tested is determined. The gene fragment can be produced, for example, by PCR, and then transcribed and translated in vitro into protein in the presence of radioactive amino acids. The binding of the antibody to the radioactively labeled antigen fragment is then determined by immunoprecipitation and gel electrophoresis.

[0148] Certain epitopes can also be identified by using large libraries of random peptide sequences displayed on the surface of phage particles (phage libraries) or yeast (yeast display). Alternatively, defined libraries of overlapping peptide fragments can be tested for binding to a test antibody in a simple binding assay. In additional examples, antigen mutagenesis, domain swapping experiments and alanine scanning mutagenesis can be performed to identify residues required, sufficient or necessary for epitope binding.

[0149] At its most detailed level, an epitope for an interaction between an antigen and an antibody can be defined by spatial coordinates that define the atomic contacts present in the antigen-antibody interaction, as well as information about their relative contribution to binding thermodynamics. At a less detailed level, an epitope can be characterized by spatial coordinates that define the atomic contacts between the antigen and the antibody. At an even less detailed level, an epitope can be characterized by the amino acid residues it contains, or by specific criteria, such as the distance between atoms (e.g., heavy atoms, i.e., non-hydrogen atoms) in the antibody and the antigen. At an even less detailed level, an epitope can be characterized via function, such as by competitive binding with other antibodies. An epitope can also be defined more generally as including amino acid residues whose replacement with another amino acid alters the characteristics of the interaction between the antibody and the antigen (e.g., using alanine scanning).

[0150] Due to the fact that epitope description and definition can be obtained at different levels of detail depending on the epitope mapping method used, it follows that comparison of epitopes for different antibodies on the same antigen can likewise be performed at different levels of detail.

[0151] Epitopes described at the amino acid level, e.g., as determined from X-ray crystallography, nuclear magnetic resonance (NMR) spectroscopy, hydrogen / deuterium exchange mass spectrometry (H / D-MS), are said to be identical if they contain the same set of amino acid residues. Epitopes are said to overlap if at least one amino acid is shared by the epitopes. Epitopes are said to be separate (unique) if no amino acid residues are shared by the epitopes.

[0152] Yet another method that can be used to characterize an antibody is to use a competitive assay with other antibodies known to bind to the same antigen to determine whether the antibody of interest binds to the same epitope as the other antibody.Competitive assays are well known to those skilled in the art.Epitopes characterized by competitive binding are said to overlap if the binding of corresponding antibodies is mutually exclusive, i.e., if the binding of one antibody excludes the simultaneous or sequential binding of the other antibody.An epitope is said to be separate (unique) if the antigen can simultaneously accept the binding of both corresponding antibodies.

[0153] Epitopes can be linear or conformational. In a linear epitope, all of the points of interaction between the protein and the interacting molecule (e.g., an antibody) occur linearly along the primary amino acid sequence of the protein. A "non-linear epitope" or "conformational epitope" comprises non-contiguous polypeptides (or amino acids) within an antigenic protein to which an antibody specific for the epitope binds.

[0154] binding affinity The term "binding affinity" refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise specified, as used herein, "binding affinity" refers to the intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X for its partner Y is generally determined by the dissociation constant (K D) Affinity can be measured by common methods known in the art. Low affinity antibodies generally bind antigens slowly and tend to dissociate easily, whereas high affinity antibodies generally bind antigens faster and tend to remain bound longer. In particular, the term "binding affinity" is intended to refer to the dissociation rate of a particular antigen-antibody interaction. K D is the "off-rate (k off )" or "k d The rate of dissociation, also called the on-rate (k on )" or "k a ". Therefore, K D is k off / k on (or k d / k a ) and is expressed as molar concentration (M). D The smaller the K, the stronger the binding affinity. Thus, a K of 1 μM D has a K of 1 nM D The K for the antibody shows a weaker binding affinity compared to the K for the antibody. D The K value can be determined using methods well established in the art. D One exemplary method for determining the K of an antibody is by using surface plasmon resonance (SPR), typically using a biosensor system, such as a BIACORE system. BIACORE kinetic analysis involves analyzing the binding and dissociation of antigens from chips having molecules (e.g., molecules containing epitope binding domains) immobilized on their surface. D Another method for determining the ion exchange potential is to use biolayer interferometry, typically OCTET technology (Octet QK e Alternatively or additionally, the KinExA (Kinetic Exclusion Assay) assay available from Sapidyne Instruments (Boise, ID) can also be used.

[0155] Monospecific antibodies A "monospecific antibody" refers to an antibody that contains one or more antigen-binding sites per molecule, such that any and all of the antibody's binding sites specifically recognize the same epitope on the antigen. Thus, when a monospecific antibody has more than one antigen-binding site, the binding sites compete with each other for binding to a single antigen molecule.

[0156] bispecific antibody "Bispecific antibodies" refer to molecules that have binding specificities for at least two different epitopes. In some embodiments, bispecific antibodies can bind to two different antigens simultaneously. In other embodiments, the two different epitopes can be present on the same antigen.

[0157] Tetrafab A tetrafab molecule refers to an antibody or antigen-binding portion thereof that contains four antigen-binding sites. The antigen-binding sites may bind to one, two, three or four different epitopes, which may be one, two, three or four different targets.

[0158] Half-maximal effective concentration (EC 50 ) The term "half maximum effective concentration (EC 50 "EC" refers to the concentration of a therapeutic agent that produces a response halfway between baseline and maximum after a specified exposure time. The therapeutic agent may produce inhibition or stimulation. As a measure of efficacy, the EC 50 The values ​​are commonly used and are used herein.

[0159] Agonist "Agonist" refers to a substance that promotes (i.e., induces, causes, enhances, or increases) the biological activity or effect of another molecule. The term agonist includes substances (e.g., antibodies) that bind to a molecule and promote the activity of that molecule.

[0160] Antagonist "Antagonist" refers to a substance that prevents, blocks, inhibits, neutralizes, or reduces the biological activity or effect of another molecule, such as a receptor. The term antagonist includes substances (e.g., antibodies) that bind to a molecule and prevent or reduce the activity of that molecule.

[0161] Competing The term "compete" as used herein with respect to antibodies means that a first antibody binds to an epitope in a manner sufficiently similar to that of a second antibody such that the result of binding of the second antibody to its cognate epitope is detectably decreased in the presence of the first antibody compared to the binding of the second antibody in the absence of the first antibody. There may be, but need not be, alternatives in which the binding of the first antibody to its epitope is also detectably decreased in the presence of the second antibody. That is, a first antibody can inhibit the binding of a second antibody to its epitope without the second antibody inhibiting the binding of the first antibody to its respective epitope. However, if each antibody detectably inhibits the binding of the other antibody to its cognate epitope or ligand, whether to the same extent, a greater extent, or a lesser extent, the antibodies are said to "cross-compete" with each other for binding of their respective epitopes. Both competing and cross-competing antibodies are encompassed by the present invention. Regardless of the mechanism by which such competition or cross-competition occurs (e.g., steric hindrance, conformational change, or binding to a common epitope or portion thereof), one of skill in the art will understand, based on the teachings provided herein, that such competing or cross-competing antibodies are encompassed and may be useful in the methods disclosed herein.

[0162] Fc receptors "Fc receptor" (FcR) refers to a receptor that binds to the Fc region of an antibody. In some embodiments, the FcR is a native human FcR. In some embodiments, the FcR is one that binds IgG antibodies (gamma receptors), including receptors of the FcgRI, FcgRII and FcgRIII subclasses, including allelic variants and alternatively spliced ​​forms of those receptors. FcgRII receptors include FcgRIIA ("activating receptor") and FcgRIIB ("inhibitory receptor"), which have similar amino acid sequences that differ primarily in their cytoplasmic domains. Activating receptor FcgRIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain. Inhibitory receptor FcgRIIB contains an immunoreceptor tyrosine-based inhibitory motif (ITIM) in its cytoplasmic domain (see, e.g., Daeron, Annu. Rev. Immunol. 1997;15:203-234). FcRs are reviewed, for example, in Ravetch and Kinet, Annu.Rev.Immunol 1991;9:457-92; Capel et al., Immunomethods 1994;4:25-34; and de Haas et al., J.Lab.Clin.Med.1995;126:330-41. Other FcRs, including those identified in the future, are encompassed herein by the term "Fc receptor". The term "Fc receptor" also includes the neonatal receptor FcRn, which is responsible for regulating maternal IgG transfer to the fetus (Guyer et al., J.Immunol.1976;117:587 and Kim et al., J.Immunol.1994;24:249) and immunoglobulin homeostasis. Methods for measuring binding to FcRn are known (see, e.g., Ghetie and Ward., Immunol. Today 1997;18(12):592-598; Ghetie et al., Nature Biotechnology 1997;15(7):637-640; Hinton et al., J. Biol. Chem. 2004;279(8):6213-6216; WO2004 / 92219).

[0163] Effector cells "Effector cell" refers to a white blood cell that expresses one or more FcR and performs effector function. In certain embodiments, the effector cell expresses at least FcgRIII and performs ADCC effector function. Examples of white blood cells that mediate ADCC include peripheral blood mononuclear cells (PBMCs), natural killer (NK) cells, monocytes, macrophages, cytotoxic T cells and neutrophils. Effector cells can be isolated from native sources, such as blood.

[0164] Antibody-dependent cell-mediated cytotoxicity (ADCC) The term "antibody-dependent cell-mediated cytotoxicity" or "ADCC" refers to a form of cytotoxicity in which secreted Ig bound to Fc receptors (FcR) present on certain cytotoxic cells (e.g., NK cells, neutrophils and macrophages) allows these cytotoxic effector cells to specifically bind to antigen-bearing target cells and subsequently kill the target cells using cytotoxins. NK cells, the primary cells mediating ADCC, express only FcgRIII, whereas monocytes express FcgRI, FcgRII and FcgRIII. To assess the ADCC activity of a molecule of interest, an in vitro ADCC assay such as that described in U.S. Pat. Nos. 5,500,362, 5,821,337 or 6,737,056 can be performed. Effector cells useful for such assays include PBMCs and NK cells. Alternatively, or additionally, ADCC activity of the molecule of interest may be assessed in vivo, e.g., in an animal model such as that disclosed in Clynes et al., Proc. Natl. Acad. Sci. (USA) 1998;95:652-656. Additional antibodies with altered Fc region amino acid sequences and increased or decreased ADCC activity are described, e.g., in U.S. Patent Nos. 7,923,538 and 7,994,290.

[0165] Enhanced ADCC activity The term "enhanced ADCC activity" refers to an antibody that is more effective in mediating ADCC in vitro or in vivo compared to a parent antibody, where the antibody and the parent antibody differ in at least one structural aspect, when essentially the same amount of such antibody and the parent antibody are used in the assay. In some embodiments, the antibody and the parent antibody have the same amino acid sequence, but the antibody is afucosylated, while the parent antibody is fucosylated. In some embodiments, the ADCC activity is determined using an in vitro ADCC assay, although other assays or methods for determining ADCC activity, such as in animal models, are contemplated. In some embodiments, an antibody with enhanced ADCC activity has enhanced affinity for FcgRIIIA.

[0166] Altered FcR binding or ADCC activity The term "altered" FcR binding affinity or ADCC activity refers to an antibody that has either enhanced or diminished activity in one or more of the FcR binding or ADCC activities compared to a parent antibody, which differs from the parent antibody in at least one structural aspect. An antibody that "exhibits increased binding" to an FcR binds at least one FcR with better affinity than the parent antibody. An antibody that "exhibits reduced binding" to an FcR binds at least one FcR with lower affinity than the parent antibody. Such an antibody that exhibits reduced binding to an FcR may possess little or no appreciable binding to an FcR, e.g., may possess 0-20 percent of binding to an FcR compared to a native sequence IgG Fc region.

[0167] Complement-dependent cytotoxicity (CDC) The term "complement-dependent cytotoxicity" or "CDC" refers to the lysis of target cells in the presence of complement. Activation of the classical complement pathway is initiated by the binding of the first component of the complement system (C1q) to antibodies (of the appropriate subclass) bound to their cognate antigen. To assess complement activation, a CDC assay, for example as described in Gazzano-Santoro et al., J.Immunol.Methods 1996;202:163, can be performed. Antibodies with altered Fc region amino acid sequences and increased or decreased C1q binding capacity are described, for example, in U.S. Pat. No. 6,194,551, U.S. Pat. No. 7,923,538, U.S. Pat. No. 7,994,290 and WO1999 / 51642.

[0168] host cell "Host cell" refers to an individual cell or cell culture that can be or has been the recipient of a vector for incorporation of a polynucleotide insert. A host cell includes the progeny of a single host cell, which 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 of the present invention.

[0169] vector "Vector" refers to a construct capable of delivering one or more genes or sequences of interest (e.g., antibody-encoding genes) into a host cell, and preferably expressing them in the host cell. Examples of vectors include, but are not limited to, plasmids and viral vectors, and may include naked nucleic acid or may include nucleic acid associated with delivery aids (e.g., cationic condensing agents, liposomes, etc.). Vectors may include DNA or RNA. "Expression vector" as used herein refers to a vector that includes at least one polypeptide-encoding gene, at least one regulatory element for transcription or translation of the gene (e.g., promoter sequence, poly(A) sequence). Typically, a vector as used herein contains at least one antibody-encoding gene, and one or more of the following regulatory elements or selectable markers. Vector components may include, for example, one or more of the following: signal sequence; origin of replication; one or more marker genes; appropriate transcription control elements (e.g., promoter, enhancer and terminator). For translation, one or more translational control elements may also be included, such as ribosome binding sites, translation initiation sites, and stop codons.

