Anti-TNFR2 antibody and method of use thereof

Abstract

To provide an antibody suitable for one or more of diagnosis, prevention, or treatment of disorders or conditions mediated by or associated with TNFR2 activity.SOLUTION: The present disclosure provides antibodies that bind to TNFR2, as well as uses and related methods thereof. The present disclosure also provides processes for making, preparing, and producing antibodies that bind to TNFR2.SELECTED DRAWING: Figure 2

Description

Background Art

[0001] TNF is a pleiotropic cytokine expressed on the cell surface of leukocytes and is subsequently released via the activity of several proteolytic enzymes. Cellular responses to TNF are mediated by two receptors: TNFR1 (TNFRSF1A), which is ubiquitously expressed and mediates pro-inflammatory responses, and TNFR2 (TNFRSF1B), which is selectively expressed on specific leukocyte subtypes and is thought to mainly mediate immunomodulatory effects (Salomon 2021). Soluble TNF can activate both receptors, but TNFR2 is more preferentially activated by membrane-associated TNF. Among the activities attributed to TNFR2 agonism, the ones most relevant for the treatment of autoimmune and autoinflammatory conditions are the expansion of regulatory T cells, activation-induced cell death and depletion of effector T cells, and the enhancement of regulatory / anti-inflammatory phenotypes in B cells, mesenchymal stem cells (MSCs), and myeloid cells, such as myeloid-derived suppressor cells (MDSCs) and glia (Faustman and Davis 2010, Salomon 2021).

Summary of the Invention

Problems to be Solved by the Invention

[0002]

Means for Solving the Problems

[0003] The present disclosure provides antibodies that bind to TNFR2, as well as uses of those antibodies and related methods. The present disclosure also provides processes 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 associated with 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), inflammatory bowel disease (IBD), autoimmune hepatitis, and systemic lupus erythematosus (SLE). The present disclosure further encompasses the expression of the antibodies, as well as the preparation and manufacture of compositions containing the antibodies of the present disclosure, such as pharmaceuticals for use of the antibodies.

[0004] Polynucleotides encoding antibodies that bind to TNFR2 are provided. Polynucleotides encoding the heavy or light chain of the antibody, or both, are also provided. Host cells expressing the antibody are provided. Methods of treatment using the antibody are provided. Such methods include, but are not limited to, one or more of methods of treating or preventing 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), inflammatory bowel disease (IBD), autoimmune hepatitis, and systemic lupus erythematosus (SLE)] associated with or mediated by TNFR2 expression and / or TNFR2 binding.

[0005] The present invention can be more easily understood by referring to the following detailed description of embodiments of the present invention and the examples included in this specification. It should be understood that the present invention is not limited to specific methods of production that can naturally vary. It should also be understood that the technical terms used in this specification are for the purpose of only describing specific embodiments and are not intended to be limiting.

[0006] Exemplary embodiments (E) of the invention provided herein include the following: E1. An isolated antibody that specifically binds to TNFR2 and comprises a heavy chain variable region (VH) and a light chain variable region (VL), the CDR-H1, CDR-H2, and CDR-H3 sequences of the 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 the CDR-L1, CDR-L2, and CDR-L3 sequences of the 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.

[0007] E2. An isolated antibody that specifically binds to TNFR2 and comprises a heavy chain variable region (VH) and a light chain variable region (VL), the CDR-H1, CDR-H2, and CDR-H3 sequences according to SEQ ID NO: 30, and the CDR-L1, CDR-L2, and CDR-L3 sequences according to SEQ ID NO: 9.

[0008] E3. An isolated antibody that specifically binds to TNFR2 and comprises a heavy chain variable region (VH) and a light chain variable region (VL), (i) the CDR-L1 sequence according to SEQ ID NO: 1; the CDR-L2 sequence according to SEQ ID NO: 7 and the CDR-L3 sequence according to SEQ ID NO: 3, and the CDR-H1 sequence according to SEQ ID NO: 10; the CDR-H2 sequence according to SEQ ID NO: 20; the CDR-H3 sequence according to SEQ ID NO: 12; or (ii) the CDR-L1 sequence according to SEQ ID NO: 1; the CDR-L2 sequence according to SEQ ID NO: 2 and the CDR-L3 sequence according to SEQ ID NO: 3, and the CDR-H1 sequence according to SEQ ID NO: 10; the CDR-H2 sequence according to SEQ ID NO: 11; the CDR-H3 sequence according to SEQ ID NO: 12 An antibody comprising

[0009] E4. An isolated antibody that specifically binds to TNFR2 and comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the CDR-L1 sequence follows SEQ ID NO: 1; the CDR-L2 sequence follows SEQ ID NO: 7, the CDR-L3 sequence follows SEQ ID NO: 3, the CDR-H1 sequence follows SEQ ID NO: 10; the CDR-H2 sequence follows SEQ ID NO: 20; the CDR-H3 sequence follows SEQ ID NO: 12.

[0010] E5. The antibody according to 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.

[0011] E6. The antibody according to any one of E1 to E5, comprising a VH framework sequence derived from the human IGHV1-46 germline sequence.

[0012] E7. The antibody according to 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.

[0013] E8. The antibody according to any one of E1 to E7, comprising a VL framework sequence derived from the human germline IGKV1-9 sequence.

[0014] E9. The antibody according to any one of E1 to 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 is 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.

[0015] An antibody according to any one of E1 to E9, comprising a VL framework region sequence and a VH framework region sequence, wherein one or both of the VL framework region sequence or the VH framework region sequence is identical to the human germline sequence from which it is derived.

[0016] An antibody according to 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.

[0017] An antibody according to any one of E1 to E11, comprising the VH sequence of SEQ ID NO: 13 and the VL of SEQ ID NO: 4.

[0018] An 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.

[0019] An antibody according to any one of E13, comprising the VH sequence of SEQ ID NO: 21 and the VL of SEQ ID NO: 8.

[0020] An antibody according to any one of E14, comprising a VH sequence encoded by the nucleic acid sequence of SEQ ID NO: 32.

[0021] An antibody according to any one of E14, comprising a VH sequence encoded by the nucleic acid sequence of SEQ ID NO: 33.

[0022] An antibody according to any one of E1 to E16, comprising a VL sequence encoded by the nucleic acid sequence of SEQ ID NO: 31.

[0023] An antibody according to any one of E1 to E17, comprising one or both of the VH sequences encoded by the plasmid deposited with ATCC under ATCC Accession No. PTA-127528 and the VH sequences encoded by the plasmid deposited with ATCC under ATCC Accession No. PTA-127529.

[0024] An antibody according to any one of E1 to E18, comprising the VL sequence encoded by the plasmid deposited with ATCC under ATCC Accession No. PTA-127531.

[0025] An antibody comprising one or both of the VH sequences encoded by the plasmid deposited with ATCC under ATCC Accession No. PTA-127528 and the VH sequences encoded by the plasmid deposited with ATCC under ATCC Accession No. PTA-127529; and the VL sequence encoded by the plasmid deposited with ATCC under ATCC Accession No. PTA-127531.

[0026] An antibody according to any one of E1 to E24, further comprising a constant heavy domain (CH1) and a constant light domain (CL).

[0027] An antibody according to E25, wherein CH1 is connected to VH and CL is connected to VL to form a Fab domain.

[0028] An antibody according to E25, comprising a first and a second Fab domain.

[0029] An antibody according to any one of E25 to E26, comprising an antibody Fc domain comprising a first Fc chain and a second Fc chain.

[0030] An antibody according to E27, wherein the first Fab domain is covalently fused to the first Fc chain and the second Fab domain is covalently fused to the second Fc chain.

[0031] E29. An antibody according to E27 - 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.

[0032] E30. An antibody according to E31, wherein the Fc domain is the Fc domain of IgA (e.g., IgA 1 or IgA 2 ), IgD, IgE, IgM or IgG (e.g., IgG 1 , IgG 2 , IgG 3 or IgG 4 ).

[0033] E31. An antibody according to E30, wherein the Fc domain is the Fc domain of IgG 1 .

[0034] E32. An antibody according to E31, wherein the CH1 domain in the first Fab domain comprises a sequence according to SEQ ID NO: 23.

[0035] E33. An antibody according to E31 - E32, wherein the CL in the first Fab domain comprises a sequence according to SEQ ID NO: 5.

[0036] E34. An antibody according to E31 - E33, comprising a light chain (LC) comprising a sequence according to SEQ ID NO: 9.

[0037] E35. An antibody according to E31 - E34, wherein the first Fab domain and the second Fab domain are identical.

[0038] E36. An antibody according to E31 - 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.

[0039] The antibody according to E36, wherein one or both of the first hinge region and the second hinge region comprise a sequence according to SEQ ID NO: 23.

[0040] The antibody according to E36 - E37, wherein one or both of the first CH2 domain and the second CH2 domain comprise a sequence according to SEQ ID NO: 25.

[0041] The antibody according to E36 - E38, wherein one or both of the first CH3 domain and the second CH3 domain comprise a sequence according to SEQ ID NO: 26.

[0042] The antibody according to E36 - E39, wherein one or both of the first Fc chain and the second Fc chain comprise a sequence according to SEQ ID NO: 37.

[0043] The antibody according to E36 - E40, wherein the first Fc chain and the second Fc chain are identical.

[0044] The antibody according to E32 - E41, comprising a heavy chain (HC) comprising a sequence according to SEQ ID NO: 22.

[0045] The antibody according to E26 - E42, further comprising a third Fab and a fourth Fab.

[0046] The antibody according to 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; a CDR - H3 sequence according to SEQ ID NO: 12.

[0047] The antibody according to E43 - E44, 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.

[0048] E46. An antibody according to any one of E43 to E45, wherein the first Fab, the second Fab, the third Fab, and the fourth Fab are identical to each other.

[0049] E47. An antibody according to any one of E43 to E46, wherein the N'-terminus of the first Fab is connected to the C'-terminus of the third Fab.

[0050] E48. An 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.

[0051] E49. An antibody according to E48, wherein the first linker comprises a sequence according to SEQ ID NO: 27.

[0052] E50. An antibody according to any one of E43 to E49, wherein the N'-terminus of the second Fab is connected to the C'-terminus of the fourth Fab.

[0053] E51. An 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.

[0054] E52. An antibody according to E51, wherein the second linker comprises a sequence according to SEQ ID NO: 27.

[0055] E53. An antibody according to E47 to E48, wherein the HC comprises a sequence according to SEQ ID NO: 30.

[0056] E54. An isolated antibody that specifically binds to TNFR2 and comprises 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.

[0057] E55. An antibody according to any one of E1 to E54, comprising a VH sequence encoded by the nucleic acid sequence of SEQ ID NO: 33.

[0058] E56. An antibody according to any one of E1 to E55, comprising a VL sequence encoded by the nucleic acid sequence of SEQ ID NO: 31.

[0059] 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.

[0060] E58. The antibody according to any one of E1 to E57, comprising an HC sequence encoded by a plasmid having ATCC accession number PTA-127530 and deposited with ATCC.

[0061] E59. The antibody according to any one of E1 to E58, comprising an LC sequence encoded by a plasmid having ATCC accession number PTA127532 and deposited with ATCC.

[0062] E60. An antibody comprising an HC sequence encoded by a plasmid having ATCC accession number PTA-127530 and deposited with ATCC, and an LC sequence encoded by a plasmid having ATCC accession number PTA-127532 and deposited with ATCC.

[0063] E61. The Fc domain is the Fc domain of IgG 2 The antibody according to E30.

[0064] E62. The antibody according to E61, wherein the CH1 domain in the first Fab domain comprises a sequence according to SEQ ID NO: 14.

[0065] E63. The antibody according to E61 to E62, wherein the CL in the first Fab domain comprises a sequence according to SEQ ID NO: 5.

[0066] E64. The antibody according to E61 to E63, wherein the first Fab domain and the second Fab domain are identical.

[0067] E65. The antibody according to E61 - 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.

[0068] E66. The antibody according to E65, wherein one or both of the first hinge region and the second hinge region comprises a sequence according to SEQ ID NO: 15.

[0069] E67. The antibody according to E65 - E66, wherein one or both of the first CH2 domain and the second CH2 domain comprises a sequence according to SEQ ID NO: 16.

[0070] E68. The antibody according to E65 - E67, wherein one or both of the first CH3 domain and the second CH3 domain comprises a sequence according to SEQ ID NO: 17.

[0071] E69. The antibody according to E65 - E68, wherein one or both of the first Fc chain and the second Fc chain comprises a sequence according to SEQ ID NO: 18.

[0072] E70. The antibody according to E65 - E69, wherein the first Fc chain and the second Fc chain are identical.

[0073] E71. The antibody according to E75 - E70, comprising a heavy chain (HC) comprising a sequence according to SEQ ID NO: 19.

[0074] E72. The antibody according to E65 - E71, comprising a light chain (LC) comprising a sequence selected from the group consisting of SEQ ID NO: 6 and SEQ ID NO: 9.

[0075] E73. The antibody according to E65 - E72, comprising a light chain (LC) comprising a sequence according to SEQ ID NO: 9.

[0076] 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.

