Agonistic tumor necrosis factor receptor superfamily polypeptides

Abstract

Agonistic TNFR2 polypeptides, such as antibodies and antigen-binding fragments thereof, and the use of these polypeptides to stimulate the proliferation of regulatory T cells (Treg cells) and / or myeloid-derived suppressor cells (MDSCs), and to inhibit the function, reduce the proliferation, and / or directly kill T effector cells, such as CD8+ T effector cells, are described. Polypeptides, such as antibodies and antigen-binding fragments thereof, of the present disclosure can be used, for example, to suppress autoimmunity and inflammation, and to promote the protection, healing, preservation, and / or regeneration of a wide variety of tissues and organs, including tissues and organs containing TNFR2+ cells.

Description

[Technical field]

[0001] Technical Field The present invention relates to agonistic tumor necrosis factor receptor superfamily polypeptides. Sequence Listing This application contains a Sequence Listing that has been submitted electronically in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy, created on November 15, 2019, is entitled 00786-582WO2_Sequence_Listing_11.15.19_ST25 and is 28,722 bytes in size. [Background technology]

[0002] Maintaining control of cellular and humoral immune responses is a critical aspect of healthy immune system activity. Aberrant regulation of immune responses driven by T and B cells plays a causative role in pathologies such as autoimmune diseases, asthma, allergic reactions, graft-versus-host disease, graft rejection, and various other immune disorders, where inappropriate elevation of immune responses to various self and foreign antigens plays a causative role. These diseases are mediated by T and B lymphocytes that exhibit reactivity to self antigens and antigens derived from non-threatening sources such as allergens or transplanted allografts. Regulatory T cells (Treg cells) have evolved to inhibit the activity of immune cells that cross-react with "self" major histocompatibility complex (MHC) proteins and other benign antigens. Treg cells represent a heterogeneous class of T cells that can be distinguished based on the presentation of unique surface proteins. The best understood populations of Treg cells include CD4+, CD25+, FoxP3+ Treg cells, and CD17+ Treg cells. Although the exact mechanisms by which these cells mediate the suppression of autoreactive T cells are the subject of ongoing research, it has been shown that a specific class of Treg cells inhibits the production of the proliferation-inducing cytokine IL-2 in target T cells and may additionally sequester IL-2 from autoreactive cells by virtue of the affinity of CD25 (a subdomain of the IL-2 receptor) for IL-2 (Josefowicz et al., Ann. Rev. Immun., 30:531-564 (2012)). Furthermore, CD4+, CD25+, FoxP3+ Treg cells are also present in B cell-rich areas and suppress the suppression of T H Independently of their ability to attenuate the activity of 2 cells, it has been shown that they can directly suppress the production of immunoglobulins (Lim et al., J. Immunol., 175:4180-4183 (2005)).

[0003] Currently, there is a need for improved therapies that can enhance the survival and proliferation of Treg cells for use in the targeted treatment of diseases such as autoimmune diseases, graft-versus-host disease, allograft rejection, allergic reactions, asthma, and inflammation, among others. Summary of the Invention

[0004] Described herein are agonistic tumor necrosis factor receptor superfamily polypeptides, e.g., single chain polypeptides, antibodies, antigen-binding fragments thereof, and constructs thereof. For example, featured are agonistic tumor necrosis factor receptor 2 (TNFR2) binding polypeptides, e.g., single chain polypeptides, antibodies, antigen-binding fragments thereof, and constructs thereof. Human TNFR2 includes four cysteine-rich domains (CRDs): CRD1 (amino acid residues 48-76 of SEQ ID NO:1), CRD2 (amino acid residues 78-120 of SEQ ID NO:1), CRD3 (amino acid residues 121-162 of SEQ ID NO:1), and CRD4 (amino acid residues 162-202 of SEQ ID NO:1). Agonistic TNFR2 polypeptides described herein include those that bind to one or more epitopes within CRD1, CRD2, and / or CRD3 of TNFR2, e.g., those that bind to one or more epitopes of TNFR2 exclusively within CRD1 and / or CRD2, without binding to TNFR2 in CRD4.

[0005] The agonistic TNFR2 polypeptides described herein include human IgG1, IgG2, IgG3, and IgG4 isotype antibodies and antigen-binding fragments thereof that specifically bind to TNFR2 at one or more of the above epitopes. In some embodiments, the TNFR2 polypeptides of the present disclosure comprise a mutant human IgG1 or IgG2 heavy chain constant 1 (CH1) domain that lacks the cysteine ​​residue at position 127 of the CH1 domain amino acid sequence, such as, for example, a TNFR2 polypeptide (e.g., an antibody or antigen-binding fragment thereof) that comprises a mutant human IgG1 or IgG2 CH1 domain with a C127S mutation (according to Kabat numbering). The present disclosure is based in part on the surprising discovery that antibodies and antigen-binding fragments thereof exhibit significantly superior TNFR2 agonistic properties when these molecules are in the form of an IgG2-B isotype, compared to other antibody isotypes.

[0006] Agonistic TNFR2 polypeptides described herein also include polypeptides that bind to TNFR2 at at least two sites (e.g., a polypeptide has two or more antigen binding sites where TNFR2 is the "antigen"). These binding sites may be spatially separated from each other by less than about 133 Å. Advantageously, agonistic TNFR2 polypeptides with antigen binding sites separated by less than 133 Å specifically bind to TNFR2 at one or more epitopes described above, but activate TNFR2 signaling with higher potency than polypeptides that contain TNFR2 binding sites separated from each other by more than about 133 Å. For example, agonistic TNFR2 polypeptides of the present disclosure include IgG1 antibodies and antigen-binding fragments thereof that contain TNFR2 binding sites separated from each other by about 117 Å, and IgG3 antibodies and antigen-binding fragments thereof that contain TNFR2 binding sites separated from each other by about 125 Å.

[0007] Agonistic TNFR2 polypeptides adopting a single disulfide bond isoform and pharmaceutical compositions comprising the same are also featured. For example, pharmaceutical compositions of the present disclosure include those comprising agonistic TNFR2 binding polypeptides, for example, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99% or more of the polypeptides in the pharmaceutical composition are present in a single disulfide bond isoform. Agonistic TNFR2 binding polypeptides adopting the human IgG2-B isoform include IgG2-A, IgG2-A / B. 1 , and IgG2-A / B 2 The TNFR2 polypeptides that employ a single disulfide bond isoform exhibit substantially superior TNFR2 agonistic effects compared to TNFR2 binding polypeptides that employ other human IgG2 isoforms such as IgG2, IgG3, IgG4, IgG5, IgG6, IgG7, IgG8, IgG9, IgG10, IgG11, IgG12, IgG13, IgG14, IgG15, IgG16, IgG17, IgG18, IgG19, IgG11, IgG11, IgG15, IgG18, IgG19, IgG11, IgG12, IgG13, IgG14, IgG15, IgG15, IgG16, IgG17, IgG18, IgG19, IgG19, IgG15, IgG16, IgG17, IgG18, IgG19, IgG19, IgG19, IgG11, IgG12, IgG13, IgG14, IgG15, IgG15, IgG16, IgG17, IgG18, IgG19, IgG19, IgG14, IgG15, IgG16, IgG17, IgG18, IgG19 ...

[0008] The agonistic TNFR2 polypeptide of the present disclosure exhibits one or more beneficial biological properties, such as the ability to promote the proliferation and stabilization of regulatory T cells (Treg cells) and / or myeloid-derived suppressor cells (MDSCs). Additionally or alternatively, agonistic TNFR2 polypeptides can be administered to promote the reciprocal contraction and / or direct killing of T effector cells, such as cytotoxic CD8+ T cells. This can occur, for example, by indirectly expanding the proliferation and activity of Treg cells, or by directly killing T effector cells. Thus, the designation of TNFR2 polypeptides as agonistic refers to the ability to promote the proliferation and activity of Treg cells and / or MDSCs, and for clarity, indicates the activation of undesired T effector cell responses. The polypeptides described herein (e.g., single chain polypeptides, antibodies, antigen-binding fragments thereof, and constructs thereof) can be used to treat a variety of medical conditions, including, inter alia, autoimmune diseases, inflammatory diseases, and the like, and promote the growth, healing, protection, and / or regeneration of tissues that express TNFR2, such as neurons and brain-related tissues, making the polypeptides of the present disclosure useful for treating neurological diseases, such as, for example, Parkinson's disease, multiple sclerosis, Alzheimer's disease, and other neurodegenerative diseases.

[0009] In a first aspect, the invention features polypeptides, e.g., single chain polypeptides, antibodies, antigen-binding fragments thereof, and constructs thereof, that specifically bind human TNFR2 at an epitope defined by one or more amino acids in cysteine-rich domain (CRD) 1 (CRD1), CRD2, and / or CRD3, and do not specifically bind human TNFR2 at an epitope defined by one or more amino acids in CRD4. In some embodiments, the polypeptide is (a) a human IgG1 or IgG2 CH1 domain having a deletion or substitution at cysteine ​​residue 127, (b) comprising antigen-binding sites that are separated from each other by a distance of less than about 133 Å; (c) (i) GZ 1 TFZ 3 Z2 YZ 3 Z 4 (SEQ ID NO:2), (ii) GYTFTDYNI (SEQ ID NO: 3), or an amino acid sequence having up to two conservative amino acid substitutions thereto; or (iii) a complementarity determining region-heavy chain 1 (CDR-H1) having an amino acid sequence of GYTFTDYNL (SEQ ID NO: 4), or an amino acid sequence having up to two conservative amino acid substitutions thereto; Each Z 1 are, independently, naturally occurring amino acids comprising a side chain that comprises an aromatic substituent; Each Z 2 are independently naturally occurring amino acids that contain an anionic side chain at physiological pH; Each Z 3 are independently naturally occurring amino acids containing a polar, uncharged side chain at physiological pH; Each Z 4 is independently leucine or isoleucine, and / or (d)TJDJSJJJX 1 YX 2 X 3 LJX 4 LJS (SEQ ID NO:5), or a framework region having an amino acid sequence having 10 or more of the residues of SEQ ID NO:5, wherein each J is independently a naturally occurring amino acid, and each X 1 is independently A, V, or F, and each X 2 is independently M or I, and each X 3 is independently E or Q, and each X 4 are independently S or R.

[0010] In some embodiments, a polypeptide, such as a single chain polypeptide, antibody, antigen-binding fragment thereof, or construct thereof, comprises a human IgG1 or IgG2 CH1 domain that lacks a cysteine ​​residue at position 127 (according to the Kabat numbering scheme) of the amino acid sequence of the IgG1 or IgG2 CH1 domain. For example, a polypeptide (e.g., a single chain polypeptide, antibody, antigen-binding fragment thereof, or construct thereof) can comprise a human IgG1 or IgG2 CH1 domain that has an amino acid other than cysteine, such as a serine residue, at position 127 of the amino acid sequence of the CH1 domain.

[0011] In some embodiments, the IgG1 CH1 domain is

[0012] ASTKGPSVFPLAPCSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKV (SEQ ID NO:6), provided, for example, that the IgG1 CH1 domain comprises a serine residue at position 127 of the amino acid sequence of the IgG1 CH1 domain (according to the Kabat numbering described herein). The IgG1 CH1 domain may, for example, have an amino acid sequence that is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO:6, provided, for example, that the IgG1 CH1 domain comprises a serine residue at position 127 of the amino acid sequence of the IgG1 CH1 domain. In some embodiments, the IgG1 CH1 domain has an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:6, provided, for example, that the IgG1 CH1 domain comprises a serine residue at position 127 of the amino acid sequence of the IgG1 CH1 domain. In some embodiments, the IgG1 CH1 domain has the amino acid sequence of SEQ ID NO:6. In some embodiments, the IgG1 CH1 domain has an amino acid sequence that differs from SEQ ID NO:6 only by a C127S substitution (according to Kabat numbering as described herein). In these embodiments, the IgG1 CH1 domain comprises ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKV It has the amino acid sequence of (SEQ ID NO:7).

[0013] In some embodiments, the IgG2 CH1 domain comprises: ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTV (SEQ ID NO: 8), provided, for example, that the IgG2 CH1 domain comprises a serine residue at position 127 of the amino acid sequence of the IgG2 CH1 domain (according to the Kabat numbering described herein). The IgG2 CH1 domain may, for example, have an amino acid sequence that is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical) to the amino acid sequence of SEQ ID NO: 8, provided, for example, that the IgG2 CH1 domain comprises a serine residue at position 127 of the amino acid sequence of the IgG2 CH1 domain. In some embodiments, the IgG2 CH1 domain has an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 8, provided, for example, that the IgG2 CH1 domain comprises a serine residue at position 127 of the amino acid sequence of the IgG2 CH1 domain. In some embodiments, the IgG2 CH1 domain has the amino acid sequence of SEQ ID NO: 8. In some embodiments, the IgG2 CH1 domain has an amino acid sequence that differs from SEQ ID NO: 8 only by a C127S substitution (according to Kabat numbering as described herein). In these embodiments, the IgG2 CH1 domain comprises ASTKGPSVFPLAPSSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTV It has the amino acid sequence of (SEQ ID NO:9).

[0014] In some embodiments, the polypeptide (eg, a single chain polypeptide, an antibody, an antigen-binding fragment thereof, or a construct thereof) has an IgG3 or IgG4 isotype.

[0015] A polypeptide (e.g., a single chain polypeptide, an antibody, an antigen-binding fragment thereof, or a construct thereof) may be spaced apart from the nucleus by a distance of less than 133 Å (e.g., a distance of about 90 Å to about 132 Å, e.g., about 90 Å, 91 Å, 92 Å, 93 Å, 94 Å, 95 Å, 96 Å, 97 Å, 98 Å, 99 Å, 100 Å, 101 Å, 102 Å, 103 Å, 104 Å, 105 Å, 106 Å, 107 Å, 108 Å, 109 Å, 110 Å, 111 Å, 112 Å, 113 Å, 114 Å, 115 Å, 116 Å, 117 Å, 118 Å, 119 Å, 120 Å, 121 Å, 122 Å, 123 Å, 124 Å, 125 Å, 126 Å, 127 Å, 128 Å, 129 Å, 130 Å, 131 Å, 132 Å, 133 Å, 134 Å, 135 Å, 136 Å, 137 Å, 138 Å, 139 Å, 140 Å, 141 Å, 142 Å, 143 Å, 144 Å, 145 Å, 146 Å, 147 Å, 148 Å, 149 Å, 150 Å, 151 Å, 152 Å 13 Å, 114 Å, 115 Å, 116 Å, 117 Å, 118 Å, 119 Å, 120 Å, 121 Å, 122 Å, 123 Å, 124 Å, 125 Å, 126 Å, 127 Å, 128 Å, 129 Å, 130 Å, 131 Å, or 132 Å). In some embodiments, the antigen binding sites are separated from one another by a distance of about 95 Å to about 130 Å, e.g., by a distance of about 95 Å, 96 Å, 97 Å, 98 Å, 99 Å, 100 Å, 101 Å, 102 Å, 103 Å, 104 Å, 105 Å, 106 Å, 107 Å, 108 Å, 109 Å, 110 Å, 111 Å, 112 Å, 113 Å, 114 Å, 115 Å, 116 Å, 117 Å, 118 Å, 119 Å, 120 Å, 121 Å, 122 Å, 123 Å, 124 Å, 125 Å, 126 Å, 127 Å, 128 Å, 129 Å, or 130 Å. In some embodiments, the antigen binding sites are separated from one another by a distance of about 98 Å to about 128 Å, e.g., by a distance of about 98 Å, 99 Å, 100 Å, 101 Å, 102 Å, 103 Å, 104 Å, 105 Å, 106 Å, 107 Å, 108 Å, 109 Å, 110 Å, 111 Å, 112 Å, 113 Å, 114 Å, 115 Å, 116 Å, 117 Å, 118 Å, 119 Å, 120 Å, 121 Å, 122 Å, 123 Å, 124 Å, 125 Å, 126 Å, 127 Å, or 128 Å.

[0016] For example, a polypeptide (e.g., a single chain polypeptide, an antibody, an antigen-binding fragment thereof, or a construct thereof) can comprise antigen-binding sites that are separated from each other by a distance of about 105 Å to about 127 Å, e.g., about 105 Å, 106 Å, 107 Å, 108 Å, 109 Å, 110 Å, 111 Å, 112 Å, 113 Å, 114 Å, 115 Å, 116 Å, 117 Å, 118 Å, 119 Å, 120 Å, 121 Å, 122 Å, 123 Å, 124 Å, 125 Å, 126 Å, or 127 Å. In some embodiments, the antigen binding sites are separated from one another by a distance of about 110 Å to about 122 Å, e.g., about 110 Å, 111 Å, 112 Å, 113 Å, 114 Å, 115 Å, 116 Å, 117 Å, 118 Å, 119 Å, 120 Å, 121 Å, or 122 Å. In some embodiments, the antigen binding sites are separated from one another by a distance of about 115 Å to about 119 Å, e.g., about 115 Å, 116 Å, 117 Å, 118 Å, or 119 Å. In some embodiments, the antigen binding sites are separated from one another by a distance of 117 Å. In some embodiments, the polypeptide (e.g., single chain polypeptide, antibody, antigen-binding fragment thereof, or construct thereof) has a spacing of the antigen binding arms as described above and has an IgG1 isotype.

[0017] In some embodiments, a polypeptide (e.g., a single chain polypeptide, an antibody, an antigen-binding fragment thereof, or a construct thereof) comprises antigen-binding sites that are separated from one another by a distance of about 115 Å to about 132 Å, e.g., about 115 Å, 116 Å, 117 Å, 118 Å, 119 Å, 120 Å, 121 Å, 122 Å, 123 Å, 124 Å, 125 Å, 126 Å, 127 Å, 128 Å, 129 Å, 130 Å, 131 Å, or 132 Å. In some embodiments, the antigen-binding sites are separated from one another by a distance of about 120 Å to about 129 Å, e.g., about 120 Å, 121 Å, 122 Å, 123 Å, 124 Å, 125 Å, 126 Å, 127 Å, 128 Å, or 129 Å. In some embodiments, the antigen binding sites are separated from each other by a distance of about 123 Å to about 127 Å, e.g., about 123 Å, 124 Å, 125 Å, 126 Å, or 127 Å. In some embodiments, the antigen binding sites are separated from each other by a distance of 125 Å. In some embodiments, the polypeptide (e.g., single chain polypeptide, antibody, antigen-binding fragment thereof, or construct thereof) has a spacing of the antigen binding arms as described above and has an IgG3 isotype.

[0018] In some embodiments, a polypeptide (e.g., a single chain polypeptide, an antibody, an antigen-binding fragment thereof, or a construct thereof) is (a) GZ 1 TFZ 3 Z 2 YZ 3 Z 4 (SEQ ID NO:2), (b) GYTFTDYNI (SEQ ID NO: 3), or an amino acid sequence having up to two conservative amino acid substitutions thereto; or (c) a CDR-H1 having an amino acid sequence of GYTFTDYNL (SEQ ID NO: 4), or an amino acid sequence having up to two conservative amino acid substitutions thereto; Each Z 1 are, independently, naturally occurring amino acids comprising a side chain that comprises an aromatic substituent; Each Z 2are independently naturally occurring amino acids that contain an anionic side chain at physiological pH; Each Z 3 are independently naturally occurring amino acids containing a polar, uncharged side chain at physiological pH; Each Z 4 are independently leucine or isoleucine.

[0019] In some embodiments, a polypeptide (e.g., a single chain polypeptide, an antibody, an antigen-binding fragment thereof, or a construct thereof) specifically binds to human TNFR2 at an epitope defined by one or more amino acids in CRD1, e.g., at an epitope defined by one or more amino acid residues 56-60 (KCSPG) in SEQ ID NO:1. In some embodiments, a polypeptide specifically binds to human TNFR2 at an epitope defined by one or more amino acids in CRD2. In some embodiments, a polypeptide specifically binds to human TNFR2 at an epitope defined by one or more amino acids in CRD3.

[0020] In some embodiments, a polypeptide (e.g., a single chain polypeptide, an antibody, an antigen-binding fragment thereof, or a construct thereof) is (a) a CDR-H2 having the amino acid sequence of INPNYDST (SEQ ID NO: 10), or an amino acid sequence having up to two conservative amino acid substitutions thereto; (b) a CDR-H3 having the amino acid sequence CARGNSWYFDV (SEQ ID NO: 11), or an amino acid sequence having up to two conservative amino acid substitutions thereto; (c) a CDR-L1 having the amino acid sequence SSVRY (SEQ ID NO: 12), or an amino acid sequence having up to two conservative amino acid substitutions thereto; (d) a CDR-L2 having the amino acid sequence LTS or an amino acid sequence having up to two conservative amino acid substitutions thereto; and (e) further comprising one or more or all of the CDRs of CDR-L3 having the amino acid sequence CQQWSSNPLT (SEQ ID NO: 13), or an amino acid sequence having up to two conservative amino acid substitutions thereto.

[0021] In some embodiments, a polypeptide (e.g., a single chain polypeptide, an antibody, an antigen-binding fragment thereof, or a construct thereof) is (a) CDR-H2 having the amino acid sequence INPNYDST (SEQ ID NO: 10); (b) CDR-H3 having the amino acid sequence CARGNSWYFDV (SEQ ID NO: 11); (c) CDR-L1 having the amino acid sequence SSVRY (SEQ ID NO: 12); (d) a CDR-L2 having the amino acid sequence LTS, and (e) comprising one or more or all of the CDRs of CDR-L3 having the amino acid sequence CQQWSSNPLT (SEQ ID NO: 13).

[0022] In some embodiments, CDR-H1 has the amino acid sequence GYTFTDYNI (SEQ ID NO:3), or an amino acid sequence having up to two conservative amino acid substitutions thereto.

[0023] In some embodiments, the polypeptide comprises: (a) CDR-H1 having the amino acid sequence GYTFTDYNI (SEQ ID NO:3); (b) CDR-H2 having the amino acid sequence INPNYDST (SEQ ID NO: 10); (c) CDR-H3 having the amino acid sequence CARGNSWYFDV (SEQ ID NO: 11); (d) CDR-L1 having the amino acid sequence SSVRY (SEQ ID NO: 12); (e) a CDR-L2 having the amino acid sequence LTS, and (f) CDR-L3 having the amino acid sequence CQQWSSNPLT (SEQ ID NO: 13).

[0024] In some embodiments, a polypeptide (e.g., a single chain polypeptide, an antibody, an antigen-binding fragment thereof, or a construct thereof) comprises a heavy chain variable domain (V) having an amino acid sequence that is at least 85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 14. H In some embodiments, the polypeptide comprises a heavy chain variable domain (V) having an amino acid sequence that is at least 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO:14. H In some embodiments, the polypeptide comprises a heavy chain variable domain (V) having an amino acid sequence that is at least 95% (e.g., at least 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO:14. H In some embodiments, the polypeptide comprises a heavy chain variable domain (V) having the amino acid sequence of SEQ ID NO: 14. H ).

[0025] In some embodiments, CDR-H1 has the amino acid sequence GYTFTDYNL (SEQ ID NO: 4), or an amino acid sequence having up to two conservative amino acid substitutions thereto.

[0026] In some embodiments, the polypeptide comprises: (a) CDR-H1 having the amino acid sequence GYTFTDYNL (SEQ ID NO: 4); (b) CDR-H2 having the amino acid sequence INPNYDST (SEQ ID NO: 10); (c) CDR-H3 having the amino acid sequence CARGNSWYFDV (SEQ ID NO: 11); (d) CDR-L1 having the amino acid sequence SSVRY (SEQ ID NO: 12); (e) a CDR-L2 having the amino acid sequence LTS, and (f) CDR-L3 having the amino acid sequence CQQWSSNPLT (SEQ ID NO: 13).

[0027] In some embodiments, a polypeptide (e.g., a single chain polypeptide, an antibody, an antigen-binding fragment thereof, or a construct thereof) comprises a heavy chain variable domain (V) having an amino acid sequence that is at least 85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 15. H In some embodiments, the polypeptide comprises a heavy chain variable domain (V) having an amino acid sequence that is at least 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO:15. H In some embodiments, the polypeptide comprises a heavy chain variable domain (V) having an amino acid sequence that is at least 95% (e.g., at least 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 15. H In some embodiments, the polypeptide comprises a heavy chain variable domain (V) having the amino acid sequence of SEQ ID NO: 15. H ).

[0028] In some embodiments, a polypeptide (e.g., a single chain polypeptide, an antibody, an antigen-binding fragment thereof, or a construct thereof) comprises a non-native constant region, such as a human constant region. In some embodiments, the polypeptide lacks all or a portion of an Fc domain, lacks all or a portion of a native Fc domain, or lacks an Fc domain altogether.

[0029] In some embodiments, the polypeptide (e.g., a single chain polypeptide, an antibody, an antigen-binding fragment thereof, or a construct thereof) is selected from the group consisting of TJDJSJJJX 1 YX 2 X 3 LJX 4LJS (SEQ ID NO:5), or an amino acid sequence having 10 or more of the residues of SEQ ID NO:5, wherein each J is independently a naturally occurring amino acid, and each X 1 is independently A, V, or F, and each X 2 is independently M or I, and each X 3 is independently E or Q, and each X 4 is independently S or R. For example, a polypeptide can be (a) TVDKSSSTAYMELRSLTS (SEQ ID NO: 16), or an amino acid sequence having up to two conservative amino acid substitutions thereto; (b) TADTSSNTAYIQLSSLTS (SEQ ID NO: 17), or an amino acid sequence having up to two conservative amino acid substitutions thereto; (c) TADTSTDTAYMELSSLRS (SEQ ID NO: 18), or an amino acid sequence having up to two conservative amino acid substitutions thereto; (d) TRDTSISTAYMELSRLTS (SEQ ID NO: 19), or an amino acid sequence having up to two conservative amino acid substitutions thereto; (e) TFYMELSSLRS (SEQ ID NO: 20), or an amino acid sequence having up to two conservative amino acid substitutions thereto; (f) TRDTSISTAYMELNRLTS (SEQ ID NO: 21), or an amino acid sequence having up to two conservative amino acid substitutions thereto; (g) TRDTSTNTVYMELTSLRS (SEQ ID NO: 22), or an amino acid sequence having up to two conservative amino acid substitutions thereto; and (h) a framework region having an amino acid sequence selected from TADTSTDRAYMELSSLRS (SEQ ID NO: 23), or an amino acid sequence having up to two conservative amino acid substitutions thereto.

[0030] In some embodiments, the framework region is located adjacent to the CDR-H2 in a polypeptide (e.g., a single chain polypeptide, an antibody, an antigen-binding fragment thereof, or a construct thereof). In some embodiments, the framework region is located adjacent to the CDR-H3 in a polypeptide. In some embodiments, the framework region is located between the CDR-H2 and CDR-H3.

[0031] In some embodiments, a polypeptide (e.g., a single chain polypeptide, an antibody, an antigen-binding fragment thereof, or a construct thereof) is (a) amino acids 56 to 60 of SEQ ID NO:1 (KCSPG); (b) amino acids 101 to 107 (CSSDQVET) of SEQ ID NO:1; (c) amino acids 115 to 142 of SEQ ID NO: 1 (NRICTCRPGWYCALSKQEGCRLCAPLRK); (d) amino acids 26 to 45 of SEQ ID NO: 1 (QTAQMCCSKCSPGQHAKVFC); (e) amino acids 90 to 109 of SEQ ID NO: 1 (REQNRICTCRPGWYCALSKQ); (f) amino acids 98 to 117 of SEQ ID NO: 1 (CRPGWYCALSKQEGCRLCAP); (g) amino acids 106 to 125 (LSKQEGCRLCAPLRKCRPGF) of SEQ ID NO:1, and / or (h) specifically binds to an epitope of TNFR2 corresponding to one or more amino acid residues among amino acids 108 to 127 (KQEGCRLCAPLRKCRPGFGV) of SEQ ID NO:1.

[0032] In some embodiments, the polypeptide (e.g., a single chain polypeptide, an antibody, an antigen-binding fragment thereof, or a construct thereof) has a K D , e.g., a K of less than about 1 nM D For example, the polypeptide (e.g., a single chain polypeptide, an antibody, an antigen-binding fragment thereof, or a construct thereof) has a K of about 1 pM to about 10 nM. DFor example, at about 1 pM, 5 pM, 10 pM, 15 pM, 20 pM, 25 pM, 30 pM, 35 pM, 40 pM, 45 pM, 50 pM, 55 pM, 60 pM, 65 pM, 70 pM, 75 pM, 80 pM, 85 pM, 90 pM, 95 pM, 100 pM, 105 pM, 110 pM, 115 pM, 120 pM, 125 pM, 130 pM, 135 pM, 140 pM, 145 pM, 150 pM, 155 pM, 160 pM, 165 pM, 170 pM, 175 pM, 180 pM, 185 pM, 190 pM, 195 pM, 200 pM, 205 pM, 2 10pM, 215pM, 220pM, 225pM, 230pM, 235pM, 240pM, 245pM, 250pM, 255pM, 260 pM, 265pM, 270pM, 275pM, 280pM, 285pM, 290pM, 295pM, 300pM, 305pM, 310pM, 315pM, 320pM, 325pM, 330pM, 335pM, 340pM, 345pM, 350pM, 355pM, 360pM, 36 5pM, 370pM, 375pM, 380pM, 385pM, 390pM, 395pM, 400pM, 405pM, 410pM, 415pM , 420pM, 425pM, 430pM, 435pM, 440pM, 445pM, 450pM, 455pM, 460pM, 465pM, 4 70pM, 475pM, 480pM, 485pM, 490pM, 495pM, 500pM, 505pM, 510pM, 515pM, 520 pM, 525pM, 530pM, 535pM, 540pM, 545pM, 550pM, 555pM, 560pM, 565pM, 570pM , 575pM, 580pM, 585pM, 590pM, 595pM, 600pM, 605pM, 610pM, 615pM, 620pM, 62 5pM, 630pM, 635pM, 640pM, 645pM, 650pM, 655pM, 660pM, 665pM, 670pM, 675p M, 680pM, 685pM, 690pM, 695pM, 700pM, 705pM, 710pM, 715pM, 720pM, 725pM, 7 30pM, 735pM, 740pM, 745pM, 750pM, 755pM, 760pM, 765pM, 770pM, 775pM, 780 pM, 785pM, 790pM, 795pM, 800pM, 805pM, 810pM, 815pM, 820pM, 825pM, 830pM,K of 835pM, 840pM, 845pM, 850pM, 855pM, 860pM, 865pM, 870pM, 875pM, 880pM, 885pM, 890pM, 895pM, 900pM, 905pM, 910pM, 915pM, 920pM, 925pM, 930pM, 935pM, 940pM, 945pM, 950pM, 955pM, 960pM, 965pM, 970pM, 975pM, 980pM, 985pM, 990pM, 995pM, 1nM, 5nM, or 10nM, D and capable of specifically binding to TNFR2.

[0033] The polypeptide (e.g., a single chain polypeptide, an antibody, an antigen-binding fragment thereof, or a construct thereof) is at least about 10 4 M -1 s -1 , or 10 5 M -1 s -1 k on For example, about 1×10 4 M -1 s -1 ~Approx. 1×10 8 M -1 s -1 k on For example, a polypeptide (e.g., a single chain polypeptide, an antibody, an antigen-binding fragment thereof, or a construct thereof) can be at about 1×10 4 M -1 s -1 , 2×10 4 M -1 s -1 , 3×10 4 M -1 s -1 , 4×10 4 M -1 s -1 , 5×10 4 M -1 s -1 , 6×10 4 M -1 s -1 , 7×10 4 M -1 s -1 , 8×10 4 M -1 s-1 、9×10 4 M -1 s -1 、1×10 5 M -1 s -1 、2×10 5 M -1 s -1 、3×10 5 M -1 s -1 、4×10 5 M -1 s -1 、5×10 5 M -1 s -1 、6×10 5 M -1 s -1 、7×10 5 M -1 s -1 、8×10 5 M -1 s -1 、9×10 5 M -1 s -1 、1×10 6 M -1 s -1 、2×10 6 M -1 s -1 、3×10 6 M -1 s -1 、4×10 6 M -1 s -1 、5×10 6 M -1 s -1 、6×10 6 M -1 s -1 、7×10 6 M -1 s -1 、8×10 6 M -1 s -1 、9×10 6 M -1 s -1 、1×10 7 M -1 s -1 、2×10 7 M -1 s -1 、3×10 7 M-1 s -1 , 4×10 7 M -1 s -1 , 5×10 7 M -1 s -1 , 6×10 7 M -1 s -1 , 7×10 7 M -1 s -1 , 8×10 7 M -1 s -1 , 9×10 7 M -1 s -1 , or 1 × 10 8 M -1 s -1 k on and can specifically bind to TNFR2 to form an antibody-antigen complex.

[0034] The polypeptide (e.g., a single chain polypeptide, an antibody, an antigen-binding fragment thereof, or a construct thereof) specifically binds to TNFR2 and is capable of inhibiting, for example, about 10 -3 s -1 or 10 -4 s -1 k off , for example, about 10 -6 s -1 ~about 10 -3 s -1 k off (For example, about 1×10 -6 s -1 , 2×10 -6 s -1 , 3×10 -6 s -1 , 4×10 -6 s -1 , 5×10 -6 s -1 , 6×10 -6 s -1 , 7×10 -6 s -1 , 8×10 -6 s -1 , 9×10 -6 s -1 , 1×10 -5 s -1 , 2×10 -5s -1 , 3×10 -5 s -1 , 4×10 -5 s -1 , 5×10 -5 s -1 , 6×10 -5 s -1 , 7×10 -5 s -1 , 8×10 -5 s -1 , 9×10 -5 s -1 , 1×10 -4 s -1 , 2×10 -4 s -1 , 3×10 -4 s -1 , 4×10 -4 s -1 , 5×10 -4 s -1 , 6×10 -4 s -1 , 7×10 -4 s -1 , 8×10 -4 s -1 , 9×10 -4 s -1 , or 1 × 10 -3 s -1 k off ) can form an antibody-antigen complex that dissociates at 100 .mu.m.

[0035] The polypeptides, e.g., single chain polypeptides, antibodies, antigen-binding fragments thereof, and constructs thereof described herein are capable of binding to cells expressing TNFR2, e.g., Treg cells (e.g., CD25 HiIn some embodiments, the polypeptides may stimulate TNFR2 signaling in TNFR2+ tissues, such as Treg cells expressing TNFR2, myeloid-derived suppressor cells (MDSCs), and / or TNFR2+ tissues, such as neurons or other cells of the central nervous system, thereby promoting cellular healing, regeneration, proliferation, and / or protection. ... The agonist TNFR2 single chain polypeptides, antibodies, antigen-binding fragments thereof, and constructs thereof may promote the expression or post-translational modification (e.g., phosphorylation) of one or more proteins involved in the activation of Tregs, MDSCs, and / or TNFR2+ cells. The polypeptides may, for example, reduce the expression of IL4.