[0170] Isolated An "isolated" molecule (e.g., an antibody) refers to a molecule that, by virtue of its origin or source of derivation, (1) is not associated with naturally associated components that accompany it in its native state, (2) is substantially free of other molecules from the same source, e.g., the species, the cell in which it is expressed, a library, etc., (3) is expressed by cells from a different species, or (4) does not exist in nature. Thus, a molecule that is chemically synthesized or expressed in a cellular system different from the system in which it naturally originates is "isolated" from its naturally associated components. A molecule can also be rendered substantially free of naturally associated components by isolation using purification techniques well known in the art.

[0171] Polypeptides / Proteins "Polypeptide" or "protein" (used interchangeably herein) refers to a chain of amino acids of any length. The chain can be linear or branched. The chain can include one or more modified amino acids. These terms also encompass amino acid chains 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. For example, polypeptides containing one or more analogs of amino acids, including, for example, unnatural amino acids, as well as other modifications known in the art, are also included within this definition. It is understood that polypeptides can exist as single chains or associated chains.

[0172] Polynucleotides / Nucleic Acids "Polynucleotide" or "nucleic acid" (used interchangeably herein) refers to a chain of nucleotides of any length, including DNA and RNA. The nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases or their analogs, or any substrate that can be incorporated into a chain by DNA or RNA polymerase. Polynucleotides can include 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 chain. The sequence of nucleotides can be interrupted by non-nucleotide components. Polynucleotides can be further modified after polymerization, such as by conjugation with a labeling component. Other types of modifications include, for example, "caps", substitution of one or more naturally occurring nucleotides with analogs, internucleotide modifications, such as those with uncharged linkages (e.g., methylphosphonates, phosphotriesters, phosphoamidates, carbamates, etc.) and those with charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.), those containing pendant moieties such as proteins (e.g., nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.), those containing intercalators (e.g., acridine, psoralen, 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 polynucleotides. Additionally, any of the hydroxyl groups normally present in the sugar can be replaced by, for example, phosphonate groups, phosphate groups, protected by standard protecting groups, or activated to provide for additional linkages to additional nucleotides, or conjugated to solid supports. The 5' and 3' terminal OH can be phosphorylated or substituted with amines or organic capping group moieties of 1 to 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, alpha- or beta-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.

[0173] conservative substitution "Conservative substitution" refers to the replacement of one amino acid with a biologically, chemically or structurally similar residue. Biologically similar means that the substitution does not destroy biological activity. Structurally similar means that the amino acids have side chains with similar length, such as alanine, glycine and serine, or have side chains with similar size. Chemical similarity means that the residues have the same charge, or are both hydrophilic or hydrophobic. Particular examples include the replacement of a hydrophobic residue, such as isoleucine, valine, leucine or methionine, with another residue, or the replacement of one polar residue with another polar residue, such as the replacement of arginine with lysine, the replacement of glutamic acid with aspartic acid or glutamine with asparagine, the replacement of serine with threonine, etc. Specific examples of conservative substitutions include substitutions of hydrophobic residues, such as isoleucine, valine, leucine, or methionine, for one another, substitutions of one polar residue for another, such as arginine for lysine, glutamic acid for aspartic acid, or glutamine for asparagine, etc. Conservative amino acid substitutions typically include, for example, substitutions within the following groups: glycine, alanine, valine, isoleucine, leucine; aspartic acid, glutamic acid; asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine.

[0174] identity The term "identity" or "identical to" refers to the overall relatedness between polymeric molecules, e.g., between nucleic acid molecules (e.g., DNA molecules or RNA molecules) or between polypeptide molecules. "Identity" measures the percent of identical matches between two or more sequences using gap alignment handled by specific mathematical models (e.g., algorithms) of computer programs well known in the art.

[0175] The terms "increase," "improve," "decrease," or "reduce" refer to values ​​compared to baseline measurements, e.g., measurements in the same individual prior to the initiation of a treatment described herein, or measurements in a control individual or subject (or control individuals or subjects) in the absence of a treatment described herein. In some embodiments, a "control individual" is an individual suffering from the same form of disease or injury as the individual being treated. In some embodiments, a "control individual" is an individual not suffering from the same form of disease or injury as the individual being treated.

[0176] Excipients The term "excipient" refers to any material that combines with a desired active ingredient (e.g., an antibody) to allow the active ingredient to retain biological activity. The choice of excipient depends to a large extent on factors such as the mode of administration, the effect of the excipient on solubility and stability, and the nature of the dosage form. As used herein, "excipient" includes any and all physiologically compatible solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, carriers, diluents, and the like. Examples of excipients include one or more of water, saline, phosphate buffer solution, dextrose, glycerol, ethanol, and the like, and combinations thereof, and may include isotonic agents, such as sugars, sodium chloride, or polyhydric alcohols, such as mannitol or sorbitol, in the composition.

[0177] Treat The terms "treating," "treat," or "treatment" refer to any type of treatment, such as to alleviate, ameliorate, or slow the progression of a patient's disease, disorder, or condition, or any tissue damage associated with a disease. In some embodiments, the disease, disorder, or condition is X.

[0178] Prevent The terms "prevent" or "prevention" refer to the treatment of a particular disease, disorder, or condition (e.g., *** ) at least one sign or symptom (e.g., specific to a particular application) of *** (a) refers to one or more of a delay in onset, a reduction in frequency, or a reduction in severity of a disease, disorder, or condition. In some embodiments, prevention is assessed on a population basis, such that an agent is considered to "prevent" a particular disease, disorder, or condition if a statistically significant reduction in the onset, frequency, or intensity of one or more symptoms of the disease, disorder, or condition is observed in a population susceptible to that disease, disorder, or condition. Prevention may be considered complete if the onset of the disease, disorder, or condition is delayed for a predefined period of time.

[0179] subject The terms "subject", "individual" or "patient" (used interchangeably herein) refer to any animal, including mammals. Mammals according to the present invention include dogs, cats, cows, goats, horses, sheep, pigs, rodents, lagomorphs, primates, humans, and the like, including mammals in utero. In certain embodiments, humans are suitable subjects. Human subjects can be of any gender and at any stage of development. In some embodiments, the subject is a patient with the following diseases: rheumatoid arthritis (RA), ankylosing spondylitis (AS), Crohn's disease (CD), ulcerative colitis (UC), graft versus host disease (GVHD), transplantation, psoriasis (PSO), psoriatic arthritis (PSA), atopic dermatitis (AD), vitiligo, alopecia areata (AA), type 1 diabetes (T1D), multiple sclerosis (MS), irritable bowel disease (IBD), autoimmune hepatitis, and systemic lupus erythematosus (SLE).

[0180] Therapeutically Effective Dose The term "therapeutically effective amount" refers to an amount of active ingredient that elicits the biological or medicinal response desired by a researcher, veterinarian, physician or other clinician in a tissue, system, animal, individual or human, which may include one or more of the following: (1) Preventing disease; e.g., preventing a disease, condition, or disorder in an individual who may be predisposed to the disease, condition, or disorder but has not yet experienced or exhibited the pathology or symptomology of the disease; (2) inhibiting a disease; e.g., inhibiting a disease, condition, or disorder (i.e., halting or slowing further development of the pathology or symptomatology) in an individual experiencing or exhibiting the pathology or symptomatology of the disease, condition, or disorder; and (3) ameliorating the disease; e.g., ameliorating the disease, condition, or disorder (i.e., reversing the pathology or symptomology) in an individual experiencing or exhibiting the pathology or symptomology of the disease, condition, or disorder.

[0181] Antibodies to TNFR2 The present disclosure provides an antibody that binds to TNFR2, also known as tumor necrosis factor receptor superfamily member 1B (TNFRSF1B), CD120b or p75. TNFR2 is one of two membrane receptors that bind to tumor necrosis factor-alpha (TNFα), the other being TNFR1. TNFR2 is a type I transmembrane receptor with an extracellular domain composed of four cysteine-rich domains CRD. Binding of TNFα to TNFR2 triggers an intracellular signaling cascade that leads to NF-KB activation and subsequently leads to cell proliferation, activation and survival.

[0182] As used herein, the term TNFR2 includes variants, isoforms, homologs, orthologs, and paralogs of TNFR2. In some embodiments, the antibodies disclosed herein cross-react with TNFR2 from species other than human, such as cynomolgus monkey TNFR2, as well as different forms of TNFR2. In some embodiments, the antibodies may be completely specific for human TNFR2 and may not exhibit species cross-reactivity (e.g., do not bind to mouse TNFR2) or other types of cross-reactivity. As used herein, TNFR2 refers to naturally occurring human TNFR2, unless the context indicates otherwise. Thus, "TNFR2 antibody," "anti-TNFR2 antibody," or other similar designation refers to any antibody (as defined herein) that binds to or reacts with TNFR2, its isoforms, fragments, or derivatives. The full-length mature form of TNFR2, as represented by UniProtKB / Swiss-Prot accession number P20333, is provided herein as SEQ ID NO: 34. The full-length mature form of mouse TNFR2, represented by UniProtKB / Swiss-Prot accession number P25119, is provided herein as SEQ ID NO: 35. The full-length mature form of cynomolgus monkey TNFR2, represented by NCBI database accession number XP_005544817, is provided herein as SEQ ID NO: 36.

[0183] In some embodiments, the antibodies of the disclosure agonize TNFR2, hi some embodiments, the antibodies of the disclosure do not inhibit TNFα binding to the TNFR2 molecule to which the antibody binds.

[0184] In some embodiments, the anti-TNFR2 antibodies of the present disclosure include antibodies that i) compete with an antibody having a heavy chain variable region amino acid sequence set forth as SEQ ID NO:21 and a light chain variable region amino acid sequence set forth as SEQ ID NO:8 for binding to human TNFR2, or ii) bind to the same epitope as such an antibody, or both.

[0185] The anti-TNFR2 antibodies of the present disclosure may be monoclonal antibodies, polyclonal antibodies, antibody fragments (e.g., Fab, Fab', F(ab') 2 , Fv, Fc, etc.), chimeric antibodies, bispecific antibodies, heteroconjugate antibodies, single chain (ScFv), variants thereof, fusion proteins comprising antibody fragments (e.g., domain antibodies), humanized antibodies, and any other modified configuration of an immunoglobulin molecule comprising an antigen-binding site of the required specificity, including glycosylation variants of antibodies, amino acid sequence variants of antibodies, and covalently modified antibodies. The antibodies may be murine, rat, human or any other origin (including chimeric or humanized antibodies). In some embodiments, the anti-TNFR2 antibody is a monoclonal antibody. In some embodiments, the anti-TNFR2 antibody is a human or humanized antibody. In some embodiments, the anti-TNFR2 antibody is a chimeric antibody.

[0186] In some embodiments, the present invention provides antibodies having a light chain variable region (VL) sequence and a heavy chain variable region (VH) sequence found in the Sequence Listing Table herein, or a variant thereof.

[0187] The present invention also provides CDR portions of antibodies against TNFR2. Determination of CDR regions is well within the skill of one of ordinary skill in the art. It is understood that in some embodiments, the CDRs can be a combination of Kabat CDRs and Chothia CDRs (also referred to as "combined CDRs" or "extended CDRs"). In another approach, referred to herein as "conformation definition" of CDRs, the positions of CDRs can be identified as residues that contribute enthalpic to antigen binding. See, for example, Makabe et al., 2008, Journal of Biological Chemistry, 283:1156-1166. In general, "conformational CDRs" include residue positions in Kabat CDRs and Vernier zones that are constrained to maintain the appropriate loop structure for the antibody to bind to a specific antigen. Determination of conformational CDRs is well within the skill of one of ordinary skill in the art. In some embodiments, the CDRs are Kabat CDRs. In other embodiments, the CDRs are Chothia CDRs. In other embodiments, the CDRs are extended, AbM, conformational or contact CDRs. In other words, in embodiments using more than one CDR, the CDRs can be any of Kabat, Chothia, extended, AbM, conformational, contact CDRs or combinations thereof.

[0188] In some embodiments, the antibody comprises one or both of i) the full-length heavy chain, with or without the C-terminal lysine, or ii) the full-length light chain, of the anti-TNFR2 antibody TF-2053.

[0189] In certain embodiments, the antibodies described herein comprise an Fc domain. The Fc domain is a domain that is associated with IgA (e.g., IgA 1 or IgA 2 ), IgG, IgE or IgG (e.g., IgG 1 , IgG 2 , IgG 3 or IgG 4 ). In some embodiments, the anti-TNFR2 antibody is an IgG1 antibody.

[0190] The present invention encompasses modifications to the CDRs and variable regions shown in Table 5. For example, the present invention includes antibodies that contain functionally equivalent variable regions and CDRs that do not significantly affect their properties, as well as variants with enhanced or decreased activity or affinity. For example, the amino acid sequence can be mutated to obtain an antibody with a desired binding affinity to TNFR2. Modification of polypeptides is a routine practice in the art and need not be described in detail herein. Examples of modified polypeptides include conservative substitution of amino acid residues, polypeptides with one or more deletions or additions of amino acids that do not significantly adversely change the functional activity or mature (increase) the affinity of the polypeptide to its ligand, or the use of chemical analogs.

[0191] Modifications or mutations may also be made in the framework or constant regions to increase the half-life of the antibodies provided herein. See, for example, PCT Publication No. WO00 / 09560. Mutations in the framework or constant regions may also be made to alter the immunogenicity of the antibody, to provide a site for covalent or non-covalent binding to another molecule, or to alter properties such as complement binding, FcR binding, and antibody-dependent cell-mediated cytotoxicity. In some embodiments, no more than one to five conservative amino acid substitutions are made in the framework or constant regions. In other embodiments, no more than one to three conservative amino acid substitutions are made in the framework or constant regions. According to the present invention, a single antibody may have mutations in any one or more of the CDRs or framework regions of the variable domain, or in the constant region.