[0077] E75. An EC of less than 5 μM in the human TNFR2 potency assay in Jurkat reporter cells 50 The antibody according to E1 to E74, characterized by

[0078] E76. An EC of less than 2 μM in the human TNFR2 potency assay in Jurkat reporter cells 50 The antibody according to E1 to E75, characterized by

[0079] E77. An EC of less than 10 μM in the human TNFR2 potency assay in human peripheral blood monocytes 50 The antibody according to E1 to E76, characterized by

[0080] E78. An EC of less than 5 μM in the human TNFR2 potency assay in human peripheral blood monocytes 50 The antibody according to E1 to E77, characterized by

[0081] E79. An EC of less than 2 μM in the human TNFR2 potency assay in human peripheral blood monocytes 50 The antibody according to E1 to E78, characterized by

[0082] E80. An EC of less than 20 μM in the cynomolgus TNFR2 potency assay in cynomolgus peripheral blood monocytes 50 The antibody according to E1 to E79, characterized by

[0083] E81. An EC of less than 15 μM in the cynomolgus TNFR2 potency assay in cynomolgus peripheral blood monocytes 50 The antibody according to E1 to E80, characterized by

[0084] E82. An EC of less than 5 μM in the human TNFR2 potency assay in Jurkat reporter cells 50 The antibody according to E1 to E81, characterized by

[0085] E83. An antibody according to any one of E1 to E82, characterized by an EC50 of less than 1 μM in a human TNFR2 potency assay in Jurkat reporter cells. 50 An antibody according to any one of E1 to E82, characterized by an EC50 of less than 1 μM in a human TNFR2 potency assay in Jurkat reporter cells.

[0086] E84. An antibody according to any one of E1 to E83, characterized by an EC50 of less than 0.5 μM in a human TNFR2 potency assay in Jurkat reporter cells. 50 An antibody according to any one of E1 to E83, characterized by an EC50 of less than 0.5 μM in a human TNFR2 potency assay in Jurkat reporter cells.

[0087] E85. An antibody according to any one of E1 to E84, characterized by an EC50 of less than 2 mg / ml for upregulation of ICAM-1 in a primary T cell population expressing human TNFR2 derived from human peripheral blood monocytes.

[0088] E86. An antibody according to any one of E1 to E85, characterized by an EC50 of less than 15 mg / ml for upregulation of ICAM-1 in a primary T cell population expressing cynomolgus TNFR2 derived from cynomolgus peripheral blood monocytes.

[0089] E87. An antibody according to any one of E1 to E86, characterized by an EC50 of less than 1 mg / ml for upregulation of ICAM-1 in a primary T cell population expressing human TNFR2 derived from human peripheral blood monocytes.

[0090] E88. An antibody according to any one of E1 to E87, characterized by an EC50 of less than 0.2 mg / ml for upregulation of ICAM-1 in a primary T cell population expressing human TNFR2 derived from human peripheral blood monocytes.

[0091] E89. An antibody according to any one of E1 to E88, characterized by an EC50 of less than 10 mg / ml for upregulation of ICAM-1 in a primary T cell population expressing cynomolgus TNFR2 derived from cynomolgus peripheral blood monocytes.

[0092] E90. An antibody according to any one of E1 to E89, characterized by an affinity KD for human TNFR2 of less than 1 nM.

[0093] An antibody according to any one of E1 to E90, characterized by an affinity KD for human TNFR2 of less than 0.5 nM.

[0094] An antibody according to any one of E1 to E91, characterized by an affinity KD for human TNFR2 of less than 0.1 nM.

[0095] An antibody according to any one of E1 to E92, characterized by an affinity KD for human TNFR2 of less than 0.07 nM.

[0096] An antibody according to any one of E1 to E93, characterized by an affinity KD for cynomolgus TNFR2 of less than 1 nM.

[0097] An antibody according to any one of E1 to E94, characterized by an affinity KD for cynomolgus TNFR2 of less than 0.1 nM.

[0098] An isolated antibody comprising the VH and VL of an antibody selected from Table 35.

[0099] An antibody according to any one of E1 to E96 for use as a medicament.

[0100] An antibody according to E97, wherein the use is for one or more treatments 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).

[0101] An antibody according to any one of E96 to E98, wherein the use is for rheumatoid arthritis (RA).

[0102] An isolated polynucleotide comprising one or more nucleotide sequences encoding an antibody as described in any one of E1 to E99.

[0103] E101. The polynucleotide according to E100, which is RNA.

[0104] E102. The polynucleotide according to E100 to E101, comprising at least one chemical modification.

[0105] E103. The polynucleotide according to 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.

[0106] E104. The polynucleotide according to E100 to E101, which does not contain a chemical modification.

[0107] E105. An isolated polynucleotide encoding the HC or LC of an antibody that binds to TNFR2, or both, 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.

[0108] An isolated polynucleotide encoding 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.

[0109] E107. An isolated polynucleotide encoding an isolated antibody comprising an HC sequence encoded by a plasmid deposited with ATCC and having ATCC accession number PTA-127530, and an LC sequence encoded by a plasmid deposited with ATCC and having ATCC accession number PTA-127532.

[0110] E108. A vector comprising the polynucleotide according to E100 - E107.

[0111] E109. An isolated host cell comprising the polynucleotide according to E100 - E107 or the vector according to E108.

[0112] E110. A method for producing an isolated antibody, comprising culturing the host cell according to E109 under conditions that result in the production of the antibody, and recovering the antibody.

[0113] E111. A pharmaceutical composition comprising a therapeutically effective amount of the antibody according to E1 - E99 and a pharmaceutically acceptable carrier.

[0114] E112. A method for treating a medical condition, comprising administering to a subject in need thereof a therapeutically effective amount of the antibody according to any one of E1 - E99, the polynucleotide according to E101, or the pharmaceutical composition according to E111.

[0115] The method according to 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).

[0116] The method according to any one of E112 to E113, comprising the step of subcutaneously administering the antibody or pharmaceutical composition.

[0117] The method according to any one of E112 to E114, wherein the antibody or pharmaceutical composition is administered twice a week, once a week, once every two weeks, once every three weeks, once every four weeks, once every five weeks, once every six weeks, once every seven weeks, once every eight weeks, once every nine weeks, once every ten weeks, twice a month, once a month, once every two months, once every three months or once every four months.

Brief Description of the Drawings

[0118]

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Mode for Carrying Out the Invention

[0119] The section headings used in this specification are for organizational purposes only and should not be construed as limiting the subject matter described.

[0120] All references cited in this specification, including patent applications, patent publications, and UniProtKB accession numbers, are incorporated herein by reference as if each individual reference were specifically and individually indicated to be incorporated in its entirety.

[0121] The techniques and procedures described and referred to in this specification are generally well understood and are commonly used by those skilled in the art using conventional methodologies such as the widely used methodologies described below: Sambrook et al., Molecular Cloning: A Laboratory Manual 3rd Edition (2001) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (ed. F.M. Ausubel et al., (2003)); series METHODS IN ENZYMOLOGY (Academic Press, Inc.): PCR 2: A PRACTICAL APPROACH (ed. M.J. MacPherson, B.D. Hames and G.R. Taylor (1995)), Harlow and Lane eds. (1988) ANTIBODIES, A LABORATORY MANUAL, and ANIMAL CELL CULTURE (ed. R.I. Freshney (1987)); Oligonucleotide Synthesis (ed. M.J. Gait, 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (ed. J.E. Cellis, 1998) Academic Press; Animal Cell Culture (ed. R.I. Freshney, 1987); Introduction to Cell and Tissue Culture (J.P. Mather and P.E. Roberts, 1998) Plenum Press; Cell and Tissue Culture Laboratory Procedures (ed. A. Doyle, J.B. Griffiths and D.G. Newell, 1993 - 8) J.Wiley and Sons; Handbook of Experimental Immunology (ed. D.M. Weir and C.C. Blackwell); Gene Transfer Vectors for Mammalian Cells (J.M. Miller and M.P.Calos, ed., 1987); PCR: The Polymerase Chain Reaction, (Mullis et al., eds., 1994); Current Protocols in Immunology (J.E. Coligan et al., eds., 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 J.D. Capra, eds., Harwood Academic Publishers, 1995); and their most recent editions.

[0122] Definitions Unless otherwise defined herein, scientific and technical terms used in conjunction with the present invention have the meanings commonly understood by one of ordinary skill in the art.

[0123] As used herein, the singular forms "a", "an", and "the" include plural references unless the context clearly dictates otherwise. For example, "an" antibody includes one or more antibodies.

[0124] When aspects or embodiments of the present invention are described in terms of Markush groups or other groupings of alternatives, the present invention encompasses the entire recited group as a whole, but not only each member of the group individually and all possible subgroups of the main group, but also main groups in which one or more of the group members are absent. The present invention also contemplates any express exclusion of one or more of any of the group members in the claimed invention.

[0125] Any examples following the terms "e.g." or "for example" are not meant to be exhaustive or limiting.

[0126] As used herein, the term "about" is used when modifying a numerically defined parameter (e.g., *** the dosage), and means that the parameter can vary to an extent that is 10% below or above the recited numerical value for that parameter. For example, a dosage of about 5 mg means 5% ± 10%, i.e., it can vary between 4.5 mg and 5.5 mg.

[0127] 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 within 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 (e.g., "antigen-binding fragments") that retain the ability to bind to a given antigen, and any other modified conformation of an immunoglobulin molecule that contains an antigen-binding site.

[0128] Antibodies include antibodies of any class, such as IgG, IgA or IgM (or their subclasses), and the antibody does not have to 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 main classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, and some of these can be further divided into subclasses (isotypes), such as IgG 1 , IgG 2 , IgG 3 , IgG 4 , IgA 1 and IgA 2 and can be further divided into. The heavy chain constant regions corresponding to 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.

[0129] Examples of antigen-binding fragments and modified configurations of antibodies include the following: (i) Fab fragment (a monovalent fragment consisting of a VL domain, a VH domain, a CL domain and a CH1 domain); (ii) F(ab’)2 fragment (a divalent fragment containing two Fab fragments linked by a disulfide bridge in the hinge region); and (iii) Fv fragment consisting of the VL domain and VH domain of a single arm of an antibody. Furthermore, the two domains of the Fv fragment: VL and VH are encoded by separate genes, but these can be joined using recombinant methods by a synthetic linker that allows them to be made as a single protein chain in which the VL region and VH region 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 diabody, are also included.

[0130] Further included are antibodies lacking a C-terminal lysine (K) amino acid residue on the heavy chain polypeptide (e.g., human IgG1 heavy chain contains a terminal lysine). As is known in the art, the C-terminal lysine may be removed during antibody production, resulting in an antibody having a heavy chain lacking the C-terminal lysine. Alternatively, the antibody heavy chain may be produced using a nucleic acid that does not contain the C-terminal lysine.

[0131] Variable region The "variable region" of an antibody refers to either the variable region of an antibody light chain or the variable region of an antibody heavy chain, either alone or in combination. As is 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 variants of a subject variable region having substitutions at amino acid residues outside the CDR regions (i.e., in the framework regions) are desired, appropriate amino acid substitutions, preferably conservative amino acid substitutions, can be identified by comparing the subject variable region to the variable regions of other antibodies containing 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).

[0132] In certain embodiments, an unambiguous delineation of the CDRs and identification of the residues constituting the binding site of an antibody is achieved by elucidating the structure of the antibody or the structure of an antibody-ligand complex. In certain embodiments, this can be achieved by any of a variety of techniques known to those of skill in the art, e.g., by X-ray crystallography. In certain embodiments, a variety of methods of analysis can be used to identify or approximate the CDR regions. In certain embodiments, a variety of methods of analysis can be used to identify or approximate the CDR regions. Examples of such methods include, but are not limited to, the Kabat definition, the Chothia definition, the AbM definition, the contact definition, the extended definition, and the conformation definition.

[0133] The Kabat definition is a standard for numbering residues in antibodies and is typically used to identify CDR regions. See, for example, Johnson and Wu, 2000, Nucleic Acids Res., 28:214-218. The Chothia definition is similar to the Kabat definition, but the Chothia definition takes into account the positions of certain structural loop regions. See, for example, Chothia et al., 1986, J. Mol. Biol., 196:901-917; Chothia et al., 1989, Nature, 342:877-883. The extended definition is a combination of the Kabat and Chothia definitions. The AbM definition uses an integrated suite of computer programs created by Oxford Molecular Group to model antibody structures. See, for example, Martin et al., 1989, Proc Natl Acad Sci (USA), 86:9268-9272; "AbM (trademark) A Computer Program for Modeling Variable Regions of Antibodies", Oxford, UK; Oxford Molecular, Ltd. The AbM definition uses a combination of a knowledge database and ab initio methods, such as those described by Samudrala et al., 1999, "Ab Initio Protein Structure Prediction Using a Combined Hierarchical Approach", PROTEINS, Structure, Function and Genetics Suppl., 3:194-198, to model the tertiary structure of an antibody from its primary sequence. The contact definition is based on the analysis of available complex crystal structures. See, for example, MacCallum et al., 1996, J. Mol. Biol., 5:732-745. In another approach, herein called the "conformational definition" of CDRs, the positions of the CDRs can be identified as residues that contribute enthalpically to antigen binding. See, for example, Makabe et al., 2008, Journal of Biological Chemistry, 283:1156-1166.Other CDR boundary definitions may not strictly follow one of the above approaches, but nevertheless may be shortened or extended in light of predictive or experimental findings that overlap at least in part with the Kabat CDR, but where certain residues or groups of residues are predicted not to significantly affect antigen binding. As used herein, CDR may refer to a CDR defined by any approach known in the art that includes a combination of approaches. The methods used herein may utilize CDRs defined according to any of these approaches. For any given embodiment containing more than one CDR, the CDRs may be defined according to any one or more of Kabat, Chothia, extended, AbM, contact or conformation definitions.

[0134] Constant region The "constant region" of an antibody refers to either 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 the hinge region between the CH1 and CH2 domains. The IgG light chain constant region contains a single immunoglobulin domain (CL).