[0036] For example, the polypeptides of the present disclosure include those that inhibit cIAP2, TRAF2, Etk, VEGFR2, PI3K, Akt, proteins involved in the angiogenesis pathway, the IKK complex, RIP, NIK, MAP3K, proteins involved in the NFkB pathway, NIK, JNK, AP-1, a MEK (e.g., MEK1, MEK7), MKK3, NEMO, IL2R, Foxp3, IL2, TNF, lymphotoxin, lymphotoxin alpha, lymphotoxin beta, TNFSF18, IL2RA, IKZF2 / 4, CTLA4, TGF-beta, CHUK, NFKBIE, NFKBIA, MAP3K11, TRAF3, relB, TNF, CXCR3, PDL1 (CD274), IL2RA, IL7R, MAP3K1, MAP, MAP3K4, NFKBIB, TANK, TBK1, TNFAIP3, NFKBIA, TNFRSF1B, TRAF2, relB, LTA, EP300, and CREBBP. The expression or post-translational modification (e.g., phosphorylation) of one or more proteins selected from the group consisting of the agonistic TNFR2 single chain polypeptides, antibodies, antigen-binding fragments thereof, or constructs thereof may be increased by, for example, about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% compared to the expression or post-translational modification (e.g., phosphorylation) of one or more of these proteins isolated from a sample not treated with the agonistic TNFR2 single chain polypeptides, antibodies, antigen-binding fragments thereof, or constructs thereof described herein. Exemplary assays that can be used to determine expression levels and phosphorylation status are known in the art and include, for example, Western blot assays to determine protein content and quantitative reverse transcription polymerase chain reaction (RT-PCR) experiments to determine mRNA content. In some embodiments, TNFR2 polypeptides (e.g., single chain polypeptides, antibodies, antigen-binding fragments thereof, and constructs thereof) are predominant TNFR2 agonists, and their agonistic activity can be further enhanced when used in combination with natural TNFR2 ligands, such as TNFα, Bacillus Calmette-Guerin (BCG), and / or growth promoters, such as IL-2.

[0037] Agonist TNFR2 polypeptides, such as single chain polypeptides, antibodies, antigen-binding fragments thereof, and constructs thereof, can stimulate a metabolic shift from glycolysis to glutaminolysis and fatty acid oxidation in peripheral lymphocytes (e.g., human peripheral lymphocytes), which is evidence of increased proliferation of Treg cells.

[0038] The agonist TNFR2 polypeptides described herein, e.g., single chain polypeptides, antibodies, antigen-binding fragments thereof, and constructs thereof, may induce an increase in itaconic acid production (e.g., about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 200%, 300%, 400%, 500%, 1,000% or more compared to cells (e.g., peripheral lymphocytes) that are not treated with the agonist TNFR2 polypeptide.

[0039] Agonist TNFR2 polypeptides described herein (eg, single chain polypeptides, antibodies, antigen-binding fragments thereof, and constructs thereof) may exhibit one or more, or all of the following properties. (a) promoting proliferation or increasing stability and persistence of Treg cells, e.g., by binding to and activating TNFR2 on the surface of Treg cells (e.g., thereby increasing the proliferation or increasing the stability and persistence of Treg cells by about 1.1 fold, 1.2 fold, 1.3 fold, 1.4 fold, 1.5 fold, 1.6 fold, 1.7 fold, 1.8 fold, 1.9 fold, 2 fold, 2.1 fold, 2.2 fold, 2.3 fold, 2.4 fold, 2.5 fold, 2.6 fold, 2.7 fold, 2.8 fold, 2.9 fold, 3 fold, 3.1 fold, 3.2 fold, 3.3 fold, 3.4 fold, 3.5 fold, 3.6 fold, 3.7 fold, 3.8 fold, 3.9 fold, 4 fold, 4.1 fold, 4.2 fold, 4.3 fold, 4.4 fold, 4.5 fold, 4.6 fold, 4.7 fold, 4.8 fold, 4.9 fold, 5 fold, 5.1 fold, 5 fold, 6 fold, 7 fold, 8 fold, 9 fold, 10 fold, 11 fold, 12 fold, 13 fold, 14 fold, 15 fold, 16 fold, 17 fold, 18 fold, 19 fold, 20 fold, 21 fold, 22 fold, 23 fold, 24 fold, 25 fold, 26 fold, 27 fold, 28 fold, 29 fold, 30 fold, 31 fold, 32 fold, 33 fold, 2x, 5.3x, 5.4x, 5.5x, 5.6x, 5.7x, 5.8x, 5.9x, 6x, 6.1x, 6.2x, 6.3x, 6.4x, 6.5x, 6.6x, 6.7x, 6 .8x, 6.9x, 7x, 7.1x, 7.2x, 7.3x, 7.4x, 7.5x, 7.6x, 7.7x, 7.8x, 7.9x, 8x, 8.1x, 8.2x, 8.3x, 8. 4-fold, 8.5-fold, 8.6-fold, 8.7-fold, 8.8-fold, 8.9-fold, 9-fold, 9.1-fold, 9.2-fold, 9.3-fold, 9.4-fold, 9.5-fold, 9.6-fold, 9.7-fold, 9.8-fold, 9.9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, or more, increase the quality or quantity of Treg cells in a population of cells); (b) promoting proliferation or increasing stability of MDSCs, e.g., by binding to and activating TNFR2 on the surface of MDSCs (e.g., by increasing the proliferation or stability of MDSCs by about 1.1 fold, 1.2 fold, 1.3 fold, 1.4 fold, 1.5 fold, 1.6 fold, 1.7 fold, 1.8 fold, 1.9 fold, 2 fold, 2.1 fold, 2.2 fold, 2.3 fold, 2.4 fold, 2.5 fold, 2.6 fold, 2.7 fold, 2.8 fold, 2.9 fold, 3 fold, 3.1 fold, 3.2 fold, 3.3 fold, 3.4 fold, 3.5 fold, 3.6 fold, 3.7 fold, 3.8 fold, 3.9 fold, 4 fold, 4.1 fold, 4.2 fold, 4.3 fold, 4.4 fold, 4.5 fold, 4.6 fold, 4.7 fold, 4.8 fold, 4.9 fold, 5 fold, 5.1 fold, 5.2 fold, 5.3x, 5.4x, 5.5x, 5.6x, 5.7x, 5.8x, 5.9x, 6x, 6.1x, 6.2x, 6.3x, 6.4x, 6.5x, 6.6x, 6.7x, 6. 8x, 6.9x, 7x, 7.1x, 7.2x, 7.3x, 7.4x, 7.5x, 7.6x, 7.7x, 7.8x, 7.9x, 8x, 8.1x, 8.2x, 8.3x, 8. 4-fold, 8.5-fold, 8.6-fold, 8.7-fold, 8.8-fold, 8.9-fold, 9-fold, 9.1-fold, 9.2-fold, 9.3-fold, 9.4-fold, 9.5-fold, 9.6-fold, 9.7-fold, 9.8-fold, 9.9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, or more, increase the quality or quantity of MDSCs in a population of cells); (c) killing or inhibiting the expansion of T effector cells, such as CD8+ T cells (e.g., thereby increasing the number of T effector cells by about 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2-fold, 2.1-fold, 2.2-fold, 2.3-fold, 2.4-fold, 2.5-fold, 2.6-fold, 2.8-fold, 2.9-fold, 3.1-fold, 3.2-fold, 3.3-fold, 3.4-fold, 3.5-fold, 3.6-fold, 3.7-fold, 3.8-fold, 3.9-fold, 3.1-fold, 3.2-fold, 3.3-fold, 3.4-fold, 6x, 2.7x, 2.8x, 2.9x, 3x, 3.1x, 3.2x, 3.3x, 3.4x, 3.5x, 3.6x, 3.7x, 3.8x, 3.9x, 4x, 4.1x , 4.2x, 4.3x, 4.4x, 4.5x, 4.6x, 4.7x, 4.8x, 4.9x, 5x, 5.1x, 5.2x, 5.3x, 5.4x, 5.5x, 5.6x, 5.7x, 5.8x, 5.9x, 6x, 6.1x, 6.2x, 6.3x, 6.4x, 6.5x, 6.6x, 6.7x, 6.8x, 6.9x, 7x, 7.1x, 7. 2x, 7.3x, 7.4x, 7.5x, 7.6x, 7.7x, 7.8x, 7.9x, 8x, 8.1x, 8.2x, 8.3x, 8.4x, 8.5x, 8.6x, 8.7 fold, 8.8-fold, 8.9-fold, 9-fold, 9.1-fold, 9.2-fold, 9.3-fold, 9.4-fold, 9.5-fold, 9.6-fold, 9.7-fold, 9.8-fold, 9.9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, or more, that reduces the amount of CD8+ effector T cells in a population of cells; and / or (d) promoting the proliferation and / or directly expanding TNFR2-expressing parenchymal cells, e.g., cells of the central and peripheral nervous systems and their associated structures (e.g., thereby increasing the proliferation rate of TNFR2-expressing parenchymal cells, e.g., cells of the central and peripheral nervous systems and their associated structures by about 1.1 fold, 1.2 fold, 1.3 fold, 1.4 fold, 1.5 fold, 1.6 fold, 1.7 fold, 1.8 fold, 1.9 fold, 2 fold, 2.1 fold, 2.2 fold, 2.3 fold, 2.4 fold, 2.5 fold, 2.6 fold, 2.7 fold, 2.8 fold, 2.9 fold, 3 fold, 3.1 fold, 3.2 fold, 3.3 fold, 3.4 fold, 3.5 fold, 3.6 fold, 3.7 fold, 3.8 fold, 3.9 fold, 4 fold, 4.1 fold, 4.2 fold, 4.3 fold, 4.4 fold, 4.5 fold, 4.6 fold, 4.7 fold, 4.8 fold, 4.9x, 5x, 5.1x, 5.2x, 5.3x, 5.4x, 5.5x, 5.6x, 5.7x, 5.8x, 5.9x, 6x, 6.1x, 6.2x, 6.3x, 6. 4x, 6.5x, 6.6x, 6.7x, 6.8x, 6.9x, 7x, 7.1x, 7.2x, 7.3x, 7.4x, 7.5x, 7.6x, 7.7x, 7.8x, 7.9 1x, 8x, 8.1x, 8.2x, 8.3x, 8.4x, 8.5x, 8.6x, 8.7x, 8.8x, 8.9x, 9x, 9.1x, 9.2x, 9.3x, 9.4x, 9.5x, 9.6x, 9.7x, 9.8x, 9.9x, 10x, 20x, 30x, 40x, 50x, 60x, 70x, 80x, 90x, 100x or more).

[0040] For example, the agonistic TNFR2 polypeptides described herein, e.g., single chain polypeptides, antibodies, antigen-binding fragments thereof, or constructs, can be used to increase the total amount of Tregs, MDSCs, and / or parenchymal cells in a patient (such as a human patient) or in a sample (e.g., a sample isolated from a patient, such as a human patient, undergoing treatment for a disease described herein), compared to a patient or sample, respectively, that is not treated with the polypeptide. For example, parenchymal cells can be one of various subsets of central nervous system-associated cells, such as lymphocytes or neurons.

[0041] In some embodiments, TNFR2 polypeptides (e.g., single chain polypeptides, antibodies, antigen-binding fragments thereof, and constructs thereof) are administered to Treg cells (e.g., CD25 Hi The present invention can increase and / or directly enhance the proliferation or expansion of activated Treg cells expressing TNFR2, MDSCs, and / or parenchymal cells expressing TNFR2. For example, the parenchymal cells can be one of various subsets of central nervous system-related cells, such as lymphocytes or neurons.

[0042] The agonistic TNFR2 polypeptides (e.g., single chain polypeptides, antibodies, antigen-binding fragments thereof, and constructs thereof) described herein can be used to inhibit or block the expression of MDSCs (e.g., B7-1 (CD80), B7-H1 (PD-L1), CCR2, CD1d, CD1d1, CD2, CD31 (PECAM-1), CD43, CD44, complement components C5a R1, F4 / 80 (EMR1), Fcγ RIII (CD16), Fcγ RII (CD32), Fcγ RIIA (CD32a), Fcγ RIIB (CD32b), Fcγ RIIB / C (CD32b / c), Fcγ RIIC (CD32c), Fcγ RIIIA (CD16A), Fcγ and TNFR2 on the surface of a cell expressing all or a subset of the proteins and small molecules selected from the group consisting of RIIIB (CD16b), galectin-3, GP130, Gr-1 (Ly-6G), ICAM-1 (CD54), IL-1RI, IL-4Rα, IL-6Rα, integrin α4 (CD49d), integrin αL (CD11a), integrin αM (CD11b), M-CSFR, MGL1 (CD301a), MGL1 / 2 (CD301a / b), MGL2 (CD301b), nitric oxide, PSGL-1 (CD162), L-selectin (CD62L), Siglec-3 (CD33), transferrin receptor (TfR), VEGFR1 (Flt-1), and VEGFR2 (KDR or Flk-1). In particular, MDSCs do not express proteins selected from the group consisting of B7-2 (CD86), B7-H4, CD11c, CD14, CD21, CD23 (FcεRII), CD34, CD35, CD40 (TNFRSF5), CD117 (c-kit), HLA-DR, and Sca-1 (Ly6). Binding of TNFR2 to MDSCs may increase proliferation or stability of MDSCs and / or directly expand MDSCs, such as by preventing apoptosis of MDSCs.The polypeptides, e.g., single chain polypeptides, antibodies, antigen-binding fragments thereof, and constructs thereof described herein may not require TNFα to promote proliferation of Treg cells, parenchymal cells (e.g., TNFR2-expressing cells), and / or MDSCs, although in some cases, TNFα or agonistic TNFα muteins (e.g., the agonistic TNFα muteins described in WO2016 / 029043, the disclosure of which is incorporated herein by reference in its entirety) can be combined with the agonistic TNFR2 polypeptides of the present disclosure, thereby further enhancing the proliferation effect.

[0043] In some embodiments, the polypeptides described herein, e.g., single chain polypeptides, antibodies, antigen-binding fragments thereof, and constructs thereof, enhance the proliferation and / or directly stabilize or expand Treg cells with greater potency in patients with autoimmune disease compared to subjects without autoimmunity. In some embodiments, the polypeptides described herein, e.g., single chain polypeptides, antibodies, antigen-binding fragments thereof, and constructs thereof, enhance the proliferation and / or directly expand Treg cells with greater potency in autoimmune microenvironments compared to sites without autoimmunity.

[0044] In some embodiments, the polypeptides described herein, e.g., single chain polypeptides, antibodies, antigen-binding fragments thereof, and constructs thereof, enhance or stabilize the proliferation and / or directly kill MDSCs in patients with graft-versus-host disease (GVHD) with greater potency compared to subjects without GVHD. In some embodiments, the polypeptides described herein, e.g., single chain polypeptides, antibodies, antigen-binding fragments thereof, and constructs thereof, enhance the proliferation and / or directly expand MDSCs with greater potency in the microenvironment of GVHD compared to sites without GVHD.

[0045] For example, the agonistic TNFR2 polypeptides (e.g., single chain polypeptides, antibodies, antigen-binding fragments thereof, and constructs thereof) described herein can bind to TNFR2 on the surface of cytotoxic T cells of patients suffering from autoimmunity or GVHD, and can increase the proliferation of MDSCs and / or promote the death of CTLs. For example, the polypeptides described herein, e.g., single chain polypeptides, antibodies, antigen-binding fragments thereof, and constructs thereof, can be used to expand the proliferation of Treg cells and / or MDSCs in the autoimmune or GVHD microenvironment, by administering IC of the polypeptides that expand the proliferation of Treg cells and / or MDSCs at sites distinct from the disease. 50 Smaller ICs 50 It can be shown that IC 50 has decreased by approximately 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, 1,000, 10,000, or more.

[0046] In some embodiments, the polypeptides described herein, e.g., single chain polypeptides, antibodies, antigen-binding fragments thereof, and constructs thereof, kill or eliminate effector T cells, such as CD8+ cytotoxic T cells, with greater potency in patients suffering from autoimmunity or GVHD than in subjects without the disease. In some embodiments, the polypeptides described herein, e.g., single chain polypeptides, antibodies, antigen-binding fragments thereof, and constructs thereof, contract T effector cells, such as CD8+ cytotoxic T cells, with greater potency in the microenvironment of an autoimmune response or GVHD compared to sites without autoimmunity or GVHD.

[0047] For example, in some embodiments, the polypeptides described herein, e.g., single chain polypeptides, antibodies, antigen-binding fragments thereof, and constructs thereof, are used to inhibit the EC of the polypeptide in killing T effector cells in a non-autoimmune subject. 50EC50 / ... 50 and directly kills T effector cells, such as CD8+ cytotoxic T cells. In some embodiments, the TNFR2 agonist polypeptide is one identified and / or produced by the disclosed methods. The disclosed methods for identifying and / or producing a TNFR2 agonist polypeptide include: (a) contacting a mixture of antibodies or fragments thereof with at least one peptide comprising five or more consecutive or non-consecutive amino acid residues within CRD1 and / or CRD2 of TNFR2; (b) separating from the mixture the antibodies or fragments thereof that specifically bind to the peptide, thereby producing an enriched antibody mixture comprising at least one TNFR2 agonist antibody or antigen-binding fragment; (c) exposing the enriched antibody mixture to at least one peptide comprising five or more consecutive or non-consecutive amino acid residues within CRD3 and / or CRD4 of TNFR2, leaving behind antibodies or fragments thereof that do not specifically bind to the peptide, thereby generating an antibody mixture comprising at least one TNFR2 agonist antibody or antigen-binding fragment thereof.

[0048] In some embodiments, the method includes determining the amino acid sequence of one or more of the antibodies or antigen-binding fragments thereof in the enriched antibody mixture. In some embodiments, the peptides of (a) and / or (c) are bound to a surface. The antibody or antigen-binding fragment thereof can be expressed on the surface of a phage, a bacterial cell, or a yeast cell. In some embodiments, the antibody or antigen-binding fragment thereof is expressed as one or more polypeptide chains that are non-covalently bound to a ribosome or covalently bound to an mRNA or cDNA. In some embodiments, the peptides of (a) and / or (c) are conjugated to a detectable label, such as a fluorescent molecule (e.g., green fluorescent protein, cyan fluorescent protein, yellow fluorescent protein, red fluorescent protein, phycoerythrin, allophycocyanin, Hoechst, 4',6-diamidino-2-phenylindole (DAPI), propidium iodide, fluorescein, coumarin, rhodamine, tetramethylrhodamine, or cyanine) or an epitope tag (e.g., maltose binding protein, glutathione-S-transferase, polyhistidine tag, FLAG tag, myc tag, human influenza hemagglutinin (HA) tag, biotin, or streptavidin). In some embodiments, steps (a) and (b) are repeated sequentially one or more times (e.g., 1 to 10 times, 2 to 9 times, 3 to 8 times, or 4 to 7 times, e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 times or more).

[0049] In some embodiments, the polypeptide is a monoclonal antibody or antigen-binding fragment thereof, a polyclonal antibody or antigen-binding fragment thereof, a human antibody or antigen-binding fragment thereof, a humanized antibody or antigen-binding fragment thereof, a primatized antibody or antigen-binding fragment thereof, a bispecific antibody or antigen-binding fragment thereof, a multispecific antibody or antigen-binding fragment thereof, a dual variable immunoglobulin domain, a monovalent antibody or antigen-binding fragment thereof, a chimeric antibody or antigen-binding fragment thereof, a single chain Fv molecule (scFv), a diabody, a triabody, a nanobody, an antibody-like protein scaffold, a domain antibody, an Fv fragment, a Fab fragment, a F(ab')2 The antibody or antigen-binding fragment thereof is selected from the group consisting of a fusion protein, a fusion protein molecule, and a tandem scFv (taFv).

[0050] In some embodiments, the polypeptide is a human antibody or antigen-binding fragment thereof, a humanized antibody or antigen-binding fragment thereof, or a chimeric antibody or antigen-binding fragment thereof.

[0051] In some embodiments, the polypeptide is a single chain polypeptide.

[0052] In some embodiments, the polypeptide is a bispecific antibody, such as a bispecific monoclonal antibody, where one arm of the antibody specifically binds to TNFR2 and the other arm specifically binds to an immune checkpoint protein, such as PD-1, PD-L1, or CTLA-4, among others described herein. An arm of a bispecific antibody that specifically binds to TNFR2 can, for example, specifically bind to an epitope of human TNFR2 defined by one or more amino acids in CRD1, CRD2, and / or CRD3, but not to an epitope of human TNFR2 defined by one or more amino acids in CRD4. In some embodiments, an arm of a bispecific antibody that specifically binds to TNFR2 can (a) amino acids 56 to 60 of SEQ ID NO:1 (KCSPG); (b) amino acids 101 to 107 (CSSDQVET) of SEQ ID NO:1; (c) amino acids 115 to 142 of SEQ ID NO: 1 (NRICTCRPGWYCALSKQEGCRLCAPLRK); (d) amino acids 26 to 45 of SEQ ID NO: 1 (QTAQMCCSKCSPGQHAKVFC); (e) amino acids 90 to 109 of SEQ ID NO: 1 (REQNRICTCRPGWYCALSKQ); (f) amino acids 98 to 117 of SEQ ID NO: 1 (CRPGWYCALSKQEGCRLCAP); (g) amino acids 106 to 125 (LSKQEGCRLCAPLRKCRPGF) of SEQ ID NO:1, and / or (h) specifically binds to an epitope of human TNFR2 corresponding to one or more of amino acids 108 to 127 (KQEGCRLCAPLRKCRPGFGV) of SEQ ID NO:1.

[0053] In some embodiments, a bispecific antibody comprises one arm that specifically binds to TNFR2, such as an epitope of human TNFR2 described above, and one arm that specifically binds to an immune checkpoint protein that specifically binds to PD-1. In some embodiments, the arm of the bispecific antibody that specifically binds to PD-1 is an agonist of PD-1 activity.

[0054] In some embodiments, a bispecific antibody comprises one arm that specifically binds to TNFR2, such as an epitope of human TNFR2 listed above, and one arm that specifically binds to PD-L1, in some embodiments, the arm of the bispecific antibody that specifically binds to PD-L1 is an agonist of PD-L1 activity.

[0055] In some embodiments, a bispecific antibody comprises one arm that specifically binds to TNFR2, such as an epitope of human TNFR2 described above, and one arm that specifically binds to CTLA-4, in some embodiments, the arm of the bispecific antibody that specifically binds to CTLA-4 is an agonist of CTLA-4 activity.

[0056] The second aspect features a construct comprising a first polypeptide domain and a second polypeptide domain.The first polypeptide domain and the second polypeptide domain are each independently an antigen-binding fragment of any of the first aspect or its embodiments.The first polypeptide domain and the second polypeptide domain can be linked together by a covalent linker, such as a linker that comprises (e.g. is) an amide bond or a disulfide bond.

[0057] A third aspect features a polynucleotide encoding a polypeptide of the first aspect (e.g., a single chain polypeptide, an antibody, an antigen-binding fragment thereof, or a construct thereof) and / or a construct of the second aspect, or any of the embodiments thereof.

[0058] The fourth aspect features a vector encoding the polynucleotide of the third aspect. The vector can be an expression vector, such as a eukaryotic expression vector. In some embodiments, the vector is a viral vector, such as an adenovirus (e.g., a serotype 1-57 adenovirus, such as a serotype 2, 5, 11, 12, 24, 26, 34, 35, 40, 48, 49, 50, 52, or Pan9 adenovirus), a retrovirus (e.g., a gamma-retrovirus or a lentivirus), a poxvirus, an adeno-associated virus, a baculovirus, a herpes simplex virus, or a vaccinia virus (e.g., a modified vaccinia Ankara virus).

[0059] A fifth aspect features an isolated host cell comprising the polynucleotide of the third aspect and / or the vector of the fourth aspect. The host cell can be a prokaryotic or eukaryotic cell, such as a mammalian cell (e.g., a Chinese Hamster Ovary (CHO) cell or a HEK cell). The host cell can be, for example, one described in Dinnis and James, Biotechnology and Bioengineering 91:180-189, 2005, the disclosure of which is incorporated herein by reference.

[0060] A sixth aspect features a pharmaceutical composition comprising a polypeptide (e.g., a single chain polypeptide, an antibody, an antigen-binding fragment thereof, or a construct thereof) that specifically binds to human TNFR2 and exhibits an agonistic effect on TNFR2 activity upon binding. The polypeptide can be, for example, an antibody or antigen-binding fragment of the first aspect or any of its embodiments. In some embodiments, at least 10% of the antibodies or antigen-binding fragments thereof in the pharmaceutical composition are present in a single disulfide-linked isoform, such as an IgG2-B or IgG2-A disulfide-linked isoform. In some embodiments, about 10% to about 99.999% of the antibody or antigen-binding fragment thereof in the pharmaceutical composition is, for example, about 11% to about 99.9%, about 12% to about 99.9%, about 13% to about 99.9%, about 14% to about 99.9%, about 15% to about 99%, about 16% to about 99.9%, about 17% to about 99.9%, about 18% to about 99.9%, about 19% to about 99.9%, or about 20% to about 99.9% of the antibody or antigen-binding fragment thereof. , about 21% to about 99.9%, about 22% to about 99.9%, about 23% to about 99.9%, about 24% to about 99.9%, about 25% to about 99.9%, about 26% to about 99.9%, about 27% to about 99.9%, about 28% to about 99.9%, about 29% to about 99.9%, about 30% to about 99.9%, about 31% to about 99.9%, about 32% to about 99.9%, about 33% to about 99.9%, about 34% to about 99.9%, about 35% to about 99.9%, about 6% to 99.9%, 37% to 99.9%, 38% to 99.9%, 39% to 99.9%, 40% to 99.9%, 41% to 99.9%, 42% to 99.9%, 43% to 99.9%, 44% to 99.9%, 45% to 99.9%, 46% to 99.9%, 47% to 99.9%, 48% to 99.9%, 49% to 99.9%, 50% to 99.9%, 51% to Approximately 99.9%, approximately 52% to approximately 99.9%, approximately 53% to approximately 99.9%, approximately 54% to approximately 99.9%, approximately 55% to approximately 99.9%, approximately 56% to approximately 99.9%, approximately 57% to approximately 99.9%, approximately 58% to approximately 99.9%, approximately 59% to approximately 99.9%, approximately 60% to approximately 99.9%, approximately 61% to approximately 99.9%, approximately 62% to approximately 99.9%, approximately 63% to approximately 99.9%, approximately 64% to approximately 99.9%, approximately 65% ​​to approximately 99.9%, approximately 66% to approximately 99.9%, about 67% to about 99.9%, about 68% to about 99.9%, about 69% to about 99.9%, about 70% to about 99.9%, about 71% to about 99.9%, about 72% to about 99.9%, about 73% to about 99.9%, about 74% to about 99.9%, about 75% to about 99.9%, about 76% to about 99.9%, about 77% to about 99.9%, about 78% to about 99.9%, about 79% to about 99.9%, about 80% to about 99.9%, about 81% to about 99.9%, about 82% to about 99.9%, about 83% to about 99.9%, about 84% to about 99.9%, about 85% to about 99.9%, about 86% to about 99.9%, about 87% to about 99.9%, about 88% to about 99.9%, about 89% to about 99.9%, about 90% to about 99.9%, about 91% to about 99.9%, about 92% to about 99.9%, about 93% to about 99.9%, about 94% to about 99.9%, about 95% to about 99.9%, about 96% to about 99.9%, about 97% to about 99.9%, about 98% to about 99.9%, about 9 ... 9.9%, about 85% to about 99.9%, about 86% to about 99.9%, about 87% to about 99.9%, about 88% to about 99.9%, about 89% to about 99.9%, about 90% to about 99.9%, about 91% to about 99.9%, about 92% to about 99.9%, about 93% to about 99.9%, about 94% to about 99.9%, about 95% to about 99.9%, about 96% to about 99.9%, about 97% to about 99.9%, about 98% to about 99.9%, or about 99% to about 99.99% are present in a single disulfide bond isoform.

[0061] In some embodiments, at least about 10% of the antibodies or antigen-binding fragments thereof in the pharmaceutical composition are present in a single disulfide bond isoform. In some embodiments, at least about 15% of the antibodies or antigen-binding fragments thereof in the pharmaceutical composition are present in a single disulfide bond isoform. In some embodiments, at least about 20% of the antibodies or antigen-binding fragments thereof in the pharmaceutical composition are present in a single disulfide bond isoform. In some embodiments, at least about 25% of the antibodies or antigen-binding fragments thereof in the pharmaceutical composition are present in a single disulfide bond isoform. In some embodiments, at least about 30% of the antibodies or antigen-binding fragments thereof in the pharmaceutical composition are present in a single disulfide bond isoform. In some embodiments, at least about 35% of the antibodies or antigen-binding fragments thereof in the pharmaceutical composition are present in a single disulfide bond isoform. In some embodiments, at least about 40% of the antibodies or antigen-binding fragments thereof in the pharmaceutical composition are present in a single disulfide bond isoform. In some embodiments, at least about 45% of the antibodies or antigen-binding fragments thereof in the pharmaceutical composition are present in a single disulfide bond isoform. In some embodiments, at least about 50% of the antibodies or antigen-binding fragments thereof in the pharmaceutical composition are present in a single disulfide bond isoform. In some embodiments, at least about 60% of the antibodies or antigen-binding fragments thereof in the pharmaceutical composition are present in a single disulfide bond isoform. In some embodiments, at least about 65% of the antibodies or antigen-binding fragments thereof in the pharmaceutical composition are present in a single disulfide bond isoform. In some embodiments, at least about 70% of the antibodies or antigen-binding fragments thereof in the pharmaceutical composition are present in a single disulfide bond isoform. In some embodiments, at least about 75% of the antibodies or antigen-binding fragments thereof in the pharmaceutical composition are present in a single disulfide bond isoform. In some embodiments, at least about 80% of the antibodies or antigen-binding fragments thereof in the pharmaceutical composition are present in a single disulfide bond isoform.In some embodiments, at least about 85% of the antibodies or antigen-binding fragments thereof in the pharmaceutical composition are present in a single disulfide bond isoform. In some embodiments, at least about 90% of the antibodies or antigen-binding fragments thereof in the pharmaceutical composition are present in a single disulfide bond isoform. In some embodiments, at least about 95% of the antibodies or antigen-binding fragments thereof in the pharmaceutical composition are present in a single disulfide bond isoform. In some embodiments, at least about 96% of the antibodies or antigen-binding fragments thereof in the pharmaceutical composition are present in a single disulfide bond isoform. In some embodiments, at least about 97% of the antibodies or antigen-binding fragments thereof in the pharmaceutical composition are present in a single disulfide bond isoform. In some embodiments, at least about 98% of the antibodies or antigen-binding fragments thereof in the pharmaceutical composition are present in a single disulfide bond isoform. In some embodiments, at least about 99% of the antibodies or antigen-binding fragments thereof in the pharmaceutical composition are present in a single disulfide bond isoform. In some embodiments, at least about 99.9% of the antibodies or antigen-binding fragments thereof in the pharmaceutical composition are present in a single disulfide bond isoform.

[0062] In some embodiments, an antibody or antigen-binding fragment thereof gives rise to only a single detectable band in gel electrophoresis analysis performed under non-reducing conditions.

[0063] In some embodiments, the single disulfide bond isoform of the antibody or antigen-binding fragment as described herein is IgG2-B. In some embodiments, the single disulfide bond isoform of the antibody or antigen-binding fragment as described herein is IgG2-A.

[0064] Additionally or alternatively, the pharmaceutical composition may comprise a construct of the second aspect or any embodiment thereof, a polynucleotide of the third aspect or any embodiment thereof, a vector of the fourth aspect or any embodiment thereof, and / or a host cell of the fifth aspect or any embodiment thereof. The pharmaceutical composition may further comprise a pharma- ceutically acceptable carrier or excipient.

[0065] In some embodiments, the polypeptide (e.g., a single chain polypeptide, an antibody, an antigen-binding fragment thereof, or a construct thereof) is present in the pharmaceutical composition in an amount of about 0.001 mg / ml to about 100 mg / ml, e.g., an amount of 0.01 mg / ml to about 10 mg / ml.

[0066] The pharmaceutical composition may further comprise an additional therapeutic agent, such as an immunotherapeutic agent. In some embodiments, the immunotherapeutic agent is an agonistic anti-CTLA-4 agent, an agonistic anti-PD-1 agent, an agonistic anti-PD-L1 agent, an agonistic anti-PD-L2 agent, an agonistic anti-CD27 agent, an agonistic anti-CD30 agent, an agonistic anti-CD40 agent, an agonistic anti-4-1BB agent, an agonistic anti-GITR agent, an agonistic anti-OX40 agent, an agonistic anti-TRAILR1 agent, an agonistic anti-TRAILR2 agent, an agonistic anti-CTLA-4 agent, an agonistic anti-PD-L2 agent, an agonistic anti-PD-L2 agent, an agonistic anti-CTLA-4 agent, an agonistic anti-PD-L1 agent, an agonistic anti-PD-L2 agent, an agonistic anti-PD-L2 agent, an agonistic anti-CD27 agent, an agonistic anti-CD30 agent, an agonistic anti-CD40 agent, an agonistic anti-4-1BB agent, an agonistic anti-GITR agent, an agonistic anti-OX40 agent, an agonistic anti-TRAILR1 agent, an agonistic anti-TRAILR2 agent, an agonistic anti-TRAILR3 agent, an agonistic anti-TRAILR4 agent, an agonistic anti-TRAILR5 agent, an agonistic anti-TRAILR6 agent, an agonistic anti-TRAILR7 agent, an agonistic anti-TRAILR8 agent, an agonistic anti-TRAILR9 agent, an agonistic anti-TRAILR10 agent, an agonistic anti-TRAILR111 agent, an agonistic anti-TRAILR122 agent, an agonistic anti-TRAILR132 agent, an agonistic anti-TRAILR142 agent, an agonistic anti-TRAILR152 agent, agonistic anti-TWEAK agents, agonistic anti-TWEAKR agents, agonistic anti-cell surface lymphocyte protein agents, agonistic anti-BRAF agents, agonistic anti-MEK agents, agonistic anti-CD33 agents, agonistic anti-CD20 agents, agonistic anti-HLA-DR agents, agonistic anti-HLA class I agents, agonistic anti-CD52 agents, agonistic anti-A33 agents, agonistic anti-GD3 agents, agonistic anti-PSMA agents, agonistic anti-Ceacan 1 agents, agonistic anti-Galedin 9 agents, agonistic anti-HVEM agents, agonistic anti-VISTA agents, agonistic anti-B7 agents H4 agents, agonistic anti-HHLA2 agents, agonistic anti-CD155 agents, agonistic anti-CD80 agents, agonistic anti-BTLA agents, agonistic anti-CD160 agents, agonistic anti-CD28 agents, agonistic anti-CD226 agents, agonistic anti-CEACAM1 agents, agonistic anti-TIM3 agents, agonistic anti-TIGIT agents, agonistic anti-CD96 agents, agonistic anti-CD70 agents, agonistic anti-CD27 agents, agonistic anti-LIGHT agents, agonistic anti-CD137 agents, agonistic anti-DR4 agents, agonistic anti-CR5 agents, agonistic The agonistic anti-TNFRS agent, agonistic anti-TNFR1 agent, agonistic anti-FAS agent, agonistic anti-CD95 agent, agonistic anti-TRAIL agent, agonistic anti-DR6 agent, agonistic anti-EDAR agent, agonistic anti-NGFR agent, agonistic anti-OPG agent, agonistic anti-RANKL agent, agonistic anti-LTβ receptor agent, agonistic anti-BCMA agent, agonistic anti-TACI agent, agonistic anti-BAFFR agent, agonistic anti-EDAR2 agent, agonistic anti-TROY agent, and agonistic anti-RELT agent.For example, the immunotherapeutic agent may be an agonistic anti-CTLA-4 agent, an agonistic anti-PD-1 agent, or an agonistic anti-PD-L1 agent.