[0192] In some embodiments, the antibody comprises a modified constant region that has increased or decreased binding affinity to human Fc gamma receptors, is immunologically inactive or partially inactive, e.g., does not induce complement-mediated lysis, does not stimulate antibody-dependent cell-mediated cytotoxicity (ADCC), or does not activate microglia; or has reduced activity (compared to unmodified antibodies) in any one or more of the following: induce complement-mediated lysis, stimulate ADCC, or activate microglia. Different modifications of the constant region can be used to achieve optimal levels or combinations of effector functions. See, e.g., Morgan et al., Immunology 86:319-324, 1995; Lund et al., J. Immunology 157:4963-9 157:4963-4969, 1996; Idusogie et al., J. Immunology 164:4178-4184, 2000; Tao et al., J. Immunology 143:2595-2601, 1989; and Jefferis et al., Immunological Reviews 163:59-76, 1998. In some embodiments, the constant region is modified as described in Eur. J. Immunol., 1999, 29:2613-2624; PCT Publication No. WO99 / 058572.

[0193] Modifications also include glycosylated and non-glycosylated polypeptides, as well as polypeptides with other post-translational modifications, such as glycosylation with different sugars, acetylation, and phosphorylation, etc. Antibodies are glycosylated at conserved positions in their constant regions (Jefferis and Lund, 1997, Chem. Immunol. 65:111-128; Wright and Morrison, 1997, TibTECH 15:26-32). The oligosaccharide side chains of immunoglobulins affect the function of the protein (Boyd et al., 1996, Mol. Immunol. 32:1311-1318; Wittwe and Howard, 1990, Biochem. 29:4175-4180) and the intramolecular interactions between moieties of the glycoprotein (Jefferis and Lund, supra; Wyss and Wagner, 1996, Current Opin. Biotech. 7:409-416), which may affect the conformation and presented three-dimensional surface of the glycoprotein. Oligosaccharides may also function to target a given glycoprotein to a particular molecule based on specific recognition structures. Glycosylation of antibodies has also been reported to affect antibody-dependent cellular cytotoxicity (ADCC). In particular, antibodies produced in CHO cells using tetracycline-regulated expression of β(1,4)-N-acetylglucosaminyltransferase III (GnTIII), a glycosyltransferase that catalyzes the formation of bisected GlcNAc, have been reported to have improved ADCC activity (Umana et al., 1999, Nature Biotech. 17:176-180).

[0194] In some aspects, the disclosure provides an isolated antibody that specifically binds to TNFR2, comprising a heavy chain variable region (VH) and a light chain variable region (VL), the antibody comprising CDR-H1, CDR-H2 and CDR-H3 sequences of a VH sequence selected from the group consisting of SEQ ID NO:13, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:29 and SEQ ID NO:30; and CDR-L1, CDR-L2 and CDR-L3 sequences of a VL sequence selected from the group consisting of SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8 and SEQ ID NO:9.

[0195] In some aspects, the disclosure provides an isolated antibody that specifically binds to TNFR2, comprising a heavy chain variable region (VH) and a light chain variable region (VL), the antibody comprising CDR-H1, CDR-H2 and CDR-H3 sequences according to SEQ ID NO: 30, and CDR-L1, CDR-L2 and CDR-L3 sequences according to SEQ ID NO: 9.

[0196] In some aspects, the disclosure provides an isolated antibody that specifically binds to TNFR2, comprising a heavy chain variable region (VH) and a light chain variable region (VL): (i) a CDR-L1 sequence according to SEQ ID NO:1; a CDR-L2 sequence according to SEQ ID NO:7 and a CDR-L3 sequence according to SEQ ID NO:3, and a CDR-H1 sequence according to SEQ ID NO:10; a CDR-H2 sequence according to SEQ ID NO:20; a CDR-H3 sequence according to SEQ ID NO:12; or (ii) a CDR-L1 sequence according to SEQ ID NO:1; a CDR-L2 sequence according to SEQ ID NO:2 and a CDR-L3 sequence according to SEQ ID NO:3, and a CDR-H1 sequence according to SEQ ID NO:10; a CDR-H2 sequence according to SEQ ID NO:11; and a CDR-H3 sequence according to SEQ ID NO:12. The present invention provides an antibody comprising:

[0197] In some aspects, the disclosure provides an isolated antibody that specifically binds to TNFR2, comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the CDR-L1 sequence is according to SEQ ID NO:1; the CDR-L2 sequence is according to SEQ ID NO:7; the CDR-L3 sequence is according to SEQ ID NO:3; the CDR-H1 sequence is according to SEQ ID NO:10; the CDR-H2 sequence is according to SEQ ID NO:20; and the CDR-H3 sequence is according to SEQ ID NO:12.

[0198] The VH framework sequences may be derived from a human germline VH sequence selected from the group consisting of IGHV1-46, IGHV4-31, IGHV4-30-4 and IGHV4-4. In some embodiments, the VH framework sequences may be derived from a human IGHV1-46 germline sequence.

[0199] The VL framework sequence may be derived from a human germline VL sequence selected from the group consisting of IGKV1-9, IGKV1-33, IGKV1-27, IGKV1-39, IGKV1-9, IGKV1-1 and IGKV1-11. In some embodiments, the VL framework sequence is derived from a human germline IGKV1-9 sequence.

[0200] In some aspects, the disclosure provides the described antibodies comprising a VL framework sequence and a VH framework sequence, wherein one or both of the VL framework sequence and the VH framework sequence are at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the human germline sequence from which it is derived.

[0201] In some aspects, the disclosure provides a described antibody comprising a VL framework sequence and a VH framework sequence, wherein one or both of the VL or VH framework sequences are identical to the human germline sequence from which it is derived.

[0202] In some aspects, the disclosure provides an antibody, wherein the VL comprises an amino acid sequence according to a sequence selected from the group consisting of SEQ ID NO:4 and SEQ ID NO:8, and the VH comprises an amino acid sequence according to a sequence selected from the group consisting of SEQ ID NO:13 and SEQ ID NO:21.

[0203] In some aspects, the disclosure provides an antibody comprising a VH sequence of SEQ ID NO:13 and a VL of SEQ ID NO:4.

[0204] In some aspects, the disclosure provides antibodies comprising a VH sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:21 and a VL sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:8.

[0205] In some aspects, the disclosure provides an antibody comprising a VH sequence of SEQ ID NO:21 and a VL of SEQ ID NO:8.

[0206] In some embodiments, the present disclosure provides anti-TNFR2 antibodies containing the variable regions shown in the sequence listing, variations of the CDRs shown in the sequence listing, where such variant polypeptides share at least 70%, at least 75%, at least 80%, at least 85%, at least 87%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity with any of the amino acid sequences disclosed in the sequence listing. These amounts are not meant to be limiting, and increments between the recited percentages are specifically contemplated as part of the present disclosure.

[0207] In some embodiments, the disclosure provides an anti-TNFR2 antibody comprising a VH sequence encoded by the nucleic acid sequence of SEQ ID NO: 32. In some embodiments, the disclosure provides an anti-TNFR2 antibody comprising a VH sequence encoded by the nucleic acid sequence of SEQ ID NO: 33. In some embodiments, the disclosure provides an anti-TNFR2 antibody comprising a VL sequence encoded by the nucleic acid sequence of SEQ ID NO: 31.

[0208] In some embodiments, the present disclosure provides an anti-TNFR2 antibody comprising a constant heavy domain (CH1) and a constant light domain (CL). The CH1 can be connected to the VH, and the CL can be connected to the VL to form a Fab domain. The antibody can comprise a first and a second Fab domain.

[0209] In some embodiments, the present disclosure provides an anti-TNFR2 antibody comprising an antibody Fc domain comprising a first Fc chain and a second Fc chain. The first Fab domain can be covalently fused to the first Fc chain, and the second Fab domain can be covalently fused to the second Fc chain. The C-terminus of the CH1 domain in the first Fab domain can be covalently fused to the N-terminus of the first Fc chain, and the C-terminus of the CH1 domain in the second Fab domain can be covalently fused to the N-terminus of the second Fc chain. The Fc domain can be covalently fused to an antibody Fc domain that is specific to IgA (e.g., IgA 1 or IgA 2 ), IgD, IgE, IgM or IgG (e.g., IgG 1 , IgG 2 , IgG 3 or IgG 4 The Fc domain may be an Fc domain of an IgG 1 The Fc domain may be

[0210] In some embodiments, the disclosure provides an anti-TNFR2 antibody, wherein the CH1 domain in the first Fab domain comprises a sequence according to SEQ ID NO:23.

[0211] In some embodiments, the disclosure provides an anti-TNFR2 antibody, wherein the CL in the first Fab domain comprises a sequence according to SEQ ID NO:5.

[0212] In some embodiments, the disclosure provides an anti-TNFR2 antibody comprising a light chain (LC) comprising a sequence according to SEQ ID NO:9.

[0213] In some embodiments, the present disclosure provides an anti-TNFR2 antibody, wherein the first Fab domain and the second Fab domain are identical. The first Fc chain may comprise, from the N-terminus to the C-terminus: a first hinge region, a first CH2 region, and a first CH3 region, and the second Fc chain may comprise, from the N-terminus to the C-terminus: a second hinge region, a second CH2 region, and a second CH3 region.

[0214] In some embodiments, the present disclosure provides an anti-TNFR2 antibody, wherein one or both of the first and second hinge regions comprise a sequence according to SEQ ID NO: 23. In some embodiments, the present disclosure provides an anti-TNFR2 antibody, wherein one or both of the first and second CH2 domains comprise a sequence according to SEQ ID NO: 25. In some embodiments, the present disclosure provides an anti-TNFR2 antibody, wherein one or both of the first and second CH3 domains comprise a sequence according to SEQ ID NO: 26. In some embodiments, the present disclosure provides an anti-TNFR2 antibody, wherein one or both of the first and second Fc chains comprise a sequence according to SEQ ID NO: 37.

[0215] In some embodiments, the present disclosure provides an anti-TNFR2 antibody, wherein the first Fc chain and the second Fc chain are identical. In some embodiments, the present disclosure provides an anti-TNFR2 antibody comprising a heavy chain (HC) comprising a sequence according to SEQ ID NO:22.

[0216] In some embodiments, the disclosure provides an anti-TNFR2 antibody further comprising a third Fab and a fourth Fab.

[0217] In some embodiments, the disclosure provides an anti-TNFR2 antibody, wherein the first Fab, the second Fab, the third Fab and the fourth Fab each comprise a CDR-L1 sequence according to SEQ ID NO:1; a CDR-L2 sequence according to SEQ ID NO:7 and a CDR-L3 sequence according to SEQ ID NO:3, and a CDR-H1 sequence according to SEQ ID NO:10; a CDR-H2 sequence according to SEQ ID NO:20; and a CDR-H3 sequence according to SEQ ID NO:12.

[0218] In some embodiments, the disclosure provides an anti-TNFR2 antibody, wherein the first Fab, the second Fab, the third Fab and the fourth Fab each comprise a VH having a sequence according to SEQ ID NO:21 and a VL having a sequence according to SEQ ID NO:8.

[0219] In some embodiments, the disclosure provides an anti-TNFR2 antibody, wherein the first Fab, the second Fab, the third Fab and the fourth Fab are identical to each other.

[0220] In some embodiments, the disclosure provides an anti-TNFR2 antibody, wherein the N'-terminus of a first Fab is joined to the C'-terminus of a third Fab.

[0221] In some embodiments, the disclosure provides an anti-TNFR2 antibody, wherein the N'-terminus of a first Fab is connected to the C'-terminus of a third Fab via a first linker.

[0222] In some embodiments, the disclosure provides an anti-TNFR2 antibody, wherein the first linker comprises a sequence according to SEQ ID NO:27.

[0223] In some embodiments, the disclosure provides an anti-TNFR2 antibody, wherein the N'-terminus of a second Fab is joined to the C'-terminus of a fourth Fab.

[0224] In some embodiments, the disclosure provides an anti-TNFR2 antibody, wherein the N'-terminus of the second Fab is connected to the C'-terminus of a fourth Fab via a second linker.

[0225] In some embodiments, the disclosure provides an anti-TNFR2 antibody, wherein the second linker comprises a sequence according to SEQ ID NO:27.

[0226] In some embodiments, the disclosure provides an anti-TNFR2 antibody, wherein the HC comprises a sequence according to SEQ ID NO:30.

[0227] E54. An isolated antibody that specifically binds to TNFR2, comprising a heavy chain (HC) comprising a sequence according to SEQ ID NO:30 and a light chain (LC) comprising a sequence according to SEQ ID NO:9.

[0228] In some embodiments, the disclosure provides an anti-TNFR2 antibody comprising a VH sequence encoded by the nucleic acid sequence of SEQ ID NO: 33. In some embodiments, the disclosure provides an anti-TNFR2 antibody comprising a VL sequence encoded by the nucleic acid sequence of SEQ ID NO: 31. In some embodiments, the disclosure provides an anti-TNFR2 antibody comprising a heavy chain (HC) sequence encoded by the nucleic acid sequence of SEQ ID NO: 33 and a light chain (LC) sequence encoded by the nucleic acid sequence of SEQ ID NO: 31.

[0229] In some embodiments, the disclosure provides an anti-TNFR2 antibody comprising an HC sequence encoded by a plasmid deposited with the ATCC and having ATCC Accession No. PTA-127530. In some embodiments, the disclosure provides an anti-TNFR2 antibody comprising an LC sequence encoded by a plasmid deposited with the ATCC and having ATCC Accession No. PTA127532. In some embodiments, the disclosure provides an anti-TNFR2 antibody comprising an HC sequence encoded by a plasmid deposited with the ATCC and having ATCC Accession No. PTA-127530 and an LC sequence encoded by a plasmid deposited with the ATCC and having ATCC Accession No. PTA-127532.