[0135] Fc domain and Fc chain "Fc domain" refers to the portion of an immunoglobulin (Ig) molecule that correlates with the crystallizable fragment obtained by papain digestion of the Ig molecule. As used herein, the term refers to the two-chain constant region of an antibody, where each chain excludes 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, an 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, a flexible hinge N-terminal to these domains.

[0136] The boundaries of the Fc chain can vary, but the human IgG heavy chain Fc chain is typically defined as including residue C226 or P230 at its carboxyl terminus, and the numbering follows the EU index of Edelman et al., Proc. Natl. Acad. Sci. USA 1969;63(1):78-85, as described by Kabat et al., 1991. Typically, the Fc chain includes approximately amino acid residues 236 to approximately 447 of the human IgG1 heavy chain constant region. "Fc chain" can refer to this polypeptide, isolated or in the context of a larger molecule (e.g., in an antibody heavy chain or an Fc fusion protein).

[0137] A "functional" Fc domain refers to an Fc domain that retains at least one effector function of the native sequence Fc domain. Exemplary "effector functions" include C1q binding; complement-dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation 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 evaluated using various assays known in the art for assessing such antibody effector functions.

[0138] A "native sequence" Fc chain refers to an Fc chain that contains an amino acid sequence identical to that of the Fc chain found in nature. A "variant" Fc chain contains an amino acid sequence that differs from that of the native sequence Fc chain by at least one amino acid modification.

[0139] Monoclonal antibody A "monoclonal antibody" (mAb) refers to an antibody derived from a single copy or clone, including, for example, any eukaryote, prokaryote, or phage clone. Monoclonal antibodies are highly specific and directed against a single antigenic site. Further, in contrast to polyclonal antibody preparations, which typically contain different antibodies against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. The modifier "monoclonal" indicates the antibody's characteristics as being derived from a substantially homogeneous population of antibodies and should not be construed as requiring antibody production by any particular method. For example, monoclonal antibodies used in accordance with the present invention can be prepared by the hybridoma method first described by Kohler and Milstein, 1975, Nature 256:495, or by recombinant DNA methods as described in U.S. Patent No. 4,816,567. In another example, monoclonal antibodies can be isolated from phage libraries such as those generated using the techniques described by McCafferty et al., 1990, Nature 348:552-554.

[0140] Human antibody A "human antibody" refers to an antibody having an amino acid sequence corresponding to that of an antibody produced by a human or prepared using any technique for making a fully human antibody. For example, fully human antibodies can be obtained by using a commercially available mouse engineered to express a particular human immunoglobulin protein or by phage, yeast, or ribosome display techniques for preparing fully human antibodies. This definition of human antibody specifically excludes humanized antibodies that contain non-human antigen-binding residues.

[0141] Chimeric antibody A "chimeric antibody" refers to an antibody in which the variable region sequence is derived from one species and the constant region sequence is derived from another species, for example, an antibody in which the variable region sequence is derived from a mouse antibody and the constant region sequence is derived from a human antibody.

[0142] Humanized antibody A "humanized" antibody refers to a chimeric antibody containing minimal sequences derived from non-human immunoglobulins, which is a non-human (e.g., mouse) antibody. Preferably, a humanized antibody is a human immunoglobulin (recipient antibody) in which residues derived from the recipient's CDRs are replaced by residues derived from the CDRs of a non-human species (donor antibody) having the desired specificity, affinity, and capacity, such as a mouse, rat, or rabbit. A humanized antibody may contain residues that are not found in the imported CDRs or framework sequences in the recipient antibody but are included to further refine and optimize antibody performance.

[0143] Antigen "Antigen" refers to a molecular entity used for immunizing an immunocompetent vertebrate to produce antibodies that recognize the antigen or for screening an expression library (e.g., especially a phage, yeast, or ribosome display library) for antibody selection. As used herein, an antigen is more broadly named and generally intended to include a target molecule specifically recognized by an antibody, and thus includes fragments or mimetics of molecules used in the immunization process to generate an antibody or in library screening to select an antibody.

[0144] Epitope An "epitope" refers to an area or region of an antigen to which an antibody specifically binds, determined by any method well-known in the art, e.g., an area or region containing residues that interact with the antibody. For example, as described in Chapter 11 of Harlow and Lane, Using Antibodies, a Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1999, there are many methods known in the art for mapping and characterizing the location of epitopes on a protein, including elucidation of the crystal structure of the antibody-antigen complex, competitive assays, gene fragment expression assays, epitope mapping, and synthetic peptide-based assays. Further or alternatively, during the discovery process, the generation and characterization of antibodies can elucidate information about the desired epitope. This information can then be used to competitively screen antibodies for binding to the same epitope.

[0145] Furthermore, the epitope to which an antibody binds can be determined in a systematic screening by using overlapping peptides derived from the antigen and determining binding by the antibody. According to a gene fragment expression assay, the open reading frame encoding the antigen can be fragmented randomly or by a specific genetic structure, and the reactivity of the expressed fragments of the antigen with the antibody being tested is determined. The gene fragments 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 radiolabeled antigen fragment is then determined by immunoprecipitation and gel electrophoresis.

[0146] 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 sufficient for, required for, or necessary for epitope binding.

[0147] At its most detailed level, an epitope for the interaction between an antigen and an antibody can be defined by the spatial coordinates that define the atomic contacts present during the antigen-antibody interaction, as well as information about their relative contributions to the binding thermodynamics. At a less detailed level, an epitope can be characterized by the spatial coordinates that define the atomic contacts between the antigen and the antibody. At an even less detailed level, an epitope can be defined by specific criteria, such as by the amino acid residues it contains, for example, by 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 through function, for example, by competitive binding with other antibodies. An epitope can also be more generally defined as including amino acid residues where substitution by another amino acid alters the characteristics of the interaction between the antibody and the antigen (e.g., using alanine scanning).

[0148] From the fact that the description and definition of an epitope are obtained at different levels of detail depending on the epitope mapping method used, it follows that the comparison of epitopes for different antibodies on the same antigen can be similarly performed at different levels of detail.

[0149] Epitopes described at the amino acid level, for example, 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 distinct (unique) if no amino acid residues are shared by the epitopes.

[0150] Yet another method that can be used to characterize an antibody is to use a competition 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 antibodies. Competition assays are well known to those skilled in the art. Epitopes characterized by competitive binding are said to overlap if the binding of the corresponding antibodies is mutually exclusive, i.e., the binding of one antibody precludes the simultaneous or sequential binding of the other antibody. Epitopes are said to be distinct (unique) if the antigen can accept the binding of both corresponding antibodies simultaneously.

[0151] Epitopes can be linear or conformational. In a linear epitope, all points of interaction between a protein and an interacting molecule (e.g., an antibody) lie linearly along the primary amino acid sequence of the protein. A "non-linear epitope" or "conformational epitope" includes non-contiguous polypeptides (or amino acids) within an antigenic protein to which an antibody specific for the epitope binds.

[0152] Binding affinity The term "binding affinity" refers to the overall strength 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 molecule X for its partner Y is generally the dissociation constant (K D) can be indicated by. Affinity can be measured by common methods known in the art. Low-affinity antibodies generally bind to antigens slowly and tend to dissociate easily, while high-affinity antibodies generally bind to antigens more quickly and tend to remain bound for a longer time. In particular, the term "binding affinity" is intended to refer to the dissociation rate of a specific antigen-antibody interaction. K D is the "off-rate (k off )" or the rate of dissociation, also called "k d ", relative to the association rate or "on-rate (k on )" or "k a ". Thus, K D is k off / k on (or k d / k a ) and is expressed as molar concentration (M). The smaller the K D , the stronger the binding affinity. Thus, a K D of 1 μM exhibits a weaker binding affinity compared to a K D of 1 nM. The K D value for an antibody can be determined using methods well established in the art. One exemplary method for determining the K D of an antibody is by using surface plasmon resonance (SPR), typically by using a biosensor system, such as a BIACORE system. BIACORE kinetic analysis involves analyzing the binding and dissociation of an antigen from a chip having molecules immobilized on their surfaces (e.g., molecules containing an epitope-binding domain). Another method for determining the K D of an antibody is by using biolayer interferometry, typically by using the OCTET® technology (Octet QK e system, ForteBio). Alternatively or additionally, the KinExA (Kinetic Exclusion Assay) assay available from Sapidyne Instruments (Boise, ID) can also be used.

[0153] Monospecific antibody 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 an antigen. Thus, if a monospecific antibody has more than one antigen - binding site, those binding sites compete with each other for binding to one antigen molecule.

[0154] Bispecific antibody A “bispecific antibody” refers to a molecule having binding specificities for at least two different epitopes. In some embodiments, the bispecific antibody can bind two different antigens simultaneously. In other embodiments, the two different epitopes can be present on the same antigen.

[0155] Tetrafab A tetrafab molecule refers to an antibody or an antigen - binding portion thereof that contains four antigen - binding sites. The antigen - binding sites can bind to one, two, three, or four different epitopes, and such epitopes can be one, two, three, or four different targets.

[0156] Maximal half - effective concentration (EC 50 ) The term “maximal half - effective concentration (EC 50 )” refers to the concentration of a therapeutic agent that causes a response halfway between the baseline and the maximum after a specified exposure time. The therapeutic agent can cause inhibition or stimulation. As a measure of potency, EC 50 values are commonly used and are used herein.

[0157] Agonist An “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 encompasses substances (e.g., antibodies) that bind to a molecule and promote its activity.

[0158] Antagonist An "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" encompasses substances (e.g., antibodies) that bind to a molecule and prevent or reduce its activity.

[0159] Competitive As used herein with respect to antibodies, the term "competitive" means that the binding of a second antibody to its cognate epitope is detectably reduced in the presence of a first antibody as compared to the binding of the second antibody in the absence of the first antibody, such that the first antibody binds to the epitope in a manner sufficiently similar to the binding of the second antibody. There may be an alternative method in which the binding of the first antibody to the epitope is also detectably reduced in the presence of the second antibody, but this is not necessary. That is, the first antibody can inhibit the binding of the second antibody to the 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, to a higher extent, or to a lower extent, those antibodies are said to "cross-compete" with each other for binding to their respective epitopes. Both competitive antibodies and cross-competitive 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 ordinary skill in the art will understand, based on the teachings provided herein, that such competitive or cross-competitive antibodies are encompassed and may be useful in the methods disclosed herein.

[0160] Fc receptor "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 binds to IgG antibodies (gamma receptors), including receptors of the FcgRI, FcgRII, and FcgRIII subclasses, including allelic variants and alternatively spliced forms of those receptors. The FcgRII receptor includes FcgRIIA ("activating receptor") and FcgRIIB ("inhibitory receptor") having similar amino acid sequences, the cytoplasmic domains of which are mainly different. The activating receptor FcgRIIA contains an immunoreceptor activation tyrosine motif (ITAM) in its cytoplasmic domain. The inhibitory receptor FcgRIIB contains an immunoreceptor inhibitory tyrosine motif (ITIM) in its cytoplasmic domain (see, for example, 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 to be identified in the future, are encompassed by the term "Fc receptor" herein. The term "Fc receptor" also includes the neonatal receptor FcRn, which is responsible for the transfer of maternal IgG to the fetus (Guyer et al., J. Immunol. 1976; 117: 587 and Kim et al., J. Immunol. 1994; 24: 249) and the regulation of immunoglobulin isotype. Methods for measuring binding to FcRn are known (see, for example, 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).

[0161] Effector cell "Effector cells" refers to leukocytes that express one or more FcRs and perform effector functions. In certain embodiments, the effector cells express at least FcgRIII and perform ADCC effector functions. Examples of leukocytes 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.

[0162] 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 (FcRs) present on certain cytotoxic cells (e.g., NK cells, neutrophils, and macrophages) enables these cytotoxic effector cells to specifically bind to antigen-bearing target cells and subsequently kill the target cells using cytotoxins. NK cells, which are the main cells that mediate ADCC, express only FcgRIII, while monocytes express FcgRI, FcgRII, and FcgRIII. To evaluate the ADCC activity of a molecule of interest, in vitro ADCC assays such as those described in U.S. Patent Nos. 5,500,362, 5,821,337, or 6,737,056 can be performed. Effector cells useful in such assays include PBMCs and NK cells. Alternatively or additionally, the ADCC activity of a molecule of interest can be evaluated in vivo, for example, in animal models such as those disclosed in Clynes et al., Proc. Natl. Acad. Sci. (USA) 1998; 95: 652-656. Additional antibodies having altered Fc region amino acid sequences and increased or decreased ADCC activity are described, for example, in U.S. Patent Nos. 7,923,538 and 7,994,290.

[0163] 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 parental antibody when the amounts of such an antibody and the parental antibody used in the assay are essentially the same, and the antibody and the parental antibody differ in at least one structural aspect. In some embodiments, the antibody and the parental antibody have the same amino acid sequence, but the antibody is afucosylated while the parental antibody is fucosylated. In some embodiments, the ADCC activity is determined using an in vitro ADCC assay, but other assays or methods for determining ADCC activity, such as in an animal model, are contemplated. In some embodiments, an antibody having enhanced ADCC activity has enhanced affinity for FcgRIIIA.

[0164] Altered FcR binding or ADCC activity The term "altered" FcR binding affinity or ADCC activity refers to an antibody having either enhanced activity or diminished activity for one or more of the FcR binding activity or ADCC activity compared to a parental antibody, and the antibody and the parental antibody differ in at least one structural aspect. An antibody that "shows increased binding" to an FcR binds to at least one FcR with better affinity than the parental antibody. An antibody that "shows decreased binding" to an FcR binds to at least one FcR with lower affinity than the parental antibody. Such an antibody that shows decreased binding to an FcR may have little or no detectable binding to the FcR, for example, may have 0 to 20 percent of the binding to the FcR compared to the native sequence IgG Fc region.