[0067] In some embodiments, the immunotherapeutic agent is an agonistic anti-CTLA-4 antibody or antigen-binding fragment thereof, an agonistic anti-PD-1 antibody or antigen-binding fragment thereof, an agonistic anti-PD-L1 antibody or antigen-binding fragment thereof, an agonistic anti-PD-L2 antibody or antigen-binding fragment thereof, an agonistic anti-CD27 antibody or antigen-binding fragment thereof, an agonistic anti-CD30 antibody or antigen-binding fragment thereof, an agonistic anti-CD40 antibody or antigen-binding fragment thereof, an agonistic anti-4-1BB antibody or antigen-binding fragment thereof, an agonistic anti-GITR antibody or antigen-binding fragment thereof, an agonistic anti-OX40 antibody or antigen-binding fragment thereof, an agonistic anti-TRAILR1 antibody or antigen-binding fragment thereof, an agonistic anti-TRAILR2 antibody or antigen-binding fragment thereof, an agonistic anti-TWEA antibody or antigen-binding fragment thereof, K antibody or antigen-binding fragment thereof, agonistic anti-TWEAKR antibody or antigen-binding fragment thereof, agonistic anti-cell surface lymphocyte protein antibody or antigen-binding fragment thereof, agonistic anti-BRAF antibody or antigen-binding fragment thereof, agonistic anti-MEK antibody or antigen-binding fragment thereof, agonistic anti-CD33 antibody or antigen-binding fragment thereof, agonistic anti-CD20 antibody or antigen-binding fragment thereof, agonistic anti-HLA-DR antibody or antigen-binding fragment thereof, agonistic anti-HLA class I antibody or antigen-binding fragment thereof, agonistic anti-CD52 antibody or antigen-binding fragment thereof, agonistic anti-A33 antibody or antigen-binding fragment thereof, agonistic anti-GD3 antibody or antigen-binding fragment thereof, agonistic anti-PSMA antibody or antigen-binding fragment thereof, agonistic anti-Ceacan 1 antibody or antigen-binding fragment thereof, agonistic anti-Galedin 9 antibody or antigen-binding fragment thereof, agonistic anti-HVEM antibody or antigen-binding fragment thereof, agonistic anti-VISTA antibody or antigen-binding fragment thereof, agonistic anti-B7 H4 antibody or antigen-binding fragment thereof, agonistic anti-HHLA2 antibody or antigen-binding fragment thereof, agonistic anti-CD155 antibody or antigen-binding fragment thereof, agonistic anti-CD80 antibody or antigen-binding fragment thereof, agonistic anti-BTLA antibody or antigen-binding fragment thereof, agonistic anti-CD160 antibody or antigen-binding fragment thereof, agonistic anti-CD28 antibody or antigen-binding fragment thereof, agonistic anti-CD226 antibody or antigen-binding fragment thereof, agonistic anti-CEACAM1 antibody or antigen-binding fragment thereof or antigen-binding fragment thereof, agonistic anti-TIM3 antibody or antigen-binding fragment thereof, agonistic anti-TIGIT antibody or antigen-binding fragment thereof, agonistic anti-CD96 antibody or antigen-binding fragment thereof, agonistic anti-CD70 antibody or antigen-binding fragment thereof, agonistic anti-CD27 antibody or antigen-binding fragment thereof, agonistic anti-LIGHT antibody or antigen-binding fragment thereof, agonistic anti-CD137 antibody or antigen-binding fragment thereof, agonistic anti-DR4 antibody or antigen-binding fragment thereof, agonistic anti-CR5 antibody or antigen-binding fragment thereof, agonistic anti-TNFRS antibody or antigen-binding fragment thereof, agonistic anti-TNFR1 antibody or antigen-binding fragment thereof, agonistic anti-FAS antibody or antigen-binding fragment thereof, agonistic anti-CD95 antibody or antigen-binding fragment thereof, agonistic The antibody is selected from the group consisting of an agonistic anti-TRAIL antibody or antigen-binding fragment thereof, an agonistic anti-DR6 antibody or antigen-binding fragment thereof, an agonistic anti-EDAR antibody or antigen-binding fragment thereof, an agonistic anti-NGFR antibody or antigen-binding fragment thereof, an agonistic anti-OPG antibody or antigen-binding fragment thereof, an agonistic anti-RANKL antibody or antigen-binding fragment thereof, an agonistic anti-LTβ receptor antibody or antigen-binding fragment thereof, an agonistic anti-BCMA antibody or antigen-binding fragment thereof, an agonistic anti-TACI antibody or antigen-binding fragment thereof, an agonistic anti-BAFFR antibody or antigen-binding fragment thereof, an agonistic anti-EDAR2 antibody or antigen-binding fragment thereof, an agonistic anti-TROY antibody or antigen-binding fragment thereof, and an agonistic anti-RELT antibody or antigen-binding fragment thereof. For example, the immunotherapeutic agent can be an agonistic anti-CTLA-4 antibody or antigen-binding fragment thereof, an agonistic anti-PD-1 antibody or antigen-binding fragment thereof, or an agonistic anti-PD-L1 antibody or antigen-binding fragment thereof.

[0068] In some embodiments, the immunotherapeutic agent is an anti-cell surface lymphocyte protein antibody or antigen-binding fragment thereof, such as, for example, CD1, CD2, CD3, CD4, CD5, CD6, CD7, CD8, CD9, CD10, CD11, CD12, CD13, CD14, CD15, CD16, CD17, CD18, CD19, CD20, CD21, CD22, CD23, CD24, CD25, CD26, CD27, CD28, CD29, CD30, CD31, CD32, CD33, CD34, CD35, CD36, CD37, CD38, CD39, CD40, CD41, CD 42, CD43, CD44, CD45, CD46, CD47, CD48, CD49, CD50, CD51, CD52, CD53, CD54, CD55, CD56, CD57, CD58, CD59, CD60, CD61, CD62, CD63, CD64, CD65, CD66, CD 67, CD68, CD69, CD70, CD71, CD72, CD73, CD74, CD75, CD76, CD77, CD78, CD79, CD80, CD81, CD82, CD83, CD84, CD85, CD86, CD87, CD88, CD89, CD90, CD91, CD 92, CD93, CD94, CD95, CD96, CD97, CD98, CD99, CD100, CD101, CD102, CD103, CD104, CD105, CD106, CD107, CD108, CD109, CD110, CD111, CD112, CD113, CD1 14, CD115, CD116, CD117, CD118, CD119, CD120, CD121, CD122, CD123, CD124, CD125, CD126, CD127, CD128, CD129, CD130, CD131, CD132, CD133, CD134, CD 135, CD136, CD137, CD138, CD139, CD140, CD141, CD142, CD143, CD144, CD145, CD146, CD147, CD148, CD149, CD150, CD151, CD152, CD153, CD154, CD155, C D156, CD157, CD158, CD159, CD160, CD161, CD162, CD163, CD164, CD165, CD166, CD167, CD168, CD169, CD170, CD171, CD172, CD173, CD174, CD175, CD176,CD177, CD178, CD179, CD180, CD181, CD182, CD183, CD184, CD185, CD186, CD187, CD188, CD189, CD190, CD191, CD192, CD193, CD194, CD195 , CD196, CD197, CD198, CD199, CD200, CD201, CD202, CD203, CD204, CD205, CD206, CD207, CD208, CD209, CD210, CD211, CD212, CD213, CD21 4, CD215, CD216, CD217, CD218, CD219, CD220, CD221, CD222, CD223, CD224, CD225, CD226, CD227, CD228, CD229, CD230, CD231, CD232, CD2 33, CD234, CD235, CD236, CD237, CD238, CD239, CD240, CD241, CD242, CD243, CD244, CD245, CD246, CD247, CD248, CD249, CD250, CD251, CD2 52, CD253, CD254, CD255, CD256, CD257, CD258, CD259, CD260, CD261, CD262, CD263, CD264, CD265, CD266, CD267, CD268, CD269, CD270, CD 271, CD272, CD273, CD274, CD275, CD276, CD277, CD278, CD279, CD280, CD281, CD282, CD283, CD284, CD285, CD286, CD287, CD288, CD289, CD 290, CD291, CD292, CD293, CD294, CD295, CD296, CD297, CD298, CD299, CD300, CD301, CD302, CD303, CD304, CD305, CD306, CD307, CD308, CD309, CD310, CD311, CD312, CD313, CD314, CD315, CD316, CD317, CD318, CD319, and / or CD320, or an antigen-binding fragment thereof that binds to and promotes activity of one or more of CD310, CD311, CD312, CD313, CD314, CD315, CD316, CD317, CD318, CD319, and / or CD320.

[0069] In some embodiments, the immunotherapeutic agent is an agent (e.g., a polypeptide, an antibody, an antigen-binding fragment thereof, a single-chain polypeptide, or a construct thereof) that binds to a chemokine or lymphokine. For example, the immunotherapeutic agent is an agent (e.g., a polypeptide, an antibody, an antigen-binding fragment thereof, a single-chain polypeptide, or a construct thereof) that binds to and activates one or more or all of CXCL1, CXCL2, CXCL3, CXCL8, CCL2, and CCL5. In some embodiments, the immunotherapeutic agent is an agent (e.g., a polypeptide, an antibody, an antigen-binding fragment thereof, a single-chain polypeptide, or a construct thereof) that binds to and activates one or more or all of CCL3, CCL4, CCL8, and CCL22.

[0070] In some embodiments, the pharmaceutical composition comprises a bispecific antibody, such as a bispecific monoclonal antibody, in which one arm of the antibody specifically binds to TNFR2 and the other arm specifically binds to an immune checkpoint protein, such as PD-1, PD-L1, or CTLA-4, among others as described herein. The arm of the bispecific antibody that specifically binds to TNFR2 can, for example, specifically bind to an epitope on human TNFR2 defined by one or more amino acids in CRD1, CRD2, and / or CRD3, but not to an epitope defined by one or more amino acids in CRD4.

[0071] In some embodiments, a bispecific antibody comprises one arm that specifically binds to TNFR2, such as an epitope of human TNFR2 described above, and one arm that specifically binds to an immune checkpoint protein that specifically binds to PD-1. In some embodiments, the arm of the bispecific antibody that specifically binds to PD-1 is an agonist of PD-1 activity.

[0072] In some embodiments, a bispecific antibody comprises one arm that specifically binds to TNFR2, such as an epitope of human TNFR2 listed above, and one arm that specifically binds to PD-L1, in some embodiments, the arm of the bispecific antibody that specifically binds to PD-L1 is an agonist of PD-L1 activity.

[0073] In some embodiments, a bispecific antibody comprises one arm that specifically binds to TNFR2, such as an epitope of human TNFR2 described above, and one arm that specifically binds to CTLA-4, in some embodiments, the arm of the bispecific antibody that specifically binds to CTLA-4 is an agonist of CTLA-4 activity.

[0074] In some embodiments, the additional therapeutic agent in the pharmaceutical composition is TNFα, an agonistic TNFα mutein (e.g., an agonistic TNFα mutein described in WO2016 / 029043, the disclosure of which is incorporated herein by reference in its entirety), or BCG.

[0075] In some embodiments, the pharmaceutical composition does not include an anti-TNFα agent, such as an anti-TNFα antibody or antigen-binding fragment thereof, hi some embodiments, the pharmaceutical composition does not include infliximab, etanercept, certolizumab, golimumab, or adalimumab.

[0076] A seventh aspect features a method of producing a polypeptide of the first aspect (e.g., a single chain polypeptide, an antibody, an antigen-binding fragment thereof, or a construct thereof) and / or a construct of the second aspect, or any of its embodiments. The method can include expressing a polynucleotide encoding the polypeptide or construct in a host cell (e.g., a host cell described herein) and recovering the polypeptide from the host cell medium.

[0077] An eighth aspect features a method of inhibiting an immune response, such as an immune response mediated by a B cell or a T cell (e.g., a CD8+ T effector cell) in a mammalian subject, by administering to the subject a polypeptide (e.g., a single chain polypeptide, an antibody, an antigen-binding fragment thereof, or a construct thereof) of the first aspect or any embodiment thereof, a construct of the second aspect or any embodiment thereof, a polynucleotide of the third aspect or any embodiment thereof, a vector of the fourth aspect or any embodiment thereof, a host cell of the fifth aspect or any embodiment thereof, and / or a pharmaceutical composition of the sixth aspect or any embodiment thereof.

[0078] A ninth aspect features a method of treating an immunological disease in a mammalian subject by administering to the subject a polypeptide (e.g., a single chain polypeptide, an antibody, an antigen-binding fragment thereof, or a construct thereof) of the first aspect or any embodiment thereof, a construct of the second aspect or any embodiment thereof, a polynucleotide of the third aspect or any embodiment thereof, a vector of the fourth aspect or any embodiment thereof, a host cell of the fifth aspect or any embodiment thereof, and / or a pharmaceutical composition of the sixth aspect or any embodiment thereof.

[0079] In some embodiments, the subject is in need of regeneration of a tissue or organ, which may be, for example, the pancreas, salivary gland, pituitary gland, kidney, heart, lung, hematopoietic system, cranial nerves, heart, aorta, olfactory gland, ear, nerves, head structures, eye, thymus, tongue, bone, liver, small intestine, large intestine, gut, lung, brain, skin, peripheral nervous system, central nervous system, spinal cord, breast, embryonic structure, embryo, or testis.

[0080] In some embodiments, the immunological disease is an autoimmune disease, a neurological disease, an allergy, asthma, macular degeneration, muscle wasting, a disease associated with miscarriage, atherosclerosis, bone loss, a musculoskeletal disease, obesity, GVHD, or allograft rejection.

[0081] In some embodiments, the autoimmune disease is selected from the group consisting of type I diabetes, alopecia areata, ankylosing spondylitis, antiphospholipid syndrome, autoimmune Addison's disease, autoimmune hemolytic anemia, autoimmune hepatitis, Behcet's disease, bullous pemphigoid, cardiomyopathy, celiac sprue dermatitis, chronic fatigue immune dysfunction syndrome (CFIDS), chronic inflammatory demyelinating polyneuropathy, Churg-Strauss syndrome, cicatricial pemphigoid, CREST syndrome, cold agglutinin disease, Crohn's disease, essential mixed cryoglobulinemia, fibromyalgia-fibromyositis, Graves' disease, Guillain-Barre, Hashimoto's thyroiditis, hypothyroidism, idiopathic pulmonary fibrosis, idiopathic thrombocytopenia ... The following conditions are considered to be present in the patient: Inflammatory Trophoblastic Purpura (ITP), IgA Nephropathy, Juvenile Arthritis, Lichen Planus, Lupus, Meniere's Disease, Mixed Connective Tissue Disease, Multiple Sclerosis, Myasthenia Gravis, Pemphigus Vulgaris, Pernicious Anemia, Polyarteritis Nodosa, Polychondritis, Polyglandular Syndrome, Polymyalgia Rheumatica, Polymyositis and Dermatomyositis, Primary Agammaglobulinemia, Primary Biliary Cirrhosis, Psoriasis, Raynaud's Phenomenon, Reiter's Syndrome, Rheumatic Fever, Rheumatoid Arthritis, Sarcoidosis, Scleroderma, Sjögren's Syndrome, Stiff-Ass Syndrome, Takayasu's Arteritis, Temporal Arteritis / Giant Cell Arteritis, Ulcerative Colitis, Uveitis, Vasculitis, Vitiligo, or Wegener's Granulomatosis.

[0082] In some embodiments, the neurological condition is a brain tumor, brain metastasis, spinal cord injury, schizophrenia, epilepsy, amyotrophic lateral sclerosis (ALS), Parkinson's disease, Alzheimer's disease, Huntington's disease, or stroke.

[0083] In some embodiments, the allergy is a food allergy, seasonal allergy, pet allergy, hives, hay fever, allergic conjunctivitis, poison ivy allergy, oak allergy, mold allergy, drug allergy, dust allergy, cosmetic allergy, or chemical allergy.

[0084] In some embodiments, the allograft rejection is skin graft rejection, bone graft rejection, vascular tissue graft rejection, ligament graft rejection, or organ graft rejection.

[0085] In some embodiments, the ligament graft rejection is cricothyroid ligament graft rejection, periodontal ligament graft rejection, crystalline suspensory ligament graft rejection, palmar radiocarpal ligament graft rejection, dorsal radiocarpal ligament graft rejection, medial collateral ligament graft rejection, lateral collateral ligament graft rejection, mammary suspensory ligament graft rejection, anterior sacroiliac ligament graft rejection, posterior sacroiliac ligament graft rejection, sacrotuberous ligament graft rejection, sacrospinous ligament graft rejection, inferior pubic ligament graft rejection, superior pubic ligament graft rejection, anterior cruciate ligament graft rejection, lateral collateral ligament graft rejection, posterior cruciate ligament graft rejection, medial collateral ligament graft rejection, cranial cruciate ligament graft rejection, caudal cruciate ligament graft rejection, or patellar ligament graft rejection.

[0086] In some embodiments, the organ transplant rejection is heart transplant rejection, lung transplant rejection, kidney transplant rejection, liver transplant rejection, pancreas transplant rejection, intestinal transplant rejection, or thymus transplant rejection.

[0087] In some embodiments, the GVHD arises from a bone marrow transplant or from one or more blood cells selected from the group consisting of hematopoietic stem cells, common myeloid progenitors, common lymphoid progenitors, megakaryocytes, monocytes, basophils, eosinophils, neutrophils, macrophages, T cells, B cells, natural killer cells, and dendritic cells.

[0088] A tenth aspect features a method of treating an inflammatory disease in a mammalian subject by administering to the subject a polypeptide (e.g., a single chain polypeptide, an antibody, an antigen-binding fragment thereof, or a construct thereof) of the first aspect or any embodiment thereof, a construct of the second aspect or any embodiment thereof, a polynucleotide of the third aspect or any embodiment thereof, a vector of the fourth aspect or any embodiment thereof, a host cell of the fifth aspect or any embodiment thereof, and / or a pharmaceutical composition of the sixth aspect or any embodiment thereof.

[0089] In some embodiments, the inflammatory disease is acute or chronic inflammation, hi some embodiments, the inflammatory disease is selected from the group consisting of osteoarthritis, fibrotic lung disease, and cardiac inflammation.

[0090] An eleventh aspect features a method of healing, protecting, growing, and / or regenerating TNFR2+ tissue in a mammalian subject by administering to the subject a polypeptide (e.g., a single chain polypeptide, an antibody, an antigen-binding fragment thereof, or a construct thereof) of the first aspect or any embodiment thereof, a construct of the second aspect or any embodiment thereof, a polynucleotide of the third aspect or any embodiment thereof, a vector of the fourth aspect or any embodiment thereof, a host cell of the fifth aspect or any embodiment thereof, and / or a pharmaceutical composition of the sixth aspect or any embodiment thereof.

[0091] In some embodiments, the TNFR2+ tissue is selected from the group consisting of tissue of the pancreas, salivary gland, pituitary gland, kidney, heart, lung, hematopoietic system, cranial nerves, heart, aorta, olfactory gland, ear, nerves, eye, thymus, tongue, bone, liver, small intestine, large intestine, gastrointestinal, lung, brain, skin, peripheral nervous system, central nervous system, spinal cord, breast, embryonic structure, embryo, or testis.

[0092] In some embodiments of the eighth, ninth, tenth, and / or eleventh aspects, the method further comprises administering an immunotherapeutic agent to the human, such as an agonistic anti-CTLA-4 agent, an agonistic anti-PD-1 agent, an agonistic anti-PD-L1 agent, an agonistic anti-PD-L2 agent, an agonistic anti-CD27 agent, an agonistic anti-CD30 agent, an agonistic anti-CD40 agent, an agonistic anti-4-1BB agent, an agonistic anti-GITR agent, an agonistic anti-OX40 agent, an agonistic anti-TRAILR1 agent, an agonistic anti-TRAILR2 agent, an agonistic anti-TW ... EAK agents, agonistic anti-TWEAKR agents, agonistic anti-cell surface lymphocyte protein agents, agonistic anti-BRAF agents, agonistic anti-MEK agents, agonistic anti-CD33 agents, agonistic anti-CD20 agents, agonistic anti-HLA-DR agents, agonistic anti-HLA class I agents, agonistic anti-CD52 agents, agonistic anti-A33 agents, agonistic anti-GD3 agents, agonistic anti-PSMA agents, agonistic anti-Ceacan 1 agents, agonistic anti-Galedin 9 agents, agonistic anti-HVEM agents, agonistic anti-VISTA agents, agonistic anti-B7 agents H4 agents, agonistic anti-HHLA2 agents, agonistic anti-CD155 agents, agonistic anti-CD80 agents, agonistic anti-BTLA agents, agonistic anti-CD160 agents, agonistic anti-CD28 agents, agonistic anti-CD226 agents, agonistic anti-CEACAM1 agents, agonistic anti-TIM3 agents, agonistic anti-TIGIT agents, agonistic anti-CD96 agents, agonistic anti-CD70 agents, agonistic anti-CD27 agents, agonistic anti-LIGHT agents, agonistic anti-CD137 agents, agonistic anti-DR4 agents, agonistic anti-CR5 agents, agonistic The agonistic anti-TNFRS agent, agonistic anti-TNFR1 agent, agonistic anti-FAS agent, agonistic anti-CD95 agent, agonistic anti-TRAIL agent, agonistic anti-DR6 agent, agonistic anti-EDAR agent, agonistic anti-NGFR agent, agonistic anti-OPG agent, agonistic anti-RANKL agent, agonistic anti-LTβ receptor agent, agonistic anti-BCMA agent, agonistic anti-TACI agent, agonistic anti-BAFFR agent, agonistic anti-EDAR2 agent, agonistic anti-TROY agent, and agonistic anti-RELT agent.For example, the immunotherapeutic agent may be an agonistic anti-CTLA-4 agent, an agonistic anti-PD-1 agent, or an agonistic anti-PD-L1 agent.

[0093] Immunotherapeutic agents administered to humans include, for example, an agonistic anti-CTLA-4 antibody or antigen-binding fragment thereof, an agonistic anti-PD-1 antibody or antigen-binding fragment thereof, an agonistic anti-PD-L1 antibody or antigen-binding fragment thereof, an agonistic anti-PD-L2 antibody or antigen-binding fragment thereof, an agonistic anti-CD27 antibody or antigen-binding fragment thereof, an agonistic anti-CD30 antibody or antigen-binding fragment thereof, an agonistic anti-CD40 antibody or antigen-binding fragment thereof, an agonistic anti-4-1BB antibody or antigen-binding fragment thereof, an agonistic anti-GITR antibody or antigen-binding fragment thereof, an agonistic anti-OX40 antibody or antigen-binding fragment thereof, an agonistic anti-TRAILR1 antibody or antigen-binding fragment thereof, an agonistic anti-TRAILR2 antibody or antigen-binding fragment thereof, an agonistic anti-TWEA antibody or antigen-binding fragment thereof, an agonistic anti-TNF-α ... K antibody or antigen-binding fragment thereof, agonistic anti-TWEAKR antibody or antigen-binding fragment thereof, agonistic anti-cell surface lymphocyte protein antibody or antigen-binding fragment thereof, agonistic anti-BRAF antibody or antigen-binding fragment thereof, agonistic anti-MEK antibody or antigen-binding fragment thereof, agonistic anti-CD33 antibody or antigen-binding fragment thereof, agonistic anti-CD20 antibody or antigen-binding fragment thereof, agonistic anti-HLA-DR antibody or antigen-binding fragment thereof, agonistic anti-HLA class I antibody or antigen-binding fragment thereof, agonistic anti-CD52 antibody or antigen-binding fragment thereof, agonistic anti-A33 antibody or antigen-binding fragment thereof, agonistic anti-GD3 antibody or antigen-binding fragment thereof, agonistic anti-PSMA antibody or antigen-binding fragment thereof, agonistic anti-Ceacan 1 antibody or antigen-binding fragment thereof, agonistic anti-Galedin 9 antibody or antigen-binding fragment thereof, agonistic anti-HVEM antibody or antigen-binding fragment thereof, agonistic anti-VISTA antibody or antigen-binding fragment thereof, agonistic anti-B7 H4 antibody or antigen-binding fragment thereof, agonistic anti-HHLA2 antibody or antigen-binding fragment thereof, agonistic anti-CD155 antibody or antigen-binding fragment thereof, agonistic anti-CD80 antibody or antigen-binding fragment thereof, agonistic anti-BTLA antibody or antigen-binding fragment thereof, agonistic anti-CD160 antibody or antigen-binding fragment thereof, agonistic anti-CD28 antibody or antigen-binding fragment thereof, agonistic anti-CD226 antibody or antigen-binding fragment thereof, agonistic anti-CEACAM1 antibody or antigen-binding fragment thereof or antigen-binding fragment thereof, agonistic anti-TIM3 antibody or antigen-binding fragment thereof, agonistic anti-TIGIT antibody or antigen-binding fragment thereof, agonistic anti-CD96 antibody or antigen-binding fragment thereof, agonistic anti-CD70 antibody or antigen-binding fragment thereof, agonistic anti-CD27 antibody or antigen-binding fragment thereof, agonistic anti-LIGHT antibody or antigen-binding fragment thereof, agonistic anti-CD137 antibody or antigen-binding fragment thereof, agonistic anti-DR4 antibody or antigen-binding fragment thereof, agonistic anti-CR5 antibody or antigen-binding fragment thereof, agonistic anti-TNFRS antibody or antigen-binding fragment thereof, agonistic anti-TNFR1 antibody or antigen-binding fragment thereof, agonistic anti-FAS antibody or antigen-binding fragment thereof, agonistic anti-CD95 antibody or antigen-binding fragment thereof, agonistic The antibody can be selected from the group consisting of an agonistic anti-TRAIL antibody or antigen-binding fragment thereof, an agonistic anti-DR6 antibody or antigen-binding fragment thereof, an agonistic anti-EDAR antibody or antigen-binding fragment thereof, an agonistic anti-NGFR antibody or antigen-binding fragment thereof, an agonistic anti-OPG antibody or antigen-binding fragment thereof, an agonistic anti-RANKL antibody or antigen-binding fragment thereof, an agonistic anti-LTβ receptor antibody or antigen-binding fragment thereof, an agonistic anti-BCMA antibody or antigen-binding fragment thereof, an agonistic anti-TACI antibody or antigen-binding fragment thereof, an agonistic anti-BAFFR antibody or antigen-binding fragment thereof, an agonistic anti-EDAR2 antibody or antigen-binding fragment thereof, an agonistic anti-TROY antibody or antigen-binding fragment thereof, and an agonistic anti-RELT antibody or antigen-binding fragment thereof. For example, the immunotherapeutic agent can be an agonistic anti-CTLA-4 antibody or antigen-binding fragment thereof, an agonistic anti-PD-1 antibody or antigen-binding fragment thereof, or an agonistic anti-PD-L1 antibody or antigen-binding fragment thereof.

[0094] In some embodiments of the eighth, ninth, tenth, and / or eleventh aspects, the method involves administering to a subject an anti-cell surface lymphocyte protein antibody or antigen-binding fragment thereof, such as CD1, CD2, CD3, CD4, CD5, CD6, CD7, CD8, CD9, CD10, CD11, CD12, CD13, CD14, CD15, CD16, CD17, CD18, CD19, CD20, CD21, CD22, CD23, CD24, CD25, CD26, CD27, CD28, CD29, CD30, CD31, CD32, CD33, CD34, CD35, CD36, CD37, CD38, CD39, CD40, CD41, CD42, CD43, CD44, CD45, CD46, CD47, CD48, CD49, CD50, CD51, CD52, CD53, CD54, CD55, CD56, CD57, CD58, CD59, CD60, CD61, CD62, CD63, CD64, CD65, CD66, CD67, CD68, CD69, CD70, CD71, CD72, CD73, CD74, CD75, CD76, CD77, CD78, CD79, CD80, CD81, CD82, CD83, CD84, CD85, CD86, CD87, CD88, CD89, CD90, CD91, CD92, CD93, CD94, CD95, CD96, CD97, CD98, CD99, CD10, CD11, CD12, CD13, CD14, CD15, CD16, CD17, CD18, CD19, CD20, CD21, CD22, CD23, CD24, CD25, CD26, CD27, CD2 D38, CD39, CD40, CD41, CD42, CD43, CD44, CD45, CD46, CD47, CD48, CD49, CD50, CD51, CD52, CD53, CD54, CD55, CD56, CD57, CD58, CD59, CD60, CD61, CD62, CD63, CD64, CD65, CD66, CD67, CD68, CD69, CD70, CD71, CD72, CD73, CD74, CD75, CD76, CD77, CD78, CD79, CD80, CD81, CD82, CD83, CD84, CD85, CD86, CD87, CD88, CD89, CD90, CD91, CD92, CD93, CD94, CD95, CD96, CD97, CD98, CD99, CD100, CD101, CD102, CD103, CD104, CD105, CD106, CD107, CD108, CD109, CD11 0, CD111, CD112, CD113, CD114, CD115, CD116, CD117, CD118, CD119, CD120, CD121, CD122, CD123, CD124, CD125, CD126, CD127, CD128, CD129, CD130, CD1 31, CD132, CD133, CD134, CD135, CD136, CD137, CD138, CD139, CD140, CD141, CD142, CD143, CD144, CD145, CD146, CD147, CD148, CD149, CD150, CD151, C D152, CD153, CD154, CD155, CD156, CD157, CD158, CD159, CD160, CD161, CD162, CD163, CD164, CD165, CD166, CD167, CD168, CD169, CD170, CD171, CD172,CD173, CD174, CD175, CD176, CD177, CD178, CD179, CD180, CD181, CD182, CD183, CD184, CD185, CD186, CD187, CD188, CD189, CD190, CD191, CD19 2, CD193, CD194, CD195, CD196, CD197, CD198, CD199, CD200, CD201, CD202, CD203, CD204, CD205, CD206, CD207, CD208, CD209, CD210, CD211, CD2 12, CD213, CD214, CD215, CD216, CD217, CD218, CD219, CD220, CD221, CD222, CD223, CD224, CD225, CD226, CD227, CD228, CD229, CD230, CD231, CD 232, CD233, CD234, CD235, CD236, CD237, CD238, CD239, CD240, CD241, CD242, CD243, CD244, CD245, CD246, CD247, CD248, CD249, CD250, CD251, C D252, CD253, CD254, CD255, CD256, CD257, CD258, CD259, CD260, CD261, CD262, CD263, CD264, CD265, CD266, CD267, CD268, CD269, CD270, CD271 , CD272, CD273, CD274, CD275, CD276, CD277, CD278, CD279, CD280, CD281, CD282, CD283, CD284, CD285, CD286, CD287, CD288, CD289, CD290, CD29 1. Administering to a mammal (e.g., a human) an antibody or antigen-binding fragment thereof that binds and activates one or more of CD292, CD293, CD294, CD295, CD296, CD297, CD298, CD299, CD300, CD301, CD302, CD303, CD304, CD305, CD306, CD307, CD308, CD309, CD310, CD311, CD312, CD313, CD314, CD315, CD316, CD317, CD318, CD319, and / or CD320.

[0095] In some embodiments of the eighth, ninth, tenth, and / or eleventh aspects, the method includes administering to a mammal (e.g., a human) an agent (e.g., a polypeptide, an antibody, an antigen-binding fragment thereof, a single-chain polypeptide, or a construct thereof) that binds to and activates a chemokine or lymphokine. For example, the immunotherapeutic agent is an agent (e.g., a polypeptide, an antibody, an antigen-binding fragment thereof, a single-chain polypeptide, or a construct thereof) that binds to and activates one or more, or all, of CXCL1, CXCL2, CXCL3, CXCL8, CCL2, and CCL5. In some embodiments, the immunotherapeutic agent is an agent (e.g., a polypeptide, an antibody, an antigen-binding fragment thereof, a single-chain polypeptide, or a construct thereof) that binds to and activates one or more, or all, of CCL3, CCL4, CCL8, and CCL22.

[0096] In some embodiments of the eighth, ninth, tenth, and / or eleventh aspects, the mammal (e.g., human) is not administered an anti-TNFα agent, such as an anti-TNFα antibody or antigen-binding fragment thereof. In some embodiments of the eighth, ninth, tenth, and / or eleventh aspects, the mammal (e.g., human) is not administered infliximab, etanercept, certolizumab, golimumab, or adalimumab.

[0097] In some embodiments of the eighth, ninth, tenth, and / or eleventh aspects, a polypeptide, e.g., a single chain polypeptide, an antibody, an antigen-binding fragment thereof, or a construct that specifically binds to TNFR2 is administered to a mammal (e.g., a human) in an amount of about 0.001 mg / kg to about 100 mg / kg, e.g., about 0.01 mg / kg to about 10 mg / kg.

[0098] A twelfth aspect features a kit comprising a polypeptide (e.g., a single chain polypeptide, an antibody, an antigen-binding fragment thereof, or a construct thereof) of the first aspect or any embodiment thereof, a construct of the second aspect or any embodiment thereof, a polynucleotide of the third aspect or any embodiment thereof, a vector of the fourth aspect or any embodiment thereof, a host cell of the fifth aspect or any embodiment thereof, and / or a pharmaceutical composition of the sixth aspect or any embodiment thereof.

[0099] In some embodiments, the kit includes instructions for transfecting the vector into a host cell. Additionally or alternatively, the kit may include instructions for expressing a polypeptide (e.g., a single chain polypeptide, an antibody, an antigen-binding fragment thereof, or a construct thereof) in a host cell. The kit may include reagents that can be used to express a polypeptide (e.g., a single chain polypeptide, an antibody, an antigen-binding fragment thereof, or a construct thereof) in a host cell. In some embodiments, the kit includes instructions for administering the agent to a mammal (e.g., a human), such as a human patient suffering from a cell proliferation disorder and / or an infectious disease described herein. In some embodiments, the kit includes instructions for a method of making or using the agent.

[0100] definition As used herein, the term "about" refers to a value that is 10% or more above or below the stated value. For example, the term "about 5 nM" indicates a range of 4.5 nM to 5.5 nM.

[0101] As used herein, the terms "agonistic TNFR2 antibody" and "agonistic TNFR2 antibody" refer to a TNFR2 antibody that can promote or increase activation of TNFR2 and / or enhance one or more signaling pathways mediated by TNFR2. For example, the agonistic TNFR2 antibody of the invention can promote or increase the proliferation of a population of Treg cells. The agonistic TNFR2 antibody of the invention can promote or increase TNFR2 activation by binding to TNFR2, e.g., by inducing a conformational change that renders the receptor biologically active. For example, the agonistic TNFR2 antibody can nucleate the trimerization of TNFR2 in a manner similar to the interaction between TNFR2 and its cognate ligand, TNFα, thus inducing TNFR2-mediated signaling. The agonistic TNFR2 antibody of the invention can induce proliferation of CD4+, CD25+, FOXP3+ Treg cells. The agonistic TNFR2 antibodies of the invention can also inhibit the proliferation of B lymphocytes and / or cytotoxic T lymphocytes (e.g., CD8+ T cells), for example, through activation of immune regulatory Treg cells or by directly binding to TNFR2 on the surface of immune effector cells (such as autoreactive cytotoxic T cells) and inducing apoptosis. Unless otherwise specified, the terms "agonistic TNFR2 antibody" and "agonistic TNFR2 antibody" also include antibody fragments that retain the ability to bind to TNFR2 and enhance TNFR2 signaling, such as those described below.