[0230] In some embodiments, provided herein is an anti-TNFR2 antibody comprising a heavy chain and a light chain, wherein the antibody heavy chain has an amino acid sequence encoded by the nucleic acid sequence of an insert of a plasmid deposited with the ATCC having ATCC Accession No. PTA-127530, and the antibody light chain has an amino acid sequence encoded by the nucleic acid sequence of an insert of a plasmid deposited with the ATCC having ATCC Accession No. PTA-127532.

[0231] In some embodiments, the Fc domain is 2 Provided herein is an anti-TNFR2 antibody, wherein the Fc domain of the first Fab domain is: In some embodiments, provided herein is an anti-TNFR2 antibody, wherein the CH1 domain in the first Fab domain comprises a sequence according to SEQ ID NO: 14. Provided herein is an anti-TNFR2 antibody, wherein the CL in the first Fab domain comprises a sequence according to SEQ ID NO: 5.

[0232] In some embodiments, provided herein are anti-TNFR2 antibodies, wherein the first Fab domain and the second Fab domain are identical.

[0233] In some embodiments, provided herein is an anti-TNFR2 antibody, wherein the first Fc chain comprises, from N-terminus to C-terminus: a first hinge region, a first CH2 region, and a first CH3 region, and the second Fc chain comprises, from N-terminus to C-terminus: a second hinge region, a second CH2 region, and a second CH3 region.

[0234] In some embodiments, provided herein is an anti-TNFR2 antibody, wherein one or both of the first hinge region and the second hinge region comprise a sequence according to SEQ ID NO: 15. In some embodiments, provided herein is an anti-TNFR2 antibody, wherein one or both of the first CH2 domain and the second CH2 domain comprise a sequence according to SEQ ID NO: 16. In some embodiments, provided herein is an anti-TNFR2 antibody, wherein one or both of the first CH3 domain and the second CH3 domain comprise a sequence according to SEQ ID NO: 17. In some embodiments, provided herein is an anti-TNFR2 antibody, wherein one or both of the first Fc chain and the second Fc chain comprise a sequence according to SEQ ID NO: 18.

[0235] In some embodiments, provided herein is an anti-TNFR2 antibody, wherein the first Fc chain and the second Fc chain are identical. In some embodiments, provided herein is an anti-TNFR2 antibody, comprising a heavy chain (HC) comprising a sequence according to SEQ ID NO: 19. In some embodiments, provided herein is an anti-TNFR2 antibody, comprising a light chain (LC) comprising a sequence according to a sequence selected from the group consisting of SEQ ID NO: 6 and SEQ ID NO: 9.

[0236] In some embodiments, provided herein is an isolated anti-TNFR2 antibody comprising a light chain (LC) comprising a sequence according to SEQ ID NO:9.

[0237] In some embodiments, provided herein is an isolated antibody that specifically binds to TNFR2, comprising a heavy chain (HC) comprising a sequence according to SEQ ID NO: 19 and a light chain (LC) comprising a sequence according to SEQ ID NO: 9.

[0238] The present invention also encompasses fusion proteins that include one or more components of the antibody disclosed herein. In some embodiments, fusion proteins can be made that include all or a portion of the anti-TNFR2 antibody of the present invention linked to another polypeptide. In another embodiment, only the variable domain of the anti-TNFR2 antibody is linked to the polypeptide. In another embodiment, the VH domain of the anti-TNFR2 antibody is linked to a first polypeptide, while the VL domain of the anti-TNFR2 antibody is linked to a second polypeptide that associates with the first polypeptide in such a manner that the VH domain and the VL domain can interact with each other to form an antigen binding site. In another embodiment, the VH domain is separated from the VL domain by a linker so that the VH domain and the VL domain can interact with each other. The VH-linker-VL antibody is then linked to a polypeptide of interest. Additionally, fusion antibodies can be created in which two (or more) single chain antibodies are linked to each other. This is useful when one wants to create a bivalent or multivalent antibody on a single polypeptide chain, or when one wants to create a bispecific antibody.

[0239] Biological activity of anti-TNFR2 antibodies In addition to binding to an epitope on TNFR2, the antibodies of the present disclosure can mediate biological activities, i.e., the present disclosure includes isolated antibodies that specifically bind to TNFR2 and mediate at least one detectable activity selected from the following: (i) specifically binds to human TNFR2; (ii) specifically binds to cynomolgus monkey TNFR2; (iii) stimulates the activity of TNFR2 (e.g., human, cynomolgus monkey); and / or (iv) while binding to TNFR2, it enables TNFα activation of TNFR2.

[0240] In some embodiments, the EC50 of the human TNFR2 potency assay in Jurkat reporter cells is less than a number selected from the group consisting of 5 μM and 2 μM. 50Provided herein is an isolated anti-TNFR2 antibody, characterized by:

[0241] In some embodiments, the EC50 of the human TNFR2 potency assay in human peripheral blood monocytes is less than a number selected from the group consisting of 10 μM, 5 μM, and 2 μM. 50 Provided herein is an isolated anti-TNFR2 antibody, characterized by:

[0242] In some embodiments, the EC50 of the cynomolgus TNFR2 potency assay in cynomolgus peripheral blood monocytes is less than a number selected from the group consisting of 20 μM and 15 μM. 50 Provided herein is an isolated anti-TNFR2 antibody, characterized by:

[0243] In some embodiments, the EC50 of the human TNFR2 potency assay in Jurkat reporter cells is less than a number selected from the group consisting of 5 μM, 1 μM, and 0.5 μM. 50 Provided herein is an isolated anti-TNFR2 antibody, characterized by:

[0244] In some embodiments, the EC50 of ICAM-1 upregulation in a human TNFR2-expressing primary T cell population derived from human peripheral blood monocytes is less than a number selected from the group consisting of 10 mg / ml, 5 mg / ml, and 2 mg / ml. 50 Provided herein is an isolated anti-TNFR2 antibody, characterized by:

[0245] In some embodiments, the EC50 / ... 50 Provided herein is an isolated anti-TNFR2 antibody, characterized by:

[0246] In some embodiments, provided herein is an isolated anti-TNFR2 antibody characterized by an affinity KD for human TNFR2 of less than a number selected from the group consisting of 1 nM, 0.5 nM, 0.1 nM and 0.07 nM.

[0247] In some embodiments, provided herein is an isolated anti-TNFR2 antibody characterized by an affinity KD for cynomolgus TNFR2 of less than a number selected from the group consisting of 1 nM and 0.1 nM.

[0248] In some embodiments, affinity is measured by surface plasmon resonance (SPR). In some embodiments, SPR was measured using BIAcore. In some embodiments, affinity was measured by SPR where biotinylated TNFR2 was captured on a streptavidin strip surface at a flow rate of 10 ml / min for 60 seconds, and antibodies were injected over the captured TNFR2 at a range of concentrations for an association phase of 50 seconds at 80 ml / min, and then the dissociation phase was initiated with running HBS-EP+ buffer injected at 80 ml / min for 600 seconds. In some embodiments, antibodies are injected at a concentration ranging from 10 to 0.625 nM. In some embodiments, particularly for standard IgG formats containing two antigen binding domains, antibodies are injected at a concentration ranging from 10 to 1.25 nM. In some embodiments, particularly for tetrafab formats containing four antigen binding domains, antibodies are injected at a concentration ranging from 5 to 1.25 nM. In some embodiments, the SPR data is analyzed using Biacore Evaluation Software.

[0249] Polynucleotides encoding anti-TNFR2 antibodies and methods of production The present disclosure also provides polynucleotides encoding any of the antibodies of the invention, including antibody portions and modified antibodies described herein. The present invention also provides methods of making any of the antibodies and polynucleotides described herein. Polynucleotides can be made and proteins expressed by procedures known in the art.

[0250] If desired, the anti-TNFR2 antibody (monoclonal or polyclonal) of interest can be sequenced, and then the polynucleotide sequence can be cloned into a vector for expression or propagation.The sequence encoding the antibody of interest can be maintained in a vector in a host cell, and then the host cell can be expanded and frozen for further use.The production of recombinant monoclonal antibody in cell culture can be carried out through the cloning of antibody genes from B cells by means known in the art.See, for example, Tiller et al., 2008, J.Immunol.Methods 329, 112; U.S. Patent No. 7,314,622.

[0251] In some embodiments, the present specification provides a polynucleotide that comprises the sequence that codes for one or both of the heavy or light chain variable regions of the anti-TFR2 antibody provided herein.The sequence that codes for the antibody of interest can be maintained in a vector in a host cell, and the host cell can then be expanded and frozen for further use.Vector (including expression vector) and host cell are further described herein.

[0252] In some embodiments, the disclosure provides a polynucleotide encoding the amino acid sequence of any of the following anti-TNFR2 antibodies: IgG2-854, IgG2-1765, and IgG1. In one embodiment, the invention provides a polynucleotide encoding the amino acid sequence of the anti-TNFR2 antibody TF-2053.

[0253] In some embodiments, the disclosure provides a polynucleotide encoding one or more anti-TNFR2 antibody heavy chain polypeptides comprising an amino acid sequence selected from the group consisting of SEQ ID NO:19, SEQ ID NO:22, and SEQ ID NO:30.

[0254] In some embodiments, the disclosure provides a polynucleotide encoding one or more anti-TNFR2 antibody light chain polypeptides comprising an amino acid sequence selected from the group consisting of SEQ ID NO:6 and SEQ ID NO:9.

[0255] In some embodiments, the disclosure provides a polynucleotide encoding one or more anti-TNFR2 antibody VH polypeptides comprising an amino acid sequence selected from the group consisting of SEQ ID NO:13, SEQ ID NO:21, SEQ ID NO:29.

[0256] In some embodiments, the disclosure provides a polynucleotide encoding one or more anti-TNFR2 antibody VL polypeptides comprising an amino acid sequence selected from the group consisting of SEQ ID NO:4 and SEQ ID NO:8.

[0257] The present invention provides a polynucleotide comprising the nucleic acid sequence of the insert of the plasmid encoding the heavy chain of the antibody Tetrafab 2053, deposited with the ATCC and having the accession number PTA-127530. The present invention also provides a polynucleotide comprising the nucleic acid sequence of the insert of the plasmid encoding the light chain of the antibody Tetrafab-2053, deposited with the ATCC and having the accession number PTA-127532. In addition, the present invention provides a polypeptide comprising the amino acid sequence encoded by the DNA insert of the plasmid encoding the VH domain of the antibody Tetrafab-2053, deposited with the ATCC and having the accession number PTA-127528. The present invention further provides a polypeptide comprising the amino acid sequence encoded by the DNA insert of the plasmid encoding the VH domain of the antibody Tetrafab-2053, deposited with the ATCC and having the accession number PTA-127529. The present invention further provides a polypeptide comprising the amino acid sequence encoded by the insert of the plasmid encoding the VL domain of antibody Tetrafab-2053, deposited with the ATCC and having accession number PTA-127531.

[0258] It will be understood by those skilled in the art that, as a result of the degeneracy of the genetic code, there are many nucleotide sequences that code for the polypeptides described herein. Some of these polynucleotides bear minimal homology to the nucleotide sequence of any native gene. Nevertheless, polynucleotides that vary due to differences in codon usage are specifically contemplated by the present invention. In addition, alleles of genes that comprise the polynucleotide sequences provided herein are within the scope of the present invention. An allele is an endogenous gene that has been altered as a result of one or more mutations, such as deletions, additions, or substitutions, of nucleotides. The resulting mRNA and protein may, but need not, have altered structure or function. Alleles can be identified using standard techniques (e.g., hybridization, amplification, or database sequence comparison).

[0259] In one embodiment, the VH and VL domains or the full length HC or LC are encoded by separate polynucleotides, or both the VH and VL, or both the HC and LC, are encoded by a single polynucleotide.

[0260] Polynucleotides complementary to any such sequences are also encompassed by the present disclosure.Polynucleotides can be single-stranded (coding or antisense) or double-stranded, and can be DNA (genomic, cDNA or synthetic) or RNA molecules.RNA molecules include HnRNA molecules, which contain introns and correspond to DNA molecules in a one-to-one manner, and mRNA molecules, which do not contain introns.Additional coding or non-coding sequences can be, but need not be, present within the polynucleotides of the present disclosure, and polynucleotides can be, but need not be, linked to other molecules or support materials.

[0261] The polynucleotide of the present invention can be obtained by chemical synthesis, recombinant method or PCR.The method of chemical polynucleotide synthesis is well known in the art and does not need to be described in detail here.Those skilled in the art can use the sequence provided herein and commercially available DNA synthesizers to produce desired DNA sequence.

[0262] As further discussed herein, to prepare polynucleotides using recombinant methods, the polynucleotides containing the desired sequences can be inserted into a suitable vector, which can then be introduced into a suitable host cell for replication and amplification. The polynucleotides can be inserted into the host cell by any means known in the art. The cells are transformed by introducing the exogenous polynucleotides by direct uptake, endocytosis, transfection, F-mating or electroporation. Once introduced, the exogenous polynucleotides can be maintained in the cell as non-integrated vectors (e.g., plasmids) or can be integrated into the host cell genome.