[0165] 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 an antibody (of the appropriate subclass) bound to its cognate antigen. To assess complement activation, for example, a CDC assay such as that described by Gazzano-Santoro et al., J. Immunol. Methods 1996;202:163 can be performed. Antibodies having an altered Fc region amino acid sequence and increased or decreased C1q binding ability are described, for example, in U.S. Patent No. 6,194,551, U.S. Patent No. 7,923,538, U.S. Patent No. 7,994,290 and WO1999 / 51642.

[0166] host cell A "host cell" refers to an individual cell or cell culture which can or has been a recipient for a vector containing a polynucleotide insert. Host cells include progeny of a single host cell, which progeny may not necessarily be identical, due to natural, accidental or deliberate mutation, to the original parent cell (either in morphology or in genomic DNA complement). Host cells include cells transfected in vivo with a polynucleotide of the invention.

[0167] 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, preferably enabling its expression in the host cell. Examples of vectors include, but are not limited to, plasmids and viral vectors, and may include naked nucleic acids or nucleic acids associated with delivery aids (e.g., cationic condensing agents, liposomes, etc.). Vectors may contain DNA or RNA. "Expression vector", as used herein, refers to a vector containing at least one polypeptide-encoding gene and at least one regulatory element (e.g., promoter sequence, poly(A) sequence) related to the transcription or translation of the gene. Typically, the vectors used herein contain at least one antibody-encoding gene, as well as one or more of regulatory elements or selectable markers. Vector components may include, for example, one or more of the following: signal sequences; origins of replication; one or more marker genes; appropriate transcriptional control elements (e.g., promoters, enhancers and terminators). For translation, one or more translational control elements such as ribosome binding sites, translation initiation sites and stop codons may also be included.

[0168] Isolated An "isolated" molecule (e.g., an antibody) is one that is (1) not associated with the naturally associated components that accompany it in its native state, by virtue of its origin or source of derivation, (2) substantially free of other molecules from the same source, e.g., species, cells in which it is expressed, library, etc., (3) expressed by cells from a different species, or (4) not naturally occurring. Thus, a molecule that is chemically synthesized or expressed in a cell line different from the system from which it is naturally derived is "isolated" from its naturally associated components. Molecules can also be rendered substantially free of naturally associated components by isolation using purification techniques well known in the art.

[0169] Polypeptide / Protein "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 contain one or more modified amino acids. These terms also include amino acid chains that are modified naturally or by intervention; for example, by 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, non-natural amino acids), as well as other modifications known in the art, are also included within this definition. It is understood that a polypeptide can exist as a single chain or as associated chains.

[0170] Polynucleotide / Nucleic Acid "Polynucleotide" or "nucleic acid" (used interchangeably herein) refers to a strand of nucleotides of any length, including DNA and RNA. Nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases or their analogs, or any substrate that can be incorporated into the strand by DNA or RNA polymerase. Polynucleotides can include modified nucleotides, such as methylated nucleotides and their analogs. Modifications to the nucleotide structure, if present, can be imparted before or after assembly of the strand. Non-nucleotide components can interrupt the nucleotide sequence. Polynucleotides can be further modified after polymerization, for example, 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 having uncharged linkages (e.g., methylphosphonate, phosphotriester, phosphoamidate, carbamate, etc.) and those having charged linkages (e.g., phosphorothioate, phosphorodithioate, etc.), those containing pendant moieties such as proteins (e.g., nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.), those having intercalators (e.g., acridine, psoralen, etc.), those containing chelators (e.g., metals, radioactive metals, boron, metal oxides, etc.), those containing alkylating agents, those having modified linkages (e.g., alpha-anomer nucleic acids, etc.), as well as polynucleotides in their unmodified form. Further, any of the hydroxyl groups normally present in the sugar can be replaced, for example, by a phosphonate group, a phosphate group, protected by a standard protecting group, or activated to provide an additional linkage to an additional nucleotide, or conjugated to a solid support. The 5' and 3' terminal OHs can be phosphorylated or substituted with an amine or organic capping group moiety of 1 to 20 carbon atoms. Other hydroxyls can also be derivatized to standard protecting groups.The polynucleotide may also contain ribose or deoxyribose sugars in similar forms 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 base-off nucleoside analogs such as methyl riboside.

[0171] 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 acid has a side chain of similar length, such as alanine, glycine and serine, or a side chain of similar size. Chemical similarity means that the residues have the same charge, or are both hydrophilic or hydrophobic. Specific examples include the substitution of a hydrophobic residue, such as isoleucine, valine, leucine or methionine, with another residue, or the substitution of one polar residue with another polar residue, such as the substitution of arginine with lysine, the substitution of glutamic acid with aspartic acid or the substitution of glutamine with asparagine, the substitution of serine with threonine, etc. Specific examples of conservative substitutions include the substitution of hydrophobic residues with each other, such as isoleucine, valine, leucine or methionine, the substitution of one polar residue with another polar residue, such as the substitution of arginine with lysine, the substitution of glutamic acid with aspartic acid, or the substitution of glutamine with asparagine, etc. Conservative amino acid substitutions typically include substitutions within the following groups, for example: glycine, alanine, valine, isoleucine, leucine; aspartic acid, glutamic acid; asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine.

[0172] Identity The term "identity" or "identical to" refers to the overall relatedness between polymeric molecules, such as between nucleic acid molecules (e.g., DNA molecules or RNA molecules) or between polypeptide molecules. "Identity" measures the percentage of identical matches between two or more sequences using gap alignment handled by a specific mathematical model (e.g., an algorithm) of a computer program well-known in the art.

[0173] The terms "increasing", "improving", "decreasing", or "reducing" refer to values compared to a baseline measurement, e.g., a measurement in the same individual before the start of a treatment described herein, or a measurement in a control individual or subject (or multiple 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 treated individual. In some embodiments, a "control individual" is an individual not suffering from the same form of disease or injury as the treated individual.

[0174] Excipient The term "excipient" refers to any material that, when combined with an active ingredient of interest (e.g., an antibody), retains the biological activity of the active ingredient. 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, etc. Examples of excipients include water, saline, phosphate buffered solutions, dextrose, glycerol, ethanol, etc., and one or more of combinations thereof, and may include isotonic agents in the composition, such as sugars, sodium chloride, or polyhydric alcohols, such as mannitol or sorbitol.

[0175] Treat The terms "treating," "treat," or "treatment" refer to any type of treatment, for example, to reduce, alleviate, or slow the progression of a patient's disease, disorder, or condition, or any tissue damage associated with the disease. In some embodiments, the disease, disorder, or condition is X.

[0176] Prevent The terms "prevent" or "prevention" refer to one or more of the delay in the onset, reduction in frequency, or reduction in severity of at least one symptom or sign (e.g., *** ) of a particular disease, disorder, or condition (e.g., *** ) specific to a particular application. In some embodiments, prevention is evaluated on a population basis such that a drug is considered to "prevent" a particular disease, disorder, or condition if a statistically significant decrease in the incidence, frequency, or intensity of one or more symptoms of that disease, disorder, or condition is observed in a population susceptible to that disease, disorder, or condition. Prevention can be considered complete if the onset of the disease, disorder, or condition is delayed over a predefined period.

[0177] 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, rabbits, primates, humans, etc., and mammals in utero are included. In certain embodiments, a human is the appropriate subject. A human subject can be of either sex and at any stage of development. In some embodiments, the subject is a patient having a disease: 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).

[0178] Therapeutically effective amount The term "therapeutically effective amount" refers to the amount of an active ingredient that elicits a biological or pharmacological response that is sought by a researcher, veterinarian, physician, or other clinician in an organization, system, animal, individual, or human, and that may include one or more of the following: (1) Prevent a disease; for example, prevent 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 general symptoms of the disease; (2) Inhibit a disease; for example, inhibit a disease, condition, or disorder in an individual who is experiencing or exhibiting the pathology or general symptoms of the disease, condition, or disorder (i.e., halt or slow further development of the pathology or general symptoms); and (3) Remit a disease; for example, remit a disease, condition, or disorder in an individual who is experiencing or exhibiting the pathology or general symptoms of the disease, condition, or disorder (i.e., reverse the pathology or general symptoms).

[0179] Antibody against 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 tumor necrosis factor-alpha (TNFα), the other being TNFR1. TNFR2 is a type I transmembrane receptor having an extracellular domain composed of four cysteine-rich domains (CRDs). Binding of TNFα to TNFR2 elicits an intracellular signaling cascade that results in NF-κB activation and subsequently leads to cell proliferation, activation, and survival.

[0180] 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 non-human species, such as cynomolgus TNFR2, as well as different forms of TNFR2. In some embodiments, the antibody may be completely specific for human TNFR2 and may not exhibit species cross-reactivity (e.g., does not bind to mouse TNFR2) or may not exhibit other types of cross-reactivity. As used herein, TNFR2 refers to native human TNFR2 unless the context indicates otherwise. Thus, the terms "TNFR2 antibody", "anti-TNFR2 antibody", or other similar designations mean any antibody (as defined herein) that binds to or reacts with TNFR2, its isoforms, fragments, or derivatives. The full-length mature form of TNFR2 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 TNFR2 represented by NCBI database accession number XP_005544817 is provided herein as SEQ ID NO: 36.

[0181] In some aspects, the antibodies of the disclosure agonize TNFR2. In some aspects, the antibodies of the disclosure do not inhibit TNFα binding to TNFR2 molecules to which the antibody is bound.

[0182] In some embodiments, the anti-TNFR2 antibodies of the disclosure include antibodies that i) compete with an antibody having the amino acid sequence of the heavy chain variable region shown as SEQ ID NO: 21 and the amino acid sequence of the light chain variable region shown as SEQ ID NO: 8 for binding to human TNFR2, or ii) bind to the same epitope as such an antibody, or both.

[0183] The anti-TNFR2 antibodies of the present disclosure include 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, as well as glycosylation variants of antibodies, amino acid sequence variants of antibodies, and covalently modified antibodies, and may encompass any other modified conformation of an immunoglobulin molecule comprising an antigen-binding site of the required specificity. The antibodies can be of mouse, 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.

[0184] In some embodiments, the present invention provides an antibody 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.

[0185] The present invention also provides the CDR portions of antibodies against TNFR2. The determination of the CDR regions is well within the skill of those 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 herein referred to as "conformation definition" of the CDRs, the positions of the CDRs can be identified as residues that contribute enthalpically to antigen binding. See, e.g., Makabe et al., 2008, Journal of Biological Chemistry, 283:1156-1166. Generally, "conformational CDRs" include the residue positions in the Kabat CDRs and the Vernier zones that are constrained to maintain an appropriate loop structure for the antibody to bind to a specific antigen. The determination of conformational CDRs is well within the skill of those 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.

[0186] In some embodiments, the antibody comprises either i) the full-length heavy chain with or without a C-terminal lysine of the anti-TNFR2 antibody TF-2053, or ii) one or both of the full-length light chains.

[0187] In certain embodiments, the antibodies described herein comprise an Fc domain. The Fc domain can be derived from 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.

[0188] The present invention encompasses modifications to the CDRs and variable regions shown in Table 5. For example, the present invention includes antibodies comprising functionally equivalent variable regions and CDRs that do not significantly affect those properties, as well as variants having enhanced or decreased activity or affinity. For example, the amino acid sequence can be mutated to obtain an antibody having the desired binding affinity for TNFR2. Modification of polypeptides is a conventional practice in the art and need not be described in detail herein. Examples of modified polypeptides include conservative substitutions of amino acid residues, polypeptides having one or more deletions or additions of amino acids that do not significantly and detrimentally change the functional activity or that mature (enhance) the affinity of the polypeptide for its ligand, or the use of chemical analogs.

[0189] Modifications or mutations can 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 can be made to alter the immunogenicity of the antibody, to provide sites for covalent or non-covalent attachment to another molecule, or to alter properties such as complement binding, FcR binding, and antibody-dependent cell-mediated cytotoxicity. In some embodiments, one or fewer to five conservative amino acid substitutions are made within the framework or constant region. In other embodiments, one or fewer to three conservative amino acid substitutions are made within the framework or constant region. According to the present invention, a single antibody can have mutations in any one or more of the CDRs or framework regions of the variable domain, or in the constant region.

[0190] In some embodiments, the antibody has an increased or decreased binding affinity for human Fc gamma receptors and is immunologically inert or partially inert, e.g., does not induce complement-mediated lysis, does not stimulate antibody-dependent cell-mediated cytotoxicity (ADCC), or does not activate microglia; or has a modified constant region with reduced activity (compared to the unmodified antibody) in any one or more of the following: inducing complement-mediated lysis, stimulating ADCC, or activating microglia. Different modifications of the constant region can be used to achieve an optimal level or combination of effector functions. See, for example, Morgan et al., Immunology 86:319-324, 1995; Lund et al., J. Immunology 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.

[0191] Modifications also include glycosylated and non-glycosylated polypeptides, as well as polypeptides having other post-translational modifications, such as glycosylation with different sugars, acetylation and phosphorylation. 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 can 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 parts of the glycoprotein that can affect the conformation and presented three-dimensional surface of the glycoprotein (Jefferis and Lund, supra; Wyss and Wagner, 1996, Current Opin. Biotech. 7:409-416). Oligosaccharides can 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 cell cytotoxicity (ADCC). In particular, antibodies produced by CHO cells using tetracycline-regulated expression of β(1,4)-N-acetylglucosaminyltransferase III (GnTIII), a glycosyltransferase that catalyzes the formation of bisecting GlcNAc, have been reported to have improved ADCC activity (Umana et al., 1999, Nature Biotech. 17:176-180).