[0102] As used herein, the term "antibody" (Ab) refers to an immunoglobulin molecule that specifically binds to or immunologically reacts with a particular antigen, and includes polyclonal, monoclonal, genetically engineered, and other modified forms of antibodies, including chimeric antibodies, humanized antibodies, heteroconjugate antibodies (e.g., bi-, tri-, and tetraspecific antibodies, diabodies, triabodies, and tetrabodies), and antibodies that are capable of binding to, for example, Fab', F(ab')2, or other antibodies. 2In addition, unless otherwise specified, the term "monoclonal antibody" (mAb) includes antigen-binding fragments of antibodies, including, but not limited to, Fab, Fv, recombinant IgG (rgG) fragments, and scFv fragments. Additionally, unless otherwise specified, the term "monoclonal antibody" (mAb) refers to intact molecules as well as antibody fragments (e.g., Fab and F(ab')) that are capable of specifically binding to a target protein. 2 The term "Fab" refers to a fragment of a human IgG antibody, such as a human IgG antibody, which is a human IgG antibody. ... 2 The fragments lack the Fc fragment of an intact antibody, clear more rapidly from an animal's circulation, and may have less non-specific tissue binding than an intact antibody (see Wahl et al., J. Nucl. Med. 24:316, 1983, incorporated herein by reference).

[0103] The term "antigen-binding fragment" as used herein refers to one or more fragments of an antibody that retain the ability to specifically bind to a target antigen. The antigen-binding function of an antibody can be performed by a fragment of a full-length antibody. Antibody fragments include, for example, Fab, F(ab') 2 Examples of binding fragments encompassed by the term "antigen-binding fragment" of an antibody include, but are not limited to, the following: (i) Fab fragments, V fragments, scFvs, SMIPs, diabodies, triabodies, affibodies, nanobodies, aptamers, or domain antibodies. L , V H , C L , and C H (ii) F(ab') 2 a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) V H and C H (iv) a V of a single arm of an antibody; L and V H (v) an Fv fragment consisting of a V H and V L (vi) a dAb containing the V H (vii) a dAb fragment consisting of the V domain (Ward et al., Nature 341:544-546, 1989); H or VL (viii) an isolated complementarity determining region (CDR), and (ix) a combination of two or more isolated CDRs, which may optionally be linked by a synthetic linker. L and V H are encoded by separate genes, L and V H The regions can be linked using recombinant techniques by a linker that allows them to be produced as a single protein chain that pairs to form a monovalent molecule (known as single-chain Fv (scFV), see, e.g., Bird et al., Science 242:423-426, 1988, and Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883, 1988). These antibody fragments can be obtained using conventional techniques known to those of skill in the art, and the fragments can be screened for utility in the same manner as intact antibodies. Antigen-binding fragments can be produced by recombinant DNA techniques, enzymatic or chemical cleavage of intact immunoglobulins, or chemical peptide synthesis procedures known in the art.

[0104] As used herein, the terms "anti-tumor necrosis factor receptor 2 antibody," "TNFR2 antibody," "anti-TNFR2 antibody portion," "anti-TNFR2 antibody fragment," and the like include proteins or peptide-containing molecules that contain at least a portion of an immunoglobulin molecule, such as at least one complementarity determining region (CDR) of a heavy or light chain or a ligand binding portion thereof, a heavy or light chain variable region, a heavy or light chain constant region, a framework region, or any portion thereof that can specifically bind to TNFR2. TNFR2 antibodies include fibronectin type III domain 10 (TNF-α), which contains structural loops BC, DE, and FG that are similar in structure and solvent accessibility to the CDRs of antibodies. 10 Also included are antibody-like protein scaffolds such as Fn3). 10 The tertiary structure of the Fn3 domain is similar to the tertiary structure of the variable region of an IgG heavy chain, and those skilled in the art will recognize, for example, 10By replacing residues in the BC, DE, and FG loops of Fn3 with residues from the CDRH-1, CDRH-2, or CDRH-3 regions of a TNFR2 monoclonal antibody, the CDRs of the TNFR2 monoclonal antibody can be grafted onto a fibronectin scaffold. 10 The use of Fn3 domains is described, for example, in WO2000 / 034784, the disclosure of which is incorporated herein by reference. Additional scaffold proteins encompassed by the term "anti-tumor necrosis factor receptor 2 antibody", "TNFR2 antibody" include peptide-Fc fusion proteins (e.g., as described in WO2012 / 122378, as well as US8,633,297, the disclosures of each of which are incorporated herein by reference).

[0105] As used herein, the term "bispecific antibody" refers to a monoclonal antibody, often a human or humanized antibody, that has binding specificity for at least two different antigens. The bispecific TNFR2 antibody of the present invention may have binding specificity for TNFR2 and any other antigen, such as a cell surface protein, a receptor, a receptor subunit, or a tissue-specific antigen. The bispecific antibody may also be an antibody or antigen-binding fragment thereof that comprises two separate antigen-binding domains (e.g., two scFvs linked by a linker). The scFvs can bind to the same antigen or different antigens.

[0106] As used herein, the term "chimeric" antibody refers to an antibody having variable domain sequences (e.g., CDR sequences) derived from the immunoglobulin of one source organism, such as a rat or mouse, and a constant region derived from the immunoglobulin of a different organism (e.g., human, another primate, pig, goat, rabbit, hamster, cat, dog, guinea pig, members of the bovine family (such as cow, bison, buffalo, elk, yak, etc.), cow, sheep, horse, or bison, among others). Methods for producing chimeric antibodies are known in the art. See, e.g., Morrison, 1985, Science 229(4719):1202-7; Oi et al, 1986, BioTechniques 4:214-221; Gillies et al, 1985, J. Immunol. Methods 125:191-202; U.S. Patent Nos. 5,807,715, 4,816,567, and 4,816,397, which are incorporated herein by reference.

[0107] As used herein, the term "complementarity determining region" (CDR) refers to the hypervariable regions found in both light and heavy chain variable domains. The more highly conserved portions of the variable domains are called framework regions (FRs). As understood in the art, the amino acid positions that delineate the hypervariable regions of an antibody may vary depending on the context and the various definitions known in the art. Some positions within a variable region may be considered hybrid hypervariable positions, where these positions are considered to be within a hypervariable region under one set of criteria, while being considered to be outside the hypervariable region under a different set of criteria. One or more of these positions may also be found in an extended hypervariable region. The present invention includes antibodies that contain modifications at these hybrid hypervariable positions. Natural heavy and light chain variable domains each primarily adopt a β-sheet structure and comprise four framework regions connected by three CDRs that form loops connecting the β-sheet structure and, in some cases, form part of the β-sheet structure. The CDRs of each chain are held together in close proximity by the FR regions in the order FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 and, together with the CDRs from the other antibody chain, contribute to the formation of the target binding site of the antibody (see Kabat et al, Sequences of Proteins of Immunological Interest (National Institute of Health, Bethesda, Md. 1987), which is incorporated herein by reference). As used herein, immunoglobulin amino acid residues are numbered according to the immunoglobulin amino acid residue numbering system of Kabat et al., unless otherwise specified.

[0108] As used herein, the terms "conservative mutation," "conservative substitution," or "conservative amino acid substitution" refer to the replacement of one or more amino acids with one or more different amino acids that exhibit similar physicochemical properties, such as polarity, electrostatic charge, and / or steric bulk. These properties are summarized for each of the 20 naturally occurring amino acids in Table 1 below. [Table 1]

[0109] From this table it can be seen that conservative amino acid families include, for example, (i) G, A, V, L, I, P, and M, (ii) D and E, (iii) C, S, and T, (iv) H, K, and R, (v) N and Q, and (vi) F, Y, and W. Thus, a conservative mutation or substitution is the replacement of one amino acid with a member of the same amino acid family (e.g., Ser with Thr, or Lys with Arg).

[0110] As used herein, the term "conjugate" refers to a compound formed by chemical bonding of a reactive functional group of one molecule with a suitable reactive functional group of another molecule.Conjugates can additionally be produced as two polypeptide domains covalently linked to each other as part of a single polypeptide chain, for example, synthesized by translation of a single RNA transcript that codes for both polypeptides in frame with each other.

[0111] As used herein with respect to TNFR2 agonists, the term "construct" refers to a fusion protein comprising a first polypeptide domain bound to a second polypeptide domain. Each polypeptide domain can be, independently, for example, an agonistic TNFR2 single-chain polypeptide as described herein. The first polypeptide domain can be covalently bound to the second polypeptide domain, for example, via a linker, such as a peptide linker or a disulfide bridge, among others. Exemplary linkers that can be used to link the polypeptide domains of an agonistic TNFR2 construct include, but are not limited to, those described in Leriche et al., Bioorg.Med.Chem.,20:571-582(2012), the disclosure of which is incorporated herein in its entirety by reference.

[0112] As used herein, the term "derivatized antibody" refers to an antibody that has been modified by chemical reaction to cleave residues or add chemical moieties not native to the isolated antibody. Derivatized antibodies can be obtained by glycosylation, acetylation, PEGylation, phosphorylation, amidation, derivatization by addition of known chemical protecting / blocking groups, proteolytic cleavage, and / or conjugation to cellular ligands or other proteins. Any of a variety of chemical modifications can be performed using established procedures by known techniques, including, but not limited to, specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, and the like. Additionally, derivatives can include one or more unnatural amino acids, for example, using amber suppression techniques (see, for example, U.S. Patent No. 6,964,859, incorporated herein by reference).

[0113] As used herein, the term "diabody" refers to a bivalent antibody comprising two polypeptide chains, each of which is a VH domain connected by a linker (e.g., a linker consisting of five amino acids) that is too short to allow intramolecular association of the VH and VL domains on the same peptide chain. H and V L domains. This configuration allows each domain to pair with a complementary domain on another polypeptide chain to form a homodimeric structure. Thus, the term "triabody" refers to a trivalent antibody that contains three peptide chains, each of which contains one VH domain and one VL domain linked by a very short linker (e.g., a linker consisting of 1-2 amino acids), allowing intramolecular association of the VH and VL domains in the same peptide chain. To fold into their native structure, peptides thus configured typically trimerize to position the VH and VL domains of adjacent peptide chains in close spatial proximity to each other and allow proper folding (see Holliger et al., Proc. Natl. Acad. Sci. USA 90:6444-48, 1993, incorporated herein by reference).

[0114] As used herein, a "disulfide bond isoform" of an antibody or antigen-binding fragment thereof is a form of an antibody or antigen-binding fragment thereof that has a particular internal disulfide bonding pattern. Disulfide bond isoforms are structural isomers of a given antibody or antigen-binding fragment thereof that do not differ from each other in amino acid sequence but exhibit different disulfide bond linkages. For example, in the context of a human IgG2 antibody or variant thereof, an antibody is referred to herein as being of the isoform IgG2-A, IgG2-B, IgG2-A / B, or IgG2-C. 1 , and IgG2-A / B 2 The disulfide bond linkages within each of these isoforms are illustrated in Figures 5A-5D.

[0115] A "dominant agonist" of TNFR2 is an agonist (e.g., an agonistic polypeptide, e.g., a single chain polypeptide, an antibody, or an antigen-binding fragment thereof) that can promote TNFR2 activation in the absence of a natural TNFR2 ligand, such as TNFα. For example, the EC 50 A TNFR2 agonist is a dominant agonist if it increases by less than 200% (e.g., less than 200%, 100%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, 1% or less) in the absence of natural TNFR2 ligands such as TNFα. TNFR2 activation can be assessed, for example, by measuring the proliferation of TNFR2+ cells, such as Treg cells, MDSCs, parenchymal cells, and cells of the central nervous system, among others, and by measuring the activation of NFκB signaling (e.g., by monitoring the expression of one or more genes selected from the group consisting of CHUK, NFKBIE, NFKBIA, MAP3K11, TRAF2, TRAF3, relB, and cIAP2 / BIRC3 in a gene expression assay).

[0116] As used herein, a "dual variable domain immunoglobulin" ("DVD-Ig") refers to an antibody that combines the target-binding variable domains of two monoclonal antibodies via a linker to create a tetravalent, dual-targeting single agent. (Gu et al., Meth. Enzymol., 502:25-41, 2012, incorporated herein by reference). Linkers suitable for use in the light chain of the DVDs described herein include those identified in Table 2.1 on page 30 of Gu et al., including the short K chain linkers ADAAP (SEQ ID NO: 24) (mouse), and TVAAP (SEQ ID NO: 25) (human); long κ chain linkers ADAAPTVSIFP (SEQ ID NO: 26) (mouse), and TVAAPSVFIFPP (SEQ ID NO: 27) (human), short λ ​​chain linker QPKAAP (SEQ ID NO: 28) (human) long λ chain linker QPKAAPSVTLFPP (SEQ ID NO: 29) (human), GS-short linker GGSGG (SEQ ID NO: 30), GS-medium linker GGSGGGGSG (SEQ ID NO: 31), GS-long linker GGSGGGSGGGGGS (SEQ ID NO: 32) (all GS linkers are mouse and human). Linkers suitable for use in the heavy chain of the DVD include those identified in Table 2.1 on page 30 of Gu & Ghayur, 2012, Methods in Enzymology 502:25-41, which is incorporated herein by reference: short linkers AKTTAP (SEQ ID NO:33) (mouse), and ASTKGP (SEQ ID NO:34) (human), long linkers AKTTAPSVYPLAP (SEQ ID NO:35) (mouse), and ASTKGPSVFPLAP (SEQ ID NO:36) (human), GS-short linker GGGGSG (SEQ ID NO:37), GS-medium linker GGGGSGGGGS (SEQ ID NO:38), GS-long linker GGGGSGGGGSGGGG (SEQ ID NO:39) (all GS linkers are mouse and human).

[0117] As used herein, the term "endogenous" refers to a molecule (e.g., a polypeptide, nucleic acid, or cofactor) that is naturally found in a particular organism (e.g., a human) or in a particular location within an organism (e.g., an organ, tissue, or cell, such as a human cell).

[0118] As used herein, the term "epitope" refers to a portion of an antigen that is recognized and bound by a polypeptide, such as an antibody, an antigen-binding fragment thereof, a single-chain polypeptide, or a construct described herein. In the context of a protein antigen (TNFR2, e.g., human TNFR2 designated by SEQ ID NO: 1, or TNFR2 of a non-human mammal, such as a non-human mammal described herein), an epitope may be a continuous epitope, which is a single uninterrupted segment of one or more amino acids covalently linked to each other by peptide bonds, and the constituent amino acids of the epitope are bound by a polypeptide (e.g., an antibody, an antigen-binding fragment thereof, a single-chain polypeptide, or a construct). An exemplary assay for determining the binding of an agonistic TNFR2 polypeptide to a specific amino acid in an antigen is described in Example 1 below. A continuous epitope may be composed of 1, 5, 10, 15, 20, or more amino acids in an antigen, such as, for example, a TNFR2 protein described herein (e.g., human TNFR2 designated by SEQ ID NO: 1). For example, a continuous epitope can be made up of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, or more amino acids within the antigen. An example of a continuous epitope on TNFR2 bound by the agonistic polypeptides described herein (e.g., single chain polypeptides, antibodies, antigen-binding fragments thereof, and constructs) includes one or more or all of the KCSPG motif (SEQ ID NO: 40) and the corresponding region of the TNFR2 protein of a non-human mammal (e.g., bison, cow, and others described herein). In some embodiments, the epitope may be a discontinuous epitope comprising two or more amino acid segments separated from each other within the amino acid sequence of the antigen by one or more intervening amino acid residues. A discontinuous epitope may be, for example, such a segment of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid residues, such as (i) the amino acids within the amino KCSPG motif of human TNFR2 (SEQ ID NO: 40), or the amino acids within the amino KCSPG motif of non-human TNFR2 (SEQ ID NO: 41). and (ii) one or more of the cysteine-rich domain 1 (CRD1 residues 48-76) of human TNFR2 or an equivalent region of a non-human TNFR2.

[0119] As used herein, the term "exogenous" refers to a molecule (e.g., a polypeptide, nucleic acid, or cofactor) that is not naturally found in a particular organism (e.g., a human) or in a particular location within an organism (e.g., an organ, tissue, or cell, such as a human cell). Exogenous materials include materials provided from an external source to an organism or culture extracted therefrom.

[0120] As used herein, the term "framework region" or "FW region" includes the amino acid residues adjacent to the CDRs. FW region residues may be present, for example, in human antibodies, rodent-derived antibodies (e.g., murine antibodies), humanized antibodies, primatized antibodies, chimeric antibodies, antibody fragments (e.g., Fab fragments), single-chain antibody fragments (e.g., scFv fragments), antibody domains, bispecific antibodies, among others.

[0121] As used herein, the term "fusion protein" refers to a protein that is linked to another molecule via a covalent bond. Fusion proteins can be chemically synthesized, for example, by an amide bond formation reaction between the N-terminus of one protein and the C-terminus of another protein. Alternatively, a fusion protein that includes one protein covalently linked to another protein can be recombinantly expressed in a cell (e.g., a eukaryotic or prokaryotic cell) by expression of a polynucleotide encoding the fusion protein, for example, from a vector or the genome of the cell. A fusion protein may include one protein covalently linked to a linker, which in turn is covalently linked to another molecule. Examples of linkers that can be used to form fusion proteins include peptide-containing linkers, such as those that include natural or unnatural amino acids. In some embodiments, it may be desirable to include D-amino acids in the linker. These residues do not occur in naturally occurring proteins and may therefore be more resistant to degradation by endogenous proteases. Linkers can be prepared using a variety of strategies known in the art and, depending on the reactive moieties of the linker, can be cleaved by enzymatic hydrolysis, photolysis, hydrolysis under acidic conditions, hydrolysis under basic conditions, oxidation, disulfide reduction, nucleophilic cleavage, or organometallic cleavage (Leriche et al., Bioorg. Med. Chem., 20:571-582, 2012).

[0122] As used herein, the term "heterospecific antibody" refers to a monoclonal antibody, preferably a human or humanized antibody, having binding specificities for at least two different antigens. Traditionally, recombinant production of heterospecific antibodies is based on the co-expression of two immunoglobulin heavy / light chain pairs, where the two heavy chains have different specificities (Milstein et al., Nature 305:537, 1983). Similar procedures are described in WO93 / 08829, U.S. Pat. Nos. 6,210,668, 6,193,967, 6,132,992, 6,106,833, 6,060,285, 6,037,453, 6,010,902, 5,989,530, 5,959,084, 5,959, and 5,970, which are incorporated herein by reference. ,083, 5,932,448, 5,833,985, 5,821,333, 5,807,706, 5,643,759, 5,601,81 No. 9, No. 5,582,996, No. 5,496,549, No. 4,676,980, WO91 / 00360, WO92 / 00373, EP03089, Traunecker et al., EMBO J. 10:3655 (1991), Suresh et al., Methods in Enzymology 121:210 (1986). Heterospecific antibodies may contain Fc mutations that enforce correct chain association in multispecific antibodies, as described in Klein et al., mAbs 4(6):653-663, 2012, which is incorporated herein by reference.

[0123] As used herein, the term "human antibody" refers to an antibody that is human and contains substantially all parts of the protein (e.g., CDRs, frameworks, C L , C H Domain (e.g., C H 1. C H 2. C H 3), Hinge (V L , V H)) are substantially non-immunogenic in humans with only minor sequence changes or alterations. Human antibodies can be produced by human cells (e.g., by recombinant expression) or by non-human animals or prokaryotic or eukaryotic cells capable of expressing functionally rearranged human immunoglobulin (e.g., heavy and / or light chain) genes. Also, when the human antibody is a single-chain antibody, it may contain a linker peptide not found in naturally occurring human antibodies. For example, an Fv may contain a linker peptide, such as 2 to about 8 glycine or other amino acid residues, connecting the variable region of the heavy chain and the variable region of the light chain. Human antibodies can be produced by a variety of methods known in the art, including phage display methods using antibody libraries derived from human immunoglobulin sequences. See U.S. Patent Nos. 4,444,887 and 4,716,111, and PCT publications WO1998 / 46645, WO1998 / 50433, WO1998 / 24893, WO1998 / 16654, WO1996 / 34096, WO1996 / 33735, and WO1991 / 10741, which are incorporated herein by reference. Human antibodies can also be produced using transgenic mice which are incapable of expressing functional endogenous immunoglobulins, but which can express human immunoglobulin genes. See, e.g., PCT Publications WO98 / 24893, WO92 / 01047, WO96 / 34096, WO96 / 33735, U.S. Patent Nos. 5,413,923, 5,625,126, 5,633,425, 5,569,825, 5,661,016, 5,545,806, 5,814,318, 5,885,793, 5,916,771, and 5,939,598, which are incorporated by reference herein.

[0124] As used herein, the term "humanized" antibody refers to a chimeric immunoglobulin, immunoglobulin chain, or fragment thereof (Fv, Fab, Fab', F(ab')) that contains minimal sequence derived from non-human immunoglobulin. 2Humanized antibodies refer to forms of non-human (e.g., murine) antibodies that are not specifically humanized (such as those that are not specifically humanized, such ... See, e.g., Riechmann et al., Nature 332:323-7, 1988, U.S. Pat. Nos. 5,530,101, 5,585,089, 5,693,761, 5,693,762, and 6,180,370 to Queen et al., EP 239400, PCT Publication WO 91 / 09967, U.S. Pat. No. 5,225,539, EP 592106, and EP 519596, which are incorporated by reference herein.

[0125] As used herein, the term "hydrophobic side chain" refers to an amino acid side chain that has a low solubility in water compared to, for example, the steric or electronic properties of the chemical moieties present in the side chain. Examples of amino acids containing hydrophobic side chains include amino acids containing unsaturated aliphatic hydrocarbons, such as alanine, valine, leucine, isoleucine, proline, and methionine, and amino acids containing aromatic ring systems that are electrostatically neutral at physiological pH, such as tryptophan, phenylalanine, and tyrosine.

[0126] As used herein, the term "immunotherapeutic agent" refers to a compound, such as an antibody, antigen-binding fragment thereof, single chain polypeptide, or construct described herein, that specifically binds to an immune checkpoint protein (e.g., immune checkpoint receptor or ligand) and exerts an agonistic effect on the receptor or ligand, thereby promoting receptor or ligand signaling, e.g., downregulating an immune response. Immunotherapeutic agents include compounds, such as antibodies, antigen-binding fragments, single chain polypeptides, and constructs, that specifically bind to receptors expressed on the surface of hematopoietic cells, such as lymphocytes (e.g., T cells), and can activate receptor- or ligand-induced signaling that results in, e.g., tolerance to endogenous ("self") antigens. Immunotherapeutic agents can enhance receptor or ligand-induced signaling, for example, by 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.9%, 100%, 200%, 300%, 400%, 500%, or more, compared to receptor or ligand-induced signaling exhibited in the absence of the immunotherapeutic agent. Exemplary assays that can be used to measure the extent of receptor or ligand signaling include, for example, enzyme-linked immunosorbent assay (ELISA) techniques to measure protein expression changes associated with a particular signaling pathway, as well as polymerase chain reaction (PCR)-based techniques, such as reverse transcription PCR, quantitative PCR, and real-time PCR experiments, which are useful for determining changes in gene expression associated with a particular signaling pathway. Examples of immunotherapeutic agents include, for example, antibodies or antigen-binding fragments thereof that specifically bind and activate one or more of OX40L, TL1A, CD40L, LIGHT, BTLA, LAG3, TIM3, Singlecs, ICOS, B7-H3, B7-H4, VISTA, TMIGD2, BTNL2, CD48, KIR, LIR, LIR antibodies, ILT, NKG2D, NKG2A, MICA, MICB, CD244, CSF1R, IDO, TGFβ, CD39, CD73, CXCR4, CXCL12, SIRPA, CD47, VEGF, and neuropilin.Specific examples of immunotherapeutic agents that can be used in conjunction with the compositions and methods described herein include agonistic anti-PD-1 antibodies and antigen-binding fragments thereof, as well as agonistic anti-PD-L1 antibodies and antigen-binding fragments thereof, and agonistic anti-CTLA-4 antibodies and antigen-binding fragments thereof.

[0127] As used herein, the term "monoclonal antibody" refers to an antibody that is derived from a single clone, including any eukaryotic, prokaryotic, or phage clone, and not the method by which it is produced.

[0128] As used herein, the term "multispecific antibody" refers to an antibody that exhibits affinity for more than one target antigen. Multispecific antibodies can have a structure similar to a complete immunoglobulin molecule and can include an Fc region, such as an IgG Fc region. Such structures include IgG-Fv, IgG-(scFv) 2 , DVD-Ig, (scFv) 2 -(scFv) 2 -Fc, and (scFv) 2 -Fc-(scFv) 2 IgG-(scFv) 2In the case of the scFv, the scFv can be attached to either the N-terminus or C-terminus of either the heavy or light chain. Exemplary multispecific molecules that include an Fc region and can incorporate TNFR2 antibodies or antigen-binding fragments thereof are reviewed in Kontermann, 2012, mAbs 4(2):182-197, Yazaki et al, 2013, Protein Engineering, Design & Selection 26(3):187- 193, and Grote et al, 2012, in Proetzel & Ebersbach (eds.), Antibody Methods and Protocols, Methods in Molecular Biology vol.901, chapter 16:247-263, which are incorporated herein by reference. In some embodiments, the antibody fragment can be a component of a multispecific molecule without an Fc region, based on a fragment of IgG or a DVD or scFv. Exemplary multispecific molecules that lack an Fc region and can incorporate antibodies or antibody fragments include scFv dimers (diabodies), trimers (trimers) and tetramers (tetramers), Fab dimers (conjugated with an adhesive polypeptide or protein domain), and Fab trimers (chemically linked), as described in Hudson and Souriau, 2003, Nature Medicine 9:129-134, incorporated herein by reference.

[0129] As used herein, the term "myeloid-derived suppressor cells" or "MDSC" refers to cells of the immune system that regulate the activity of various effector and antigen-presenting cells, such as T cells, NK cells, dendritic cells, and macrophages, among others. Myeloid-derived suppressor cells are distinguished by their gene expression profile and include B7-1 (CD80), B7-H1 (PD-L1), CCR2, CD1d, CD1d1, CD2, CD31 (PECAM-1), CD43, CD44, complement components C5a R1, F4 / 80 (EMR1), Fcγ RIII (CD16), Fcγ RII (CD32), Fcγ RIIA (CD32a), Fcγ RIIB (CD32b), Fcγ RIIB / C (CD32b / c), Fcγ RIIC (CD32c), Fcγ RIIIA (CD16A), Fcγ RIIIB (CD16b), Galectin-3, GP130, Gr-1 (Ly-6G), ICAM-1 (CD54), IL-1RI, IL-4Rα, IL-6Rα, integrin α4 (CD49d), integrin αL (CD11a), integrin αM (CD11b), M-CSFR, MGL1 (CD301a), MGL1 / 2 (CD301a / b), MGL2 (CD301b), nitric oxide, PSGL-1 (CD162), L-selectin (CD62L), Siglec-3 (CD33), transferrin receptor (TfR), VEGFR1 (Flt-1), and VEGFR2 (KDR or Flk-1). In particular, MDSCs do not express proteins selected from the group consisting of B7-2 (CD86), B7-H4, CD11c, CD14, CD21, CD23 (FcεRII), CD34, CD35, CD40 (TNFRSF5), CD117 (c-kit), HLA-DR, and Sca-1 (Ly6).

[0130] As used herein, a "non-native constant region" refers to an antibody constant region that is derived from a source other than the antibody variable region, or that is a human-generated synthetic polypeptide having an amino acid sequence different from that of the constant region of the antibody. For example, an antibody that includes a non-native constant region may have a variable region derived from a non-human source (e.g., mouse, rat, or rabbit) and a constant region derived from a human source (e.g., a human antibody constant region), or from another primate, such as pig, goat, rabbit, hamster, cat, dog, guinea pig, member of the bovine family (such as cow, bison, buffalo, moose, yak, etc.), cow, sheep, horse, bison, among others.

[0131] As used herein, the term "percent (%) sequence identity" refers to the percentage of amino acid (or nucleic acid) residues of a candidate sequence that are identical to the amino acid (or nucleic acid) residues of a reference sequence after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity (e.g., gaps can be introduced in one or both of the candidate and reference sequences for optimal alignment, and non-homologous sequences can be ignored for comparison purposes). Alignment for purposes of determining percent sequence identity can be accomplished in a variety of ways that are within the skill of the art, for example, using publicly available computer software such as BLAST, ALIGN, or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms required to achieve maximum alignment over the entire length of the sequences being compared. For example, a reference sequence aligned for comparison with a candidate sequence may show that the candidate sequence exhibits 50% to 100% sequence identity over the entire length of the candidate sequence or over a selected portion of consecutive amino acid (or nucleic acid) residues of the candidate sequence. The length of the candidate sequence aligned for comparison can be, for example, at least 30% (e.g., 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%) of the length of the reference sequence. When a position in the candidate sequence is occupied by an amino acid residue that is identical to the corresponding position in the reference sequence, then the molecules are identical at that position.

[0132] As used herein, the term "primatized antibody" refers to an antibody that comprises framework regions derived from an antibody of primate origin and other regions, such as CDRs and / or constant regions, derived from an antibody of non-primate origin. Methods for generating primatized antibodies are known in the art. See, for example, U.S. Patent Nos. 5,658,570, 5,681,722, and 5,693,780, which are incorporated herein by reference. For example, the primatized antibody or antigen-binding fragment thereof described herein can be generated by inserting the CDRs of a non-primate antibody or antigen-binding fragment thereof into an antibody or antigen-binding fragment thereof that comprises one or more framework regions of a primate.

[0133] As used herein, the term "operably linked" in the context of polynucleotide fragments is intended to mean that two polynucleotide fragments are joined such that the amino acid sequences encoded by the two polynucleotide fragments remain in-frame.

[0134] As used herein, the term "pharmacokinetic profile" refers to the absorption, distribution, metabolism, and clearance of a therapeutic agent (e.g., a polypeptide of the disclosure, such as an agonistic TNFR2 antibody, antigen-binding fragment thereof, single chain polypeptide, or construct) over time following drug administration to a patient.

[0135] As used herein, the term "regulatory sequence" includes promoters, enhancers and other expression control elements (e.g., polyadenylation signals) that control the transcription or translation of, for example, antibody chain genes. Such regulatory sequences are described, for example, in Goeddel, Gene Expression Technology: Methods in Enzymology 185 (Academic Press, San Diego, Calif., 1990), which is incorporated herein by reference.

[0136] As used herein, the term "scFv" refers to a single chain Fv antibody in which the variable domains of the heavy and light chains of an antibody are combined to form one chain. The scFv fragment contains a single polypeptide chain that includes the variable region of the antibody light chain (VL) (e.g., CDR-L1, CDR-L2, and / or CDR-L3) and the variable region of the antibody heavy chain (VH) (e.g., CDR-H1, CDR-H2, and / or CDR-H3) separated by a linker. The linker connecting the VL and VH regions of the scFv fragment can be a peptide linker composed of proteinogenic amino acids. Alternative linkers can be used to increase the resistance of the scFv fragment to proteolysis (e.g., linkers containing D amino acids), to increase the solubility of the scFv fragment (e.g., hydrophilic linkers such as polyethylene glycol-containing linkers or polypeptides containing repeating glycine and serine residues), to improve the biophysical stability of the molecule (e.g., linkers containing cysteine ​​residues that form intra- or intermolecular disulfide bonds), or to reduce the immunogenicity of the scFv fragment (e.g., glycosylation site-containing linkers). scFv molecules are known in the art and are described, for example, in U.S. Patent No. 5,892,019, Flo et al., (Gene 77:51, 1989), Bird et al., (Science 242:423, 1988), Pantoliano et al., (Biochemistry 30:10117, 1991), Milenic et al., (Cancer Research 51:6363, 1991), and Takkinen et al., (Protein Engineering 4:837, 1991). The VL and VH domains of scFv molecules can be derived from one or more antibody molecules. It will also be understood by those skilled in the art that the variable regions of the scFv molecules described herein can be modified to differ in amino acid sequence from the antibody molecule from which they are derived. For example, in one embodiment, nucleotide or amino acid substitutions can be made that result in conservative substitutions or changes in amino acid residues (e.g., in CDR and / or framework residues).Alternatively, or in addition, mutations are made to CDR amino acid residues to optimize antigen binding using art-recognized techniques. scFv fragments are described, for example, in WO2011 / 084714, which is incorporated herein by reference.

[0137] As used herein, the phrase "specifically binds" refers to a binding reaction that determines the presence of an antigen in a heterogeneous population of proteins and other biomolecules that is specifically recognized, for example, by an antibody or antigen-binding fragment thereof. An antibody or antigen-binding fragment thereof that specifically binds to an antigen has a K D For example, an antibody or antigen-binding fragment thereof that specifically binds to an antigen may have a K of up to 100 nM (e.g., 1 pM to 100 nM). D An antibody or antigen-binding fragment thereof that does not exhibit specific binding to a particular antigen or epitope thereof will have a K of greater than 100 nM (e.g., greater than 500 nm, 1 μM, 100 μM, 500 μM, or 1 mM) for that particular antigen or epitope. D A variety of immunoassay formats can be used to select antibodies specifically immunoreactive with a particular protein or carbohydrate. For example, solid-phase ELISA immunoassays are routinely used to select antibodies specifically immunoreactive with a protein or carbohydrate. For a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity, see Harlow & Lane, Antibodies, A Laboratory Manual, Cold Spring Harbor Press, New York (1988) and Harlow & Lane, Using Antibodies, A Laboratory Manual, Cold Spring Harbor Press, New York (1999).

[0138] As used herein, the terms "subject" and "patient" refer to an organism receiving treatment (e.g., by administration of an agonistic TNFR2 polypeptide, such as an antibody, antigen-binding fragment thereof, single-chain polypeptide, or construct described herein) for a particular disease or condition, such as an immunological disorder (e.g., an autoimmune disease). Examples of subjects and patients include mammals, such as humans, primates, pigs, goats, rabbits, hamsters, cats, dogs, guinea pigs, members of the bovine family (such as cows, bison, buffalo, yaks), sheep, and cattle, among others, that are receiving treatment for an immune disease or condition, such as an autoimmune disease, graft-versus-host disease, allograft rejection, allergic reactions, and asthma. Patients that can be treated using the compositions and methods described herein may have an established disease (e.g., an established immune disease, such as an autoimmune disease), where the patient has been diagnosed with the disease and has been showing symptoms of the disease for an extended period of time (e.g., over days, weeks, months, or years). Alternatively, a patient may be showing symptoms of a particular disease, such as an immune disorder described herein, but has not yet been diagnosed with the disease by a physician. Other patients that can be treated using the compositions and methods described herein include patients who have been diagnosed with an immunological disorder, and may or may not currently be showing symptoms of the disease. For example, a patient eligible for treatment with the compositions and methods described herein may be described as diagnosed but asymptomatic if the patient has been diagnosed with an immunological disorder, such as multiple sclerosis, by detection of depletion of myelin around one or more of the patient's neurons due to the activity of autoreactive T cells, even if the patient has not yet shown symptoms of multiple sclerosis (e.g., loss of balance, decreased cognitive ability, blurred vision, decreased coordination, etc.). Another example of a patient who has been diagnosed with an immunological condition but is asymptomatic includes a patient who has been diagnosed with rheumatoid arthritis, such as by detection of autoreactive T cells in a lymph sample isolated from the patient, even if the patient has not yet shown symptoms associated with the disease, such as joint pain, joint stiffness, reduced muscle range or movement, among others.