[0263] A suitable cloning vector can be constructed according to standard techniques or selected from a large number of cloning vectors available in the art. The cloning vector selected can vary according to the host cell intended for use, but a useful cloning vector will generally have one or more features, such as i) the ability to replicate autonomously, ii) a single target for a specific restriction endonuclease, or iii) a marker gene that can be used in selecting clones containing the vector. Suitable examples include plasmids and bacterial viruses, such as pUC18, pUC19, Bluescript (e.g., pBS SK+) and its derivatives, mp18, mp19, pBR322, pMB9, ColE1, pCR1, RP4, phage DNA, and shuttle vectors, such as pSA3 and pAT28. These and many other cloning vectors are available from commercial suppliers, such as BioRad, Strategene, and Invitrogen.

[0264] Further provided is an expression vector. An expression vector is generally a replicable polynucleotide construct that contains a polynucleotide according to the present invention. It is implied that an expression vector must be replicable in a host cell, either as an episome or as an integrated part of chromosomal DNA. Suitable expression vectors include, but are not limited to, plasmids, viral vectors, including adenoviruses, adeno-associated viruses, retroviruses, cosmids and expression vectors disclosed in PCT Publication No. WO87 / 04462. Vector components may generally include, but are not limited to, one or more of the following: signal sequence; origin of replication; one or more marker genes; appropriate transcription control elements (e.g., promoter, enhancer and terminator). For expression (i.e., translation), one or more translation control elements are also usually required, such as ribosome binding sites, translation initiation sites and stop codons.

[0265] A vector containing a polynucleotide of interest can be introduced into a host cell by any of several suitable means, including electroporation, transfection using calcium chloride, rubidium chloride, calcium phosphate, DEAE-dextran or other substances; particle bombardment; lipofection; and infection (e.g., where the vector is an infectious agent, e.g., vaccinia virus). The choice of introducing a vector or polynucleotide often depends on the characteristics of the host cell.

[0266] The present invention also provides a host cell comprising any of the polynucleotides described herein. Any host cell capable of overexpressing heterologous DNA can be used to isolate genes encoding antibodies, polypeptides or proteins of interest. Non-limiting examples of mammalian host cells include, but are not limited to, COS, HeLa and CHO cells. See also PCT Publication No. WO87 / 04462. Suitable non-mammalian host cells include prokaryotes (e.g., E. coli or B. subtilis) and yeast (e.g., S. cerevisiae, S. pombe; or K. lactis).

[0267] Additionally, any number of commercially available and non-commercially available cell lines that express polypeptides or proteins may be utilized in accordance with the present invention. Those skilled in the art will understand that different cell lines may have different nutritional requirements or require different culture conditions for optimal growth and polypeptide or protein expression, and will be able to modify the conditions as necessary.

[0268] Pharmaceutical Compositions In another embodiment, the present invention comprises a pharmaceutical composition.

[0269] A "pharmaceutical composition" refers to a mixture of an antibody of the invention and one or more excipients.

[0270] The pharmaceutical compositions of the present invention can be in various forms. These include, for example, liquid, semi-solid and solid dosage forms, such as liquid solutions (e.g., injectable and infusible solutions), dispersions or suspensions, and lyophilized powders. The form depends on the intended mode of administration and therapeutic application.

[0271] Other excipients and modes of administration known in the pharmaceutical art can also be used.The pharmaceutical composition of the present invention can be prepared by any of the well-known techniques of pharmacy, such as effective formulation and administration procedures.The above considerations regarding effective formulation and administration procedures are well-known in the art and are described in standard textbooks.Drug formulation is discussed, for example, in Hoover, John E., Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pennsylvania, 1975; Liberman et al., eds., Pharmaceutical Dosage Forms, Marcel Decker, New York, NY, 1980; and Kibbe et al., eds., Handbook of Pharmaceutical Excipients (3rd ed.), American Pharmaceutical Association, Washington, 1999.

[0272] Acceptable excipients are nontoxic to recipients at the dosages and concentrations employed and include, but are not limited to, buffers, such as phosphate, citric acid and other organic acids; salts, such as sodium chloride; antioxidants, including ascorbic acid and methionine; preservatives (e.g., octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens, such as methylparaben or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, For example, it may include serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates including glucose, mannose or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); or non-ionic surfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG).

[0273] Therapeutic, Diagnostic and Other Methods The antibodies and antibody conjugates of the invention are useful in a variety of applications, including, but not limited to, therapeutic and diagnostic treatment methods.

[0274] In some embodiments, the antibodies of the invention may stimulate the activity of TNFR2 and, by stimulating TNFR2, may be useful in the treatment, prevention, suppression and amelioration of the following diseases: rheumatoid arthritis (RA), ankylosing spondylitis (AS), Crohn's disease (CD), ulcerative colitis (UC), graft versus host disease (GVHD), transplantation, psoriasis (PSO), psoriatic arthritis (PSA), atopic dermatitis (AD), vitiligo, alopecia areata (AA), type 1 diabetes (T1D), multiple sclerosis (MS), irritable bowel disease (IBD), autoimmune hepatitis and systemic lupus erythematosus (SLE) or diseases, disorders and conditions.

[0275] In one aspect, the present invention provides a method for treating one or more selected from the group consisting of rheumatoid arthritis (RA), ankylosing spondylitis (AS), Crohn's disease (CD), ulcerative colitis (UC), graft versus host disease (GVHD), transplantation, psoriasis (PSO), psoriatic arthritis (PSA), atopic dermatitis (AD), vitiligo, alopecia areata (AA), type 1 diabetes (T1D), multiple sclerosis (MS), irritable bowel disease (IBD), autoimmune hepatitis, and systemic lupus erythematosus (SLE). In some embodiments, a method for treating one or more selected from the group consisting of rheumatoid arthritis (RA), ankylosing spondylitis (AS), Crohn's disease (CD), ulcerative colitis (UC), graft-versus-host disease (GVHD), transplantation, psoriasis (PSO), psoriatic arthritis (PSA), atopic dermatitis (AD), vitiligo, alopecia areata (AA), type 1 diabetes (T1D), multiple sclerosis (MS), irritable bowel disease (IBD), autoimmune hepatitis, and systemic lupus erythematosus (SLE) in a subject comprises administering to a subject in need thereof an effective amount of a pharmaceutical composition comprising any of the TNFR2 antibodies described herein. In some embodiments, a method for reducing inflammation and / or activating or expanding immunoregulatory cell types in a subject is provided, comprising administering to a subject in need thereof an effective amount of a composition comprising an antibody provided herein.

[0276] In another aspect, the present invention further provides an antibody or pharmaceutical composition as described herein for use in the described methods of treating rheumatoid arthritis (RA), ankylosing spondylitis (AS), Crohn's disease (CD), ulcerative colitis (UC), graft versus host disease (GVHD), transplantation, psoriasis (PSO), psoriatic arthritis (PSA), atopic dermatitis (AD), vitiligo, alopecia areata (AA), type 1 diabetes (T1D), multiple sclerosis (MS), irritable bowel disease (IBD), autoimmune hepatitis and systemic lupus erythematosus (SLE) or other autoimmune and inflammatory conditions. The invention also provides for the use of an antibody as described herein in the manufacture of a medicament for treating one or more selected from the group consisting of rheumatoid arthritis (RA), ankylosing spondylitis (AS), Crohn's disease (CD), ulcerative colitis (UC), graft versus host disease (GVHD), transplantation, psoriasis (PSO), psoriatic arthritis (PSA), atopic dermatitis (AD), vitiligo, alopecia areata (AA), type 1 diabetes (T1D), multiple sclerosis (MS), irritable bowel disease (IBD), autoimmune hepatitis and systemic lupus erythematosus (SLE) or other autoimmune and inflammatory conditions.

[0277] In another aspect, one or more methods are provided for detecting, diagnosing or monitoring one or more selected from the group consisting of rheumatoid arthritis (RA), ankylosing spondylitis (AS), Crohn's disease (CD), ulcerative colitis (UC), graft-versus-host disease (GVHD), transplantation, psoriasis (PSO), psoriatic arthritis (PSA), atopic dermatitis (AD), vitiligo, alopecia areata (AA), type 1 diabetes (T1D), multiple sclerosis (MS), irritable bowel disease (IBD), autoimmune hepatitis and systemic lupus erythematosus (SLE) or other autoimmune and inflammatory conditions.For example, the anti-TNFR2 antibody described herein can be labeled with detectable moieties, such as imaging agents and enzyme-substrate labels.The antibody described herein can be used for in vivo diagnostic assays, such as in vivo imaging (e.g., PET or SPECT), or staining reagents.

[0278] With respect to all of the methods described herein, reference to an anti-TNFR2 antibody also includes a pharmaceutical composition comprising an anti-TNFR2 antibody and one or more additional agents.

[0279] Administration and Dosing Typically, the antibodies of the invention are administered in an amount effective to treat the conditions described herein. The antibodies of the invention can be administered as the antibodies themselves, or alternatively, as a pharmaceutical composition containing the antibodies.

[0280] The antibodies of the present invention are administered by any suitable route in the form of a pharmaceutical composition adapted to such route, and in a dosage effective for the intended treatment.

[0281] In some embodiments, the antibody can be administered parenterally, for example, directly into the bloodstream, muscle, or internal organs. Suitable means for parenteral administration include intravenous, intraarterial, intraperitoneal, intrathecal, intraventricular, intraurethral, ​​intrasternal, intracranial, intramuscular, and subcutaneous. Suitable devices for parenteral administration include needle (including microneedle) injectors, needle-free injectors, and infusion techniques.

[0282] In another embodiment, the compounds of the invention may also be administered topically to the skin or mucosa, i.e., dermally or transdermally. In another embodiment, the compounds of the invention may also be administered intranasally or by inhalation. In another embodiment, the compounds of the invention may also be administered rectally or vaginally. In another embodiment, the compounds of the invention may also be administered directly to the eye or ear.

[0283] Dosing regimens for the antibodies of the invention or compositions containing said antibodies are based on a variety of factors, including the type, age, weight, sex, and medical condition of the subject; the severity of the condition; the route of administration; and the activity of the particular antibody used. Thus, dosing regimens can vary widely. In one embodiment, the total daily dose of the antibodies of the invention is typically about 0.01 to about 100 mg / kg (i.e., mg of the antibodies of the invention per kg of body weight) for treatment of the indicated conditions discussed herein. In another embodiment, the total daily dose of the antibodies of the invention is about 0.1 to about 50 mg / kg, and in another embodiment, about 0.5 to about 30 mg / kg.

[0284] Coadministration The antibody of the present invention may be used alone or in combination with one or more other therapeutic agents. The present invention provides any of the uses, methods or compositions defined herein, in which the antibody of the present invention is used in combination with one or more other therapeutic agents discussed herein.

[0285] The administration of two or more drugs in "combination" means that all drugs are administered close enough in time to affect the treatment of the subject.Two or more drugs can be administered simultaneously or sequentially.Furthermore, simultaneous administration can be carried out by mixing the drugs before administration, or by administering the drugs as separate dosage forms at the same time in time but at the same or different administration sites.

[0286] kit Another aspect of the present invention provides a kit comprising the antibody of the present invention or a pharmaceutical composition comprising the antibody. The kit may comprise a diagnostic or therapeutic agent in addition to the antibody of the present invention or a pharmaceutical composition thereof. The kit may also comprise instructions for use in a diagnostic or therapeutic method. In some embodiments, the kit comprises the antibody or a pharmaceutical composition thereof and a diagnostic agent.

[0287] In yet another embodiment, the present invention includes a kit suitable for use in carrying out the method of treatment described herein.In one embodiment, the kit contains a first dosage form comprising one or more antibodies of the present invention in an amount sufficient to carry out the method of the present invention.In another embodiment, the kit comprises one or more antibodies of the present invention in an amount sufficient to carry out the method of the present invention, and at least a first container for the first dosage and a second container for the second dosage.

[0288] biological deposit Representative material of the present invention was deposited on March 3, 2023 at the American Type Culture Collection, 10801 University Boulevard, Manassas, VA 20110-2209, USA.

[0289] [Table 1]

[0290] The deposit was made under the terms of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purpose of Patent Procedure and Regulations thereunder (Budapest Treaty), which assures maintenance of a viable culture of the deposit for 30 years from the date of deposit. The deposit will be made available by ATCC under the terms of the Budapest Treaty, pursuant to an agreement between Pfizer Inc. and ATCC to assure perpetual and unlimited availability to the public of the progeny of the culture of the deposit upon issuance of the relevant U.S. patent or upon the public publication of any U.S. or foreign patent application, whichever occurs first, and to guarantee availability of the progeny to persons determined by the Director of the U.S. Patent and Trademark Office to be entitled pursuant to 35 U.S.C. Section 122 and the Director's regulations thereunder (including 37 CFR Section 1.14, which specifically pursuant to 886 OG 638).

[0291] The assignee of the present application has agreed that in the event that cultures of the deposited materials die when cultivated under appropriate conditions or are lost or destroyed, such materials will be replaced without undue delay with identical ones upon notice. Availability of the deposited materials is not to be construed as a license to practice the invention in violation of rights granted under the authority of any government pursuant to its patent laws. EXAMPLES

[0292] The following examples of specific modes for carrying out the present invention are offered for illustrative purposes only and are not intended to limit the scope of the invention in any way.

[0293] The above description and the following examples detail certain specific embodiments of the present disclosure and describe the best mode contemplated by the inventors. However, no matter how detailed the above appears in the text, it is understood that the present disclosure can be implemented in many ways and that the present disclosure should be interpreted according to the appended claims and any equivalents thereof.

[0294] Although the disclosed teachings have been described with respect to various applications, methods, kits and compositions, it is understood that various changes and modifications can be made without departing from the teachings herein and the following claimed disclosure. The following examples are provided to better illustrate the disclosed teachings and are not intended to limit the scope of the teachings set forth herein. Although the present teachings have been described with respect to these exemplary embodiments, those skilled in the art will readily appreciate that numerous variations and modifications of these exemplary embodiments are possible without undue experimentation. All such variations and modifications are within the scope of the present teachings.