[0192] In some embodiments, the present disclosure provides an isolated antibody that specifically binds to TNFR2 and comprises a heavy chain variable region (VH) and a light chain variable region (VL), the CDR-H1, CDR-H2, and CDR-H3 sequences of the 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 the CDR-L1, CDR-L2, and CDR-L3 sequences of the 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.

[0193] In some embodiments, the present disclosure provides an isolated antibody that specifically binds to TNFR2 and comprises a heavy chain variable region (VH) and a light chain variable region (VL), the CDR-H1, CDR-H2, and CDR-H3 sequences according to SEQ ID NO: 30, and the CDR-L1, CDR-L2, and CDR-L3 sequences according to SEQ ID NO: 9.

[0194] In some embodiments, the present disclosure provides an isolated antibody that specifically binds to TNFR2 and comprises a heavy chain variable region (VH) and a light chain variable region (VL), (i) the CDR-L1 sequence according to SEQ ID NO: 1; the CDR-L2 sequence according to SEQ ID NO: 7; and the CDR-L3 sequence according to SEQ ID NO: 3, and the CDR-H1 sequence according to SEQ ID NO: 10; the CDR-H2 sequence according to SEQ ID NO: 20; the CDR-H3 sequence according to SEQ ID NO: 12; or (ii) the CDR-L1 sequence according to SEQ ID NO: 1; the CDR-L2 sequence according to SEQ ID NO: 2; and the CDR-L3 sequence according to SEQ ID NO: 3, and the CDR-H1 sequence according to SEQ ID NO: 10; the CDR-H2 sequence according to SEQ ID NO: 11; the CDR-H3 sequence according to SEQ ID NO: 12 and provides an antibody.

[0195] In some embodiments, the present disclosure provides an isolated antibody that specifically binds to TNFR2 and comprises 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.

[0196] The VH framework sequence 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 sequence may be derived from the human IGHV1-46 germline sequence.

[0197] 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 the human germline IGKV1-9 sequence.

[0198] In some embodiments, the present disclosure provides an antibody as described above, 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.

[0199] In some embodiments, the present disclosure provides an antibody as described above, 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 identical to the human germline sequence from which it is derived.

[0200] In some embodiments, the present 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.

[0201] In some embodiments, the present disclosure provides an antibody comprising the VH sequence of SEQ ID NO: 13 and the VL of SEQ ID NO: 4.

[0202] In some embodiments, the present disclosure provides an antibody 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.

[0203] In some embodiments, the present disclosure provides an antibody comprising the VH sequence of SEQ ID NO: 21 and the VL of SEQ ID NO: 8.

[0204] In some embodiments, the present disclosure provides an anti-TNFR2 antibody containing the variable regions shown in the sequence listing and variations of the CDRs shown in the sequence listing, wherein 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.

[0205] In some embodiments, the present 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 present 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 present disclosure provides an anti-TNFR2 antibody comprising a VL sequence encoded by the nucleic acid sequence of SEQ ID NO: 31.

[0206] In some embodiments, the present disclosure provides an anti-TNFR2 antibody comprising a constant heavy domain (CH1) and a constant light domain (CL). CH1 can be connected to VH, and CL can be connected to VL to form a Fab domain. The antibody can comprise a first and a second Fab domain.

[0207] 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 an Fc domain of 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 can be an Fc domain of IgG 1 .

[0208] In some embodiments, the present disclosure provides an anti-TNFR2 antibody, wherein the CH1 domain in the first Fab domain comprises a sequence according to SEQ ID NO: 23.

[0209] In some embodiments, the present disclosure provides an anti-TNFR2 antibody, wherein the CL in the first Fab domain comprises a sequence according to SEQ ID NO: 5.

[0210] In some embodiments, the present disclosure provides an anti-TNFR2 antibody comprising a light chain (LC) comprising a sequence according to SEQ ID NO: 9.

[0211] In some embodiments, the present disclosure provides an anti-TNFR2 antibody in which 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.

[0212] In some embodiments, the present disclosure provides an anti-TNFR2 antibody in which one or both of the first hinge region and the second hinge region comprise a sequence according to SEQ ID NO: 23. In some embodiments, the present disclosure provides an anti-TNFR2 antibody in which one or both of the first CH2 domain and the second CH2 domain comprise a sequence according to SEQ ID NO: 25. In some embodiments, the present disclosure provides an anti-TNFR2 antibody in which one or both of the first CH3 domain and the second CH3 domain comprise a sequence according to SEQ ID NO: 26. In some embodiments, the present disclosure provides an anti-TNFR2 antibody in which one or both of the first Fc chain and the second Fc chain comprise a sequence according to SEQ ID NO: 37.

[0213] In some embodiments, the present disclosure provides an anti-TNFR2 antibody in which 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.

[0214] In some embodiments, the present disclosure provides an anti-TNFR2 antibody further comprising a third Fab and a fourth Fab.

[0215] In some embodiments, the present 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; a CDR-H3 sequence according to SEQ ID NO: 12.

[0216] In some embodiments, the present 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.

[0217] In some embodiments, the present 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.

[0218] In some embodiments, the present disclosure provides an anti-TNFR2 antibody, wherein the N'-terminus of the first Fab is connected to the C'-terminus of the third Fab.

[0219] In some embodiments, the present disclosure provides an anti-TNFR2 antibody, wherein the N'-terminus of the first Fab is connected to the C'-terminus of the third Fab via a first linker.

[0220] In some embodiments, the present disclosure provides an anti-TNFR2 antibody, wherein the first linker comprises a sequence according to SEQ ID NO: 27.

[0221] In some embodiments, the present disclosure provides an anti-TNFR2 antibody, wherein the N'-terminus of the second Fab is connected to the C'-terminus of the fourth Fab.

[0222] In some embodiments, the present disclosure provides an anti-TNFR2 antibody, wherein the N'-terminus of the second Fab is connected to the C'-terminus of the fourth Fab via a second linker.

[0223] In some embodiments, the present disclosure provides an anti-TNFR2 antibody, wherein the second linker comprises a sequence according to SEQ ID NO: 27.

[0224] In some embodiments, the present disclosure provides an anti-TNFR2 antibody, wherein the HC comprises a sequence according to SEQ ID NO: 30.

[0225] E54. An isolated antibody that specifically binds to TNFR2 and comprises 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.

[0226] In some embodiments, the present 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 present 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 present 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.

[0227] In some embodiments, the present 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 present 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 present 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.

[0228] 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 the insert of the plasmid having ATCC accession number PTA-127530 deposited with the ATCC, and the antibody light chain has an amino acid sequence encoded by the nucleic acid sequence of the insert of the plasmid having ATCC accession number PTA-127532 deposited with the ATCC.

[0229] In some embodiments, provided herein is an anti-TNFR2 antibody wherein the Fc domain is the Fc domain of IgG 2 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. In some embodiments, provided herein is an anti-TNFR2 antibody wherein the CL in the first Fab domain comprises a sequence according to SEQ ID NO: 5.

[0230] In some embodiments, provided herein is an anti-TNFR2 antibody wherein the first Fab domain and the second Fab domain are identical.

[0231] In some embodiments, provided herein is an anti-TNFR2 antibody 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.

[0232] In some embodiments, provided herein are anti-TNFR2 antibodies, 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 are anti-TNFR2 antibodies, 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 are anti-TNFR2 antibodies, 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 are anti-TNFR2 antibodies, wherein one or both of the first Fc chain and the second Fc chain comprise a sequence according to SEQ ID NO: 18.

[0233] In some embodiments, provided herein are anti-TNFR2 antibodies, wherein the first Fc chain and the second Fc chain are identical. In some embodiments, provided herein are anti-TNFR2 antibodies, comprising a heavy chain (HC) comprising a sequence according to SEQ ID NO: 19. In some embodiments, provided herein are anti-TNFR2 antibodies, 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.

[0234] 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.

[0235] 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.

[0236] The present invention also encompasses fusion proteins comprising one or more components of the antibodies 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 and VL domains 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 such that the VH and VL domains can interact with each other. The VH-linker-VL antibody is then linked to the 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 it is desired to create a bivalent or multivalent antibody in a single polypeptide chain, or when it is desired to create a bispecific antibody.

[0237] Biological activity of the anti-TNFR2 antibody In addition to binding to an epitope on TNFR2, the antibodies of the present disclosure can mediate a biological activity. That is, the present disclosure includes isolated antibodies that specifically bind to TNFR2 and mediate at least one detectable activity selected from: (i) specifically binds to human TNFR2; (ii) specifically binds to cynomolgus TNFR2; (iii) stimulates the activity of TNFR2 (e.g., human, cynomolgus); and / or (iv) enables TNFα activation of TNFR2 while binding to TNFR2.

[0238] In some embodiments, an EC less than a number selected from the group consisting of 5 μM and 2 μM in a human TNFR2 potency assay in Jurkat reporter cells 50An isolated anti-TNFR2 antibody, characterized by, is provided herein.

[0239] In some embodiments, an EC less than a number selected from the group consisting of 10 μM, 5 μM, and 2 μM in a human TNFR2 potency assay in human peripheral blood monocytes 50 An isolated anti-TNFR2 antibody, characterized by, is provided herein.

[0240] In some embodiments, an EC less than a number selected from the group consisting of 20 μM and 15 μM in a cynomolgus TNFR2 potency assay in cynomolgus peripheral blood monocytes 50 An isolated anti-TNFR2 antibody, characterized by, is provided herein.

[0241] In some embodiments, an EC less than a number selected from the group consisting of 5 μM, 1 μM, and 0.5 μM in a human TNFR2 potency assay in Jurkat reporter cells 50 An isolated anti-TNFR2 antibody, characterized by, is provided herein.

[0242] In some embodiments, an EC less than a number selected from the group consisting of 10 mg / ml, 5 mg / ml, and 2 mg / ml for ICAM-1 upregulation in a human TNFR2-expressing primary T cell population derived from human peripheral blood monocytes 50 An isolated anti-TNFR2 antibody, characterized by, is provided herein.

[0243] In some embodiments, an EC less than a number selected from the group consisting of 50 mg / ml, 20 mg / ml, 15 mg / ml, and 10 mg / ml for ICAM-1 upregulation in a cynomolgus TNFR2-expressing primary T cell population derived from cynomolgus peripheral blood monocytes 50 An isolated anti-TNFR2 antibody, characterized by, is provided herein.

[0244] In some embodiments, provided herein are isolated anti-TNFR2 antibodies characterized by an affinity KD for human TNFR2 that is less than a number selected from the group consisting of 1 nM, 0.5 nM, 0.1 nM, and 0.07 nM.

[0245] In some embodiments, provided herein are isolated anti-TNFR2 antibodies characterized by an affinity KD for cynomolgus TNFR2 that is less than a number selected from the group consisting of 1 nM and 0.1 nM.

[0246] In some aspects, the affinity is measured by surface plasmon resonance (SPR). In some aspects, SPR is measured using a BIAcore. In some aspects, the affinity is measured by SPR in which biotinylated TNFR2 is captured onto a streptavidin strip surface at a flow rate of 10 μl / min for 60 seconds, the antibody is injected onto the captured TNFR2 at concentrations ranging for a 50-second association phase at 80 μl / min, and then the dissociation phase is initiated with running HBS-EP+ buffer injected at 80 μl / min for 600 seconds. In some aspects, the antibody is injected at concentrations in the range of 10 to 0.625 nM. In some aspects, particularly for a standard IgG format comprising two antigen-binding domains, the antibody is injected at concentrations in the range of 10 to 1.25 nM. In some aspects, particularly for a tetrafab format comprising four antigen-binding domains, the antibody is injected at concentrations in the range of 5 to 1.25 nM. In some aspects, the SPR data is analyzed using Biacore Evaluation Software.

[0247] Polynucleotides encoding anti-TNFR2 antibodies, and methods of manufacture The present disclosure also provides polynucleotides encoding any of the antibodies of the invention, including the 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.

[0248] If desired, the desired anti-TNFR2 antibody (monoclonal or polyclonal) can be sequenced, and then the polynucleotide sequence can be cloned into a vector for expression or propagation. The sequence encoding the desired antibody can be maintained in a vector in a host cell, and then the host cell can be expanded and frozen for further use. Production of recombinant monoclonal antibodies in cell culture can be carried out via 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.

[0249] In some embodiments, polynucleotides are provided herein that include sequences encoding one or both of the heavy or light chain variable regions of the anti-TFR2 antibodies provided herein. The sequence encoding the desired antibody can be maintained in a vector in a host cell, and then the host cell can be expanded and frozen for further use. Vectors (including expression vectors) and host cells are further described herein.

[0250] In some embodiments, the present disclosure provides polynucleotides encoding the amino acid sequences of any of the following anti-TNFR2 antibodies: IgG2-854, IgG2-1765, and IgG1. In one embodiment, the present invention provides a polynucleotide encoding the amino acid sequence of the anti-TNFR2 antibody TF-2053.

[0251] In some embodiments, the present disclosure provides polynucleotides encoding one or more anti-TNFR2 antibody heavy chain polypeptides comprising amino acid sequences selected from the group consisting of SEQ ID NO: 19, SEQ ID NO: 22, and SEQ ID NO: 30.

[0252] In some embodiments, the present disclosure provides polynucleotides encoding one or more anti-TNFR2 antibody light chain polypeptides comprising amino acid sequences selected from the group consisting of SEQ ID NO: 6 and SEQ ID NO: 9.