[0139] As used herein, the various forms of the term "transfection" refer to any of a wide variety of techniques commonly used to introduce exogenous DNA into prokaryotic or eukaryotic host cells, such as electroporation, lipofection, calcium phosphate precipitation, DEAE-dextran transfection, etc.

[0140] As used herein, the term "treat" or "treatment" refers to therapeutic procedures aimed at inhibiting or slowing down (alleviating) undesirable physiological changes or disorders, such as immunological disorders (e.g., autoimmune diseases, allergic reactions, graft-versus-host disease, host-versus-host disease, or allograft rejection). Beneficial or desired clinical results of treatment include, but are not limited to, alleviation of disease symptoms, reduction in the extent of disease, stabilization of the disease state (i.e., not worsening), delay or slowing of disease progression, remission or reversal of the disease state, and remission (partial or total), whether detectable or undetectable. Those in need of treatment include those already with a condition or disorder, as well as those prone to having the condition or disorder, or those in which the condition or disorder is to be inhibited.

[0141] As used herein, the term "tumor necrosis factor receptor superfamily," "TNFR," or "TNFRS" refers to a group of type I transmembrane proteins that have a carboxy-terminal intracellular domain and an amino-terminal extracellular domain characterized by a common cysteine-rich domain (CRD). The TNFR superfamily includes receptors that mediate cell signaling as a result of binding to one or more ligands of the TNF superfamily. The TNFR superfamily can be divided into two subgroups: receptors that contain an intracellular death domain and receptors that do not have this domain. The death domain is an 80 amino acid motif that propagates the signaling cascade of apoptosis following receptor activation. Exemplary TNFR superfamily members that contain an intracellular death domain include TNFR1, while TNFR2 represents a TNFR superfamily protein that does not contain this domain. Members of the TNFR superfamily include TNFR1, TNFR2, RANK, CD30, CD40, lymphotoxin beta receptor (LT-βR), OX40, Fas receptor, decoy receptor 3 (DCR3), CD27, 4-1BB, death receptor 4 (DR4), death receptor 5 (DR5), decoy receptor 1 (DCR1), decoy receptor 2 (DCR2), osteoprotegerin, TWEAK receptor, TACI, BAFF receptor, herpes virus entry mediator, nerve growth factor receptor, B-cell maturation antigen, glucocorticoid-induced TNFR-related, TROY, death receptor 6 (DR6), death receptor 3 (DR3), and ectodysplasia receptor A2.

[0142] As used herein, the term "variable region CDR" includes amino acids within a CDR or complementarity determining region that are identified using sequence or structure-based methods. As used herein, the term "CDR" or "complementarity determining region" refers to the non-contiguous antigen-binding sites found within the variable regions of both heavy and light chain polypeptides. These particular regions are described in Kabat et al., J. Biol. Chem. 252:6609-6616, 1977 and Kabat, et al., Sequences of Proteins of Immunological Interest, Fifth Edition, USDepartment of Health and Human Services, NIH Publication No. 91 -3242, 1991, Chothia et al., (J. Mol. Biol. 196:901-917, 1987), and MacCallum et al., (J. Mol. Biol. 262:732-745, 1996), where the definitions include overlapping or subsets of amino acid residues when compared to each other. In certain embodiments, the term "CDR" refers to the CDRs defined by Kabat based on sequence comparisons.

[0143] As used herein, the term "vector" includes nucleic acid vectors, e.g., DNA vectors such as plasmids, RNA vectors, viruses or other suitable replicons (e.g., viral vectors). A variety of vectors have been developed to deliver polynucleotides encoding exogenous proteins into prokaryotic or eukaryotic cells. Examples of such expression vectors are disclosed, for example, in WO1994 / 11026, which is incorporated herein by reference. The expression vectors described herein include polynucleotide sequences and additional sequence elements, etc., used for protein expression and / or integration of these polynucleotide sequences into the genome of mammalian cells. Particular vectors that can be used for expression of the antibodies and antibody fragments described herein include plasmids that include regulatory sequences, such as promoter and enhancer regions, that direct gene transcription. Other useful vectors for expression of antibodies and antibody fragments include polynucleotide sequences that enhance the translation rate of these genes or improve the stability or nuclear transport of the mRNA resulting from gene transcription. These sequence elements include, for example, 5' and 3' untranslated regions, internal ribosome entry sites (IRES), and polyadenylation signal sites to direct efficient transcription of genes carried in the expression vector. The expression vectors described herein may also include a polynucleotide encoding a marker for the selection of cells containing such a vector. Examples of suitable markers include genes encoding resistance to antibiotics such as ampicillin, chloramphenicol, kanamycin, or nourseothricin.

[0144] As used herein, the term "VH" refers to the variable region of an immunoglobulin heavy chain of an antibody, including the heavy chain of an Fv, scFv, or Fab. Reference to "VL" refers to the variable region of an immunoglobulin light chain, including the light chain of an Fv, scFv, dsFv, or Fab. Antibodies (Ab) and immunoglobulins (Ig) are glycoproteins with identical structural characteristics. While antibodies exhibit binding specificity to a specific target, immunoglobulins include both antibodies and other antibody-like molecules that lack target specificity. Natural antibodies and immunoglobulins are usually heterotetrameric glycoproteins of about 150,000 daltons, composed of two identical light (L) chains and two identical heavy (H) chains. Each heavy chain of a natural antibody has a variable domain (VH) at the amino terminus followed by several constant domains. Each light chain of a natural antibody has a variable domain at the amino terminus (VL) and a constant domain at the carboxy terminus. [Brief description of the drawings]

[0145] [Figure 1] 1 shows the amino acid sequence of human TNFR2, with the highlighted regions indicating exemplary epitopes to which the TNFR2 agonistic antibodies of the present disclosure may bind. Human TNFR2 is numbered herein starting with the N-terminal methionine at position 1 and ending with the C-terminal serine at position 461 (SEQ ID NO:1). All references to amino acid positions within TNFR2 are made relative to the TNFR2 numbering scheme shown in FIG. 1. [Diagram 2] 1 is a graph showing the proliferative effect of an exemplary TNFR2 agonistic antibody of the present disclosure on Treg cells. The results shown in this figure were obtained by incubating the agonistic TNFR2 antibody TNFRAG1 described herein at various concentrations (shown along the x-axis) with Treg cells for 48-72 hours. Values ​​along the y-axis represent the percentage of Treg cells in the cell population examined. Data represent the average of 35 separate experiments. [Diagram 3]1 is a graph showing the effect of the natural TNFR2 agonist TNFα on the expansion of Treg cells induced by the TNFR2 agonist antibody TNFRAG1 of the present disclosure. Values ​​along the x-axis indicate the presence of increasing concentrations of either IL-2, TNFα, or antibody TNFRAG1 alone, or antibody TNFRAG1 in combination with 20 ng / ml of TNFα, in an in vitro population of Treg cells. Values ​​along the y-axis represent the percentage of Treg cells in the cell population examined. These data indicate that the presence of TNFα further enhances the ability of TNFR2 agonist antibodies such as TNFRAG1 of the present disclosure to stimulate Treg cell proliferation. These data are consistent with the ability of the TNFR2 agonist antibodies of the present disclosure to promote TNFR2 activation by binding to TNFR2 monomers to form a trimeric structure. This binding event brings individual TNFR2 molecules into close proximity with each other, sterically exposing sites on the TNFR2 surface to which endogenous ligands such as TNFα may bind. Without being limited by mechanism, this sequence of events represents a pathway by which the TNFR2 agonist polypeptides of the present disclosure (e.g., single chain polypeptides, antibodies, antigen-binding fragments thereof, and constructs) can result in TNFR2 activation in vivo. [Figure 4] 1 is a graph demonstrating the inhibitory effect of anti-TNFα antibodies on the ability of TNFRAG1, an agonistic TNFR2 antibody of the present disclosure, to stimulate Treg cell proliferation. Values ​​along the x-axis represent the concentration of anti-TNFα antibodies in μg / ml present in an in vitro population of Treg cells. All conditions shown along the x-axis contain 2.5 μg / ml TNFRAG1. Values ​​along the y-axis represent the percentage of Treg cells in the cell population examined. [Figure 5-1]Figures 5A-5D: A series of schematic diagrams comparing the disulfide bond arrangements present in each of the IgG2-A (Figure 5A), IgG2-B (Figure 5B), IgG2-A / B1 (Figure 5C), and IgG2-A / B2 (Figure 5D) isoforms of human IgG2 isotype antibodies. Thin lines represent disulfide bonds connecting various portions of the heavy or light chains of each antibody, represented by shaded rectangles. Heavy chains are represented by the outermost long rectangles of each antibody. Within each heavy chain, black shading indicates the constant region and light shading indicates the variable region. Light chains are represented by the innermost short rectangles of each antibody. Within each light chain, dark shading indicates the constant region and light shading indicates the variable region. [Figure 5-2] This is a continuation of Figure 5-1. [Figure 6-1]Figure 6A: Graph showing the expression density of TNFR2 in Treg cells of cancer patients, healthy subjects, and human type 1 diabetes patients, as assessed by immunofluorescence. The mean fluorescence intensity (MFI) of TNFR2 in Treg cells obtained from cancer patients was 147.435 (SEM 29.053, n = 10). The MFI of TNFR2 in Treg cells obtained from healthy subjects was 59.055 (SEM 9.545, n = 10). The MFI of TNFR2 in Treg cells obtained from type 1 diabetes patients was 29.313 (SEM 2.894, n = 24). Outliers of the Grubbs test were removed. Figure 6B: Graph showing the expression density of TNFR2 in T effector (Teff) cells of cancer patients, healthy subjects, and human type 1 diabetes patients, as assessed by immunofluorescence. The mean fluorescence intensity (MFI) of TNFR2 in Teff cells obtained from cancer patients was 32.070 (SEM 6.290, n = 10). The MFI of TNFR2 in Teff cells obtained from healthy subjects was 40.811 (SEM 6.466, n = 10). The MFI of TNFR2 in Teff cells obtained from type 1 diabetes patients was 66.600 (SEM 1.815, n = 10). Outliers of the Grubbs test were removed. Figure 6C: Graph showing the effect of the agonistic TNFR2 antibody TNFRAG1 on the proliferation of Treg cells when TNFRAG1 was incubated for 48 h with a population of freshly isolated peripheral human CD4+ cells (n = 13). Values ​​along the x-axis represent the concentration of the antibody. Values ​​along the y-axis represent the percentage of Treg cells within the population of lymphocytes. Data are mean ± SEM. FIG. 6D: Graph showing the effect of agonistic TNFR2 antibody TNFRAG1 on Treg cell proliferation when TNFRAG1 was incubated with a population of freshly isolated peripheral human CD4+ cells for 48 hours in the presence of the endogenous TNFR2 ligand TNFα (20 ng / ml) (n=2). Values ​​along the x-axis represent the concentration of antibody. Values ​​along the y-axis represent the percentage of Treg cells within the population of lymphocytes. Data are mean±SEM. [Figure 6-2] This is a continuation of Figure 6-1. [Figure 7-1] FIG. 7A: Graph showing the effect of F(ab')2 antibody fragments containing the complementarity determining regions (CDRs) of TNFRAG1 on the proliferation of Treg cells. Values ​​along the x-axis represent the concentration of the antibody fragment. Values ​​along the y-axis represent the relative increase in Treg cell mass, normalized to Treg cells treated with interleukin-2 (IL-2). Data are mean ± SEM. These data indicate that a functional Fc region, absent in the F(ab')2 antibody fragment, is not required for TNFRAG1 to induce Treg proliferation. FIG. 7B: Graph showing the effect of TNFRAG1 in the presence of a bridging antibody (2.5 μg / ml) on the proliferation of Treg cells. The bridging antibody ab9165 (Abcam) binds to the Fc region of the adjacent TNFRAG1 antibody, thus increasing the local density of TNFRAG1 on the Treg cell surface. Data are mean ± SEM. Figure 7C: Two graphs comparing the effect of an agonistic TNFR2 antibody, MR2-1 (Abcam), on Treg cell proliferation in the presence of TNFα (20 mg / ml) (left) and the effect of TNFα alone on Treg cell proliferation (right). Values ​​along the x-axis represent the concentration of TNFR2 agonist. Values ​​along the y-axis represent the relative increase in Treg cell mass. Figure 7D: Graph showing the effect of IL-2, TNFα, and TNFRAG1 on the secretion of soluble TNFR2 in a population of peripheral human CD4+ cells. Values ​​along the y-axis are fluorescence intensity measurements obtained from an enzyme-linked immunosorbent assay (ELISA) quantifying soluble TNFR2. [Figure 7-2] This is a continuation of Figure 7-1. [Figure 8-1]8A-8E: Graphs showing the effect of TNFRAG1 on Treg proliferation in peripheral human lymphocytes. FIG. 8A shows the overall trend of CD8+ Teff cell reduction observed when peripheral lymphocytes were incubated with TNFRAG1. FIG. 8B shows the effect of TNFRAG1 on Teff cells alone. FIG. 8C shows the effect of TNFRAG1 on Teff cell mass when incubated with a mixture of Teff and Treg cells at a 4:1 ratio either in the absence of rapamycin and TNFα (left), in the absence of rapamycin and the presence of TNFα (center), or in the presence of rapamycin and the absence of TNFα (right). Figure 8D shows the effect of TNFRAG1 on Teff cell mass when incubated with a mixture of Teff and Treg cells at a ratio of 2:1 in either the absence of rapamycin and TNFα (left), the absence of rapamycin and the presence of TNFα (middle), or the presence of rapamycin and the absence of TNFα (right). Figure 8E shows the effect of TNFRAG1 on Teff cell mass when incubated with a mixture of Teff and Treg cells at a ratio of 1:1 in either the absence of rapamycin and TNFα (left), the absence of rapamycin and the presence of TNFα (middle), or the presence of rapamycin and the absence of TNFα (right). In all experiments, cells were incubated with soluble anti-CD3 antibody (Hit3a) and IL-2 (50U / ml) in 10% FBS RPMI for 4 days. [Figure 8-2] This is a continuation of Figure 8-1. [Figure 9]Figure 9A: Graph showing the effect of TNFRAG1 on gene expression in peripheral human CD4+ cells. Expression of genes characteristic of Treg cells (indicated with an asterisk) is selectively upregulated. Figure 9B: Illustrates specific genes that are upregulated upon incubation of peripheral CD4+ cells with TNFRAG1. Figure 9C: Graphs demonstrating the effect of TNFRAG1 on expression of genes characteristic of Treg cells ETS (left), EP300 (middle), and CREB (right). For comparison purposes, cells were treated with either IL-2 alone ("none"), TNFRAG1 ("TNFR2 agonist"), or a TNFR2 antagonist antibody ("TNFR2 antagonist"). [Figure 10-1] FIG. 10A: A diagram showing the glycolysis system and various genes expressed in the glycolysis system, and the glutaminolysis system and various genes expressed in the glutaminolysis system. FIG. 10B: A graph showing the effect of TNFRAG1 on the expression of genes involved in glycolysis by incubation of TNFRAG1 with peripheral human CD4+ cells. Treg cells are characterized by decreased glycolysis. These data demonstrate that TNFRAG1 induces an increase in the relative amount of Treg cells upon incubation with peripheral human CD4+ cells. FIG. 10C: A graph showing the effect of TNFRAG1 on the expression of genes involved in glutaminolysis by incubation of TNFRAG1 with peripheral human CD4+ cells. Treg cells are characterized by increased glutaminolysis. These data demonstrate that TNFRAG1 induces an increase in the relative amount of Treg cells upon incubation with peripheral human CD4+ cells. [Figure 10-2] This is a continuation of Figure 10-1. [Figure 11-1]FIG. 11A: A diagram showing the Krebs cycle and the effect of the early steps of this cycle on upregulation of itaconic acid, a suppressor of autoimmunity. FIG. 11B: A graph showing the effect of TNFRAG1 on the expression of genes involved in the Krebs cycle upon incubation of TNFRAG1 with peripheral human CD4+ cells. These data show that TNFRAG1 upregulates the expression of early genes of the Krebs cycle, resulting in increased itaconic acid. FIG. 11C: A graph showing the effect of TNFRAG1 on the expression of KEAP1, a known inducer of inflammation and autoimmunity, upon incubation of TNFRAG1 with peripheral human CD4+ cells. [Figure 11-2] This is a continuation of Figure 11-1. [Figure 12-1] Figure 12A: Metabolic preferences of Treg cells. Treg cells show decreased glycolysis, increased glutaminolysis, and increased fatty acid consumption. Figures 12B-12D: A series of graphs showing the effect of TNFRAG1 on the expression of CPT1A (Figure 12B), CPT1B (Figure 12C), and AMPK (Figure 12D), genes involved in fatty acid metabolism, upon incubation of TNFRAG1 with peripheral human CD4+ cells. These data further demonstrate the ability of TNFRAG1 to selectively induce Treg cell proliferation. [Figure 12-2] This is a continuation of Figure 12-1. [Figure 13-1] Figures 13A and 13B: Tables showing the results of ELISA experiments identifying the epitopes on TNFR2 to which TNFRAG1 binds. Epitopes within cysteine-rich domain 2 (CRD2) are indicated in brackets. The amino acid sequences shown in Figures 13A and 13B correspond to the first through last sequences of SEQ ID NOs: 49-70, respectively. [Figure 13-2] This is a continuation of Figure 13-1. [Figure 14] Structural model of a TNFR2 agonist antibody bound to the CRD2 of TNFR2. [Figure 15]15 is a table showing the results of an ELISA experiment to further confirm the epitope on TNFR2 to which TNFRAG1 binds.The amino acid sequences shown in Figure 15 correspond to the first to last sequences of SEQ ID NOs: 49 to 71, respectively. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0146] The agonistic TNFR2 polypeptides described herein, such as single chain polypeptides, antibodies, antigen-binding fragments thereof, and constructs thereof, can promote the proliferation, protection, healing, and / or regeneration of TNFR2-expressing cells. This is accomplished, for example, by binding to TNFR2 (e.g., on the surface of parenchymal cells, such as regulatory T cells (Treg cells), myeloid-derived suppressor cells (MDSCs), or TNFR2+ somatic cells (e.g., cells of the central nervous system (e.g., neurons), among others) and inducing trimerization of the receptor. The activity of TNFR2 agonist polypeptides can be enhanced by the natural ligand TNFα, such as the membrane-bound trimer TNFα. Without being limited by mechanism, this trimerization event is thought to promote the proliferation, protection, healing, and / or regeneration of TNFR2-expressing cells by bringing individual TNFR2 proteins into close proximity with each other. R2 activation, which can promote cell proliferation via the MAPK / NFκB / TRAF2 / 3 pathway. TNFR2 signaling is also characterized by the secretion of soluble TNFR2 (sTNFR2) in a trimeric state and the dual activation of p300 and CBP, two intracellular proteins that bind to Foxp3 transcription sites, thereby enhancing cell stability and maintenance. Importantly, activation of TNFR2 signaling exerts an inhibitory effect on T effector cells, such as CD8+ T cells. In these cells, TNFR2 agonism triggers cell death.

[0147] The agonistic TNFR2 polypeptides described herein can be used to promote this trimerization event in a wide variety of TNFR2+ tissues. Thus, the compositions and methods described herein provide important physiological benefits, since the agonistic TNFR2 polypeptides of the present disclosure can be used to increase Treg cell and / or MDSC activity and decrease T effector cell activity, thereby suppressing autoimmunity, host-versus-disease (GVHD), inflammation, and graft rejection, among others, as described herein. The compositions and methods of the present disclosure can also be used to directly kill effector T cells, such as CD8+ T cells, including those that react with endogenous ("self") antigens. The agonistic TNFR2 polypeptides described herein additionally provide the beneficial feature of being able to stimulate regeneration of TNFR2+ tissues and organs, and to treat a wide variety of diseases characterized by tissue degeneration and organ failure.

[0148] The TNFR2 polypeptides (e.g., antibodies, antigen-binding fragments thereof, single chain polypeptides, and constructs) described herein specifically bind to epitopes within TNFR2 to promote receptor activation and the various beneficial biological activities described above. Human TNFR2 comprises four cysteine-rich domains (CRDs): CRD1 (amino acid residues 48-76 of SEQ ID NO:1), CRD2 (amino acid residues 78-120 of SEQ ID NO:1), CRD3 (amino acid residues 121-162 of SEQ ID NO:1), and CRD4 (amino acid residues 162-202 of SEQ ID NO:1). The agonistic TNFR2 polypeptides described herein specifically bind to TNFR2 at one or more epitopes within CRD1, CRD2, and / or CRD3. In some embodiments, the agonistic TNFR2 polypeptides do not bind to an epitope within CRD4. For example, a polypeptide of the disclosure (e.g., a single chain polypeptide, an antibody, an antigen-binding fragment thereof, or a construct thereof) can bind to human TNFR2 at an epitope corresponding to one or more of the following residues: (a) amino acids 56 to 60 of SEQ ID NO:1 (KCSPG); (b) amino acids 101 to 107 (CSSDQVET) of SEQ ID NO:1; (c) amino acids 115 to 142 of SEQ ID NO: 1 (NRICTCRPGWYCALSKQEGCRLCAPLRK); (d) amino acids 26 to 45 of SEQ ID NO: 1 (QTAQMCCSKCSPGQHAKVFC); (e) amino acids 90 to 109 of SEQ ID NO: 1 (REQNRICTCRPGWYCALSKQ); (f) amino acids 98 to 117 of SEQ ID NO: 1 (CRPGWYCALSKQEGCRLCAP); (g) amino acids 106 to 125 (LSKQEGCRLCAPLRKCRPGF) of SEQ ID NO:1, and / or (h) amino acids 108 to 127 of SEQ ID NO: 1 (KQEGCRLCAPLRKCRPGFGV);

[0149] Alternatively, an equivalent epitope within TNFR2 of a non-human mammal, such as a non-human mammal described herein.

[0150] The present disclosure is based, in part, on the discovery that agonistic TNFR2 polypeptides (e.g., antibodies, antigen-binding fragments thereof, single-chain polypeptides, and constructs) exhibit substantially improved TNFR2 activation efficacy when these molecules are in the form of an IgG2-B isotype. As described in the Examples below, it has now been discovered that this class of TNFR2 polypeptides exhibits surprisingly superior abilities to activate TNFR2 signaling, promote Treg cell and MDSC proliferation, and heal, protect, and regenerate TNFR2-expressing tissues and organs compared to TNFR2 polypeptides of other isotypes.

[0151] Another discovery underlying the present disclosure is the discovery that agonistic TNFR2 polypeptides that specifically bind to TNFR2 at one or more of the epitopes described above, but that exhibit unexpectedly superior TNFR2 activation effects compared to polypeptides that contain antigen-binding sites that are spaced apart by about 133 Å or more. Examples of such polypeptides include IgG1, IgG2, IgG3, and IgG4 antibodies and antigen-binding fragments thereof that contain antigen-binding sites that are spaced apart by about 90 Å to about 133 Å, and particularly include IgG1 antibodies and antigen-binding fragments thereof that contain antigen-binding sites that are spaced apart by about 117 Å, and IgG3 antibodies and antigen-binding fragments thereof that contain antigen-binding sites that are spaced apart by about 125 Å.

[0152] The agonistic TNFR2 polypeptides of the present disclosure can be formulated into pharmaceutical compositions. Preferably, the polypeptides present in the pharmaceutical composition adopt a single disulfide bond isoform. For example, the pharmaceutical compositions of the present disclosure include those that contain an agonistic TNFR2 polypeptide, for example, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99% or more of the polypeptides in the pharmaceutical composition are present in a single disulfide bond isoform. The agonistic TNFR2 polypeptides of the present disclosure include IgG2-A, IgG2-A / B, IgG2-C, IgG2-D, IgG2-E, IgG2-F, IgG2-G, IgG2-H, IgG2-I, IgG2-I / B, IgG2-I / C ... 1 , and IgG2-A / B 2The IgG2-B disulfide bond isoform, which has been surprisingly found to promote substantially stronger levels of TNFR2 activation compared to other IgG2 disulfide bond isoforms such as the IgG2-B isoforms. These isoforms are illustrated in Figures 5A-5D. The polypeptides of the present disclosure may be engineered to predominantly adopt the IgG2-B isoform, for example, by introducing a mutation into the IgG2 heavy chain constant 1 (CH1) domain that prohibits the formation of other disulfide bond isoforms. Exemplary mutations in the amino acid sequence of the human IgG2 CH1 domain that promote the formation of the IgG2-B isoform to the exclusion of the remaining isoforms described above include deletion and / or substitution of the cysteine ​​residue at position 127 of the amino acid sequence of the human IgG2 CH1 domain set forth in SEQ ID NO:8 (according to the Kabat numbering scheme described herein). For example, an IgG2 antibody or antigen-binding fragment thereof may be engineered to predominantly adopt the IgG2-B isoform by introducing a conservative amino acid substitution at cysteine ​​residue 127 of SEQ ID NO:8 to predominantly adopt the IgG2-B isoform. An exemplary IgG2 CH1 domain present predominantly in the IgG2-B isoform has SEQ ID NO:9, which contains a C127S substitution relative to SEQ ID NO:8.

[0153] The following biological activities are examples of agonistic TNFR2 phenotypes that are conferred to a greater degree by polypeptides of the present disclosure compared to TNFR2 binding polypeptides that (i) contain antigen-binding sites that are separated from each other by more than 133 Å and / or (ii) are predominantly absent in single disulfide-bonded isoforms (e.g., IgG2-B isoforms): (a) Enhancement of proliferation and / or direct expansion of Treg cells, for example, by binding and activating TNFR2 on the surface of Treg cells. (b) enhancing proliferation and / or direct expansion of MDSCs, for example, by binding and activating TNFR2 on the surface of MDSCs; (c) depletion of T effector cells, such as CD8+ T cells (e.g., autoreactive CD8+ T cells); and / or (d) Enhanced proliferation and / or direct expansion of TNFR2-expressing parenchymal cells.

[0154] The following sections provide a description of exemplary features of the agonistic TNFR2 polypeptides, eg, single chain polypeptides, antibodies, antigen-binding fragments thereof, and constructs thereof, described herein, as well as their uses in therapeutic methods.

[0155] Agonistic TNFR2 Polypeptides IgG isotype antibodies promote superior TNFR2 agonism As described above and herein, potent TNFR2 activation is achieved by the TNFR2 agonist polypeptides (e.g., antibodies, antigen-binding fragments thereof, single-chain polypeptides, and constructs) described herein. Such polypeptides may have one or more or all of the CDRs of the agonistic TNFR2 antibody TNFRAG1 described herein, or one or more CDRs having at least 85% sequence identity (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% sequence identity) to the corresponding CDR(s) of TNFRAG1 with human IgG1, IgG2, IgG3, or IgG4 isotype. Polypeptides with modified IgG1 and IgG2 isotypes, such as those that contain a CH1 domain that favors the IgG2-B disulfide bond isoform, are particularly advantageous. The disulfide bonding patterns of various isoforms of human IgG2 antibodies are shown in Figures 5A-5D. As shown in Figure 5B, the IgG2-B isoform exhibits a disulfide bonding pattern that generates a narrower and more conformationally constrained structure compared to other IgG2 isoforms. This structure is ideal for binding TNFR2 on the surface of cells (e.g., Treg cells, MDSCs, or parenchymal cells expressing TNFR2) and bringing receptor monomers into close proximity to each other to promote receptor trimerization and thus receptor activation.

[0156] To stabilize the IgG2-B disulfide bond isoform, mutations can be introduced into the IgG2 CH1 domain to prevent or reduce the occurrence of disulfide bonds between cysteine ​​residues that are present as unbound thiols in the IgG2-B isoform. Mutations can also be introduced into the IgG2 CH1 domain to prevent the formation of all four isoform variants, which may be caused by cysteine ​​oxidation and reduction processes in vivo after administration of the antibody to a patient (e.g., a mammalian patient such as a human). An example of such a mutation is an amino acid substitution or deletion at residue C127 of the human IgG2 CH1 domain. By deleting this residue and / or modifying this site by amino acid substitution (e.g., conservative amino acid substitution), the disulfide bond pattern in a population of IgG2 isoforms can be biased towards the IgG2-B isoform. A particularly advantageous mutation in the CH1 domain that can be used to favor the IgG2-B isoform is the C127S mutation.

[0157] Spacing between antigen binding sites Agonistic TNFR2 polypeptides (e.g., single chain polypeptides, antibodies, antigen-binding fragments thereof, or constructs thereof) described herein may contain antigen-binding sites (i.e., antigen-binding arms) that are separated from each other by a distance less than 133 Å, the spacing observed between antigen-binding arms in human IgG2 isotype antibodies. As described in the Examples below, it has been discovered that this spacing results in antibodies with optimal TNFR2 agonistic properties. TNFR2 agonist polypeptides of the disclosure include those that contain antigen-binding arms that are separated from each other by a distance of, for example, less than about 133 Å, e.g., 90 Å, 91 Å, 92 Å, 93 Å, 94 Å, 95 Å, 96 Å, 97 Å, 98 Å, 99 Å, 100 Å, 101 Å, 102 Å, 103 Å, 104 Å, 105 Å, 106 Å, 107 Å, 108 Å, 109 Å, 110 Å, 111 Å, 112 Å, 113 Å, 114 Å, 115 Å, 116 Å, 117 Å, 118 Å, 119 Å, 120 Å, 121 Å, 122 Å, 123 Å, 124 Å, 125 Å, 126 Å, 127 Å, 128 Å, 129 Å, 130 Å, 131 Å, or 132 Å. In some embodiments, the antigen binding sites are separated from one another by a distance of about 95 Å to about 130 Å, e.g., by a distance of about 95 Å, 96 Å, 97 Å, 98 Å, 99 Å, 100 Å, 101 Å, 102 Å, 103 Å, 104 Å, 105 Å, 106 Å, 107 Å, 108 Å, 109 Å, 110 Å, 111 Å, 112 Å, 113 Å, 114 Å, 115 Å, 116 Å, 117 Å, 118 Å, 119 Å, 120 Å, 121 Å, 122 Å, 123 Å, 124 Å, 125 Å, 126 Å, 127 Å, 128 Å, 129 Å, or 130 Å. In some embodiments, the antigen binding sites are separated from one another by a distance of about 98 Å to about 128 Å, e.g., by a distance of about 98 Å, 99 Å, 100 Å, 101 Å, 102 Å, 103 Å, 104 Å, 105 Å, 106 Å, 107 Å, 108 Å, 109 Å, 110 Å, 111 Å, 112 Å, 113 Å, 114 Å, 115 Å, 116 Å, 117 Å, 118 Å, 119 Å, 120 Å, 121 Å, 122 Å, 123 Å, 124 Å, 125 Å, 126 Å, 127 Å, or 128 Å.

[0158] For example, a polypeptide (e.g., a single chain polypeptide, an antibody, an antigen-binding fragment thereof, or a construct thereof) can comprise antigen-binding sites that are separated from each other by a distance of about 105 Å to about 127 Å, e.g., about 105 Å, 106 Å, 107 Å, 108 Å, 109 Å, 110 Å, 111 Å, 112 Å, 113 Å, 114 Å, 115 Å, 116 Å, 117 Å, 118 Å, 119 Å, 120 Å, 121 Å, 122 Å, 123 Å, 124 Å, 125 Å, 126 Å, or 127 Å. In some embodiments, the antigen binding sites are separated from one another by a distance of about 110 Å to about 122 Å, e.g., about 110 Å, 111 Å, 112 Å, 113 Å, 114 Å, 115 Å, 116 Å, 117 Å, 118 Å, 119 Å, 120 Å, 121 Å, or 122 Å. In some embodiments, the antigen binding sites are separated from one another by a distance of about 115 Å to about 119 Å, e.g., about 115 Å, 116 Å, 117 Å, 118 Å, or 119 Å. In some embodiments, the antigen binding sites are separated from one another by a distance of 117 Å. In some embodiments, the polypeptide (e.g., single chain polypeptide, antibody, antigen-binding fragment thereof, or construct thereof) has a spacing of the antigen binding arms as described above and has an IgG1 isotype.

[0159] In some embodiments, a polypeptide (e.g., a single chain polypeptide, an antibody, an antigen-binding fragment thereof, or a construct thereof) comprises antigen-binding sites that are separated from one another by a distance of about 115 Å to about 132 Å, e.g., about 115 Å, 116 Å, 117 Å, 118 Å, 119 Å, 120 Å, 121 Å, 122 Å, 123 Å, 124 Å, 125 Å, 126 Å, 127 Å, 128 Å, 129 Å, 130 Å, 131 Å, or 132 Å. In some embodiments, the antigen-binding sites are separated from one another by a distance of about 120 Å to about 129 Å, e.g., about 120 Å, 121 Å, 122 Å, 123 Å, 124 Å, 125 Å, 126 Å, 127 Å, 128 Å, or 129 Å. In some embodiments, the antigen binding sites are separated from each other by a distance of about 123 Å to about 127 Å, e.g., about 123 Å, 124 Å, 125 Å, 126 Å, or 127 Å. In some embodiments, the antigen binding sites are separated from each other by a distance of 125 Å. In some embodiments, the polypeptide (e.g., single chain polypeptide, antibody, antigen-binding fragment thereof, or construct thereof) has a spacing of the antigen binding arms as described above and has an IgG3 isotype.

[0160] The TNFR2 agonistic polypeptides described herein can have, for example, two, three, four, five, or more antigen-binding arms separated by the distances specified above. Examples of antibody fragments having two or more antigen-binding arms include diabodies, triabodies, F(ab')2 fragments, among others. 2 Antibody fragments include, but are not limited to, scFv (taFv) molecules, and tandem scFv (taFv) molecules. Methods for producing these antibody fragments include peptide synthesis and recombinant protein expression techniques described herein and known in the art.

[0161] There are various methods for measuring the distance between the antigen-binding arms of an antibody or antibody fragment. For example, the distance between the antigen-binding arms of an antibody can be generated by analyzing the three-dimensional structure of an antibody or antibody fragment using computer software, such as using PYMOL® and other molecular imaging software. The three-dimensional structure of a polypeptide, such as an antibody and an antibody fragment, can be calculated using data obtained from X-ray crystallography experiments and nuclear magnetic resonance (NMR) techniques known in the art. Examples of X-ray crystallography and NMR methods that can be used to obtain three-dimensional polypeptide structures are described, for example, in Eigenbrot et al., Journal of Molecular Biology, 229:969-995, 1993, and Huang et al., Science, 317:1930-1934, 2007, the disclosures of each of which are incorporated herein by reference in their entirety.