[0295] [Table 2-1]

[0296] [Table 2-2]

[0297] Example 1 Isolation of a mouse monoclonal antibody that binds to human and cynomolgus monkey TNFR2 SJL mice were immunized with multiple IP injections of Enbrel (HuTNFR2-IgG1-Fc) with ribi as an adjuvant. Sera were screened for binding to human and cynomolgus monkey (Cyno) TNFR2 overexpressed on the surface of CHO cells. Mice showing strong binding to CHO cells were euthanized and isolated B cells were fused with P3X myeloma by electrofusion.

[0298] After hybridoma supernatant screening using TNFR2 CHO binding, 14 clones were selected and reformatted into HuIgG1 and HuIgG2 (see Example 2). Of these 28 HuIgG1 and HuIgG2, after confirmation screening with CHO cells overexpressing human and cyno TNFR2 (see Table 1), 16 (clones 813, 817, 836, 846, 851, 854, 1201, 1070, 1211, 1215, 1296, 1302, 1304, 1310, 1327 and 1329) showing a range of binding to human and cyno TNFR2 were selected for further characterization.

[0299] [Table 3]

[0300] Example 2 Cloning of the heavy and light chain variable regions of mouse anti-TNFR2 antibody The heavy and light chain variable regions of the anti-TNFR2 antibody were cloned using the SMART® cDNA synthesis system (Takara Bio Inc., Shiga, Japan) followed by PCR amplification. cDNA was synthesized from 1 ug of total RNA isolated from approximately 500,000 hybridoma cells using the RNEasy kit (Qiagen) and template switching oligos with SuperscriptIV™ reverse transcriptase (Invitrogen). The cDNA was then amplified by PCR using a primer annealing to the SMART® IIA oligo sequence and a mouse constant region specific primer (mouse kappa for light chain, mouse IgG for heavy chain) with Q5 High-Fidelity 2× Master Mix (New England Biolabs). The variable heavy and light chain regions were cloned into mouse pTT5 mammalian expression vectors containing mouse IgG2a and kappa constant regions, respectively, using the injection cloning method (Takara Bio), and the nucleic acid sequences were determined.

[0301] The variable heavy region was then cloned into a pTT5 expression vector containing a human IgG1 constant region mutated to abolish effector function (Leu234Ala, Leu235Ala and Gly237Ala, EU numbering; U.S. Patent No. 5,624,821) to produce a chimeric heavy chain. The variable light region was cloned into a pTT5 mammalian expression vector containing a constant human kappa region to produce a chimeric light chain.

[0302] Example 3 Some anti-TNFR2 antibodies agonize human TNFR2 in an NF-KB-GFP Jurkat cell reporter cell assay NF-KB-GFP Jurkat cells (System Biosciences, Palo Alto, CA) were stably transfected by transduction using a lentiviral vector (pLVX-puro, Takara Bio US) containing a full-length human TNFR2 insert. For the assay, 100000 cells / well were plated in 96-well plates in RPMI1640 medium supplemented with 10% FBS, 1x sodium pyruvate, 1x Glutamax and Pen / Strep. The cells were then incubated overnight at 37°C with serial dilutions of anti-TNFR2 antibodies. The following day, the cells were prepared for flow cytometry analysis as follows: after centrifugation to remove the supernatant, the cells were treated with 0.25% trypsin to break up clumps. Finally, the cells were centrifuged again, resuspended in 75 ml of flow buffer containing 1 / 1000 dilution of DAPI reagent, and analyzed on a flow cytometer for GFP expression.

[0303] Table 2 summarizes the results of the previous TNFR2 CHO cell assay and potency in the Jurkat cell reporter assay. We selected nine clones, all IgG2, based on a range of EC50 in the Jurkat assay and sequence diversity to proceed with further characterization.

[0304] [Table 4]

[0305] Example 4 Biophysical characteristics and agonism in primary cells (human PBMC activation assay) of selected agonist clones The nine previously selected clones were then tested for their ability to induce ICAM-1 upregulation in CD3+CD4+TIGIT+ human PBMCs (see Example 11 for methods). We also evaluated several biophysical properties, such as sequence liability and non-specificity (AC-SINS and polyreactivity).

[0306] [Table 5]

[0307] As can be seen in Table 3, we observed a range of agonism on human PBMCs and decided to move forward with clones showing some potency on primary cells (clones 836, 846, 854, 1201 and 1310), with a non-specificity level that we could manage by subsequent optimization if necessary.

[0308] Example 6 Epitope binning of anti-TNFR2 antibodies Epitope binning was also performed using yeast expressing human TNFR1 / TNFR2 domain chimeras. Yeast cells were stably transfected with various constructs in which one or two CRDs of TNFR2 were replaced with the corresponding human TNFR1 CRD. Similarly, the reverse constructs in which one or two CRDs of TNFR1 were replaced with the corresponding TNFR2 CRD were also tested. Loss or gain of binding indicated that a particular CRD was required for binding to TNFR2 and contained the epitope, respectively.

[0309] As seen in Table 4, most clones bind to CRD3-4, except for clone 836 (CRD1) and clone 1310 (CRD1-3-4).

[0310] [Table 6]

[0311] Example 7 Humanization of mouse anti-TNFR2 antibody Clone 854 was the more agonistic antibody and was therefore selected for further humanization. Humanization of clone 854 was performed by grafting the murine hypervariable regions into various human frameworks.

[0312] The hypervariable regions of the heavy chain were defined using the SDR definition (specificity determining residues) whereas the hypervariable regions of the light chain were defined using the CDR definition (complementarity determining region).

[0313] [Table 7]

[0314] Because the SDR definitions rather than the CDRs were used for grafting, murine amino acids important for binding and affinity are preserved during the humanization process.

[0315] The human heavy chain framework selected for grafting of the 854 mouse heavy chain SDRs was: IGHJ4 * 01 IGHV4-31 with J-gene * 02, IGHV4-30-4 * 07, IGHV4-4 * 08 (the three germline families most closely related to clone 854), IGHV3-23 * 01, IGHV1-46 * 01 and IGHV3-7 *01 (three commonly used human frameworks). The human light chain framework selected for grafting of the 854 mouse light chain CDRs was: IGKJ4 * 01 IGKV3-11 in combination with the J-gene * 01, IGKV1-9 * 01, IGKV1-33 * 01, IGKV1-27 * 01, IGKV1-39 * 01 and IGKV4-1 * 01.

[0316] Example 8 Binding of human TNFR2 CHO cells by humanized TNFR2 antibodies All combinations of heavy and light chains were expressed and tested for binding to human TNFR2 CHO cells:

[0317] [Table 8]

[0318] After grafting into various human frameworks, a significant loss of binding was observed, as summarized in Table 6. Some clones, such as grafts 31, 33, 34 and 35, showed EC50 values ​​close to 854, but the maximum signal was significantly lower and their AC-SINS scores were high. All DNA / insulin polyreactivity scores were low (data not shown).

[0319] Example 9 Introduction of back mutations to restore binding to TNFR2 To restore binding to TNFR2 to the levels of the parental 854 clone, various backmutations were introduced into a selected number of heavy chain graft constructs:

[0320] [Table 9]

[0321] Each heavy chain was paired with the following humanized light chains: VK4-1, VK3-11, VK1-39 and VK1-9, as described in Example 7, but also with the same humanized light chains in which CDR2-L2 was germlined.

[0322] Example 10 Binding to human TNFR2 by humanized TNFR2 antibodies A total of 114 clones were expressed and purified and their binding to human TNFR2 CHO cells was compared to the parental 854 clone. As seen in Table 8 below, most of the clones showed EC50s close to or better than the parental clone 854. Clones 1847, 1765, 1753 and 1747 were selected for further characterization.

[0323] [Table 10-1]

[0324] [Table 10-2]

[0325] [Table 10-3]

[0326] Example 11 The humanized variants show a range of potency in human and cyno PBMC assays.

[0327] Selected humanized clones were first tested in the human TNFR2 Jurkat reporter cell assay. The results in Table 9 show that clone 1765 exhibits a similar EC50 as parent clone 854. Both clones 1847 and 1753 lost potency, and interestingly, clone 1747 was not an agonist in the Jurkat reporter cell assay, despite being the strongest binder in the human TNFR2 CHO binding assay.

[0328] Following this result, clones 1847, 1765 and 1753 were tested in human and cyno PBMC assays.

[0329] Briefly, 250000 human PBMCs from different donors were plated in wells of a 96-well plate in Optimizer CST medium. Each well was treated with 50 ml of 5ng / ml IL-2 and 50 ml of 4x anti-TNFR2 antibody or TetraFab for 72 hours at 37°C. Subsequently, cells were prepared for flow cytometry analysis. After two washes in PBS, cells were first resuspended in 100 ml of Live Near IR staining buffer for 30 minutes at 4°C. After two additional washes in flow buffer, cells were incubated with 1 / 40 diluted CD3 BV421, ICAM APC CD4 PE and TIGIT BUV395 for 1 hour at 4°C and then fixed with 1% PFA for 15 minutes at room temperature. After two additional washes in flow buffer, cells were finally analyzed by flow cytometry. Doublets were excluded using light scattering and low Near IR staining was an indicator of viability. Single viable cells were gated on the CD3+ / CD4+ / TIGIT+ population and ICAM-1 mean fluorescence intensity (MFI) was measured for ICAM-1. A four-parameter curve fit in Graph Pad Prism was used to calculate the EC50 of TNFR2 agonists for ICAM-1 induction. ICAM-1 was chosen because it is known to be regulated by NF-KB, a marker of acute T cell activation, and has a known role in Treg-mediated suppression. TIGIT was used to identify populations of mature T cells and Tregs enriched for TNFR2 expression without the need to stain for the intracellular marker FoxP3. A similar strategy was used to determine T cell activation in cyno PBMCs, but T cell activation was measured as the percentage of CD4+ cells double positive for CD25+ / Ki67+ due to differences in activation marker expression between human and cyno and antibody cross-reactivity.

[0330] The results in Table 9 confirmed that humanized clone 1765 exhibited similar potency in human PBMC assays as parental clone 854, with EC50s of 1.308 and 1.402 nM, respectively. Potency against cyno cells was 10-fold lower for both clones, with EC50s of 13.63 and 15.02 nM, respectively. Clone 1753 showed lower potency (56 nM), especially against cyno PBMC cells, and clone 1847 showed no potency against both cell types. Clone 1765 (heavy chain sequence, SEQ ID NO:22 and light chain sequence, SEQ ID NO:21) was selected for further investigation.

[0331] [Table 11]

[0332] Example 12 Aggregated anti-TNFR2 antibodies show increased potency in the Jurkat NF-KB-GFP reporter cell assay. To assess the effect of aggregates on TNFR2 agonism, we compared anti-TNFR2 antibodies purified by the one-step method (ProA purification only, containing 15-20% aggregates on average) with the same antibodies purified by the two-step method (ProA followed by SEC purification, no aggregates) in a Jurkat NFKB-GFP reporter cell assay.

[0333] Briefly, 100000 NF-KB-GFP Jurkat cells / well were plated in 96-well plates in RPMI1640 medium supplemented with 10% FBS, 1x sodium pyruvate, 1x Glutamax and Pen / Strep. The cells were then incubated overnight at 37°C with serial dilutions of anti-TNFR2 antibodies. The following day, the cells were prepared for flow cytometry analysis as follows: after centrifugation to remove the supernatant, the cells were treated with 0.25% trypsin to break up clumps. Finally, the cells were centrifuged again and resuspended in 75 ml of flow buffer containing 1 / 1000 dilution of DAPI reagent and analyzed on a flow cytometer for GFP expression.

[0334] [Table 12]

[0335] Clone 162, selected as a representative result, was compared with the commercially available TNFR2 agonist MR2-1 (Hycult Biotech, ref. HM2007).

[0336] When purified on a ProA column, clone 162 showed similar potency as MR2-1 (EC50 of 347.3 and 119.6 ng / ml, respectively). Interestingly, we found that both clone 162 and MR2-1 contained significant amounts of aggregates, as seen below in the analytical SEC profile in Figure 1.

[0337] When both clone 162 and MR2-1 lacked aggregates, their ability to agonize TNFR2 in Jurkat cell assays after purification by SEC was significantly impaired, with clone 162 showing a 20-fold loss in potency (EC50=7.02 mg / ml) and MR2-1 showing a 50-fold loss (EC50=5.75 mg / ml), suggesting that aggregates may artificially increase anti-TNFR2 antibody potency.

[0338] This result prompted us to investigate whether increasing valence could result in increased potency.

[0339] (Example 13) Reformat to TetraFab To test our hypothesis, we sought to increase the valency of clone 1765 by adding an additional paratope onto its existing IgG scaffold and reformatting it into TetraFab 2053 (see FIG. 2). As depicted in the schematic, TetraFab is composed of a double Fab heavy chain and typical human IgG1 CH2 and CH3, with the outer Fab domain composed of a variable domain and a constant CH1 domain linked by a short G4S linker to an inner Fab composed of the same variable domain followed by the same constant CH1. This longer heavy chain can associate with four light chains instead of two, creating a molecule composed of four paratopes and theoretically capable of binding four molecules of TNFR2. See SEQ ID NO: 30 and SEQ ID NO: 9 for the heavy and light chains of TetraFab 2053, respectively.

[0340] The rationale behind the reformatting into TetraFab is to mimic TNFR2 oligomerization by membrane-bound TNFα, which in fact forms trimers that can potently agonize TNFR2 on the surface of Tregs by simultaneously binding to multiple TNFR2 molecules.