[0253] In some embodiments, the present 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, and SEQ ID NO: 29.

[0254] In some embodiments, the present 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.

[0255] The present invention provides a polynucleotide comprising the nucleic acid sequence of the insert of the plasmid encoding the heavy chain of antibody Tetrafab 2053, deposited with the ATCC and having 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 antibody Tetrafab-2053, deposited with the ATCC and having accession number PTA-127532. Further, the present invention provides a polypeptide comprising the amino acid sequence encoded by the DNA insert of the plasmid encoding the VH domain of antibody Tetrafab-2053, deposited with the ATCC and having 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 antibody Tetrafab-2053, deposited with the ATCC and having 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.

[0256] As a result of the degeneracy of the genetic code, it will be appreciated by those skilled in the art that there are many nucleotide sequences that encode the polypeptides described herein. Some of these polynucleotides possess minimal homology to the nucleotide sequences of any native gene. Nevertheless, polynucleotides that vary due to differences in codon usage are specifically contemplated by the present invention. Furthermore, alleles of the genes containing the polynucleotide sequences provided herein are within the scope of the present invention. Alleles are endogenous genes that have been altered as a result of one or more nucleotide mutations, such as deletions, additions or substitutions. The resulting mRNA and proteins may or may not have an altered structure or function. Alleles can be identified using standard techniques (e.g., hybridization, amplification or database sequence comparison).

[0257] In one embodiment, the VH and VL domains or the full-length HC or LC are encoded by separate polynucleotides. Alternatively, both VH and VL, or both HC and LC, are encoded by a single polynucleotide.

[0258] Polynucleotides that are complementary to any such sequences are also encompassed by the present disclosure. The 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 that contain introns and correspond to DNA molecules in a one-to-one manner, and mRNA molecules that do not contain introns. Additional coding or non-coding sequences may or may not be present within the polynucleotides of the present disclosure, and the polynucleotides may or may not be linked to other molecules or support materials.

[0259] The polynucleotides of the present invention can be obtained using chemical synthesis, recombinant methods or PCR. Methods of chemical polynucleotide synthesis are well known in the art and need not be described in detail herein. One of ordinary skill in the art can use the sequences provided herein and commercially available DNA synthesizers to produce the desired DNA sequences.

[0260] As further discussed herein, to prepare a polynucleotide using recombinant methods, a polynucleotide containing the desired sequence can be inserted into an appropriate vector, which can then be introduced into a host cell suitable for replication and amplification. The polynucleotide can be inserted into the host cell by any means known in the art. The cell is transformed by introducing an exogenous polynucleotide by direct uptake, endocytosis, transfection, F-mating or electroporation. Once introduced, the exogenous polynucleotide can be maintained intracellularly as a non-integrated vector (e.g., plasmid) or can be integrated into the host cell genome.

[0261] A suitable cloning vector can be constructed according to standard techniques or selected from a number of cloning vectors available in the art. The cloning vector selected may vary according to the host cell intended for use, but useful cloning vectors generally have one or more features, such as: i) the ability to self-replicate; ii) a single target for a particular restriction endonuclease; or iii) the gene for a marker that can be used to select 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.

[0262] An expression vector is further provided. An expression vector is generally a replicable polynucleotide construct containing a polynucleotide according to the invention. It is implied that the expression vector must be replicable in the host cell, either as an episome or as an integrated part of the chromosomal DNA. Suitable expression vectors include, but are not limited to, plasmids, viral vectors, such as those disclosed in PCT Publication No. WO87 / 04462, adenovirus, adeno-associated virus, retrovirus, cosmid, and expression vectors. Vector components generally may include, but are not limited to, one or more of the following: a signal sequence; an origin of replication; one or more marker genes; suitable transcriptional control elements (e.g., a promoter, an enhancer, and a terminator). For expression (i.e., translation), one or more translational control elements, such as a ribosome binding site, a translation initiation site, and a stop codon, are also usually required.

[0263] Vectors containing the polynucleotide of interest can be introduced into host cells 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 such as vaccinia virus). The choice of means for introducing the vector or polynucleotide often depends on the characteristics of the host cell.

[0264] The present invention also provides host cells containing any of the polynucleotides described herein. Any host cell capable of overexpressing heterologous DNA can be used for the purpose of isolating a gene encoding an antibody, polypeptide or protein 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., Escherichia coli or Bacillus subtilis) and yeast (e.g., Saccharomyces cerevisiae, Schizosaccharomyces pombe; or Kluyveromyces lactis).

[0265] In addition, numerous commercially available and non-commercially available cell lines expressing polypeptides or proteins can 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 may require different culture conditions for optimal growth and polypeptide or protein expression, and can modify the conditions as needed.

[0266] Pharmaceutical Compositions In another embodiment, the present invention includes pharmaceutical compositions.

[0267] "Pharmaceutical composition" refers to a mixture of an antibody of the invention and one or more excipients.

[0268] The pharmaceutical composition 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.

[0269] Other excipients and modes of administration known in the pharmaceutical art may also be used. The pharmaceutical composition of the present invention can be prepared by any of the well-known techniques of pharmacy, for example, by 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. The formulation of drugs is discussed, for example, in Hoover, John E., Remington’s Pharmaceutical Sciences, Mack Publishing Co., Easton, Pennsylvania, 1975; Liberman et al., Pharmaceutical Dosage Forms, Marcel Decker, New York, N.Y., 1980; and Kibbe et al., Handbook of Pharmaceutical Excipients (3rd Edition), American Pharmaceutical Association, Washington, 1999.

[0270] Excipients that are acceptable are non-toxic to the recipient at the dosages and concentrations employed, and include buffers such as phosphoric, citric and other organic acids; salts such as sodium chloride; antioxidants including ascorbic acid and methionine; preservatives (e.g., octadecyl dimethyl benzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzetonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl paraben or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins such as 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 dextrin; 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 nonionic surfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG).

[0271] Methods of treatment, diagnosis and other methods The antibodies and antibody conjugates of the invention are useful in a variety of applications including, but not limited to, methods of therapeutic treatment and methods of diagnostic treatment.

[0272] In some embodiments, the antibodies of the invention can stimulate the activity of TNFR2, and by stimulating TNFR2, can be useful in the treatment, prevention, suppression, and remission of 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), inflammatory bowel disease (IBD), autoimmune hepatitis, and systemic lupus erythematosus (SLE) or diseases, disorders, and conditions.

[0273] In one aspect, the 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), inflammatory bowel disease (IBD), autoimmune hepatitis, and systemic lupus erythematosus (SLE). In some embodiments, the 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), inflammatory bowel disease (IBD), autoimmune hepatitis, and systemic lupus erythematosus (SLE) in a subject comprises administering to the subject in need thereof an effective amount of a pharmaceutical composition comprising any of the TNFR2 antibodies described herein. In some embodiments, provided is a method for reducing inflammation and / or activating or expanding immunomodulatory cell types in a subject, the method comprising administering to the subject in need thereof an effective amount of a composition comprising an antibody provided herein.

[0274] In another aspect, the present invention further provides the antibodies or pharmaceutical compositions described herein for use in the methods described for 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), inflammatory bowel disease (IBD), autoimmune hepatitis and systemic lupus erythematosus (SLE) or other autoimmune and inflammatory conditions. The present invention also provides the use of the antibodies 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), inflammatory bowel disease (IBD), autoimmune hepatitis and systemic lupus erythematosus (SLE) or other autoimmune and inflammatory conditions.

[0275] 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), inflammatory bowel disease (IBD), autoimmune hepatitis and systemic lupus erythematosus (SLE) or other autoimmune and inflammatory conditions. For example, the anti-TNFR2 antibodies described herein can be labeled with a detectable moiety, such as a contrast agent and an enzyme-substrate label. The antibodies described herein can be used for in vivo diagnostic assays, such as in vivo imaging (e.g., PET or SPECT), or as a staining reagent.

[0276] For all methods described herein, reference to an anti-TNFR2 antibody also includes a pharmaceutical composition comprising the anti-TNFR2 antibody and one or more additional agents.

[0277] Administration and Dosage 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 antibody itself, or alternatively, as a pharmaceutical composition containing the antibody.

[0278] The antibodies of the invention are administered in a dosage effective for the intended treatment by any suitable route in the form of a pharmaceutical composition adapted to such route.

[0279] In some embodiments, the antibody can be administered parenterally, for example, directly into the bloodstream, muscle, or viscera. Means suitable for parenteral administration include intravenous, arterial, intraperitoneal, intrathecal, intraventricular, intraurethral, intrasternal, intracranial, intramuscular, and subcutaneous. Devices suitable for parenteral administration include needles (including microneedles) syringes, needleless syringes, and infusion techniques.

[0280] In another embodiment, the compounds of the invention can also be administered topically to the skin or mucosa, i.e., dermally or transdermally. In another embodiment, the compounds of the invention can also be administered intranasally or by inhalation. In another embodiment, the compounds of the invention can be administered rectally or vaginally. In another embodiment, the compounds of the invention can also be administered directly to the eye or ear.

[0281] The dosing regimen for the antibody of the present invention or a composition containing said antibody is based on various 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 being used. Thus, the dosing regimen can vary widely. In one embodiment, the total daily dose of the antibody of the present invention is typically from about 0.01 to about 100 mg / kg (i.e., the number of mg of the antibody of the present invention per kg of body weight) for the treatment of the indicated conditions discussed herein. In another embodiment, the total daily dose of the antibody of the present invention is from about 0.1 to about 50 mg / kg, and in another embodiment, from about 0.5 to about 30 mg / kg.

[0282] Co-administration The antibody of the present invention can be used alone or in combination with one or more other therapeutic agents. The present invention provides any of the uses, methods or compositions as defined herein, wherein the antibody of the present invention is used in combination with one or more other therapeutic agents discussed herein.

[0283] The administration of two or more "combined" agents means that all agents are administered in close temporal proximity so as to affect the treatment of the subject. The two or more agents can be administered simultaneously or sequentially. Further, co-administration can be effected by mixing the agents prior to administration or by administering the agents as separate dosage forms at the same time point in time but at the same or different sites of administration.

[0284] 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 can include a diagnostic or therapeutic agent in addition to the antibody of the present invention or its pharmaceutical composition. The kit can also include instructions regarding use in a diagnostic or therapeutic method. In some embodiments, the kit includes the antibody or its pharmaceutical composition and a diagnostic agent.

[0285] In yet another embodiment, the present invention includes a kit suitable for use in practicing the methods 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 practice the methods of the present invention. In another embodiment, the kit comprises one or more antibodies of the present invention in an amount sufficient to practice the methods of the present invention, and at least a first container for a first dosage and a second container for a second dosage.

[0286] Biological deposit Representative materials of the present invention were deposited with the American Type Culture Collection, 10801 University Boulevard, Manassas, VA 20110-2209, USA on March 3, 2023.

[0287] [Table 1]

[0288] The deposit was made under the provisions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purpose of Patent Procedure and Regulations thereunder (Budapest Treaty). This guarantees the maintenance of viable cultures of the deposited material for 30 years from the date of deposit. The deposited material is made available by the ATCC under the terms of the Budapest Treaty, guaranteeing permanent and unrestricted availability of progeny of the deposited material cultures upon the earlier of the issuance of the relevant U.S. patent or the publication of any U.S. or foreign patent application, and compensating for the availability of progeny to those determined by the Commissioner of the U.S. Patent and Trademark Office to be entitled thereto in accordance with 35 U.S.C. Section 122 and the Commissioner's regulations thereunder (including 37 C.F.R. Section 1.14, which specifically conforms to 886 OG 638), in accordance with the agreement between Pfizer Inc. and the ATCC.

[0289] The assignee of this application agrees that in the event the cultures of the deposited materials die, are lost or destroyed, if cultivated under appropriate conditions, the materials will be replaced promptly upon notice with other like materials. The availability of the deposited materials should not be construed as a license to practice the invention in violation of any rights granted under the authority of any government pursuant to its patent laws.

Examples

[0290] The following examples of specific embodiments for carrying out the invention are provided for illustrative purposes only and are in no way intended to limit the scope of the invention.

[0291] The foregoing description and the following examples detail certain specific embodiments of the present disclosure and describe the best mode contemplated by the inventors. However, it is understood that no matter how detailed the foregoing appears in the text, the present disclosure may be practiced in many ways and should be construed in accordance with the appended claims and any equivalents thereof.

[0292] The disclosed teachings have been described with respect to various applications, methods, kits, and compositions, but it is understood that various changes and modifications can be made without departing from the teachings of this specification 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 presented 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.

[0293] [Table 2-1]

[0294] [Table 2-2]

[0295] (Example 1) Isolation of Mouse Monoclonal Antibodies That Bind to Human and Cynomolgus Monkey TNFR2 SJL mice were immunized with several IP injections of Enbrel (HuTNFR2-IgG1-Fc) using ribi as an adjuvant. Serum was screened for binding to human and cynomolgus (Cyno) TNFR2 overexpressed on the surface of CHO cells. Mice showing strong binding to CHO cells were euthanized, and the isolated B cells were fused with P3X myeloma by electrofusion.

[0296] 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 cell overexpressed 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.