[0162] Structural features of the variable and constant regions of agonistic TNFR2 polypeptides Among the molecular features of agonistic TNFR2 polypeptides (e.g., single chain polypeptides, antibodies, antigen-binding fragments thereof, and constructs), the complementarity determining region heavy chain 1 (CDR-H1) can dictate the TNFR2 binding properties of the molecule. In particular, TNFR2 polypeptides having the CDR-H1 described below bind to TNFR2 at one or more of the epitopes described herein as conferring agonistic activity. For example, agonistic TNFR2 polypeptides of the present disclosure include: (a) GZ 1 TFZ 3 Z 2 YZ 3 Z 4 (SEQ ID NO:2), (b) GYTFTDYNI (SEQ ID NO: 3), or an amino acid sequence having up to two conservative amino acid substitutions thereto; or (c) a CDR-H1 having an amino acid sequence of GYTFTDYNL (SEQ ID NO: 4), or an amino acid sequence having up to two conservative amino acid substitutions thereto; Each Z 1 are, independently, naturally occurring amino acids comprising a side chain that comprises an aromatic substituent; Each Z 2 are independently naturally occurring amino acids that contain an anionic side chain at physiological pH; Each Z 3 are independently naturally occurring amino acids containing a polar, uncharged side chain at physiological pH; Each Z 4 are independently leucine or isoleucine.

[0163] The presence of the CDR-H1 described above confers on a polypeptide (e.g., a single chain polypeptide, an antibody, an antigen-binding fragment thereof, or a construct thereof) the ability to specifically bind to human TNFR2 at an epitope defined by one or more amino acids in CRD1, e.g., at an epitope defined by one or more amino acid residues 56-60 (KCSPG) in SEQ ID NO:1. In some embodiments, the polypeptide specifically binds to human TNFR2 at an epitope defined by one or more amino acids in CRD2 in the presence of CDR-H1. In some embodiments, the polypeptide specifically binds to human TNFR2 at an epitope defined by one or more amino acids in CRD3 in the presence of CDR-H1.

[0164] The agonistic TNFR2 polypeptides of the present disclosure include (a) a CDR-H2 having the amino acid sequence of INPNYDST (SEQ ID NO: 10), or an amino acid sequence having up to two conservative amino acid substitutions thereto; (b) a CDR-H3 having the amino acid sequence CARGNSWYFDV (SEQ ID NO: 11), or an amino acid sequence having up to two conservative amino acid substitutions thereto; (c) a CDR-L1 having the amino acid sequence SSVRY (SEQ ID NO: 12), or an amino acid sequence having up to two conservative amino acid substitutions thereto; (d) a CDR-L2 having the amino acid sequence LTS or an amino acid sequence having up to two conservative amino acid substitutions thereto; and (e) CDR-L3 having the amino acid sequence CQQWSSNPLT (SEQ ID NO: 13), or an amino acid sequence having up to two conservative amino acid substitutions thereto.

[0165] In some embodiments, a polypeptide (e.g., a single chain polypeptide, an antibody, an antigen-binding fragment thereof, or a construct thereof) comprises the above-mentioned CDR-H1 as follows: (a) CDR-H2 having the amino acid sequence INPNYDST (SEQ ID NO: 10); (b) CDR-H3 having the amino acid sequence CARGNSWYFDV (SEQ ID NO: 11); (c) CDR-L1 having the amino acid sequence SSVRY (SEQ ID NO: 12); (d) a CDR-L2 having the amino acid sequence LTS, and (e) in combination with one or more, or all, of the CDRs of CDR-L3 having the amino acid sequence CQQWSSNPLT (SEQ ID NO: 13).

[0166] In some embodiments, CDR-H1 has the amino acid sequence GYTFTDYNI (SEQ ID NO:3), or an amino acid sequence having up to two conservative amino acid substitutions thereto. For example, the agonistic TNFR2 polypeptides described herein include (a) CDR-H1 having the amino acid sequence GYTFTDYNI (SEQ ID NO:3); (b) CDR-H2 having the amino acid sequence INPNYDST (SEQ ID NO: 10); (c) CDR-H3 having the amino acid sequence CARGNSWYFDV (SEQ ID NO: 11); (d) CDR-L1 having the amino acid sequence SSVRY (SEQ ID NO: 12); (e) a CDR-L2 having the amino acid sequence LTS, and (f) CDR-L3 may have the amino acid sequence CQQWSSNPLT (SEQ ID NO: 13).

[0167] In some embodiments, a polypeptide (e.g., a single chain polypeptide, an antibody, an antigen-binding fragment thereof, or a construct thereof) comprises a heavy chain variable domain (V) having an amino acid sequence that is at least 85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 14. H In some embodiments, the polypeptide comprises a heavy chain variable domain (V) having an amino acid sequence that is at least 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO:14. H In some embodiments, the polypeptide comprises a heavy chain variable domain (V) having an amino acid sequence that is at least 95% (e.g., at least 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO:14. H In some embodiments, the polypeptide comprises a heavy chain variable domain (V) having the amino acid sequence of SEQ ID NO: 14. H ).

[0168] In some embodiments, CDR-H1 has the amino acid sequence GYTFTDYNL (SEQ ID NO:4), or an amino acid sequence having up to two conservative amino acid substitutions thereto.

[0169] In some embodiments, the polypeptide comprises: (a) CDR-H1 having the amino acid sequence GYTFTDYNL (SEQ ID NO: 4); (b) CDR-H2 having the amino acid sequence INPNYDST (SEQ ID NO: 10); (c) CDR-H3 having the amino acid sequence CARGNSWYFDV (SEQ ID NO: 11); (d) CDR-L1 having the amino acid sequence SSVRY (SEQ ID NO: 12); (e) a CDR-L2 having the amino acid sequence LTS, and (f) CDR-L3 having the amino acid sequence CQQWSSNPLT (SEQ ID NO: 13).

[0170] In some embodiments, a polypeptide (e.g., a single chain polypeptide, an antibody, an antigen-binding fragment thereof, or a construct thereof) comprises a heavy chain variable domain (V) having an amino acid sequence that is at least 85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 15. H In some embodiments, the polypeptide comprises a heavy chain variable domain (V) having an amino acid sequence that is at least 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO:15. H In some embodiments, the polypeptide comprises a heavy chain variable domain (V) having an amino acid sequence that is at least 95% (e.g., at least 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 15. H In some embodiments, the polypeptide comprises a heavy chain variable domain (V) having the amino acid sequence of SEQ ID NO: 15. H ).

[0171] In some embodiments, a polypeptide (e.g., a single chain polypeptide, an antibody, an antigen-binding fragment thereof, or a construct thereof) comprises a non-native constant region, such as a human constant region. In some embodiments, the polypeptide lacks all or a portion of an Fc domain, lacks all or a portion of a native Fc domain, or lacks an Fc domain altogether.

[0172] Framework Regions of Agonistic TNFR2 Polypeptides Agonistic TNFR2 polypeptides (e.g., single chain polypeptides, antibodies, antigen-binding fragments thereof, or constructs thereof) of the present disclosure can be used in combination with any of the following: 1 YX 2 X 3 LJX 4 LJS (SEQ ID NO:5), or a framework region having an amino acid sequence having 10 or more of the residues of SEQ ID NO:5, wherein each J is independently a naturally occurring amino acid and each X 1 is independently A, V, or F, and each X 2 is independently M or I, and each X 3 is independently E or Q, and each X 4 is independently S or R. For example, a polypeptide can be (a) TVDKSSSTAYMELRSLTS (SEQ ID NO: 16), or an amino acid sequence having up to two conservative amino acid substitutions thereto; (b) TADTSSNTAYIQLSSLTS (SEQ ID NO: 17), or an amino acid sequence having up to two conservative amino acid substitutions thereto; (c) TADTSTDTAYMELSSLRS (SEQ ID NO: 18), or an amino acid sequence having up to two conservative amino acid substitutions thereto; (d) TRDTSISTAYMELSRLTS (SEQ ID NO: 19), or an amino acid sequence having up to two conservative amino acid substitutions thereto; (e) TFYMELSSLRS (SEQ ID NO: 20), or an amino acid sequence having up to two conservative amino acid substitutions thereto; (f) TRDTSISTAYMELNRLTS (SEQ ID NO: 21), or an amino acid sequence having up to two conservative amino acid substitutions thereto; (g) TRDTSTNTVYMELTSLRS (SEQ ID NO: 22), or an amino acid sequence having up to two conservative amino acid substitutions thereto; and (h) a framework region having an amino acid sequence selected from TADTSTDRAYMELSSLRS (SEQ ID NO: 23), or an amino acid sequence having up to two conservative amino acid substitutions thereto.

[0173] The aforementioned amino acid sequences recognize and bind to MHC class II proteins, including inter alia human leukocyte antigen (HLA) DR and DQ. Typically, antibodies known to contain amino acid sequences that bind to MHC proteins have been engineered to remove such motifs. This is because antibodies that bind to MHC proteins, when administered to a patient (e.g., a mammalian patient, such as a human patient), are prone to degradation and are located outside of antigen-presenting cells of the immune system, thereby eliciting an inappropriate immune response against the administered antibody. Methods for determining whether a particular amino acid sequence has a propensity to bind to an MHC molecule are known in the art and are described, for example, in Wang et al., BMC Bioinformatics 11:568, 2010; Nielsen et al., BMC Bioinformatics 8:238, 2007; Gonzalez-Galarza et al., Nucleic Acid Research 39:D913-D919, 2011; and Greenbaum et al., Immunogenetics 63:325, 2011, the disclosures of each of which are incorporated herein by reference in their entireties.

[0174] Although the above-mentioned framework regions tend to bind to MHC class II molecules, they are not immunogenic peptides. Rather, these amino acid sequences confer high affinity TNFR2 binding. The agonistic TNFR2 polypeptide of the present disclosure that comprises any of the above-mentioned framework region sequences exhibits the unexpected advantageous property of high TNFR2 affinity without inducing an immune response against the polypeptide when the polypeptide is administered to a patient (e.g., a mammalian patient, such as a human).

[0175] Homogeneity of the population of TNFR2 agonistic polypeptides Pharmaceutical compositions can be produced in which the TNFR2 agonistic polypeptides described herein (e.g., antibodies, antigen-binding fragments thereof, single chain polypeptides, or constructs thereof) are present as a single disulfide bond isoform. For example, at least 10% or more of the polypeptides in the pharmaceutical composition can be present as a single disulfide bond isoform (e.g., IgG2-B isoform). This can be achieved, for example, by amino acid substitution or deletion at cysteine ​​residue 127 of wild-type human IgG1 or IgG2 CH1 domain, thereby preventing or reducing the occurrence of disulfide bonds that can generate isoforms other than IgG2-B (see, for example, Figures 5A-5D). Pharmaceutical compositions of the present disclosure include, for example, those in which about 10% to about 99.999% of the agonistic TNFR2 polypeptides in the pharmaceutical composition are present as a single disulfide bond isoform, such as the IgG2-B isoform. For example, pharmaceutical compositions of the disclosure include those that contain an agonist TNFR2 polypeptide, e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99% or more of the polypeptide in the pharmaceutical composition is present in a single disulfide-linked isoform.

[0176] Techniques for measuring the relative amounts of various disulfide-bonded isoforms present in a sample of an agonist TNFR2 polypeptide include liquid chromatography techniques known in the art and described herein, such as those exemplified in Wypych et al., The Journal of Biological Chemistry, 283:16194-16205, 2008, the disclosure of which is incorporated herein by reference in its entirety.

[0177] Influence of TNFR2 on signaling cascades The agonistic TNFR2 polypeptides (e.g., single chain polypeptides, antibodies, and antigen-binding fragments thereof) described herein can interact with TNFR2 and enhance the activity of TNFR2. The TNFR2 polypeptides of the present disclosure can bind to TNFR2 at one or more specific epitopes that favor the formation of TNFR2 trimers by bringing adjacent TNFR2 monomers into close proximity with each other. Trimerization of TNFR2 is particularly beneficial for receptor activation because it places TNFR2 monomers in a conformation where the TNFα binding site is sterically available. This trimerization event can activate intracellular signaling by TNFR2, which in turn can promote the proliferation of TNFR2-expressing cells, such as Treg cells, MDSCs, and / or TNFR2+ parenchymal cells. Activation of TNFR2 can also result in increased expression levels of CD274 (PDL1), PDCD1LG2 (PDL2), CD137 (4-1BB), and IKZF2.

[0178] TNFR2 activation leads to the expression of a variety of genes that can exert important therapeutic effects, but the TNFR2 agonist polypeptides described herein can promote the expression of several proteins of particular therapeutic importance. Without being limited by mechanism, the TNFR2 polypeptides described herein can induce TNFR2-mediated cellular healing, protection, proliferation, and / or regeneration by stimulating the expression of EP300 and CREB, two histone acetyltransferases that promote Foxp3 transcription. This biological activity is particularly important because it can promote the flux of TNFR2+ cells through the cell cycle while causing the death of T effector cells, such as CD8+ T cells. The selective expansion of TNFR2+ cells and inhibition of T effector cells are part of the phenotype underlying the ability of agonistic TNFR2 polypeptides to treat autoimmunity, GVHD, inflammation, and other indications described herein.

[0179] For example, activation of TNFR2 can be monitored by measuring the expression of genes associated with the MAPK and TRAF2 / 3 signaling cascades, such as the expression of genes whose transcription is mediated by NFκB (Faustman, et al., Nat. Rev. Drug Disc., 9:482-493, 2010, the disclosure of which is incorporated herein by reference).

[0180] Dual proliferation and cell killing effects of TNFR2 agonist polypeptides The agonistic TNFR2 polypeptides disclosed herein, e.g., single chain polypeptides, antibodies, antigen-binding fragments thereof, and constructs thereof, can not only promote the proliferation of Treg cells, MDSCs, and / or TNFR2+ parenchymal cells, but also induce the death of T effector cells, such as CD8+ T effector cells (e.g., in a patient, such as a human patient). The agonistic TNFR2 polypeptides described herein can increase the total amount of Treg cells, MDSCs, and / or TNFR2+ parenchymal cells in a sample treated with an agonistic TNFR2 antibody or antigen-binding fragment thereof (e.g., a sample isolated from a human patient being treated for autoimmunity, GVHD, transplant rejection, allergy, chronic inflammatory disease, asthma, or another disorder described herein) by, e.g., 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100% or more, as compared to a sample not treated with an agonistic TNFR2 antibody or antigen-binding fragment thereof. The ability of the agonistic TNFR2 polypeptides (e.g., single chain polypeptides, antibodies, and antigen-binding fragments) described herein to enhance Treg, MDSC, and / or parenchymal cell proliferation may be due, in part, to the fact that these polypeptides increase the amount of soluble TNFR2 within a sample (e.g., a sample isolated from a human patient undergoing treatment for autoimmunity, GVHD, transplant rejection, allergy, chronic inflammatory disease, asthma, or another disorder described herein).

[0181] Stimulation of Treg and MDSC proliferation produces important therapeutic benefits, as these cells suppress the activity of T effector cells, such as autoreactive CD8+ T cells, that initiate inappropriate immune responses against self-tissues. Similarly, direct killing of T effector cells provides therapeutic activity, as TNFR2 agonist polypeptides can be used to reduce the amount of autoreactive T cells in patients suffering from autoimmunity, GVHD, transplant rejection, allergy, chronic inflammatory disease, asthma, or another disorder described herein. Through either or both of these mechanisms, the TNFR2 agonist polypeptides of the present disclosure can be used to suppress an immunological condition in a patient, such as a mammalian patient (eg, a human patient).

[0182] Additionally, the ability of the TNFR2 agonist polypeptides of the present disclosure to stimulate the proliferation of TNFR2+ parenchymal cells provides beneficial effects in inducing tissue and organ regeneration.Exemplary TNFR2+ cells that can be induced to proliferate using the TNFR2 agonist polypeptides of the present disclosure include, but are not limited to, cells of the pancreas, salivary gland, pituitary gland, kidney, heart, lung, hematopoietic system, cranial nerves, heart, aorta, olfactory gland, ear, nerves, head structure, eye, thymus, tongue, bone, liver, small intestine, large intestine, intestine, lung, brain, skin, peripheral nervous system, central nervous system, spinal cord, breast, embryonic structure, embryo, and testis.Therefore, the TNFR2 agonist polypeptides described herein can be used to treat disorders in which regeneration, protection, and / or cure of one or more of these cell types is desired.Exemplary diseases that can be treated using the TNFR2 agonist polypeptides of the present disclosure are described herein.

[0183] Active (CD25 Hi and CD45RA Low) Selective modification of Treg cells The agonistic TNFR2 polypeptides (e.g., single chain polypeptides, antibodies, antigen-binding fragments, and constructs thereof) described herein can enhance the proliferation or increase in the total amount of Treg cells in a sample (e.g., a sample isolated from a human patient being treated for autoimmunity, GVHD, transplant rejection, allergy, chronic inflammatory disease, asthma, or another disorder described herein), and can selectively act on Treg cells that are in an actively dividing state. For example, the agonistic TNFR2 antibodies or antigen-binding fragments thereof described herein can inhibit the proliferation or increase in the total amount of Treg cells in a sample (e.g., a sample isolated from a human patient being treated for autoimmunity, GVHD, transplant rejection, allergy, chronic inflammatory disease, asthma, or another disorder described herein), and can selectively act on Treg cells that are in an actively dividing state. Hi and CD45RA Low The proliferation of Treg cells expressing, for example, CD25 Hi and CD45RA Low Treg cells that do not express proteins, e.g., CD25 Med and CD45RA Hi by 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100% or more compared to Treg cells expressing the protein or non-Treg T cells.

[0184] TNFR2 agonist polypeptides are not dependent on additional TNFR2 binding agents for activity Importantly, the agonistic TNFR2 polypeptides described herein, such as single chain polypeptides, antibodies, or antigen-binding fragments thereof, can bind to TNFR2 and enhance TNFR2-mediated signaling without the need for the natural TNFR2 ligand TNFα. Such agonists are referred to herein as dominant TNFR2 agonists. The agonistic TNFR2 polypeptides described herein, such as antibodies, antigen-binding fragments thereof, single chain polypeptides, and constructs, do not require TNFα to expand Treg cells, MDSCs, and / or TNFR2+ parenchymal cells. Without being limited by mechanism, the agonistic TNFR2 polypeptides of the present disclosure may exhibit this property due to the ability of these molecules to bind to TNFR2 at a specific epitope that stabilizes the trimeric conformation of the receptor and its adjacent monomers upon binding. This structural configuration enhances NFκB signaling and enhances part of the fundamental signaling cascade associated with the activation of Tregs, MDSCs, and TNFR2+ parenchymal cells.

[0185] For example, the agonistic TNFR2 polypeptides described herein, such as single chain polypeptides, antibodies, antigen-binding fragments thereof, and constructs thereof, bind to TNFR2 on the surface of TNFR2+ cells, such as Treg cells, MDSCs, and / or TNFR2+ parenchymal cells, in the presence or absence of TNFα and enhance the proliferation of such cells. For example, the agonistic TNFR2 polypeptides described herein increase the proliferation of such cells by 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 200%, 300%, 400%, 500%, or more, for example, compared to such cells not treated with the TNFR2 polypeptide. Agonistic TNFR2 polypeptides (e.g., single chain polypeptides, antibodies, or antigen-binding fragments thereof) induce EC2 activity in cell proliferation assays that is not significantly altered by the presence or absence of TNFα. 50Values ​​are shown (e.g., EC 50 but not the EC50 of an agonistic TNFR2 polypeptide (e.g., a single chain polypeptide, an antibody, or an antigen-binding fragment thereof) in the same cell proliferation assay in the absence of TNFα. 50 (less than 50%, 45%, 40%, 35%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or less than 1% change compared to the value.) Examples of Treg assays that can be used to measure the agonistic effect of TNFR2 antibodies are described herein, e.g., in Example 2 below. Similarly, the agonistic TNFR2 polypeptides, e.g., single chain polypeptides, antibodies, antigen-binding fragments thereof, and constructs thereof described herein can be used to treat or inhibit the TNFR2 agonist, such as Foxp3, TNFSFSF18, IL2RA, IKZF2 / 4, CTLA4, TGF-beta, CHUK, NFKBIE, NFKBIA, MAP3K11, TRAF2, TRAF3, relB, TNF, CXCR3, PDL1 (CD274), IL2RA, IL7R, MAP3K1, MAP, MAP3K4, NFKBIB, TANK, TBK1, TNF, TNFAIP3, NFKBIA, MAP3K11, TNFRSF1B, TRAF2, relB, LTA, EP300, CREBB, P, NFkB signaling proteins, can enhance TNFR2 signaling, for example, by 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, or more, as compared to such cells not treated with a TNFR2 agonist polypeptide. An agonistic TNFR2 polypeptide (e.g., a single chain polypeptide, an antibody, or an antigen-binding fragment thereof) can enhance ECs that are little changed or not enhanced by the presence or absence of TNFα in a gene expression assay. 50 Values ​​are shown (e.g., EC 50but not the EC50 of an agonistic TNFR2 polypeptide (e.g., a single chain polypeptide, an antibody, or an antigen-binding fragment thereof) in the same gene expression assay in the absence of TNFα. 50 (less than 50%, 45%, 40%, 35%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% change compared to the value).

[0186] Activity of antigen-binding fragments of full-length TNFR2 agonist antibodies The agonistic TNFR2 antibodies described herein enhance the proliferation of Treg cells, MDSCs, and / or parenchymal cells, and / or promote T effector cell death, with similar potency as that exhibited by antigen-binding fragments of such antibodies. For example, removal of the Fc region of the agonistic TNFR2 antibodies described herein may not alter the ability of the molecule to promote the proliferation of Treg cells, MDSCs, and / or parenchymal cells, and / or directly kill T effector cells (e.g., CD8+ autoreactive T cells) in a sample (e.g., a sample isolated from a human patient undergoing treatment for autoimmunity, GVHD, allergy, transplant rejection, or another condition described herein). The agonistic TNFR2 antibodies and antigen-binding fragments thereof described herein may function, for example, by a pathway different from antibody-dependent cellular cytotoxicity (ADCC), in which the Fc region is required to recruit effector proteins to induce cell death. Additionally, the agonistic TNFR2 antibody or antigen-binding fragment thereof can be, among others, a single chain polypeptide (e.g., a single chain polypeptide comprising one or more CDRs covalently linked to each other, e.g., by an amide bond, a thioether bond, a carbon-carbon bond, or a disulfide bridge), a monoclonal antibody or antigen-binding fragment thereof, a polyclonal antibody or antigen-binding fragment thereof, a humanized antibody or antigen-binding fragment thereof, a primatized antibody or antigen-binding fragment thereof, a bispecific antibody or antigen-binding fragment thereof, a multispecific antibody or antigen-binding fragment thereof, a dual variable immunoglobulin domain, a monovalent antibody or antigen-binding fragment thereof, a chimeric antibody or antigen-binding fragment thereof, a single chain Fv molecule (scFv), a diabody, a triabody, a nanobody, an antibody-like protein scaffold, a domain antibody, an Fv fragment, a Fab fragment, a F(ab') 2 The therapeutic activity may be exhibited in a variety of forms, such as single scFvs, and tandem scFvs (taFvs).

[0187] Specific Binding Properties of Agonistic TNFR2 Polypeptides Specific binding of a polypeptide, e.g., a single chain polypeptide, antibody, or antibody fragment described herein, to human TNFR2 can be determined by any of a variety of established methods. Affinity can be measured by measuring the half-maximal activation (EC) of the TNFR2 receptor in vitro or in vivo. 50 ) and the equilibrium constant for dissociation of the antibody-TNFR2 complex (K D The equilibrium constant K, which describes the interaction between TNFR2 and the antibodies described herein, can be expressed quantitatively by various measurements, such as the concentration of antibody required to achieve the desired binding activity. D is the chemical equilibrium constant for the dissociation of the TNFR2-antibody complex into solvent-separated TNFR2 and antibody molecules that do not interact with each other.

[0188] The polypeptides (e.g., single chain polypeptides, antibodies, and antigen-binding fragments) described herein have a K of less than 100 nM (e.g., 95 nM, 90 nM, 85 nM, 80 nM, 75 nM, 70 nM, 65 nM, 60 nM, 55 nM, 50 nM, 45 nM, 40 nM, 35 nM, 30 nM, 25 nM, 20 nM, 15 nM, 10 nM, 5 nM, 4 nM, 3 nM, 2 nM, or 1 nM). DIn some embodiments, the polypeptides (e.g., single chain polypeptides, antibodies, antigen-binding fragments, and constructs thereof) described herein specifically bind to TNFR2 at a concentration of less than 1 nM (e.g., 990 pM, 980 pM, 970 pM, 960 pM, 950 pM, 940 pM, 930 pM, 920 pM, 910 pM, 900 pM, 890 pM, 880 pM, 870 pM, 860 pM, 850 pM, etc.). M, 840pM, 830pM, 820pM, 810pM, 800pM, 790pM, 780pM, 770pM, 760pM, 750pM, 740pM, 730pM, 720pM, 710pM , 700pM, 690pM, 680pM, 670pM, 660pM, 650pM, 640pM, 630pM, 620pM, 610pM, 600pM, 590pM, 580pM, 570pM, 560pM, 550pM, 540pM, 530pM, 520pM, 510pM, 500pM, 490pM, 480pM, 470pM, 460pM, 450pM, 440pM, 430pM, 4 20pM, 410pM, 400pM, 390pM, 380pM, 370pM, 360pM, 350pM, 340pM, 330pM, 320pM, 310pM, 300pM, 290pM, 28 K of 0pM, 270pM, 260pM, 250pM, 240pM, 230pM, 220pM, 210pM, 200pM, 190pM, 180pM, 170pM, 160pM, 150pM, 140pM, 130pM, 120pM, 110pM, 100pM, 90pM, 80pM, 70pM, 60pM, 50pM, 40pM, 30pM, 20pM, 10pM, 5pM, or 1pM) D It specifically binds to TNFR2 at 100 ng / mL.

[0189] The polypeptides described herein may also be characterized by various in vitro binding assays. The K D or EC 50Examples of experiments that can be used to determine include surface plasmon resonance, isothermal titration calorimetry, fluorescence anisotropy, ELISA-based assays, gene expression assays, and protein expression assays, among others. ELISA is a particularly useful method for analyzing the activity of an antibody, and such assays usually require a minimal concentration of antibody. The general signal analyzed in a typical ELISA assay is luminescence, which is usually the result of the activity of a peroxidase conjugated to a secondary antibody that specifically binds to the primary antibody (e.g., the TNFR2 antibody described herein). The polypeptides (e.g., single-chain polypeptides, antibodies, and antigen-binding fragments) described herein can bind to TNFR2 and epitopes therein, such as epitopes that include one or more consecutive or discontinuous residues within CRD1, CRD2, and / or CRD3 of human TNFR2. The agonistic polypeptides described herein can additionally bind to isolated peptides derived from TNFR2 that structurally pre-organize various residues in a manner that simulates the conformation of the aforementioned epitopes in the native protein. In a direct ELISA experiment, this binding can be quantified, for example, by analyzing the luminescence generated upon incubation of an HRP substrate (e.g., 2,2'-azino-di-3-ethylbenzthiazoline sulfonate) with an antigen-antibody complex bound to an HRP-conjugated secondary antibody.

[0190] Kinetic properties of agonistic TNFR2 polypeptides In addition to the thermodynamic parameters of TNFR2-polypeptide interactions, it is also possible to quantitatively characterize the dynamic association and dissociation of the polypeptides described herein with TNFR2. This can be done, for example, by monitoring the rate of polypeptide-antigen (e.g., antibody-antigen) complex formation according to established procedures. For example, surface plasmon resonance (SPR) can be used to measure the formation of antibody-TNFR2 complexes (k on ) and dissociation (k off These data allow the determination of the equilibrium constant for dissociation of the antibody-TNFR2 complex (K D), which means that the equilibrium constant for this unimolecular dissociation is k off Value and k on SPR is a particularly advantageous technique for determining the kinetic and thermodynamic parameters of receptor-antibody interactions because the experiment does not require one component to be modified by the attachment of a chemical label. Rather, the receptor is typically immobilized on a solid metal surface that is pulsed with solutions of increasing concentrations of antibody. Binding of the antibody to the receptor causes a distortion of the angle of reflection of the incident light at the metal surface, and this change in refractive index over time as the antibody is introduced into the system can be fitted to established regression models to calculate the association and dissociation rate constants of the antibody-receptor interaction.

[0191] The polypeptides described herein (e.g., single chain polypeptides, antibodies, antigen-binding fragments, and constructs thereof) exhibit high k α -α and β -α upon interaction with TNFR2, consistent with high affinity receptor binding. on and low k off For example, the polypeptides described herein may exhibit a 10 4 M -1 s -1 (e.g., 1.0×10 4 M -1 s -1 , 1.5×10 4 M -1 s -1 , 2.0×10 4 M -1 s -1 , 2.5×10 4 M -1 s -1 , 3.0×10 4 M -1 s -1 , 3.5×10 4 M -1 s -1 , 4.0×10 4 M -1 s -1 , 4.5×10 4 M -1 s -1 , 5.0×10 4 M-1 s -1 、5.5×10 4 M -1 s -1 、6.0×10 4 M -1 s -1 、6.5×10 4 M -1 s -1 、7.0×10 4 M -1 s -1 、7.5×10 4 M -1 s -1 、8.0×10 4 M -1 s -1 、8.5×10 4 M -1 s -1 、9.0×10 4 M -1 s -1 、9.5×10 4 M -1 s -1 、1.0×10 5 M -1 s -1 、1.5×10 5 M -1 s -1 、2.0×10 5 M -1 s -1 、2.5×10 5 M -1 s -1 、3.0×10 5 M -1 s -1 、3.5×10 5 M -1 s -1 、4.0×10 5 M -1 s -1 、4.5×10 5 M -1 s -1 、5.0×10 5 M -1 s -1 、5.5×10 5 M -1 s -1 、6.0×10 5 M -1 s -1 、6.5×105 M -1 s -1 , 7.0×10 5 M -1 s -1 , 7.5×10 5 M -1 s -1 , 8.0×10 5 M -1 s -1 , 8.5×10 5 M -1 s -1 , 9.0×10 5 M -1 s -1 , 9.5×10 5 M -1 s -1 , or 1.0 × 10 6 M -1 s -1 )k on The polypeptides described herein (e.g., single chain polypeptides, antibodies, antigen-binding fragments, and constructs thereof) can exhibit low k values ​​upon binding to TNFR2 because these polypeptides can interact with different TNFR2 epitopes with high affinity. off Residues within these epitopes can form strong intermolecular contacts with TNFR2 and delay dissociation of the antibody-TNFR2 complex. This high receptor affinity is associated with a low k off For example, the polypeptides described herein may exhibit a 10 -3 s -1 Less than (e.g., 1.0×10 -3 s -1 , 9.5×10 -4 s -1 , 9.0×10 -4 s -1 , 8.5×10 -4 s -1 , 8.0×10 -4 s -1 , 7.5×10 -4 s -1 , 7.0×10 -4 s -1 , 6.5×10 -4 s -1 , 6.0×10 -4 s-1 , 5.5×10 -4 s -1 , 5.0×10 -4 s -1 , 4.5×10 -4 s -1 , 4.0×10 -4 s -1 , 3.5×10 -4 s -1 , 3.0×10 -4 s -1 , 2.5×10 -4 s -1 , 2.0×10 -4 s -1 , 1.5×10 -4 s -1 , 1.0×10 -4 s -1 , 9.5×10 -5 s -1 , 9.0×10 -5 s -1 , 8.5×10 -5 s -1 , 8.0×10 -5 s -1 , 7.5×10 -5 s -1 , 7.0×10 -5 s -1 , 6.5×10 -5 s -1 , 6.0×10 -5 s -1 , 5.5×10 -5 s -1 , 5.0×10 -5 s -1 , 4.5×10 -5 s -1 , 4.0×10 -5 s -1 , 3.5×10 -5 s -1 , 3.0×10 -5 s -1 , 2.5×10 -5 s -1 , 2.0×10 -5 s -1 , 1.5×10 -5 s -1 , or 1.0 × 10 -5 s -1 ) k off The value may be indicated.

[0192] Epitopes within TNFR2 bound by agonistic TNFR2 polypeptides The agonistic TNFR2 polypeptides described herein, e.g., dominant agonistic TNFR2 polypeptides (e.g., single chain polypeptides, antibodies, antigen-binding fragments, and constructs thereof), can be used to: (a) amino acids 56 to 60 of SEQ ID NO: 1 in human TNFR2 (KCSPG); (b) amino acids 101 to 107 of SEQ ID NO: 1 in human TNFR2 (CSSDQVET); (c) amino acids 115 to 142 of SEQ ID NO: 1 in human TNFR2 (NRICTCRPGWYCALSKQEGCRLCAPLRK); (d) amino acids 26 to 45 of SEQ ID NO: 1 in human TNFR2 (QTAQMCCSKCSPGQHAKVFC); (e) amino acids 90 to 109 of SEQ ID NO: 1 in human TNFR2 (REQNRICTCRPGWYCALSKQ); (f) amino acids 98 to 117 of SEQ ID NO: 1 in human TNFR2 (CRPGWYCALSKQEGCRLCAP); (g) amino acids 106 to 125 of SEQ ID NO: 1 in human TNFR2 (LSKQEGCRLCAPLRKCRPGF); (h) amino acids 108 to 127 (KQEGCRLCAPLRKCRPGFGV) of SEQ ID NO: 1 in human TNFR2; and / or (i) capable of specifically binding to one or more of the following: an equivalent epitope in TNFR2 of a non-human mammal, e.g., a non-human mammal as described herein; an epitope that exhibits at least 85% sequence identity (e.g., 85%, 90%, 95%, 97%, 99%, or 100% sequence identity) to any of the above-mentioned epitopes; and / or an epitope that includes one or more conservative amino acid substitutions to any of the above-mentioned epitopes.

[0193] In some embodiments, agonistic TNFR2 polypeptides, such as the dominant agonistic TNFR2 polypeptides described herein (e.g., single chain polypeptides, antibodies, antigen-binding fragments, and constructs thereof), do not bind to an epitope that includes one or more or all of residues 142-146 of SEQ ID NO:1 (KCRPG, SEQ ID NO:41) within human TNFR2.

[0194] One exemplary procedure that can be used to predict the growth, regeneration, healing, or protective activity of the TNFR2 polypeptides described herein is to determine the affinity of an antibody or antibody fragment to a peptide that contains the KCSPG motif (SEQ ID NO: 40), e.g., a linear or cyclic peptide that contains this motif. The peptide may be structurally pre-organized by one or more conformational constraints (e.g., backbone or side chain to side chain cyclization), e.g., in a manner that simulates the three-dimensional orientation of the KCSPG motif (SEQ ID NO: 40). For example, the agonistic TNFR2 polypeptides described herein bind to a peptide containing a KCSPG motif (SEQ ID NO: 40) with an affinity that is, for example, 10-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 200-fold, 300-fold, 400-fold, 500-fold, 600-fold, 700-fold, 800-fold, 900-fold, 1000-fold, or more than 1000-fold higher than the affinity of the agonistic polypeptide for a peptide containing the amino acid sequence of KCRPG (SEQ ID NO: 41).