[0341] The increase in binding force was confirmed by surface plasmon resonance (SPR) using BIAcore. Briefly, biotinylated human-TNFR2 or cyno-TNFR2 was captured on the surface of a streptavidin chip for 60 seconds at a flow rate of 10 ml / min. Antibodies or TetraFabs were then injected over the captured TNFR2 for a 50 second association phase at 80 ml / min, at concentrations ranging from 10 to 1.25 nM for antibodies and 5 to 0.625 nM for TetraFabs. The dissociation phase was then initiated with running buffer (HBS-EP+) injected for 600 seconds at 80 ml / min. Data was analyzed using Biacore Evaluation Software.

[0342] [Table 13]

[0343] The data in Table 11 show that both the parental 854 clone and the humanized 1765 clone have similar apparent affinities for human TNFR2, with KDs of 0.11 and 0.32 nM, respectively. TetraFab shows stronger apparent affinity, with a KD of 0.06 nM, a 5-fold improvement compared to its IgG counterparts 854 or 1765.

[0344] A similar improvement in affinity, although less dramatic, could be observed for cyno TNFR2: the affinity of TetraFab for cyno TNFR2 was 10-fold lower than for human TNFR2 (KD of 0.61 and 0.06 nM, respectively).

[0345] Example 14 Anti-TNFR2 antibodies and TetraFab agonize TNFR2 in Jurkat NF-KB-GFP reporter cell assays TetraFab was first tested for its ability to induce NF-KB-GFP expression in the TNFR2-expressing Jurkat cell reporter assay used previously. Similarly, 100,000 cells / well were plated in 96-well plates in RPMI1640 medium supplemented with 10% FBS, 1x sodium pyruvate, 1x Glutamax and Pen / Strep, and incubated overnight at 37°C with serial dilutions of anti-TNFR2 antibodies or TetraFab. The next day, cells were prepared for flow cytometry analysis as described above and analyzed for GFP expression on a flow cytometer.

[0346] The results summarized in Table 12 show that the parent clone 854 and its humanized variant 1765 have similar EC50 of 0.526 mg / ml and 1.009 mg / ml, respectively. On the other hand, the EC50 of TetraFab 2053 shows a greater than 10-fold improvement compared to both 854 and 1765, with an EC50 of 0.037 mg / ml. This result suggests that increased valency promotes stronger potency.

[0347] [Table 14]

[0348] Example 15 Anti-TNFR2 antibody and TetraFab induce ICAM-1 upregulation in CD3+CD4+TIGIT+ human PBMCs and Ki67 / CD25 co-expression on CD4+ cells in cyno PBMCs TetraFab was then tested for its ability to upregulate ICAM-1 in TNFR2-expressing primary T cell populations (CD3+CD4+TIGIT+) as described below. ICAM-1 was chosen because it is regulated by NF-KB, known to be a marker of acute T cell activation, and has a known role in Treg-mediated suppression. TIGIT was used to identify populations of mature T cells and Tregs enriched for TNFR2 expression without the need to stain for the intracellular marker FoxP3.

[0349] Briefly, 250000 human PBMCs from different donors were plated in wells of a 96-well plate in Optimizer CST medium. Each well was treated with 50ml of 5ng / ml IL-2 and 50ml of 4x anti-TNFR2 antibody or TetraFab for 72 hours at 37°C. Subsequently, cells were prepared for flow cytometry analysis. After two washes in PBS, cells were first resuspended in 100ml of Live Near IR staining buffer for 30 minutes at 4°C. After two additional washes in flow buffer, cells were incubated with 1 / 40 diluted CD3 BV421, ICAM APC CD4 PE and TIGIT BUV395 for 1 hour at 4°C and then fixed with 1% PFA for 15 minutes at room temperature (all reagents are summarized in Table 13). After two additional washes in flow buffer, cells were finally analyzed by flow cytometry. Light scatter was used to exclude doublets and low Near IR staining was an indication of viability. Single viable cells were gated on the CD3+ / CD4+ / TIGIT+ population and ICAM-1 MFI was measured for ICAM-1. A four-parameter curve fit in Graph Pad Prism was used to calculate the EC50 of TNFR2 agonists for ICAM-1 induction. A similar strategy was used to determine T cell activation in cyno PBMCs, but T cell activation was measured as the percent of CD4+ cells double positive for CD25+ / Ki67+ due to differences in activation marker expression between human and cyno and antibody cross-reactivity.

[0350] [Table 15]

[0351] As seen in Table 14, similar to the NF-KB-GFP Jurkat assay results, the EC50 for upregulation of human ICAM-1 on the surface of human CD3+CD4+TIGIT+ cells is similar between the parental 854 and humanized 1765 antibodies (1.402 and 1.308 mg / ml, respectively). The EC50 for upregulation of ICAM-1 is improved 10-fold after treatment with TetraFab 2053, confirming that increased valency improves potency even when the number of TNFR2 cell surface receptors is low (11000 in Jurkat and <500 in human Treg).

[0352] [Table 16]

[0353] Example 16 TetraFab 2053 induces TNFR2 agonism-induced IKBa degradation in cultured human and cyno Tregs The following assay was used as a surrogate for NF-KB activation resulting from TNFR2 agonism, which also allowed for a direct comparison of anti-TNFR2 antibody pharmacology between human and cyno Tregs using the same endpoints.

[0354] Briefly, human or cyno natural Tregs sorted from PBMCs of different donors and expanded via multiple cycles of anti-CD3 / 28 stimulation in the presence of IL-2 and rapamycin were stimulated overnight at 37°C in medium containing 10ng / ml recombinant human or cyno IL2, respectively. The following day, cells were seeded in 96-deep well plates (>50000 cells / well) and treated with various concentrations of TetraFab 2053, PMA / ionomycin (PMA 40ng / ml; ionomycin 2mM, positive control for IKBa) or medium (unstimulated control) for 20 minutes at 37°C. After incubation, cells were washed, fixed, permeabilized and stained with anti-IKBa-PE antibody for 30 minutes at 4°C. Finally, cells were analyzed by flow cytometer and the degradation level of IKBa was evaluated by MFI.

[0355] [Table 17]

[0356] As detailed above in Table 15, the average EC50 for IKBa in expanded human Tregs was 26 + / - 17 pM (n=5), whereas that for expanded cyno Tregs was 145 + / - 154 pM (n=5). Despite significant variability in results between Tregs expanded from individual human and cyno donors, these data indicate consistent activation of the NF-KB pathway in Tregs, and a more than 5-fold loss of potency in cynos compared to humans.

[0357] (Example 17) Anti-TNFR2 TetraFab induces OX-40 upregulation in human and cyno splenocytes in the absence of IL-2 Since pharmacology studies in cyno revealed activation of tissue Tregs after in vivo administration as indicated by upregulation of the TNF superfamily member OX-40, an ex vivo assay was set up to generate EC50 values ​​for tissue resident Treg activation using TeraFab 2053. In this assay, frozen human or cyno splenocytes from different donors were thawed and treated with various concentrations of TetraFab 2053 for 22 hours at 37°C. Samples were then fixed, permeabilized and stained with the following antibodies: CD4-BV786, FoxP3-AF647, OX40-BV421, CD3-BV605, Tigit-A488, CD25-PE-Cy5 and CD45-PE-Cy7, and then analyzed by flow cytometry. Tissue-resident Tregs were identified as single / live / CD45+ / CD3+ / CD4+ / CD25+ / FoxP3+, and the MFI of OX-40 was used to generate EC50 curves in GraphPad Prism using a 4-parameter curve fit. Individual EC50 values ​​generated for each donor of human or cyno frozen splenocytes are shown in this table. Table 16 summarizes the results obtained.

[0358] [Table 18]

[0359] The mean EC50 for upregulation of OX-40 on human splenic Tregs was 33 + / - 21 pM (n = 2), similar to the EC50 for IKBa degradation in expanded human blood Tregs, whereas the mean EC50 for upregulation of OX-40 in cyno splenic Tregs was 958 + / - 455 pM (n = 5).

[0360] (Example 18) In vivo pharmacology of TetraFab 2053 in cynomolgus monkeys Male and female cynomolgus monkeys of Mauritian origin, aged >2.5 years, were acclimated for a minimum of 30 days before dosing was initiated. Dose groups containing one male (M) and one female (F) animal were administered either vehicle control or 20 mg / kg, 60 mg / kg or 180 mg / kg of TetraFab 2053 by intravenous (IV) injection on days 1, 4, 8, 11 and 15. After administration of TetraFab 2053 on day 11, blood was collected at various time points over 96 hours for toxicokinetic analysis. On day 16, all animals were euthanized and a portion of each spleen was collected for Treg phenotype analysis. Single cell splenocyte suspensions were prepared by routine methods, and leukocytes were then stained with a panel of fluorescently labeled monoclonal antibodies that recognize surface and intracellular markers used to identify and characterize Tregs. The stained cells were analyzed by flow cytometry for the following phenotypes: OX40-expressing Treg: CD45+CD3+CD4+FoxP3+OX40+ TIGIT-expressing Treg: CD45+CD3+CD4+FoxP3+TIGIT+ Proliferative Treg: CD45+CD3+CD4+FoxP3+Ki-67+

[0361] The percentage and mean fluorescence intensity (MFI) of OX40 or TIGIT expressing Tregs, as well as the percentage of proliferating Tregs, were determined using flow cytometry analysis software. The fold increase in Treg parameters in TetraFab 2053 treated animals compared to gender-matched vehicle controls is shown in Table 17 below. Total TetraFab 2053 concentrations in serum were determined using a ligand binding assay, and the area under the curve (AUC) concentrations from days 11 to 15 were assessed for each individual animal, as shown in Table 17 below.

[0362] [Table 19]

[0363] Administration of TetraFab 2053 at doses ranging from 20 to 180 mg / kg / dose IV for 16 days to male and female cynomolgus monkeys increased OX-40 and TIGIT expression on splenic Tregs by 3.14- and 2.44-fold, respectively, and increased the percentage of splenic Tregs undergoing proliferation by 2.27-fold compared to vehicle-treated controls. Increases in these markers of Treg activation were observed at all dose levels in both male and female animals and generally did not follow a dose-response relationship, which most likely reflected maximal pharmacological activity.

[0364] Systemic exposure increased with increasing dose in male cynomolgus monkeys in an approximately dose-proportional manner at 20 to 60 mg / kg and greater than dose-proportional at 60 to 180 mg / kg. Systemic exposure generally increased with increasing dose in female cynomolgus monkeys in a greater than dose-proportional manner at 20 to 180 mg / kg.

[0365] (Example 19) TetraFab 2053 demonstrates acceptable development potential To confirm that TetraFab 2053 is a suitable candidate for further development as a biotherapeutic, a range of bioanalytical assays were completed (see Table 18).

[0366] [Table 20]

[0367] The results summarized in the table above indicated that TetraFab 2053 exhibited acceptable non-specific properties. Furthermore, the molecule was stable when tested at high concentrations for 6 weeks at 4°C and 4 weeks at 40°C. Finally, TetraFab 2053 exhibited some viscosity at high concentrations, but this undesirable feature could be managed with buffer formulation. Overall, these results suggested that TetraFab 2053 is suitable for manufacturing as a biological drug product.

[0368] Example 16 TetraFab 2053 Optimization The following amino acids shown in Table 19 were targeted for optimization to remove sequence fidelity (DE deamidation site, W oxidation site, predicted T cell epitope) and reduce hydrophobicity.

[0369] [Table 21]

[0370] Optimized constructs were made in IgG and TetraFab formats and the various mutations described above were tested alone or in combination for binding to human TNFR2 CHO, as summarized in Table 20:

[0371] [Table 22-1]

[0372] [Table 22-2]

[0373] [Table 22-3]

[0374] [Table 22-4]

[0375] Surprisingly, all clones except two pairs of IgG and TetraFab completely lost their ability to bind human TNFR2 expressed on the surface of CHO cells. Clones TetraFab 2208 / IgG 2204 and TetraFab 2209 / IgG 2205 were still able to bind TNFR2, as were IgG and TetraFab, but the Emax was significantly reduced compared to the parent clone 2053 / 1765. These clones were mutated only in CDR3-VL to remove predicted T cell epitopes. In vitro analysis (not shown) showed that the predicted T cell epitopes of TetraFab 2053 were not presented on the surface of antigen-presenting cells, and peptides overlapping with the predicted T cell epitopes did not activate CD4+ T cells, suggesting that the risk of immunogenicity posed by the predicted T cell epitopes was low, and therefore these optimized variants were not taken forward.

[0376] (Example 17) TF-2053 provides long-term survival benefit in NSG-GVHD models Objective: To test the prophylactic efficacy of human TetraFab-2053 in preventing xenograft GvHD Transfer of human peripheral blood mononuclear cells (PBMCs) into immunodeficient mice lacking endogenous lymphoid cell differentiation (NOD-SCID gamma common chain knockout, NSG) results in graft-versus-host disease (GVHD) mediated primarily by transferred human CD4+ and CD8+ lymphocytes [1]. Transferred human lymphocytes are not educated in the thymus in recipient mice and are therefore not tolerized to mouse proteins presented in association with mouse major histocompatibility complex (MHC) class I and II molecules [2]. Mouse-reactive human CD4+ and CD8+ cells mature and expand over the course of several weeks into effector memory cells that release large amounts of inflammatory cytokines, e.g., tumor necrosis factor alpha (TNFa) and interferon gamma (IFNg), resulting in extensive tissue inflammatory infiltrates as well as organ dysfunction, weight loss, and death [1]. The NSG GVHD model is often used by the field to demonstrate the role of proteins expressed by human lymphocytes in mediating T cell activation, expansion and effector function. This model was chosen to demonstrate the role of agonizing TNFR2 in reducing human effector T cell expansion, activation and pathophysiological function in a model independent of human regulatory T cell (Treg) survival. Effector T cell activation-induced cell death via TNFR2 agonism has been previously demonstrated in vitro for human T cells [3], but not in vivo, although in mice, TNFR2 has been demonstrated to be responsible for suppressing some autoreactive CD8 T cell activity [4].