[0297]

Table 3

[0298] (Example 2) Cloning of Mouse Anti-TNFR2 Antibody Heavy and Light Chain Variable Regions 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 μg of total RNA isolated from approximately 500,000 hybridoma cells using the RNEasy kit (Qiagen) and template switching oligo with SuperscriptIV™ reverse transcriptase (Invitrogen). The cDNA was then amplified by PCR using primers annealing to the SMART® IIA oligo sequence and mouse constant region specific primers (mouse kappa for the light chain and mouse IgG for the heavy chain) with Q5 High-Fidelity 2× Master Mix (New England Biolabs). The variable heavy and light chain regions were cloned into the mouse pTT5 mammalian expression vector containing the mouse IgG2a constant region and kappa constant region respectively using the injection cloning method (Takara Bio), and the nucleic acid sequences were determined.

[0299] Next, the variable heavy region was cloned into the pTT5 expression vector containing a human IgG1 constant region (Leu234Ala, Leu235Ala, and Gly237Ala, EU numbering; U.S. Patent No. 5,624,821) mutated to disable effector function 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.

[0300] (Example 3) Some anti-TNFR2 antibodies agonize human TNFR2 in the NF-κB-GFP Jurkat cell reporter cell assay NF-κB-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, 100,000 cells / well were plated in a 96-well plate in RPMI 1640 medium supplemented with 10% FBS, 1× sodium pyruvate, 1× Glutamax, and Pen / Strep. Next, the cells were incubated overnight at 37°C with serial dilutions of anti-TNFR2 antibodies. The next 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 dissociate the clumps. Finally, the cells were centrifuged again and resuspended in 75 μl of flow buffer containing 1 / 1000 dilution of DAPI reagent and analyzed by flow cytometry for GFP expression.

[0301] Table 2 summarizes the results of the previous TNFR2 CHO cell assay and the potencies in the Jurkat cell reporter assay. The inventors selected nine clones, all of which are IgG2, based on a range of EC50s and sequence diversity in the Jurkat assay for further characterization.

[0302] [Table 4]

[0303] (Example 4) Biophysical characteristics and agonism in primary cells of selected agonist clones (human PBMC activation assay) Nine previously selected clones were next tested for their ability to induce upregulation of ICAM-1 in CD3+CD4+TIGIT+ human PBMCs (see Example 11 for methods). The inventors also evaluated several biophysical properties, such as sequence liability and nonspecificity (AC-SINS and polyreactivity).

[0304] [Table 5]

[0305] As seen in Table 3, the inventors observed a range of agonism against human PBMCs and decided to advance clones (clones 836, 846, 854, 1201 and 1310) that showed some potency against primary cells. Using the nonspecificity levels, the inventors were able to manage by later optimization if necessary.

[0306] (Example 6) Epitope binding of anti-TNFR2 antibodies Epitope binding was also performed using yeast expressing the human TNFR1 / TNFR2 domain chimera. Yeast cells were stably transfected with various constructs in which one or two CRDs of TNFR2 were replaced with the corresponding human TNFR1 CRDs. Similarly, reverse constructs in which one or two CRDs of TNFR1 were replaced with the corresponding TNFR2 CRDs were also tested. Loss or gain of binding indicated that the specific CRD was required for binding to TNFR2 and contained the epitope.

[0307] As shown in Table 4, most clones except clone 836 (CRD1) and clone 1310 (CRD1-3-4) bind to CRD3-4.

[0308]

Table 6

[0309] (Example 7) Humanization of mouse anti-TNFR2 antibody Clone 854 was a stronger agonistic antibody and was selected for further humanization. Humanization of clone 854 was performed by grafting the mouse hypervariable regions into various human frameworks.

[0310] The hypervariable regions of the heavy chain were defined using the SDR definition (specificity-determining residues), while the hypervariable regions of the light chain were defined using the CDR definition (complementary-determining regions).

[0311]

Table 7

[0312] Since the SDR definition rather than the CDR was used for grafting, mouse amino acids important for binding and affinity are preserved during the humanization process.

[0313] The human heavy chain frameworks selected for grafting of the 854 mouse heavy chain SDR were as follows: IGHJ4 * 01 In combination with the J-gene, IGHV4-31 * 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 frameworks selected for grafting of the 854 mouse light chain CDR were as follows: IGKJ4 * 01 In combination with the J-gene, IGKV3-11 * 01, IGKV1-9 * 01, IGKV1-33 * 01, IGKV1-27 * 01, IGKV1-39 * 01 and IGKV4-1 * 01.

[0314] (Example 8) Binding of the humanized TNFR2 antibody to human TNFR2 CHO cells All combinations of heavy and light chains were expressed and tested for binding to human TNFR2 CHO cells:

[0315]

Table 8

[0316] After grafting into various human frameworks, a significant loss of binding was observed, as summarized in Table 6. Some clones, e.g., 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).

[0317] (Example 9) Introduction of reverse mutations to restore binding to TNFR2 To restore binding to TNFR2 to the level of the parental 854 clone, various reverse mutations were introduced into a selected number of heavy chain graft constructs:

[0318]

Table 9

[0319] 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, and also with the same humanized light chains with germlined CDR2-L2.

[0320] (Example 10) Binding of humanized TNFR2 antibody to human TNFR2 A total of 114 clones were expressed, purified, and their binding to human TNFR2 CHO cells was compared to that of the parental 854 clone. As seen in Table 8 below, most of the clones showed an EC50 similar to or better than that of the parental clone 854. Clones 1847, 1765, 1753, and 1747 were selected for further characterization.

[0321]

Table 10-1

[0322]

Table 10-2

[0323]

Table 10-3

[0324] (Example 11) The humanized variants show a range of potencies in human and cyno PBMC assays.

[0325] The selected humanized clones were first tested in the human TNFR2 Jurkat reporter cell assay. The results in Table 9 show that clone 1765 exhibits an EC50 similar to that of the parental 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.

[0326] According to this result, clones 1847, 1765, and 1753 were tested in human and cyno PBMC assays.

[0327] Briefly, 250,000 human PBMCs from various donors were plated in wells of a 96-well plate in Optimizer CST medium. Each well was treated with 50 μl of 5 ng / ml IL-2 and 50 μl of 4× anti-TNFR2 antibody or TetraFab at 37 °C for 72 hours. Subsequently, the cells were prepared for flow cytometry analysis. After two washes in PBS, the cells were first resuspended in 100 μl of Live Near IR staining buffer at 4 °C for 30 minutes. After two additional washes in flow buffer, the cells were incubated with 1 / 40 diluted CD3 BV421, ICAM APC CD4 PE, and TIGIT BUV395 at 4 °C for 1 hour and then fixed with 1% PFA at room temperature for 15 minutes. After two additional washes in flow buffer, the cells were finally analyzed by flow cytometry. Doublets were excluded using light scatter, and low Near IR staining was used as an indicator of viability. Single live cells were gated against the CD3+ / CD4+ / TIGIT+ population, and the ICAM-1 mean fluorescence intensity (MFI) was measured for ICAM-1. The EC50 of the TNFR2 agonist for ICAM-1 induction was calculated using 4-parameter curve fitting in GraphPad Prism. ICAM-1 was selected because it is known to be regulated by NF-κB, 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 that were double positive for CD25+ / Ki67+ due to differences in activation marker expression and antibody cross-reactivity between human and cyno.

[0328] From the results in Table 9, it was confirmed that the humanized clone 1765 exhibited efficacy similar to that of the parental clone 854 in the human PBMC assay with EC50 values of 1.308 and 1.402 nM, respectively. The efficacy against cyno cells was 10-fold lower for both clones, with EC50 values of 13.63 and 15.02 nM, respectively. Clone 1753 showed particularly lower efficacy (56 nM) against cyno PBMC cells, and clone 1847 showed no efficacy against either cell type. Clone 1765 (heavy chain sequence, SEQ ID NO: 22 and light chain sequence, SEQ ID NO: 21) was selected for further investigation.

[0329]

Table 11

[0330] (Example 12) Aggregated anti-TNFR2 antibodies show increased efficacy in the Jurkat NF-KB-GFP reporter cell assay. To evaluate the effect of aggregates on TNFR2 agonism, the inventors compared anti-TNFR2 antibodies purified by a 1-step method (ProA purification only, containing on average 15 - 20% aggregates) with the same antibodies purified by a 2-step method (ProA followed by SEC purification, no aggregates) in the Jurkat NFKB-GFP reporter cell assay.

[0331] Briefly, 100,000 NF-KB-GFP Jurkat cells / well were plated in a 96-well plate in RPMI 1640 medium supplemented with 10% FBS, 1× sodium pyruvate, 1× Glutamax and Pen / Strep. Next, the cells were incubated overnight at 37 °C with serial dilutions of the anti-TNFR2 antibody. The next 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 dissociate the clumps. Finally, the cells were centrifuged again and resuspended in 75 ml of flow buffer containing 1 / 1000 diluted DAPI reagent and analyzed on a flow cytometer for GFP expression.

[0332]

Table 12

[0333] Clone 162, selected as a representative result, was compared with MR2-1 (Hycult Biotech, reference number HM2007), a commercially available TNFR2 agonist.

[0334] When purified on a ProA column, clone 162 showed similar potency to MR2-1 (EC50s were 347.3 and 119.6 ng / ml, respectively). Interestingly, the inventors found that both clone 162 and MR2-1 contained significant amounts of aggregates, as seen in the analytical SEC profiles in Figure 1 below.

[0335] If both clone 162 and MR2-1 were devoid of aggregates, their ability to agonize TNFR2 in the Jurkat cell assay was significantly impaired after purification by SEC. Clone 162 showed a 20-fold loss of potency (EC50 = 7.02 mg / ml), and MR2-1 showed a 50-fold loss (EC50 = 5.75 mg / ml), suggesting that aggregates can artificially increase anti-TNFR2 antibody potency.

[0336] This result prompted the inventors to investigate whether an increase in valency could lead to an increase in efficacy.

[0337] (Example 13) Reformatting to TetraFab To test the inventors' hypothesis, the inventors sought to increase the valency of Clone 1765 by adding additional paratopes onto its existing IgG scaffold and reformat it into TetraFab 2053 (see Figure 2). As depicted in the schematic, TetraFab is composed of a dual Fab heavy chain as well as CH2 and CH3 of a typical human IgG1, and the outer Fab domains are 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 and the same subsequent constant CH1. This longer heavy chain can associate with four light chains instead of two, creating a molecule composed of four paratopes that can theoretically bind four molecules of TNFR2. See SEQ ID NO: 30 and SEQ ID NO: 9 for the heavy and light chains of TetraFab 2053, respectively.

[0338] The rationale behind the reformatting to TetraFab is to mimic TNFR2 oligomerization by membrane-bound TNFα. Indeed, membrane-bound TNFα forms a trimer that can strongly agonize TNFR2 on the surface of Tregs by binding multiple TNFR2 molecules simultaneously.

[0339] 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 at a flow rate of 10 ml / min for 60 seconds. Next, the antibody or TetraFab was injected onto the captured TNFR2 at a concentration in the range of 10 - 1.25 nM for the antibody and 5 - 0.625 nM for TetraFab, over an association phase of 50 seconds at 80 ml / min. Subsequently, the dissociation phase was initiated with running buffer (HBS-EP+) injected at 80 ml / min for 600 seconds. The data was analyzed using Biacore Evaluation Software.

[0340]

Table 13

[0341] The data in Table 11 shows that both the parental 854 clone and the humanized 1765 clone have similar apparent affinities for human TNFR2, with KD values of 0.11 and 0.32 nM, respectively. TetraFab shows a stronger apparent affinity with a KD of 0.06 nM, which is a 5-fold improvement compared to its IgG counterparts 854 or 1765.

[0342] A similar but less significant improvement in affinity can be observed for cyno TNFR2. The affinity of TetraFab for cyno TNFR2 was one-tenth that for human TNFR2 (KD values of 0.61 and 0.06 nM, respectively).

[0343] (Example 14) Anti-TNFR2 antibodies and TetraFab agonize TNFR2 in the Jurkat NF-κB-GFP reporter cell assay TetraFab was first tested for its ability to induce NF-κB-GFP expression in the previously used TNFR2-expressing Jurkat cell reporter assay. Similarly, 100,000 cells / well were plated in 96-well plates in RPMI 1640 medium supplemented with 10% FBS, 1× sodium pyruvate, 1× Glutamax and Pen / Strep and incubated overnight at 37 °C with serial dilutions of anti-TNFR2 antibody or TetraFab. The next day, the cells were prepared for flow cytometry analysis as described above and analyzed on a flow cytometer for GFP expression.

[0344] The results summarized in Table 12 show that the parental clone 854 and its humanized variant 1765 have similar EC50s of 0.526 mg / ml and 1.009 mg / ml, respectively. In contrast, the EC50 of TetraFab 2053 is 0.037 mg / ml, showing a greater than 10-fold improvement compared to both 854 and 1765. This result suggests that the increase in valency promotes stronger efficacy.

[0345] [Table 14]

[0346] (Example 15) Anti-TNFR2 antibody and TetraFab induce upregulation of ICAM-1 in CD3+CD4+TIGIT+ human PBMC and induce co-expression of Ki67 / CD25 on CD4+ cells in cyno PBMC Next, TetraFab was tested for its ability to upregulate ICAM-1 in a TNFR2-expressing primary T cell population (CD3+CD4+TIGIT+) as described below. ICAM-1 was selected because it is regulated by NF-KB, which is known to be a marker of acute T cell activation, and has a known role in Treg-mediated suppression. TIGIT was used to identify a population of mature T cells and Tregs enriched for TNFR2 expression without the need to stain for the intracellular marker FoxP3.