[0195] Agonistic TNFR2 antibody TNFRAG1 Exemplary agonistic TNFR2 polypeptides, such as single-chain polypeptides, antibodies, antigen-binding fragments, and constructs thereof, described herein may include one or more, or all, of the CDRs of TNFRAG1, a monoclonal antibody that specifically binds to TNFR2 at an epitope and promotes the signal transduction of TNFR2, thus promoting the proliferation of Treg cells and MDSCs, the regeneration, healing, and protection of TNFR2+ parenchymal cells, and the death of T effector cells (e.g., CD8+ T effector cells). For example, the CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and / or CDR-L3 of TNFRAG1, and variants of these CDRs (e.g., variants that exhibit conservative amino acid substitutions with respect to these CDR sequences) can be used to generate additional agonistic TNFR2 polypeptides (e.g., single-chain polypeptides, antibodies, antigen-binding fragments, and constructs thereof) of the present disclosure, for example, by antibody humanization methods described herein or known in the art.

[0196] Agonistic TNFR2 polypeptides (e.g., single chain polypeptides, antibodies, and antigen-binding fragments) of the present disclosure may exhibit binding characteristics that are the same as or similar to those of TNFRAG1. In particular, TNFRAG1 is (a) amino acids 56 to 60 of SEQ ID NO:1 (KCSPG); (b) amino acids 101 to 107 (CSSDQVET) of SEQ ID NO:1; (c) amino acids 115 to 142 of SEQ ID NO: 1 (NRICTCRPGWYCALSKQEGCRLCAPLRK); (d) amino acids 26 to 45 of SEQ ID NO: 1 (QTAQMCCSKCSPGQHAKVFC); (e) amino acids 90 to 109 of SEQ ID NO: 1 (REQNRICTCRPGWYCALSKQ); (f) amino acids 98 to 117 of SEQ ID NO: 1 (CRPGWYCALSKQEGCRLCAP); (g) amino acids 106 to 125 (LSKQEGCRLCAPLRKCRPGF) of SEQ ID NO:1, and / or (h) specifically binds to human TNFR2 at one or more of amino acids 108 to 127 (KQEGCRLCAPLRKCRPGFGV) of SEQ ID NO:1.

[0197] TNFRAG1 may comprise one or both of the heavy chain variable regions set forth below in SEQ ID NOs: 14 and 15. The CDRs of each of these regions are shown in bold. [Sequence Table 1] TIFF0007652690000002.tif22154[Sequence Table 2] TIFF0007652690000003.tif21154

[0198] Additionally, TNFRAG1 comprises a light chain variable region as set forth below in SEQ ID NO: 42. The CDRs of this region are shown in bold. [Sequence Table 3] TIFF0007652690000004.tif22154

[0199] The following sections provide detailed descriptions of methods that can be used to prepare human, humanized, primatized, chimeric, and other polypeptides (e.g., single chain polypeptides, antibodies, antigen-binding fragments thereof, and constructs) based, for example, on the molecular characteristics of TNFRAG1.

[0200] Fully human, humanized, primatized, and chimeric antibodies The antibodies described herein include fully human, humanized, primatized, and chimeric antibodies that contain one or more or all of the CDR sequences of TNFRAG1. Additionally, the antibodies described herein include fully human, humanized, primatized, and chimeric antibodies that contain one or more or all of CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3, where one or more or all of the CDR sequences exhibit at least 85% sequence identity (e.g., 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity) to the corresponding CDR sequences of TNFRAG1. The agonistic TNFR2 antibodies described herein further include fully human, humanized, primatized, and chimeric antibodies comprising one or more or all of CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3, where one or more or all of the CDR sequences contain one or more (e.g., up to three) amino acid substitutions (e.g., one or more conservative amino acid substitutions) relative to the corresponding CDR sequence or a shared heavy chain framework sequence that is also found in a version of a significant human heavy chain sequence.

[0201] As an example, one strategy that can be used to design the humanized antibodies described herein is to align the sequences of the heavy and light chain variable regions of TNFRAG1 with the heavy and light chain variable regions of a consensus human antibody. The heavy and light chain sequences of consensus human antibodies are known in the art (see, for example, the "VBASE" human germline sequence database; see also Kabat, et al., Sequences of Proteins of Immunological Interest, Fifth Edition, USDepartment of Health and Human Services, NIH Publication No. 91 -3242, 1991; Tomlinson et al., J. Mol. Biol. 227: 776-98, 1992, and Cox et al., Eur. J. Immunol. 24: 827-836, 1994, the disclosures of which are incorporated herein by reference). In this way, variable domain framework residues and CDRs can be identified by sequence alignment (see Kabat, supra). To generate a humanized TNFR2 agonist antibody, for example, one or more CDRs of a consensus human antibody can be replaced with the corresponding CDR(s) of TNFRAG1. Exemplary variable domains of consensus human antibodies include a heavy chain variable domain: [Sequence Table 4] TIFF0007652690000005.tif21154 and the light chain variable domain: [Sequence Table 5] TIFF0007652690000006.tif14155 is included, (CDRs were determined according to the method of Chothia, et al., J. Mol. Biol, 196:901-917, 1987, and are shown in bold.) These amino acid substitutions can be made, for example, by recombinant expression of polynucleotides encoding the heavy and light chains of the humanized antibody in a host cell using methods known in the art or described herein.

[0202] Similarly, this strategy can be used to generate primatized agonistic TNFR2 antibody.For example, one or more or all of the CDRs of primate antibody consensus sequence can be replaced with one or more or all of the CDRs of TNFRAG1.Consensus primate antibody sequences are known in the art (see, for example, U.S. Patent Nos. 5,658,570, 5,681,722 and 5,693,780, the disclosures of each of which are incorporated herein by reference).

[0203] In some embodiments, it may be desirable to import certain framework residues into the heavy and / or light chain variable domains of a human antibody in addition to the CDR sequences from an agonistic TNFR2 antibody, such as TNFRAG1. For example, U.S. Patent No. 6,054,297 identifies several examples where it may be advantageous to retain certain framework residues from certain antibody heavy or light chain variable regions in the resulting humanized antibody. In some embodiments, framework residues may engage antigens through non-covalent interactions and contribute to the affinity of the antibody for the target antigen. In some embodiments, individual framework residues may modulate the conformation of the CDRs and indirectly affect the antibody-antigen interaction. Certain framework residues may form the interface between the VH and VL domains and thus contribute to the global antibody structure. In some cases, framework residues may constitute functional glycosylation sites (e.g., Asn-X-Ser / Thr), which upon attachment to carbohydrate moieties may determine antibody structure and antigen affinity. In such cases, it may be beneficial to retain certain framework residues of a TNFR2 agonist antibody (e.g., TNFRAG1) in, for example, a humanized or primatized agonist antibody or antigen-binding fragment thereof, since different framework residues may promote higher epitope affinity and improved biochemical activity of the antibody or antigen-binding fragment thereof.

[0204] The antibodies described herein include antibody fragments, Fab domains, F(ab') molecules, F(ab') fragments, and / or Fab domains that contain one or more or all of the CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 sequences that (i) contain one or more or all of the CDRs of TNFRAG1, and / or (ii) contain one or more or all of the CDR sequences whose CDRs exhibit at least 85% sequence identity (e.g., 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity) to the corresponding CDR sequences of TNFRAG1. 2 Also included are antibodies, single chain variable fragments (scFv), tandem scFv fragments, diabodies, triabodies, dual variable domain immunoglobulins, multispecific antibodies, bispecific antibodies, and heterospecific antibodies. These molecules can be recombinantly expressed, for example, by incorporating polynucleotides encoding these proteins into expression vectors and transfecting eukaryotic or prokaryotic cells using techniques described herein or known in the art, or can be chemically synthesized, for example, by solid phase peptide synthesis methods described herein or known in the art.

[0205] The polypeptides described herein additionally include antibody-like scaffolds, for example, comprising one or more or all of the CDRs of TNFRAG1, or in which one or more or all of the CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 sequences exhibit at least 85% sequence identity (e.g., 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity) to the corresponding CDR sequences of TNFRAG1, and / or comprise one or more (e.g., up to three) amino acid substitutions (e.g., one or more conservative amino acid substitutions) compared to the corresponding CDR sequences of TNFRAG1. Examples of antibody-like scaffolds include the 10th fibronectin III domain ( 10 Fn3) are included. 10 The tertiary structure of the Fn3 domain resembles the tertiary structure of the variable region of an IgG heavy chain, and one of skill in the art will recognize that 10For example, one or more or all of the CDR sequences of TNFRAG1, or a sequence having at least 85% sequence identity (e.g., 90%, 95%, 97%, 99%, or 100% sequence identity) to any one or more of these CDR sequences, or a sequence containing amino acid substitutions, e.g., conservative or non-conservative amino acid substitutions (e.g., up to three amino acid substitutions) to one or more of these CDR sequences, can be grafted onto a fibronectin scaffold by replacing residues of the BC, DE, and FG loops of Fn3 with residues of the corresponding CDR sequences of TNFRAG1. This allows for the grafting of a fibronectin scaffold with ... 10 This can be accomplished by recombinant expression of the Fn3 domain (e.g., using the vectors and techniques described herein) as an antibody-like scaffold for grafting CDRs from an antibody into the BC, DE, and FG structural loops. 10 Examples of using Fn3 domains are reported in WO2000 / 034784, WO2009 / 142773, WO2012 / 088006, and U.S. Patent No. 8,278,419, the disclosures of each of which are incorporated herein by reference.

[0206] Molecular determinants of TNFR2 affinity and agonist action The polypeptides of the present disclosure may exhibit a set of shared structural features that give rise to a TNFR2 agonist phenotype (e.g., a dominant TNFR2 agonist phenotype). For example, alignment of the amino acid sequences of the respective CDR-H1 of TNRAG1 with two additional agonistic TNFR2 monoclonal antibodies, TNFRAG2 and TNFRAG3, demonstrates that these antibodies feature a conserved consensus CDR-H1 sequence, as shown below: GYTFTDYN (I / L) (TNFRAG1 CDR-H1, SEQ ID NO: 45) GFTFTDYD (TNFRAG2 CDR-H1, SEQ ID NO:46) GYTFTDY-I (TNFRAG3 CDR-H1, SEQ ID NO: 47) G(Y / F)TFTDY(N / D)- (consensus sequence, SEQ ID NO: 48)

[0207] By sequence alignment, GZ 1 TFZ 3 Z 2 YZ 3 Z 4 A shared CDR-H1 motif, such as that set forth in (SEQ ID NO:2), was revealed, and each Z 1 are independently naturally occurring amino acids containing a side chain that contains an aromatic substituent, and each Z 2 are independently naturally occurring amino acids that contain an anionic side chain at physiological pH, and each Z 3 are independently naturally occurring amino acids containing a polar, uncharged side chain at physiological pH, and each Z 4 are independently leucine or isoleucine. Agonistic TNFR2 polypeptides of the present disclosure may have a CDR-H1 sequence set forth in any one of SEQ ID NOs: 45-47 above, and / or a CDR-H1 represented by the conserved motif of SEQ ID NO: 2.

[0208] Nucleic acids and expression systems The agonistic TNFR2 polypeptides (e.g., single-chain peptides, antibodies, antigen-binding fragments thereof, and constructs thereof) described herein can be prepared by any of a variety of established techniques. For example, the agonistic TNFR2 antibodies or antigen-binding fragments thereof described herein can be prepared by recombinant expression of immunoglobulin light and heavy chain genes in host cells. To express the antibody recombinantly, one or more recombinant expression vectors each containing DNA fragments encoding the immunoglobulin light and heavy chains of the antibody are transfected into the host cell, and the light and heavy chains are expressed in the host cell and can be optionally secreted into the medium in which the host cell is cultured, from which the antibody can be recovered. Standard recombinant DNA techniques are used to obtain antibody heavy and light chain genes, incorporate these genes into recombinant expression vectors, and introduce the vectors into host cells, as described in Molecular Cloning; A Laboratory Manual, Second Edition (Sambrook, Fritsch and Maniatis (eds), Cold Spring Harbor, NY, 1989), Current Protocols in Molecular Biology (Ausubel et al., eds., Greene Publishing Associates, 1989), and U.S. Pat. No. 4,816,397, the disclosures of each of which are incorporated herein by reference.

[0209] Vectors for the expression of agonistic TNFR2 polypeptides Viral genomes provide a rich source of vectors that can be used to efficiently deliver exogenous genes into the genome of cells (e.g., eukaryotic or prokaryotic cells). Viral genomes are particularly useful vectors for gene delivery because the polynucleotides contained in such genomes are typically integrated into the genome of target cells by generalized or specialized transduction. These processes occur as part of the natural viral replication cycle and do not require additional proteins or reagents to induce gene integration. Examples of viral vectors include retroviruses, adenoviruses (e.g., Ad5, Ad26, Ad34, Ad35, and Ad48), parvoviruses (e.g., adeno-associated viruses), coronaviruses, negative strand RNA viruses such as orthomyxoviruses (e.g., influenza virus), rhabdoviruses (e.g., rabies and vesicular stomatitis viruses), paramyxoviruses (e.g., measles and Sendai), positive strand RNA viruses such as picornaviruses and alphaviruses, and double-stranded DNA viruses including adenoviruses, herpesviruses (e.g., herpes simplex virus types 1 and 2), Epstein-Barr virus, cytomegalovirus), and poxviruses (e.g., vaccinia, modified vaccinia Ankara (MVA), fowlpox, and canarypox). Other viruses useful for delivering polynucleotides encoding the light and heavy chains or antibody fragments of the antibodies described herein include, for example, Norwalk virus, togavirus, flavivirus, reovirus, papovavirus, hepadnavirus, and hepatitis virus. Examples of retroviruses include avian leukosis sarcoma, mammalian C, B, and D viruses, HTLV-BLV group, lentivirus, and spumavirus (Coffin, JM, Retroviridae: The viruses and their replication, In Fundamental Virology, Third Edition, BN Fields, et al., Eds., Lippincott-Raven Publishers, Philadelphia, 1996). Other examples include juniper viruses, Examples of vectors include myeloid leukemia virus, murine sarcoma virus, mouse mammary tumor virus, bovine leukemia virus, feline leukemia virus, feline sarcoma virus, avian leukemia virus, human T-cell leukemia virus, baboon endogenous virus, gibbon ape leukemia virus, Mason-Pfizer monkey virus, simian immunodeficiency virus, simian sarcoma virus, Rous sarcoma virus, and lentivirus. Other examples of vectors are described, for example, in McVey et al., (U.S. Patent No. 5,801,030), the disclosure of each of which is incorporated herein by reference.

[0210] Non-viral vectors such as plasmids are also known in the art, including, but not limited to, prokaryotic and eukaryotic vectors (e.g., yeast and bacterial-based plasmids) and plasmids for expression in mammalian cells.Methods for introducing vectors into host cells and isolating and purifying expressed proteins are also known in the art (e.g., Molecular Cloning; A Laboratory Manual, Second Edition (Sambrook, Fritsch and Maniatis (eds), Cold Spring Harbor, NY, 1989)).

[0211] Genome editing technology In addition to viral vectors, antibody light and heavy chains, single chain polypeptides, single chain variable fragments (scFv), tandem scFv, Fab domains, F(ab') 2Various additional methods have been developed for the integration of genes encoding domains, diabodies, and triabodies into the genome of target cells. One such method that can be used to integrate polynucleotides encoding agonistic TNFR2 polypeptides (e.g., single-chain polypeptides, antibodies, antigen-binding fragments thereof, or constructs thereof) into prokaryotic or eukaryotic cells includes the use of transposons. Transposons are polynucleotides that encode transposase enzymes and contain a polynucleotide sequence or gene of interest flanked by excision sites at 5' and 3' positions. Once the transposon is delivered to a cell, expression of the transposase gene begins, resulting in an active enzyme that cuts the gene of interest from the transposon. This activity is mediated by the site-specific recognition of the transposon excision site by the transposase. In some embodiments, these excision sites can be terminal repeats or inverted terminal repeats. Once excised from the transposon, the gene of interest can be integrated into the genome of a prokaryotic or eukaryotic cell by transposase-catalyzed cleavage of similar excision sites present in the nuclear genome of the cell. This allows the gene encoding the agonistic TNFR2 polypeptide described herein to be inserted into the excision site of the cleaved nuclear DNA, followed by ligation of the phosphodiester bond that binds the gene of interest to the DNA of the prokaryotic or eukaryotic genome, completing the incorporation process.In some embodiments, the transposon is a retrotransposon, and the gene encoding the polypeptide is first transcribed into an RNA product, then reverse transcribed into DNA, and then integrated into the genome of the prokaryotic or eukaryotic cell.Exemplary transposon systems include the piggyback transposon (described in detail in WO2010 / 085699) and the Sleeping Beauty transposon (described in detail in US2005 / 0112764), the disclosures of each of which are incorporated herein by reference.

[0212] Another useful method for integrating nucleic acid molecules encoding agonistic TNFR2 polypeptides (e.g., single chain polypeptides, antibodies, or antigen-binding fragments thereof) into the genome of prokaryotic or eukaryotic cells is the clustered regularly interspaced short round repeats (CRISPR) / Cas system, which originally evolved as an adaptive defense mechanism in bacteria and archaea against viral infection. The CRISPR / Cas system consists of palindromic repeat sequences in plasmid DNA and the associated Cas9 nuclease. This DNA and protein assembly directs site-specific DNA cleavage of a target sequence by first integrating foreign DNA into the CRISPR locus. Polynucleotides containing these foreign sequences and the repeat spacer elements of the CRISPR locus are in turn transcribed in the host cell to create guide RNAs that can subsequently anneal to the target sequence and localize the Cas9 nuclease to this site. In this way, the foreign polynucleotide can trigger highly site-specific cas9-mediated DNA cleavage, since the interaction that brings cas9 within the vicinity of the target DNA molecule is governed by RNA:DNA hybridization. As a result, one can theoretically design a CRISPR / Cas system that cuts a target DNA molecule of interest. This technology has been utilized to edit eukaryotic genomes (Hwang et al., Nat. Biotech., 31:227-229, 2013) and can be used as an efficient means of site-specifically editing eukaryotic or prokaryotic genomes to cut DNA prior to the incorporation of a polynucleotide encoding an agonistic TNFR2 polypeptide (e.g., a single chain polypeptide, an antibody, or an antigen-binding fragment thereof) as described herein. The use of CRISPR / Cas to regulate gene expression is described in U.S. Pat. No. 8,697,359, the disclosure of which is incorporated herein by reference.

[0213] Alternative methods for site-specific cleavage of genomic DNA prior to integration of polynucleotides encoding TNFR2 antibodies or antibody fragments described herein include the use of zinc finger nucleases and transcription activator-like effector nucleases (TALENs). Unlike the CRISPR / Cas system, these enzymes do not contain guide polynucleotides for localization to specific target sequences. Instead, target specificity is controlled by DNA binding domains within these enzymes. Zinc finger nucleases and TALENs for use in genome editing applications are described in Urnov et al. (Nat.Rev.Genet.,11:636-646,2010), and Joung et al., (Nat.Rev.Mol.Cell.Bio.14:49-55,2013), which are incorporated herein by reference. Additional genome editing techniques that can be used to integrate the polynucleotides encoding the antibodies described herein into the genome of prokaryotic or eukaryotic cells include the use of ARCUS™ meganucleases, which can be rationally designed to site-specifically cleave genomic DNA. The use of these enzymes to integrate the polynucleotides encoding the agonistic TNFR2 polypeptides (e.g., single-chain polypeptides, antibodies, antigen-binding fragments thereof, or constructs thereof) described herein into the genome of prokaryotic or eukaryotic cells is particularly advantageous in view of the structure-activity relationship established for such enzymes. Thus, single-chain meganucleases can be modified at specific amino acid positions to create nucleases that selectively cleave DNA at desired positions. These single-chain nucleases are described in detail, for example, in U.S. Patent Nos. 8,021,867 and 8,445,251, the disclosures of each of which are incorporated herein by reference.

[0214] Polynucleotide sequence elements To express the agonistic TNFR2 polypeptide (e.g., a single-chain polypeptide, an antibody, an antigen-binding fragment thereof, or a construct thereof) described herein, a polynucleotide encoding a partial or full-length light chain and a heavy chain, for example, a polynucleotide encoding one or more or all of the CDR sequences of an antibody or an antigen-binding fragment thereof described herein, can be inserted into an expression vector such that the gene is operably linked to a transcriptional and translational control sequence. The expression vector and the expression control sequence are selected to be compatible with the expression host cell used. Using established techniques described herein or known in the art, the light chain gene and the polynucleotide encoding the heavy chain of the TNFR2 polypeptide can be inserted into separate vectors, or, optionally, both polynucleotides can be incorporated into the same expression vector.

[0215] In addition to polynucleotides encoding the antibody heavy and light chains (or antibody fragments such as single chain polypeptides, scFv molecules, or constructs described herein), the recombinant expression vectors described herein can carry regulatory sequences that control the expression of the antibody chain genes in a host cell. The design of the expression vector, including the selection of regulatory sequences, may depend on factors such as the choice of the host cell to be transformed or the desired level of expression of protein. For example, suitable control sequences for mammalian host cell expression include viral elements that direct high levels of protein expression in mammalian cells, such as promoters and / or enhancers from cytomegalovirus (CMV) (such as the CMV promoter / enhancer), simian virus 40 (SV40) (such as the SV40 promoter / enhancer), adenovirus (e.g., the adenovirus major late promoter (AdMLP)) and polyoma. Viral regulatory elements and sequences thereof are described in detail in, for example, US Pat. No. 5,168,062, US Pat. No. 4,510,245, and US Pat. No. 4,968,615, the disclosures of each of which are incorporated herein by reference.

[0216] In addition to the antibody chain genes and control sequences, the recombinant expression vectors described herein may carry additional sequences, such as sequences regulating replication of the vector in a host cell (e.g., origins of replication) and selectable marker genes. The selectable marker gene facilitates selection of host cells into which the vector has been introduced (see, e.g., U.S. Pat. Nos. 4,399,216, 4,634,665 and 5,179,017). For example, typically the selectable marker gene confers resistance to cytotoxic drugs, such as G418, puromycin, blasticidin, hygromycin or methotrexate, on the host cell into which the vector has been introduced. Suitable selectable marker genes include the dihydrofolate reductase (DHFR) gene (DHFR with methotrexate selection / amplification), and the selectable marker gene (DHFR with methotrexate selection / amplification). ” The gene includes a gene encoding the heavy and light chains of a TNFR2 antibody or TNFR2 antibody fragment, and a gene encoding the light and light chains of the TNFR2 antibody or TNFR2 antibody fragment.

[0217] Polynucleotides encoding modified agonistic TNFR2 polypeptides Using the compositions and methods of the disclosure, agonistic TNFR2 polypeptides can be produced, for example, by introducing one or more CDRs from a given TNFR2 agonist polypeptide into a germline antibody sequence, e.g., a human germline antibody sequence in the case of generating a human or humanized antibody. By way of example, an agonistic TNFR2 polypeptide (e.g., a single chain polypeptide, an antibody, an antigen-binding fragment thereof, or a construct thereof) described herein may comprise one or more or all of the CDRs of TNFRAG1 (set forth in SEQ ID NOs:2-4 and 10-13), or one or more or all of the CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 sequences, in which one or more or all of the CDRs exhibit at least 85% sequence identity (e.g., 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity) to the corresponding CDR sequence of TNFRAG1 or contain one or more (e.g., up to three) amino acid substitutions (e.g., one or more conservative amino acid substitutions) compared to the corresponding CDR sequence of TNFRAG1. Optionally, the TNFR2 agonist polypeptide can be characterized by differences in one or more framework regions compared to those of TNFRAG1, such as the framework region located between CDR-H1 and CDR-H2. For example, one or more framework regions of TNFRAG1 can be replaced with framework regions of human antibodies. Exemplary framework regions include, for example, the human framework regions described in US 7,829,086 and the primate framework regions described in EP1945668, the disclosures of each of which are incorporated herein by reference. To generate nucleic acids encoding such TNFR2 antibodies, for example, DNA fragments encoding at least one or both of the light chain variable region and the heavy chain variable region can be generated by chemical synthesis (e.g., solid-phase polynucleotide synthesis technology), in vitro gene amplification (e.g., by polymerase chain reaction technology), or by replicating polynucleotides in a host organism.

[0218] In some embodiments, a humanized agonistic TNFR2 antibody may comprise one or more or all of the CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 sequences, in which one or more or all of the CDR sequences exhibit at least 85% sequence identity (e.g., 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity) to the corresponding CDR sequence of TNFRAG1 or contain one or more (e.g., up to three) amino acid substitutions (e.g., one or more conservative amino acid substitutions) compared to the corresponding CDR sequence of TNFRAG1. This can be accomplished, for example, by site-directed mutagenesis of germline DNA or cDNA and amplifying the resulting polynucleotide using polymerase chain reaction (PCR) according to established procedures. Germline DNA sequences of human heavy and light chain variable region genes are known in the art (see, e.g., "VBASE" human germline sequence database; see also Kabat, et al., Sequences of Proteins of Immunological Interest, Fifth Edition, USDepartment of Health and Human Services, NIH Publication No. 91-3242, 1991; Tomlinson et al., J. Mol. Biol. 227:776-798, 1992, and Cox et al., Eur. J. Immunol. 24:827-836, 1994, which are incorporated herein by reference). Chimeric nucleic acid constructs encoding human heavy and light chain variable regions comprising one or more or all of the CDRs of TNFRAG1 or similar sequences described above can be generated, for example, using established cloning techniques known in the art. Additionally, polynucleotides encoding heavy chain variable regions containing one or more of the CDRs of TNFRAG1 or similar sequences described above can be synthesized and used as templates for mutagenesis to generate the variants described herein using conventional mutagenesis techniques. Alternatively, DNA fragments encoding the variants can be directly synthesized (e.g., by established solid-phase nucleic acid chemical synthesis procedures).

[0219] A DNA fragment encoding a VH segment containing one or more or all of the CDR-H1, CDR-H2, and CDR-H3 sequences of TNFRAG1 can be further manipulated by standard recombinant DNA techniques, for example, to convert the variable region genes into full-length antibody chain genes, Fab fragment genes, or scFv genes, in which the VL- or VH-encoding DNA fragment is operably linked to another DNA fragment encoding another protein, such as an antibody constant region or a flexible linker.

[0220] The isolated DNA encoding the VH region of the agonistic TNFR2 antibody described herein can be converted into a full-length heavy chain gene (and a Fab heavy chain gene) by, for example, operably linking the VH-encoding DNA to another DNA molecule encoding the heavy chain constant region domains (CH1, CH2, CH3, and optionally CH4). The sequences of human heavy chain constant region genes are known in the art (see, for example, Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, USDepartment of Health and Human Services, NIH Publication No. 91-3242, 1991), and DNA fragments encompassing these regions can be obtained by standard PCR amplification. The heavy chain constant region can be an IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM, or IgD constant region, and in a particular embodiment, is an IgG1 constant region. In the case of a Fab fragment heavy chain gene, the VH-encoding DNA can be operably linked to another DNA molecule encoding only the heavy chain CH1 domain.

[0221] The isolated DNA encoding the VL region of the agonistic TNFR2 antibody can be converted into a full-length light chain gene by operably linking the DNA encoding the VL to another DNA molecule encoding the light chain constant region, CL. The sequences of human light chain constant region genes are known in the art (see, for example, Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, USDepartment of Health and Human Services, NIH Publication No. 91-3242, 1991), and DNA fragments containing these regions can be obtained, for example, by amplifying polynucleotides encoding these regions in prokaryotic or eukaryotic cells, PCR amplification, or chemical polynucleotide synthesis. The light chain constant region can be a kappa (κ) or lambda (λ) constant region, but in certain embodiments is a kappa constant region. To create an scFv gene, the DNA fragments encoding the VH and VL are operably linked to another fragment encoding a flexible linker, for example, the amino acid sequence (Gly 4 Ser) 3 operably linked to a polynucleotide encoding a flexible hydrophilic amino acid sequence, such as V H and V L The sequence is V L and V H This allows the regions to be expressed as a contiguous single-chain protein joined by a linker (see, e.g., Bird et al., Science 242:423-426, 1988; Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883, 1988; McCafferty et al., Nature 348:552-554, 1990).

[0222] Recombinant DNA technology can also be used to remove some or all of the DNA encoding either or both of the light and heavy chains that are not necessary for binding to TNFR2. Molecules expressed from such truncated DNA molecules are also encompassed by the antibodies described herein. In addition, bifunctional antibodies can be generated in which one heavy chain comprises one or more or all of the CDRs of TNFRAG1 or similar CDR sequences described above, and the other heavy and / or light chain is specific for a particular antigen other than TNFR2. Such antibodies can be generated, for example, by crosslinking the heavy and light chains comprising the CDRs of TNFRAG1 or one or more or all of the similar CDR sequences described above to the heavy and light chains of a second antibody that is specific for a different antigen, for example, by using standard chemical crosslinking methods (e.g., by disulfide bond formation). Bifunctional antibodies can also be generated by expressing engineered nucleic acid molecules encoding bifunctional antibodies in prokaryotic or eukaryotic cells.

[0223] Bispecific antibodies, i.e., antibodies that use the same binding site to bind TNFR2 and different antigens, can be generated by mutating amino acid residues in the CDRs of the light and / or heavy chains. In some embodiments, bispecific antibodies that bind to two antigens, such as TNFR2 and a second cell surface receptor, can be generated by mutating amino acid residues surrounding the antigen binding site (Bostrom et al., Science 323:1610-1614, 2009). Bifunctional antibodies can be generated by expressing polynucleotides engineered to encode the bispecific antibodies.

[0224] The modified agonistic TNFR2 antibody and antibody fragment described herein can also be produced by chemical synthesis (for example, by the method described in Solid Phase Peptide Synthesis, 2nd ed., 1984 The Pierce Chemical Co., Rockford, 111, which is incorporated herein by reference).Variant antibodies can also be produced using cell-free synthesis platforms (for example, see Chu et al., Biochemia No. 2, 2001 (Roche Molecular Biologicals), which is incorporated herein by reference).

[0225] Host cells for expression of agonistic TNFR2 polypeptides The polypeptides described herein (e.g., single chain polypeptides, antibodies, antigen-binding fragments thereof, and constructs thereof) can be expressed in prokaryotic or eukaryotic host cells. In some embodiments, expression of the polypeptides (e.g., single chain polypeptides, antibodies, or antigen-binding fragments thereof) is in eukaryotic cells, such as mammalian host cells, for optimal secretion of properly folded, immunologically active antibodies. Exemplary mammalian host cells for expressing the recombinant antibodies or antigen-binding fragments thereof described herein include Chinese Hamster Ovary (CHO) cells (DHFR-selectable markers as described in Urlaub and Chasin (1980, Proc. Natl. Acad. Sci. USA 77:4216-4220, used with the DHFR selectable marker as described in Kaufman and Sharp (1982, Mol. Biol. 159:601-621)). -Cell types that may be useful for expressing antibodies and fragments thereof include bacterial cells, such as BL-21(DE3) E. coli cells, which can be transformed with vectors containing foreign DNA according to established protocols. Additional eukaryotic cells that may be useful for expressing antibodies include yeast cells, such as auxotrophic strains of S. cerevisiae, which can be transformed and selectively grown in incomplete medium according to established procedures known in the art. Once a recombinant expression vector encoding an antibody gene has been introduced into a mammalian host cell, the antibody is produced by culturing the host cell for a period of time sufficient to allow for expression of the antibody in the host cell or secretion of the antibody into the medium in which they are grown.

[0226] Polypeptides (e.g., single chain polypeptides, antibodies, antigen-binding fragments thereof, and constructs thereof) can be recovered from the culture medium using standard protein purification methods. Host cells can also be used to produce portions of intact antibodies, such as Fab fragments or scFv molecules. Also included herein are methods in which the above procedures are modified according to established protocols known in the art. For example, to produce antigen-binding fragments of antibodies, it may be desirable to transfect host cells with DNA encoding either the light or heavy chain (but not both) of the agonistic TNFR2 antibodies described herein.

[0227] Once an agonistic TNFR2 polypeptide described herein (e.g., a single chain polypeptide, an antibody, an antigen-binding fragment thereof, or a construct thereof) is produced by recombinant expression, it can be purified by any method known in the art, such as, for example, chromatography (e.g., ion exchange, affinity, particularly affinity for TNFR2 after Protein A or Protein G selection, and sizing column chromatography), centrifugation, differential solubility, or other standard techniques for purifying proteins. Additionally, an agonistic TNFR2 polypeptide described herein or a fragment thereof can be fused to a heterologous polypeptide sequence described herein or known in the art to facilitate purification or to generate a therapeutic conjugate.

[0228] Once isolated, the agonistic TNFR2 single chain polypeptides, antibodies, antigen-binding fragments thereof, or constructs can be further purified, if desired, by, for example, high performance liquid chromatography (see, e.g., Fisher, Laboratory Techniques in Biochemistry and Molecular Biology (Work and Burdon, eds., Elsevier, 1980); incorporated herein by reference), or gel filtration chromatography, such as on a Superdex™ 75 column (Pharmacia Biotech AB, Uppsala, Sweden).

[0229] A platform for generating and affinity maturing agonistic TNFR2 polypeptides Mapping epitopes on TNFR2 that promote receptor agonism Agonistic TNFR2 polypeptides (e.g., single chain polypeptides, antibodies, antigen binding thereof, and constructs thereof) described herein can be generated by screening libraries of polypeptides (e.g., single chain polypeptides, antibodies, antigen binding thereof, and constructs thereof) for functional molecules that can bind to epitopes within TNFR2 that selectively promote receptor agonism rather than receptor inhibition. Such epitopes can be modeled by screening antibodies or antigen binding fragments thereof against a series of linear or cyclic peptides that contain residues corresponding to the desired epitope within TNFR2.

[0230] By way of example, peptides comprising individual fragments isolated from TNFR2 that promote receptor agonism can be synthesized by peptide synthesis techniques described herein or known in the art. These peptides can be immobilized on a solid surface and molecules that bind to agonistic TNFR2 polypeptides (e.g., single chain polypeptides, antibodies, antigen-binding fragments thereof, and constructs thereof) can be screened, for example, using an ELISA-based screening platform using established procedures. Using this assay, for example, peptides that specifically bind to TNFRAG1 with high affinity thus contain residues within the epitope of TNFR2 that preferentially bind to these antibodies. Peptides identified in this manner can be used to screen libraries of polypeptides (e.g., single chain polypeptide fragments, antibodies, antigen-binding fragments thereof, and constructs thereof) to identify agonistic TNFR2 polypeptides. Furthermore, because these peptides serve as surrogates for epitopes within TNFR2 that promote receptor agonism, polypeptides generated using this screening technique may bind to the corresponding epitopes of TNFR2, and are expected to be agonistic in receptor activity.