[0377] method: Summary: 8-9 week old NSG mice were purchased from Jackson Labs and acclimated in a Pfizer vivarium. All animal use and handling was performed under IACUC approved protocols. TetraFab-2053 or isotype control tetraFab was administered at 3 mg / kg, 10 ml / kg, biweekly throughout the experiment, starting 1 day prior to human PBMC administration. Previously cryopreserved human PBMC were thawed on day 0 of the experiment, washed in PBS, and diluted at 10 × 10 per mouse. 6 Mice were dosed IV with 0.1 ml PBS / mL, n=20 mice per group. Cohorts were formed based on randomization of pre-study body weight. Body weight was measured twice weekly until sacrifice on day 78. Blood was collected at 2-week intervals to measure human cell engraftment / expansion, CD4 / CD8 distribution, and markers of T-cell anergy, exhaustion and senescence by flow cytometry, and circulating human cytokine levels by electrochemiluminescence MSD assay. Mice that lost >20% BW were euthanized according to IACUC protocol, and all remaining mice were killed on day 78. At the time of death, spleen and liver weights were recorded and flow cytometric analysis was performed on splenocytes as well as blood.

[0378] result: TF2053 provides long-term survival benefit in the NSG-GVHD model (Figure 3, Table 21). TF2053 reduces weight loss in the NSG-GVHD model (Figure 4, Table 22). Additionally, in the NSG GVHD model, TF2053 reduces circulating human cytokines IFNγ, IL10, IL17A, TNFα (Figures 5-8 and Tables 23-26). TF2053 reduces liver and spleen weights at sacrifice in the NSG GVHD model (Figures 9 and 10, Tables 27 and 28). TF2053 reduces human cell engraftment over the course of the NSG GVHD model as measured by CD45+, CD4+, and CD8+ (Figures 11-13, Tables 27-29). TF2053 reduces anergy and increases senescent and exhausted CD8+ cells over the course of the NSG GVHD model (Figures 14-16, Tables 32-34). TF2053 alters CD4:CD8 engraftment ratios (Figure 12, Table 30).

[0379] [Table 23]

[0380] [Table 24]

[0381] [Table 25]

[0382] [Table 26]

[0383] [Table 27]

[0384] [Table 28]

[0385]

Table 29

[0386]

Table 30

[0387]

Table 31

[0388]

Table 32

[0389]

Table 33

[0390]

Table 34

[0391]

Table 35

[0392]

Table 36

[0393] Conclusion: 10 7Treatment of NSG mice with 3 mg / kg TF2053 beginning one day prior to transfer of human PBMCs provided significant protection from GVHD. Body weight loss was reduced while survival was improved. Furthermore, increases in liver and spleen weights were reduced. Circulating human inflammatory cytokines and human CD45 cell engraftment were significantly higher in TF2053 at day 14 but significantly lower at all later time points. TF2053 also altered the CD4 to CD8 ratio in favor of CD8+ cells, increasing the percentage of these CD8+ cells that were exhausted and senescent over time while decreasing percent anergy. In summary, the hypothesis that agonism of TNFR2 may provide benefit in a humanized NSG-GVHD in vivo model through effects on effector T cells in the absence of regulatory T cell expansion was confirmed.

[0394] References: 1. Pino, S. et al., "Development of novel major histocompatibility complex class I and class II deficient NOD-SCID IL2R gamma chain knockout mice to model human xenograft graft-versus-host disease," class II-deficient NOD-SCID IL2R gamma chainout knock mice for modeling human xenogeneic graft-versus-host disease)”. Methods Mol Biol. 2010. 602: p. 105-17. 2. Brehm, MA et al., "Lack of acute xenogeneic graft- versus-host disease, but retention of T cell function following engraftment of human peripheral blood mononuclear cells in NSG mice deficient in MHC class I and II expression." of T-cell function following engraftment of human peripheral blood mononuclear cells in NSG mice deficient in MHC class I and II expression)”. Faseb j, 2019. 33(3): p. 3137-3151. 3. Ban, L. et al., "Selective death of autoreactive T cells in human diabetes by TNF or TNF receptor 2 agonism," TNF receptor 2 agonism)”. Proc Natl Acad Sci USA, 2008. 105(36): p. 13644-9. 4. Punit, S. et al., "Tumor Necrosis Factor Receptor 2 Restricts the Pathogenicity of CD8(+) T Cells in Mice with Colitis" CD8(+) T Cells in Mice With Colitis)”. Gastroenterology, 2015. 149(4): p. 993-1005.e2.

[0395] [Table 37-1]

[0396] [Table 37-2]

[0397] [Table 37-3]

[0398]

Table 37-4

Claims

1. An isolated antibody that specifically binds to TNFR2, comprising a heavy chain variable region (VH) and a light chain variable region (VL), the antibody comprising CDR-H1, CDR-H2 and CDR-H3 sequences of the VH sequence selected from the group consisting of SEQ ID NO:30, SEQ ID NO:13, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:22 and SEQ ID NO:29; and CDR-L1, CDR-L2 and CDR-L3 sequences of the VL sequence selected from the group consisting of SEQ ID NO:9, SEQ ID NO:6, SEQ ID NO:8 and SEQ ID NO:

4.

2. 1. An isolated antibody that specifically binds to TNFR2, comprising a heavy chain variable region (VH) and a light chain variable region (VL), (i) a CDR-L1 sequence according to SEQ ID NO:1; a CDR-L2 sequence according to SEQ ID NO:7 and a CDR-L3 sequence according to SEQ ID NO:3, and a CDR-H1 sequence according to SEQ ID NO:10; a CDR-H2 sequence according to SEQ ID NO:20; a CDR-H3 sequence according to SEQ ID NO:12; or (ii) a CDR-L1 sequence according to SEQ ID NO:1; a CDR-L2 sequence according to SEQ ID NO:2 and a CDR-L3 sequence according to SEQ ID NO:3, and a CDR-H1 sequence according to SEQ ID NO:10; a CDR-H2 sequence according to SEQ ID NO:11; a CDR-H3 sequence according to SEQ ID NO:

12. An antibody comprising:

3. (i) a VH framework sequence derived from a human germline VH sequence selected from the group consisting of IGHV1-46, IGHV4-31, IGHV4-30-4, and IGHV4-4; and (ii) a VL framework sequence derived from a human germline VL sequence selected from the group consisting of IGKV1-9, IGKV1-33, IGKV1-27, IGKV1-39, IGKV1-9, IGKV1-1, and IGKV1-11. The antibody of claim 1 , comprising one or both of the following:

4. The antibody of any one of claims 1 to 3, wherein the VL comprises an amino acid sequence according to a sequence selected from the group consisting of SEQ ID NO: 8 and SEQ ID NO: 4, and the VH comprises an amino acid sequence according to a sequence selected from the group consisting of SEQ ID NO: 21 and SEQ ID NO:

13.

5. An antibody described in any one of claims 1 to 4, comprising a VH sequence of SEQ ID NO:21 and a VL sequence of SEQ ID NO:

8.

6. An antibody comprising one or both of a VH sequence encoded by a plasmid deposited with the ATCC having ATCC Accession No. PTA-127528 and a VH sequence encoded by a plasmid deposited with the ATCC having ATCC Accession No. PTA-127529; and a VL sequence encoded by a plasmid deposited with the ATCC having ATCC Accession No. PTA-127531.

7. 7. The antibody of claim 1 , further comprising an Fc domain, wherein the Fc domain is an IgA, IgD, IgE, IgM or IgG Fc domain.

8. 8. The antibody of claim 1 , further comprising a heavy chain (HC) comprising the sequence according to SEQ ID NO:

22.

9. 9. The antibody of claim 1 , further comprising a light chain (LC) comprising the sequence according to SEQ ID NO:

9.

10. 10. The antibody of any one of claims 1 to 9, comprising a first Fab, a second Fab, a third Fab and a fourth Fab, wherein the first Fab, the second Fab, the third Fab and the fourth Fab each comprise a CDR-L1 sequence according to SEQ ID NO: 1; a CDR-L2 sequence according to SEQ ID NO: 7 and a CDR-L3 sequence according to SEQ ID NO: 3, and a CDR-H1 sequence according to SEQ ID NO: 10; a CDR-H2 sequence according to SEQ ID NO: 20; and a CDR-H3 sequence according to SEQ ID NO:

12.

11. The antibody of claim 10, wherein the first Fab, the second Fab, the third Fab and the fourth Fab each comprise a VH having a sequence according to SEQ ID NO:21 and a VL having a sequence according to SEQ ID NO:

8.

12. 12. The antibody of any one of claims 10 to 11, wherein the N'-terminus of the first Fab is connected to the C'-terminus of the third Fab via a first linker, and the N'-terminus of the second Fab is connected to the C'-terminus of the fourth Fab via a second linker, optionally wherein one or both of the first and second linkers comprise a sequence according to SEQ ID NO:

27.

13. 13. An antibody according to any one of claims 1 to 12, comprising a heavy chain (HC) comprising a sequence according to SEQ ID NO: 30 and a light chain (LC) comprising a sequence according to SEQ ID NO:

9.

14. 14. The antibody of claim 1 , comprising a heavy chain (HC) sequence encoded by the nucleic acid sequence of SEQ ID NO: 33 and a light chain (LC) sequence encoded by the nucleic acid sequence of SEQ ID NO:

31.

15. An antibody comprising an HC sequence encoded by a plasmid deposited with the ATCC having ATCC Accession No. PTA-127530, and an LC sequence encoded by a plasmid deposited with the ATCC having ATCC Accession No. PTA-127532.

16. (i) an EC of less than 5 μM in the human TNFR2 potency assay in Jurkat reporter cells ５０ ; (ii) an EC value of less than 10 μM in the human TNFR2 potency assay in human peripheral blood monocytes; ５０ ; (iii) an EC of less than 5 μM in the human TNFR2 potency assay in Jurkat reporter cells; ５０ ; (iv) an EC50 of less than 2 mg / ml for ICAM-1 upregulation in human TNFR2-expressing primary T cell populations derived from human peripheral blood monocytes; (v) an EC50 of less than 1 mg / ml for ICAM-1 upregulation in a human TNFR2-expressing primary T cell population derived from human peripheral blood monocytes; and (vi) an affinity KD for human TNFR2 of less than 1 nm The antibody according to any one of claims 1 to 15, characterized by one or more selected from the group consisting of:

17. 17. An antibody according to any one of claims 1 to 16 for use as a medicament.

18. 18. The antibody of claim 17, wherein the use is for the treatment of one or more selected from the group consisting of rheumatoid arthritis (RA), ankylosing spondylitis (AS), Crohn's disease (CD), ulcerative colitis (UC), graft versus host disease (GVHD), transplantation, psoriasis (PSO), psoriatic arthritis (PSA), atopic dermatitis (AD), vitiligo, alopecia areata (AA), type 1 diabetes (T1D), multiple sclerosis (MS), irritable bowel disease (IBD), autoimmune hepatitis, and systemic lupus erythematosus (SLE).

19. 20. A pharmaceutical composition comprising a therapeutically effective amount of an antibody according to any one of claims 1 to 18 and a pharma- ceutically acceptable carrier.

20. A method for treating a medical condition, comprising administering to a subject in need thereof a therapeutically effective amount of an antibody of any one of claims 1 to 18 or a pharmaceutical composition of claim 19.

21. 21. The method of claim 20, wherein the condition is selected from the group consisting of rheumatoid arthritis (RA), ankylosing spondylitis (AS), Crohn's disease (CD), ulcerative colitis (UC), graft versus host disease (GVHD), transplantation, psoriasis (PSO), psoriatic arthritis (PSA), atopic dermatitis (AD), vitiligo, alopecia areata (AA), type 1 diabetes (T1D), multiple sclerosis (MS), irritable bowel disease (IBD), autoimmune hepatitis, and systemic lupus erythematosus (SLE).

22. 19. An isolated polynucleotide comprising one or more nucleotide sequences encoding the antibody of any one of claims 1 to 18.

23. An isolated polynucleotide encoding the HC and / or LC of an antibody that binds to TNFR2, wherein the nucleic acid comprises one or more selected from the group consisting of the nucleic acid sequence of SEQ ID NO:31, the nucleic acid sequence of SEQ ID NO:32, and the nucleic acid sequence of SEQ ID NO:

33.

24. An isolated polynucleotide encoding an isolated antibody that specifically binds to TNFR2, the antibody comprising a heavy chain (HC) sequence encoded by the nucleic acid sequence of SEQ ID NO:33 and a light chain (LC) sequence encoded by the nucleic acid sequence of SEQ ID NO:

31.

25. An isolated polynucleotide encoding an isolated antibody comprising an HC sequence encoded by a plasmid deposited with the ATCC having ATCC Accession No. PTA-127530, and an LC sequence encoded by a plasmid deposited with the ATCC having ATCC Accession No. PTA-127532.

26. 26. A vector comprising a polynucleotide according to any one of claims 22 to 25.

27. 27. An isolated host cell comprising a polynucleotide according to any one of claims 1 to 25 or a vector according to claim 26.

28. 30. A method of producing an isolated antibody, comprising culturing a host cell according to claim 27 under conditions resulting in the production of said antibody, and recovering said antibody.

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