[0347] Briefly, 250,000 human PBMCs from various donors were plated into wells of a 96-well plate in Optimizer CST medium. Each well was treated with 50 μl of 5 ng / ml IL-2 and 50 μl of 4× anti-TNFR2 antibody or TetraFab at 37 °C for 72 hours. Subsequently, the cells were prepared for flow cytometry analysis. After two washes in PBS, the cells were first resuspended in 100 μl of Live Near IR staining buffer at 4 °C for 30 minutes. After two additional washes in flow buffer, the cells were incubated with 1 / 40 diluted CD3 BV421, ICAM APC CD4 PE, and TIGIT BUV395 at 4 °C for 1 hour and then fixed with 1% PFA at room temperature for 15 minutes (all reagents are summarized in Table 13). After two additional washes in flow buffer, the cells were finally analyzed by flow cytometry. Doublets were excluded using light scatter, and low Near IR staining was used as an indicator of viability. Single viable cells were gated against the CD3+ / CD4+ / TIGIT+ population, and the ICAM-1 MFI was measured for ICAM-1. The EC50 of the TNFR2 agonist for ICAM-1 induction was calculated using 4-parameter curve fitting in GraphPad Prism. 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 that were double positive for CD25+ / Ki67+ due to differences in activation marker expression and antibody cross-reactivity between human and cyno.

[0348]

Table 15

[0349] As seen in Table 14, similar to the NF-KB-GFP Jurkat assay results, the EC50 for the upregulation of human ICAM-1 on the surface of human CD3+CD4+TIGIT+ cells is similar between the parental 854 antibody and the humanized 1765 antibody (1.402 and 1.308 mg / ml, respectively). The EC50 for the upregulation of ICAM-1 was improved 10-fold after treatment with TetraFab 2053, confirming that the increase in valency improves efficacy even in cases where the number of TNFR2 cell surface receptors is low (<11,000 in Jurkat and <500 in human Treg).

[0350]

Table 16

[0351] (Example 16) TetraFab 2053 elicits TNFR2 agonism-induced IKBa degradation in cultured human and cyno Treg The following assay was used as a surrogate for NF-KB activation resulting from TNFR2 agonism. This also enabled a direct comparison of anti-TNFR2 antibody pharmacology between human and cyno Treg using the same endpoint.

[0352] Briefly stated, human or cyno natural Tregs sorted from PBMCs of various 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 media containing 10 ng / ml of recombinant human or cyno IL2, respectively. The next day, the cells were seeded into 96-deep well plates (>50000 cells / well) and treated with various concentrations of TetraFab 2053, PMA / ionomycin (PMA 40 ng / ml; ionomycin 2 mM, positive control for IKBa) or media (unstimulated control) at 37°C for 20 minutes. After incubation, the cells were washed, fixed, permeabilized, and stained with anti-IKBa-PE antibody at 4°C for 30 minutes. Finally, the cells were analyzed by flow cytometry and the level of IKBa degradation was evaluated by MFI.

[0353]

Table 17

[0354] As detailed in Table 15 above, the mean EC50 for IKBa in expanded human Tregs was 26+ / -17 pM (n = 5), while that for expanded cyno Tregs was 145+ / -154 pM (n = 5). Despite the significant variability in the results between Tregs expanded from individual human and cyno donors, these data indicate consistent activation of the NF-KB pathway in Tregs and a loss of potency in cyno of up to one-fifth compared to humans.

[0355] (Example 17) Anti-TNFR2 TetraFab induces OX-40 upregulation in human and cyno splenocytes in the absence of IL-2 Pharmacological studies in cyno have revealed the activation of tissue Tregs after in vivo administration, as indicated by the upregulation of the TNF superfamily member OX-40. Therefore, 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 various donors were thawed and treated with various concentrations of TetraFab 2053 at 37 °C for 22 hours. Next, the samples were 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 an EC50 curve was generated in GraphPad Prism using a 4-parameter curve fit using the MFI of OX-40. The individual EC50 values generated for each donor of human or cyno frozen splenocytes are shown in this table. Table 16 summarizes the results obtained.

[0356]

Table 18

[0357] The mean EC50 for OX-40 upregulation on human splenic Tregs was 33 + / - 21 pM (n = 2), similar to the EC50 for IkBa degradation in expanded human blood Tregs, while the mean EC50 for OX-40 upregulation in cyno splenic Tregs was 958 + / - 455 pM (n = 5).

[0358] (Example 18) In Vivo Pharmacology of TetraFab 2053 in Cynomolgus Monkeys Male and female cynomolgus monkeys of Mauritian origin, older than 2.5 years, were acclimated for at least 30 days before starting dosing. Each dose group containing 1 male (M) and 1 female (F) animal was administered either vehicle control or TetraFab 2053 at 20 mg / kg, 60 mg / kg or 180 mg / kg 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 pharmacokinetic analysis. On day 16, all animals were euthanized and a portion of each spleen was collected for Treg phenotype analysis. Single cell spleen cell suspensions were prepared by conventional methods and then leukocytes were 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 Tregs: CD45+CD3+CD4+FoxP3+OX40+ · TIGIT-expressing Tregs: CD45+CD3+CD4+FoxP3+TIGIT+ · Proliferative Tregs: CD45+CD3+CD4+FoxP3+Ki-67+

[0359] The percentage and mean fluorescence intensity (MFI) of OX40 or TIGIT-expressing Tregs, and the percentage of proliferative Tregs were determined using flow cytometry analysis software. The fold increase in Treg parameters in TetraFab 2053-treated animals compared to sex-matched vehicle controls is shown in Table 17 below. The total TetraFab 2053 concentration in serum was determined using a ligand binding assay, and the area under the curve (AUC) concentration from days 11 - 15 was evaluated for each individual animal as shown in Table 17 below.

[0360]

Table 19

[0361] Administration of TetraFab 2053 at doses of 20 - 180 mg / kg / dose IV over 16 days to male and female cynomolgus monkeys increased OX - 40 and TIGIT expression on splenic Tregs by up to 3.14 - fold and 2.44 - fold, respectively, compared to vehicle - treated controls, and increased the percentage of proliferating splenic Tregs by 2.27 - fold. These increases in 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 was most likely reflective of maximal pharmacological activity.

[0362] Total body exposure increased with increasing dose in male cynomolgus monkeys in an approximately dose - proportional manner at 20 - 60 mg / kg and supra - dose - proportionally at 60 - 180 mg / kg. Total body exposure generally increased with increasing dose above a dose - proportional pattern at 20 - 180 mg / kg in female cynomolgus monkeys.

[0363] (Example 19) TetraFab 2053 shows acceptable developability 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).

[0364]

Table 20

[0365] The results summarized in the above table showed that TetraFab 2053 exhibited acceptable non - specific characteristics. Furthermore, this molecule was stable when tested at high concentrations at 4°C for 6 weeks and at 40°C for 4 weeks. Finally, TetraFab 2053 showed 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 manufacture as a biotherapeutic.

[0366] (Example 16) Optimization of TetraFab 2053 The following amino acids shown in Table 19 were targeted for optimization to remove sequence reliability (DE deamidation site, W oxidation site, predicted T cell epitope) and reduce hydrophobicity.

[0367] [Table 21]

[0368] Optimized constructs were made in IgG and TetraFab formats and the various mutations were tested alone or in combination for binding to human TNFR2 CHO as summarized in Table 20:

[0369] [Table 22-1]

[0370] [Table 22-2]

[0371] [Table 22-3]

[0372] [Table 22-4]

[0373] Surprisingly, all clones except for the two pairs of IgG and TetraFab completely lost their ability to bind to human TNFR2 expressed on the surface of CHO cells. Clones TetraFab 2208 / IgG 2204 and TetraFab 2209 / IgG 2205 were still able to bind to TNFR2, similar to IgG and TetraFab, but the Emax was significantly decreased compared to the parental clone 2053 / 1765. These clones were mutated only in CDR3-VL to remove the predicted T cell epitopes. In vitro analysis (not shown) indicated that the predicted T cell epitope of TetraFab 2053 was not presented on the surface of antigen-presenting cells and that a peptide overlapping with the predicted T cell epitope did not activate CD4+ T cells, suggesting a low immunogenic risk posed by its predicted T cell epitope. Therefore, these optimized variants could not be advanced previously.

[0374] (Example 17) TF-2053 provides long-term survival benefits in the NSG-GVHD model Objective: To test the preventive efficacy of human TetraFab-2053 in preventing xenogeneic graft-versus-host disease 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) that is mediated primarily by the transferred human CD4+ and CD8+ lymphocytes [1]. The transferred human lymphocytes do not undergo thymic education in the recipient mouse and thus are 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, such as tumor necrosis factor alpha (TNFa) and interferon gamma (IFNg), which result in organ dysfunction, weight loss, and death in addition to extensive tissue inflammatory infiltrates [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 selected to demonstrate the role of agonizing TNFR2 in reducing the expansion, activation, and pathophysiological function of human effector T cells 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 shown to be responsible for suppressing some autoreactive CD8 T cell activity [4].

[0375] Methods: Summary: 8- to 9-week-old NSG mice were purchased from Jackson Labs and acclimated at Pfizer's vivarium. All animal use and handling was performed under an IACUC-approved protocol. TetraFab-2053 or isotype control tetraFab was initiated 1 day prior to human PBMC administration and administered biweekly throughout the experiment at 3 mg / kg, 10 ml / kg. Previously cryopreserved human PBMCs were thawed on day 0 of the experiment, washed in PBS, and administered IV to n = 20 mice per group at 10×10 6 / 0.1 ml PBS. 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, depletion and senescence by flow cytometry, and circulating human cytokine levels by electrochemiluminescence MSD assay. Mice that lost more than 20% of their BW were euthanized according to the IACUC protocol and all remaining mice were sacrificed on day 78. At the time of death, spleen and liver weights were recorded and flow cytometry analysis was performed on splenocytes as well as blood.

[0376] Results: TF2053 provides long-term survival benefits in the NSG-GVHD model (Figure 3, Table 21). TF2053 reduces weight loss in the NSG-GVHD model (Figure 4, Table 22). Furthermore, in the NSG GVHD model, TF2053 reduces the circulating human cytokines IFNγ, IL10, IL17A, and 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 the CD4:CD8 engraftment ratio (Figure 12, Table 30).

[0377]

Table 23

[0378]

Table 24

[0379]

Table 25

[0380]

Table 26

[0381]

Table 27

[0382]

Table 28

[0383]

Table 29

[0384]

Table 30

[0385]

Table 31

[0386]

Table 32

[0387]

Table 33

[0388]

Table 34

[0389]

Table 35

[0390]

Table 36

[0391] Conclusion: 10 7Treatment of NSG mice with 3 mg / kg TF2053 starting 1 day prior to transfer of individual human PBMCs provided significant protection from GVHD. Survival was improved while weight loss was reduced. 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 ratio of CD4 to CD8 to favor CD8+ cells, reducing percent anergy while increasing the percentage of these CD8+ cells that were exhausted and senescent over time. In summary, the hypothesis that TNFR2 agonism can provide benefit in the humanized NSG-GVHD in vivo model through effects on effector T cells in the absence of regulatory T cell expansion was confirmed.

[0392] 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 for modeling human xenogeneic graft-versus-host disease". Methods Mol Biol, 2010. 602: p. 105-17. class II-deficient NOD-SCID IL2R gamma chain knockout mice for modeling human xenogeneic graft-versus-host disease)”. Methods Mol Biol, 2010. 602: p. 105-17. 2. Brehm, M.A. et al., "Lack of acute xenogeneic graft-versus-host disease, but retention of T cell function after engraftment of human peripheral blood mononuclear cells in NSG mice lacking 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." 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.

[0393] [Table 37-1]

[0394] [Table 37-2]

[0395] [Table 37-3]

[0396]

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 one or more selected from the group consisting of: (i) comprising CDR-H1, CDR-H2 and CDR-H3 sequences of a 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 a 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; (ii)(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; and (iii) 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.

23. A pharmaceutical composition comprising:

2. (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 pharmaceutical composition of claim 1 , comprising one or both of the following:

3. 2. The pharmaceutical composition of claim 1, 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.

4. The antibody of claim 1, comprising a VH sequence of SEQ ID NO:21 and a VL sequence of SEQ ID NO:

8.

5. 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.

6. 2. The pharmaceutical composition of claim 1, comprising one or both of a heavy chain (HC) comprising a sequence according to SEQ ID NO:22 and a light chain (LC) comprising a sequence according to SEQ ID NO:

9.

7. 2. The pharmaceutical composition of claim 1, wherein the antibody comprises a first Fab, a second Fab, a third Fab and a fourth Fab, each of the first Fab, the second Fab, the third Fab and the fourth Fab comprising 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.

8. 8. The pharmaceutical composition of claim 7, 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.

9. 8. The pharmaceutical composition of claim 7, 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.

10. The pharmaceutical composition of claim 1 , wherein the antibody comprises 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.

11. 2. The pharmaceutical composition of claim 1, wherein the antibody comprises 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.

12. (i) one or both of the VH sequence encoded by the plasmid deposited with the ATCC having ATCC Accession No. PTA-127528 and the VH sequence encoded by the plasmid deposited with the ATCC having ATCC Accession No. PTA-127529; and the VL sequence encoded by the plasmid deposited with the ATCC having ATCC Accession No. PTA-127531. (ii) 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. A pharmaceutical composition comprising an antibody comprising:

13. The antibody, (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 2. The pharmaceutical composition of claim 1, characterized by one or more selected from the group consisting of:

14. 10. The pharmaceutical composition of claim 1 for use as a medicament.

15. 15. The pharmaceutical composition of claim 14, 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).

16. 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 one or more selected from the group consisting of: (i) comprising CDR-H1, CDR-H2 and CDR-H3 sequences of a 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 a 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; (ii)(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; and (iii) 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. and a pharma- ceutically acceptable carrier.

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