[0231] Screening a library of agonist TNFR2 polypeptides High throughput screening of libraries of polypeptides (e.g., single chain polypeptides, antibodies, antibody fragments, or constructs thereof) for molecules capable of binding to epitopes within TNFR2 (e.g., amino acids 56-60, 101-107, and / or 115-142 of SEQ ID NO:1) includes, but is not limited to, 1) phage display, bacterial display, yeast display, mammalian display, ribosome display, mRNA display, and cDNA display. The use of phage display to isolate ligands that bind to biologically relevant molecules has been reviewed, for example, by Felici et al. (Biotechnol. Annual Rev. 1:149-183, 1995), Katz (Annual Rev. Biophys. Biomol. Struct. 26:27-45, 1997), and Hoogenboom et al. (Immunotechnology 4:1-20, 1998). Several randomized combinatorial peptide libraries have been constructed to select polypeptides that bind to different targets such as cell surface receptors or DNA (reviewed by Kay (Perspect. Drug Discovery Des. 2, 251-268, 1995), Kay et al., (Mol. Divers. 1:139-140, 1996). Proteins and multimeric proteins have been successfully phage displayed as functional molecules (see EP0349578A, EP4527839A, EP0589877A, Chiswell and McCafferty (Trends Biotechnol. 10, 80-84 1992)). In addition, functional antibody fragments (e.g., Fab, single chain Fv [scFv]) have been expressed (McCafferty et al. (Nature 348:552- 554, 1990), Barbas et al. (Proc. Natl. Acad. Sci. 2010, 111:117-119, 1996). Sci. USA 88:7978-7982, 1991), Clackson et al. (Nature 352:624-628, 1991), which references are hereby incorporated by reference in their entireties.

[0232] (i) Phage display technology As an example, phage display technology can be used to screen libraries of polypeptides (e.g., single chain polypeptides, antibodies, antigen-binding fragments thereof, and constructs) for functional molecules that can bind to cyclic or polycyclic peptides that contain epitopes within TNFR2 that promote receptor activation (e.g., amino acids 56-60, 101-107, and / or 115-142 of SEQ ID NO:1). For example, libraries of polynucleotides encoding single chain antibody fragments, such as scFv fragments, can be obtained using established procedures (e.g., solid-phase polynucleotide synthesis or error-prone PCR techniques, see McCullum et al. (Meth. Mol. Biol., 634:103-109, 2010), incorporated herein by reference). These randomized polynucleotides can be incorporated into viral genomes such that the randomized antibody chains encoded by these genes are expressed on the surface of filamentous phage, for example, by covalent bonds between the antibody chains and coat proteins (e.g., the pIII coat protein on the surface of M13 phage). This provides a physical link between the genotype and phenotype of the antibody chain. In this way, libraries of phage displaying diverse antibody chains containing random mutations in the hypervariable regions can be screened for the ability of the external antibody chains to bind to TNFR2 epitopes (e.g., amino acids 56-60, 101-107, and / or 115-142 of SEQ ID NO: 1) immobilized on the surface using established procedures. For example, such peptides can be physically attached to the surface of a microtiter plate by forming a covalent bond between the peptide and the epitope tag (e.g., biotin) and incubating the peptide in a well of the microtiter plate coated with a complementary tag (e.g., avidin) that binds with high affinity to the tag previously attached to the peptide. Suitable epitope tags include, but are not limited to, maltose binding protein, glutathione-S-transferase, polyhistidine tag, FLAG tag, myc tag, human influenza hemagglutinin (HA) tag, biotin, streptavidin. The epitopes displayed by these molecules can be used to identify the phenotype of the antibody chain. The peptides containing the tope can be immobilized on a surface containing a complementary molecule, such as maltose, glutathione, nickel-containing complexes, anti-FLAG antibodies, anti-myc antibodies, anti-HA antibodies, streptavidin, or biotin, respectively. Thus, the phage can be incubated with the surface containing the immobilized TNFR2-derived peptide in the presence of a suitable buffer system (e.g., one containing physiological salt concentration, ionic strength, and maintained at physiological pH by a buffer) for a suitable time to allow binding of the antibody to the constrained peptide. The surface is then washed (e.g., with phosphate buffer containing 0.1% Tween-20) to remove phage that do not display antibody chains that interact with the TNFR2-derived peptide with an affinity above a certain threshold.

[0233] The affinity of the polypeptides remaining after this initial panning (i.e., screening) step can be adjusted by adjusting the conditions of the washing step (e.g., by including weakly acidic or basic components, or by including low concentrations of other TNFR2-derived peptides to compete with the immobilized peptides for antigen-binding sites). In this way, the population of phages that remain bound to the surface of the microtiter plate after the washing step is enriched for phages that bind to TNFR2-derived peptide epitopes that promote receptor activation. The remaining phages can be amplified by reducing the strength of protein-protein interactions to elute the phages from the surface containing these peptides (e.g., by changing the surrounding pH, ionic strength, or temperature). The isolated phages can then be amplified, for example, by infecting bacterial cells, and the resulting phages can be optionally subjected to panning with additional iterations of screening to further enrich the population of phages bearing higher affinity agonistic TNFR2 polypeptides. Following these panning steps, phages displaying high affinity antibodies or antigen-binding fragments thereof can be isolated, and the genomes of these phages can be sequenced to identify the polynucleotide and polypeptide sequences of the encoded antibodies. Such phage display techniques can be used, for example, to generate antibody chains such as scFv fragments, tandem scFv fragments, and other antigen-binding fragments described herein that can be used as agonists of TNFR2. Exemplary phage display protocols for identifying antibody chains and antigen-binding fragments thereof that bind with high affinity to a particular antigen are well established and are described, for example, in U.S. Patent No. 7,846,892, WO1997 / 002342, U.S. Patent No. 8,846,867, and WO2007 / 132917, the disclosures of each of which are incorporated herein by reference. Similar phage display techniques can be used to generate antibody-like scaffolds (e.g., antibody-like scaffolds described herein) that bind to epitopes within TNFR2 (e.g., amino acids 56-60, 101-107, and / or 115-142 of SEQ ID NO:1) that promote receptor activation. 10Fn3 domain) Exemplary phage display protocols for the identification of antibody-like scaffold proteins are described, for example, in WO2009 / 086116, the disclosure of which is incorporated herein by reference.

[0234] (ii) Cell-based display technology Other in vitro display techniques that utilize the linkage between the genotype and phenotype of solvent-exposed polypeptides include yeast and bacterial display. Yeast display technology is well established in the art and has the advantage that it can display large numbers of antibodies (often up to 30,000) on the surface of individual yeast cells (see, for example, Boder et al. (Nat Biotechno. 15:553, 1997), which are incorporated herein by reference). The larger size of yeast cells than filamentous phages allows additional screening strategies, since yeast libraries can be analyzed and sorted using flow cytometry. For example, established procedures can be used to generate libraries of bacterial or yeast cells that express polypeptides (e.g., single-chain polypeptides, antibodies, and antigen-binding fragments thereof) that contain randomized hypervariable regions (see, for example, U.S. Patent No. 7,749,501 and US2013 / 0085072, the teachings of each of which are incorporated herein by reference). For example, large libraries of yeast cells expressing polynucleotides encoding naive scFv fragments can be made using established procedures (de Bruin et al., Nat Biotechnol 17:397, 1999, incorporated herein by reference). Yeast cells expressing these polynucleotides can be incubated with two different fluorescent molecules during the panning step. One binds to a conserved residue in the antibody and reflects the amount of antibody displayed, and another dye fluoresces at a different wavelength and binds to the antigen, thus indicating the amount of antigen bound. For example, one skilled in the art could use a TNFR2-derived peptide containing amino acids 56-60, 101-107, and / or 115-142 of SEQ ID NO:1, optionally conjugated to an epitope tag (e.g., biotin) at the N- or C-terminus of the peptide, or at a residue not expected to interfere with antibody-antigen binding. This allows a fluorescent dye labeled with a complementary tag (e.g., avidin) to localize to the antibody-antigen complex. This allows for great flexibility and immediate feedback on the progress of your selections.In contrast to phage display, normalizing to antibody display levels allows one to easily select for antibodies with higher affinity rather than higher expression levels. Indeed, it is possible to distinguish and classify antibodies that differ only by a factor of two in affinity (VanAntwerp and Wittrup (Biotechnol Prog 16:31, 2000)).

[0235] (iii) Nucleotide display technology Display techniques utilizing in vitro translation of randomized polynucleotide libraries also provide a powerful approach to generate agonistic TNFR2 polypeptides as described herein. For example, randomized DNA libraries encoding polypeptides (e.g., single chain polypeptides, antibodies, and antigen-binding fragments thereof) containing mutations within designated hypervariable regions can be obtained, for example, using established PCR-based mutagenesis techniques as described herein. The polynucleotides of these libraries can include transcriptional regulatory sequences, such as promoters and transcription termination sequences, and can additionally encode sequences that enhance the translation rate of the resulting mRNA construct (e.g., IRES sequences, 5' and 3' UTRs, poly-adenylation tracts, etc.). These polynucleotide libraries can be incubated in a suitable buffer containing RNA polymerase and RNA nucleoside triphosphates (NTPs) to transcribe the DNA sequences into competent mRNA molecules, which can then be translated by the large and small ribosomal subunits, aminoacyl-tRNA molecules, and translation initiation and elongation factors present in solution (e.g., using the PURExpress® In Vitro Protein Synthesis Kit, New England Biolab®). Designed mRNA modifications allow the antibody product to remain covalently attached to the mRNA template by chemical conjugation to puromycin (see, e.g., Keefe (Curr. Protoc. Mol. Biol., Chapter 24, Unit 24.5, 2001), incorporated herein by reference). Thus, this genotype-phenotype link can be used to select antibodies that bind to TNFR2-derived peptides by incubating mRNA:antibody fusion constructs with surface-immobilized peptides and panning in a manner similar to phage display techniques (see, e.g., WO2006 / 072773, incorporated herein by reference).

[0236] Optionally, the polypeptides (e.g., single chain polypeptides, antibodies, and antigen-binding fragments thereof) described herein can be generated using similar techniques, except that the antibody product can be non-covalently linked to the ribosome-mRNA complex, rather than covalently linked via a puromycin linker. This platform, known as ribosome display, is described, for example, in U.S. Patent No. 7,074,557, which is incorporated herein by reference. Alternatively, antibodies can be generated using cDNA display, a technique similar to mRNA display, except that cDNA, rather than mRNA, is covalently linked to the antibody product via a puromycin linker. cDNA display technology can offer the advantage that the panning step can be performed under increasingly stringent conditions, such as conditions in which the salt concentration, ionic strength, pH, and / or temperature of the environment are adjusted, particularly to screen for antibodies with high affinity to TNFR2-derived peptides. This is due to the natural greater stability of double-stranded cDNA than single-stranded mRNA. cDNA display screening techniques are described, for example, in Ueno et al. (Methods Mol. Biol., 805:113-135, 2012), which is incorporated herein by reference.

[0237] In addition to generating agonistic TNFR2 polypeptides (e.g., single chain polypeptides, antibodies, antigen-binding fragments thereof, and constructs thereof) described herein, in vitro display techniques (e.g., those described herein and known in the art) also provide methods for improving the affinity of agonistic TNFR2 polypeptides described herein. For example, rather than screening a library of antibodies and fragments thereof that contain fully randomized hypervariable regions, one can screen a narrower library of antibodies and antigen-binding fragments thereof that feature targeted mutations at specific sites within the hypervariable regions. This can be accomplished, for example, by assembling a library of polynucleotides encoding antibodies or antigen-binding fragments thereof that encode random mutations only at specific sites within the hypervariable regions. These polynucleotides can then be expressed, for example, in filamentous phage, bacterial cells, yeast cells, mammalian cells, or in vitro, using, for example, ribosome display, mRNA display, or cDNA display techniques, to screen for antibodies or antigen-binding fragments thereof that specifically bind to TNFR2 epitopes with improved binding affinity. For example, yeast display is well suited for affinity maturation, previously demonstrating the affinity of a single chain antibody at a K of 48 fM. D It has been used to improve the quality of life of mice (Boder et al. (Proc Natl Acad Sci USA 97:10701, 2000)).

[0238] Additional in vitro techniques that can be used for the generation and affinity maturation of agonistic TNFR2 polypeptides (e.g., single chain polypeptides, antibodies, and antigen-binding fragments thereof) described herein include screening combinatorial libraries of antibodies or antigen-binding fragments thereof for functional molecules capable of specifically binding to TNFR2-derived peptides. Combinatorial antibody libraries can be obtained, for example, by expression of polynucleotides encoding randomized hypervariable regions of antibodies or antigen-binding fragments thereof in eukaryotic or prokaryotic cells. This can be accomplished, for example, using gene expression techniques described herein or known in the art. Heterogeneous mixtures of antibodies can be purified, for example, by protein A or protein G selection, sizing column chromatography), centrifugation, differential solubility, and / or other standard techniques for protein purification. The resulting combinatorial library library can be screened, for example, by incubating these heterogeneous mixtures of antibodies with peptides derived from TNFR2 immobilized on a surface for a time sufficient to allow binding of the antibodies to the antigen. Unbound antibodies or fragments thereof can be removed by washing the surface with an appropriate buffer (e.g., a solution buffered at physiological pH (about 7.4), containing physiological salt concentrations and ionic strength, and optionally containing a surfactant such as TWEEN-20). Antibodies that remain bound can then be detected using, for example, an ELISA-based detection protocol (see, e.g., U.S. Pat. No. 4,661,445, the disclosure of which is incorporated herein by reference).

[0239] Additional techniques for screening combinatorial libraries of polypeptides (e.g., single chain polypeptides, antibodies, antigen-binding fragments thereof, and constructs thereof) that specifically bind to TNFR2-derived peptides include screening one-bead one-compound libraries of antibody fragments. Antibody fragments can be chemically synthesized on solid beads composed of hydrophilic, water-swellable materials (e.g., using established split-and-pool solid-phase peptide synthesis protocols) such that each bead displays a single antibody fragment. The heterogeneous bead mixture can then be incubated with TNFR2-derived peptides that are optionally labeled with a detectable moiety (e.g., a fluorescent dye) or conjugated to an epitope tag (e.g., biotin, avidin, FLAG tag, HA tag) that can be subsequently detected by treatment with a complementary tag (e.g., avidin, biotin, anti-FLAG antibody, anti-HA antibody, respectively). Beads that contain antibody fragments that specifically bind to TNFR2-derived peptides can be identified by analyzing the fluorescent properties of beads after incubation with fluorescently labeled antigen or complementary tag (e.g., by confocal fluorescence microscopy or fluorescence-activated bead sorting, see, e.g., Muller et al. (J.Biol.Chem., 16500-16505, 1996), which is incorporated herein by reference). Thus, beads that contain antibody fragments that specifically bind to TNFR2-derived peptides can be separated from beads that do not contain high affinity antibody fragments. The sequence of antibody fragments that specifically bind to TNFR2-derived peptides can be determined by techniques known in the art, including, for example, Edman degradation, tandem mass spectrometry, matrix-assisted laser desorption time-of-flight mass spectrometry (MALDI-TOF MS), nuclear magnetic resonance (NMR), and 2D gel electrophoresis (see, e.g., WO2004 / 062553, the disclosures of each of which are incorporated herein by reference).

[0240] Negative screening of polypeptides In addition to the above-mentioned methods for screening single-chain polypeptides, antibodies, or antibody fragments that specifically bind to epitopes derived from human TNFR2 that promote receptor activation, negative screening can be additionally performed to eliminate antibodies or antibody fragments that may also bind to epitopes that promote receptor antagonism, such as the KCRPG sequence. This can be achieved using any of the above-mentioned methods or variations thereof, for example, such that the antibody or antibody fragment to be screened is one that has previously been identified as having agonistic TNFR2 activity. Examples of techniques useful for negative screening include phage display, yeast display, bacterial display, ribosome display, mRNA display, cDNA display, or surface-based combinatorial library screening (e.g., ELISA format). This screening technique represents a useful strategy for identifying agonistic TNFR2 antibodies or antibody fragments, as antibodies or antibody fragments that can bind to TNFR2 epitopes that include the KCRPG sequence, lack or significantly reduce agonistic activity.

[0241] Immunization of Non-Human Mammals Another strategy that can be used to generate the agonistic TNFR2 antibodies and antigen-binding fragments thereof described herein involves immunizing non-human mammals. Examples of non-human mammals that can be immunized to generate the agonistic TNFR2 antibodies and fragments thereof described herein include rabbits, mice, rats, goats, guinea pigs, hamsters, horses, and sheep, as well as non-human primates. For example, established procedures for immunizing primates are known in the art (see, for example, WO1986 / 6004782, which is incorporated herein by reference). Immunization is a robust method of generating monoclonal antibodies that exploits the antigen specificity of B lymphocytes. For example, monoclonal antibodies can be prepared by the Kohler-Milstein method (e.g., as described in EP0110716, which is incorporated herein by reference), in which spleen cells from a non-human animal (e.g., a primate) immunized with a peptide present an antigen derived from TNFR2 that promotes receptor activation. The clonally expanded B lymphocytes produced by immunization can be isolated from the serum of the animal and then fused with myeloma cells to form hybridomas. Hybridomas are particularly useful entities for antibody production, since these immortalized cells can provide a continuous supply of antigen-specific antibodies. Antibodies from such hybridomas can then be isolated by purifying the antibodies from cell culture medium by affinity chromatography using techniques known in the art, for example, using reagents such as protein A or protein G.

[0242] Agonistic TNFR2 Polypeptide Conjugates Before administering the agonistic TNFR2 polypeptides (e.g., single-chain polypeptides, antibodies, and antigen-binding fragments thereof) described herein to a mammalian subject (e.g., a human), it may be desirable to conjugate the antibody or a fragment thereof to a second molecule, for example, to modulate the activity of the antibody in vivo. The agonistic TNFR2 antibodies and fragments thereof can be conjugated to other molecules at either the N-terminus or C-terminus of the light or heavy chain of the antibody using any of a variety of established conjugation strategies well known in the art. Examples of pairs of reactive functional groups that can be used to covalently bind the agonistic TNFR2 antibodies or fragments thereof to another molecule include thiol pairs, carboxylic acids and amino groups, ketones and amino groups, aldehydes and amino groups, thiols and α,β-unsaturated moieties (such as maleimides or dehydroalanines), thiols and α-haloamides, carboxylic acids and hydrazides, aldehydes and hydrazides, and ketones and hydrazides.

[0243] Agonistic TNFR2 polypeptides (e.g., single-chain peptides, antibodies, antigen-binding fragments thereof, and constructs thereof) can be directly covalently linked to another molecule by chemical bonds, as described. Alternatively, fusion proteins comprising agonistic TNFR2 antibodies and fragments thereof can be recombinantly expressed from cells (e.g., eukaryotic or prokaryotic cells). This can be accomplished, for example, by integrating a polynucleotide encoding the fusion protein into the nuclear genome of the cell (e.g., using techniques described herein or known in the art). Optionally, the antibodies and fragments thereof described herein can be linked to a second molecule by forming a covalent bond between the antibody and a linker. This linker can then be conjugated to another molecule, or the linker can be conjugated to another molecule and then linked to the agonistic TNFR2 antibodies or fragments thereof. Examples of linkers that can be used to form conjugates include polypeptide linkers, such as those that contain natural or unnatural amino acids. In some embodiments, it may be desirable to include D-amino acids in the linker. These residues are not present in naturally occurring proteins and therefore are more resistant to degradation by endogenous proteases. Fusion proteins containing polypeptide linkers can be made using chemical synthesis techniques as described herein or through recombinant expression of a polynucleotide encoding the fusion protein in a cell (e.g., a prokaryotic or eukaryotic cell). Linkers can be prepared using a variety of strategies well known in the art and can be cleaved by enzymatic hydrolysis, photolysis, hydrolysis under acidic conditions, hydrolysis under basic conditions, oxidation, disulfide reduction, nucleophilic cleavage, or organometallic cleavage, depending on the reactive components of the linker (Leriche et al., Bioorg. Med. Chem., 20:571-582, 2012).

[0244] Drug-polypeptide conjugates The agonistic TNFR2 polypeptides described herein (e.g., single chain polypeptides, antibodies, and antigen-binding fragments thereof) can additionally be conjugated, mixed, or administered separately with therapeutic agents, such as agents used to treat autoimmunity and / or inflammation. Exemplary agents that can be conjugated, mixed, or administered separately with the agonistic TNFR2 polypeptides described herein include Bacillus Calmette-Guerin (BCG). Additional drugs that can be conjugated, mixed, or administered separately to the agonistic TNFR2 polypeptides described herein include nonsteroidal anti-inflammatory drugs (NSAIDs), such as (i) salicylic acid derivatives including acetylsalicylic acid (aspirin), diflunisal, and sulfasalazine, (ii) paraaminophenol derivatives including acetaminophen, (iii) fenamates including mefenamic acid, meclofenamic acid, and flufenamic acid, (iv) propionic acid derivatives including ibuprofen, naproxen, fenoprofen, ketoprofen, flurbiprofen, and oxaprozin, and (v) enolic acid (oxicam) derivatives including piroxicam and tenoxicam. Additional drugs that can be conjugated, mixed, or administered separately to the agonistic TNFR2 polypeptides described herein include glucocorticoids. The agonistic TNFR2 polypeptides described herein may additionally or alternatively be conjugated, mixed, or administered separately with disease-modifying antirheumatic drugs (DMARDs), such as methotrexate, leflunomide, gold compounds, sulfasalazine, azathioprine, cyclophosphamide, antirheumatic drugs, D-penicillamine, cyclosporine, etc. Additional agents that may be conjugated, mixed, or administered separately with the agonistic TNFR2 polypeptides described herein include, among others, infliximab, etanercept, adalimumab, golimumab, and certolizumab pegol. Additional agents that may be conjugated, mixed, or administered separately with the agonistic TNFR2 polypeptides of the present disclosure are described, for example, in Li et al., Front.Pharmacol.8:460,2017, the disclosure of which is incorporated herein by reference.

[0245] In some embodiments, the agonistic TNFR2 polypeptides described herein are, for example, agonistic anti-CTLA-4 agents, agonistic anti-PD-1 agents, agonistic anti-PD-L1 agents, agonistic anti-PD-L2 agents, agonistic anti-CD27 agents, agonistic anti-CD30 agents, agonistic anti-CD40 agents, agonistic anti-4-1BB agents, agonistic anti-GITR agents, agonistic anti-OX40 agents, agonistic anti-TRAILR1 agents, agonistic anti-TR AILR2 agents, agonistic anti-TWEAK agents, agonistic anti-TWEAKR agents, agonistic anti-cell surface lymphocyte protein agents, agonistic anti-BRAF agents, agonistic anti-MEK agents, agonistic anti-CD33 agents, agonistic anti-CD20 agents, agonistic anti-HLA-DR agents, agonistic anti-HLA class I agents, agonistic anti-CD52 agents, agonistic anti-A33 agents, agonistic anti-GD3 agents, agonistic anti-PSMA agents, agonistic anti-Ceacan 1 agents, agonistic anti-Galedin 9 agents, agonistic anti-HVEM agents, agonistic anti-VISTA agents, agonistic anti-B7 agents H4 agents, agonistic anti-HHLA2 agents, agonistic anti-CD155 agents, agonistic anti-CD80 agents, agonistic anti-BTLA agents, agonistic anti-CD160 agents, agonistic anti-CD28 agents, agonistic anti-CD226 agents, agonistic anti-CEACAM1 agents, agonistic anti-TIM3 agents, agonistic anti-TIGIT agents, agonistic anti-CD96 agents, agonistic anti-CD70 agents, agonistic anti-CD27 agents, agonistic anti-LIGHT agents, agonistic anti-CD137 agents, agonistic anti-DR4 agents, agonistic anti-CR5 agents, agonistic anti-TNFRS agents, agonistic The immunotherapeutic agent is conjugated, mixed, or administered separately with an immunotherapeutic agent, such as an agonistic anti-TNFR1 agent, an agonistic anti-FAS agent, an agonistic anti-CD95 agent, an agonistic anti-TRAIL agent, an agonistic anti-DR6 agent, an agonistic anti-EDAR agent, an agonistic anti-NGFR agent, an agonistic anti-OPG agent, an agonistic anti-RANKL agent, an agonistic anti-LTβ receptor agent, an agonistic anti-BCMA agent, an agonistic anti-TACI agent, an agonistic anti-BAFFR agent, an agonistic anti-EDAR2 agent, an agonistic anti-TROY agent, and an agonistic anti-RELT agent. For example, the immunotherapeutic agent is an agonistic anti-CTLA-4 agent, an agonistic anti-PD agent, an agonistic anti-PD-L1 agent, an agonistic anti-PD-L2 agent, an agonistic anti-PD-L1 ... -1 agent, or an agonistic anti-PD-L1 agent.

[0246] In some embodiments, the immunotherapeutic agent is an agonistic anti-CTLA-4 antibody or antigen-binding fragment thereof, an agonistic anti-PD-1 antibody or antigen-binding fragment thereof, an agonistic anti-PD-L1 antibody or antigen-binding fragment thereof, an agonistic anti-PD-L2 antibody or antigen-binding fragment thereof, an agonistic anti-CD27 antibody or antigen-binding fragment thereof, an agonistic anti-CD30 antibody or antigen-binding fragment thereof, an agonistic anti-CD40 antibody or antigen-binding fragment thereof, an agonistic anti-4-1BB antibody or antigen-binding fragment thereof, an agonistic anti-GITR antibody or antigen-binding fragment thereof, an agonistic anti-OX40 antibody or antigen-binding fragment thereof, an agonistic anti-TRAILR1 antibody or antigen-binding fragment thereof, an agonistic anti-TRAILR2 antibody or antigen-binding fragment thereof, an agonistic anti-TWEA antibody or antigen-binding fragment thereof, K antibody or antigen-binding fragment thereof, agonistic anti-TWEAKR antibody or antigen-binding fragment thereof, agonistic anti-cell surface lymphocyte protein antibody or antigen-binding fragment thereof, agonistic anti-BRAF antibody or antigen-binding fragment thereof, agonistic anti-MEK antibody or antigen-binding fragment thereof, agonistic anti-CD33 antibody or antigen-binding fragment thereof, agonistic anti-CD20 antibody or antigen-binding fragment thereof, agonistic anti-HLA-DR antibody or antigen-binding fragment thereof, agonistic anti-HLA class I antibody or antigen-binding fragment thereof, agonistic anti-CD52 antibody or antigen-binding fragment thereof, agonistic anti-A33 antibody or antigen-binding fragment thereof, agonistic anti-GD3 antibody or antigen-binding fragment thereof, agonistic anti-PSMA antibody or antigen-binding fragment thereof, agonistic anti-Ceacan 1 antibody or antigen-binding fragment thereof, agonistic anti-Galedin 9 antibody or antigen-binding fragment thereof, agonistic anti-HVEM antibody or antigen-binding fragment thereof, agonistic anti-VISTA antibody or antigen-binding fragment thereof, agonistic anti-B7 H4 antibody or antigen-binding fragment thereof, agonistic anti-HHLA2 antibody or antigen-binding fragment thereof, agonistic anti-CD155 antibody or antigen-binding fragment thereof, agonistic anti-CD80 antibody or antigen-binding fragment thereof, agonistic anti-BTLA antibody or antigen-binding fragment thereof, agonistic anti-CD160 antibody or antigen-binding fragment thereof, agonistic anti-CD28 antibody or antigen-binding fragment thereof, agonistic anti-CD226 antibody or antigen-binding fragment thereof, agonistic anti-CEACAM1 antibody or antigen-binding fragment thereof or antigen-binding fragment thereof, agonistic anti-TIM3 antibody or antigen-binding fragment thereof, agonistic anti-TIGIT antibody or antigen-binding fragment thereof, agonistic anti-CD96 antibody or antigen-binding fragment thereof, agonistic anti-CD70 antibody or antigen-binding fragment thereof, agonistic anti-CD27 antibody or antigen-binding fragment thereof, agonistic anti-LIGHT antibody or antigen-binding fragment thereof, agonistic anti-CD137 antibody or antigen-binding fragment thereof, agonistic anti-DR4 antibody or antigen-binding fragment thereof, agonistic anti-CR5 antibody or antigen-binding fragment thereof, agonistic anti-TNFRS antibody or antigen-binding fragment thereof, agonistic anti-TNFR1 antibody or antigen-binding fragment thereof, agonistic anti-FAS antibody or antigen-binding fragment thereof, agonistic anti-CD95 antibody or antigen-binding fragment thereof, agonistic The antibody is selected from the group consisting of an agonistic anti-TRAIL antibody or antigen-binding fragment thereof, an agonistic anti-DR6 antibody or antigen-binding fragment thereof, an agonistic anti-EDAR antibody or antigen-binding fragment thereof, an agonistic anti-NGFR antibody or antigen-binding fragment thereof, an agonistic anti-OPG antibody or antigen-binding fragment thereof, an agonistic anti-RANKL antibody or antigen-binding fragment thereof, an agonistic anti-LTβ receptor antibody or antigen-binding fragment thereof, an agonistic anti-BCMA antibody or antigen-binding fragment thereof, an agonistic anti-TACI antibody or antigen-binding fragment thereof, an agonistic anti-BAFFR antibody or antigen-binding fragment thereof, an agonistic anti-EDAR2 antibody or antigen-binding fragment thereof, an agonistic anti-TROY antibody or antigen-binding fragment thereof, and an agonistic anti-RELT antibody or antigen-binding fragment thereof. For example, the immunotherapeutic agent can be an agonistic anti-CTLA-4 antibody or antigen-binding fragment thereof, an agonistic anti-PD-1 antibody or antigen-binding fragment thereof, or an agonistic anti-PD-L1 antibody or antigen-binding fragment thereof.

[0247] In some embodiments, the immunotherapeutic agent is an anti-cell surface lymphocyte protein antibody or antigen-binding fragment thereof, such as, for example, CD1, CD2, CD3, CD4, CD5, CD6, CD7, CD8, CD9, CD10, CD11, CD12, CD13, CD14, CD15, CD16, CD17, CD18, CD19, CD20, CD21, CD22, CD23, CD24, CD25, CD26, CD27, CD28, CD29, CD30, CD31, CD32, CD33, CD34, CD35, CD36, CD37, CD38, CD39, CD40, CD41, CD 42, CD43, CD44, CD45, CD46, CD47, CD48, CD49, CD50, CD51, CD52, CD53, CD54, CD55, CD56, CD57, CD58, CD59, CD60, CD61, CD62, CD63, CD64, CD65, CD66, CD 67, CD68, CD69, CD70, CD71, CD72, CD73, CD74, CD75, CD76, CD77, CD78, CD79, CD80, CD81, CD82, CD83, CD84, CD85, CD86, CD87, CD88, CD89, CD90, CD91, CD 92, CD93, CD94, CD95, CD96, CD97, CD98, CD99, CD100, CD101, CD102, CD103, CD104, CD105, CD106, CD107, CD108, CD109, CD110, CD111, CD112, CD113, CD1 14, CD115, CD116, CD117, CD118, CD119, CD120, CD121, CD122, CD123, CD124, CD125, CD126, CD127, CD128, CD129, CD130, CD131, CD132, CD133, CD134, CD 135, CD136, CD137, CD138, CD139, CD140, CD141, CD142, CD143, CD144, CD145, CD146, CD147, CD148, CD149, CD150, CD151, CD152, CD153, CD154, CD155, C D156, CD157, CD158, CD159, CD160, CD161, CD162, CD163, CD164, CD165, CD166, CD167, CD168, CD169, CD170, CD171, CD172, CD173, CD174, CD175, CD176,CD177, CD178, CD179, CD180, CD181, CD182, CD183, CD184, CD185, CD186, CD187, CD188, CD189, CD190, CD191, CD192, CD193, CD194, CD195 , CD196, CD197, CD198, CD199, CD200, CD201, CD202, CD203, CD204, CD205, CD206, CD207, CD208, CD209, CD210, CD211, CD212, CD213, CD21 4, CD215, CD216, CD217, CD218, CD219, CD220, CD221, CD222, CD223, CD224, CD225, CD226, CD227, CD228, CD229, CD230, CD231, CD232, CD2 33, CD234, CD235, CD236, CD237, CD238, CD239, CD240, CD241, CD242, CD243, CD244, CD245, CD246, CD247, CD248, CD249, CD250, CD251, CD2 52, CD253, CD254, CD255, CD256, CD257, CD258, CD259, CD260, CD261, CD262, CD263, CD264, CD265, CD266, CD267, CD268, CD269, CD270, CD 271, CD272, CD273, CD274, CD275, CD276, CD277, CD278, CD279, CD280, CD281, CD282, CD283, CD284, CD285, CD286, CD287, CD288, CD289, CD 290, CD291, CD292, CD293, CD294, CD295, CD296, CD297, CD298, CD299, CD300, CD301, CD302, CD303, CD304, CD305, CD306, CD307, CD308, CD309, CD310, CD311, CD312, CD313, CD314, CD315, CD316, CD317, CD318, CD319, and / or CD320, or an antigen-binding fragment thereof that binds to and promotes activity of one or more of CD310, CD311, CD312, CD313, CD314, CD315, CD316, CD317, CD318, CD319, and / or CD320.

[0248] In some embodiments, the immunotherapeutic agent is an agent (e.g., a polypeptide, an antibody, an antigen-binding fragment thereof, a single-chain polypeptide, or a construct thereof) that binds to a chemokine or lymphokine. For example, the immunotherapeutic agent is an agent (e.g., a polypeptide, an antibody, an antigen-binding fragment thereof, a single-chain polypeptide, or a construct thereof) that binds to and activates one or more or all of CXCL1, CXCL2, CXCL3, CXCL8, CCL2, and CCL5. In some embodiments, the immunotherapeutic agent is an agent (e.g., a polypeptide, an antibody, an antigen-binding fragment thereof, a single-chain polypeptide, or a construct thereof) that binds to and activates one or more or all of CCL3, CCL4, CCL8, and CCL22.

[0249] Labeled TNFR2 Polypeptide In some embodiments, the agonistic TNFR2 single chain polypeptide, antibody, antigen-binding fragment thereof, or construct described herein is conjugated to another molecule (e.g., epitope tag) for purification or detection purposes. Examples of such molecules useful for protein purification include molecules that present a structural epitope that can be recognized by a second molecule. This is a common strategy used in protein purification by affinity chromatography, where a molecule is immobilized on a solid support and exposed to a heterogeneous mixture that contains a target protein conjugated to a molecule that can bind to the immobilized compound. Examples of epitope tag molecules that can be conjugated to agonistic TNFR2 polypeptides for molecular recognition purposes include, but are not limited to, maltose binding protein, glutathione-S-transferase, polyhistidine tag, FLAG tag, myc tag, human influenza hemagglutinin (HA) tag, biotin, and streptavidin. Conjugates containing epitopes presented by these molecules can be recognized by complementary molecules such as maltose, glutathione, nickel-containing complexes, anti-FLAG antibodies, anti-myc antibodies, anti-HA antibodies, streptavidin, or biotin. For example, the agonistic TNFR2 antibody or fragment thereof conjugated to an epitope tag described herein can be purified from a complex mixture of other proteins and biomolecules (e.g., DNA, RNA, carbohydrates, phospholipids, etc.) by treating the mixture with a solid-phase resin containing a complementary molecule that can selectively recognize and bind to the epitope tag of the agonistic TNFR2 antibody or fragment thereof. Examples of solid-phase resins include agarose beads, which are suitable for purification in aqueous solution.

[0250] The agonistic TNFR2 polypeptides described herein can also be covalently linked to fluorescent molecules to detect the antibody or its antigen-binding fragments, for example, by fluorometry and / or direct visualization using a fluorescent microscope.Exemplary fluorescent molecules that can be conjugated to the antibodies described herein include green fluorescent protein, cyan fluorescent protein, yellow fluorescent protein, red fluorescent protein, phycoerythrin, allophycocyanin, Hoechst, 4',6-diamidino-2-phenylindole (DAPI), propidium iodide, fluorescein, coumarin, rhodamine, tetramethylrhodomine, and cyanine.Additional examples of fluorescent molecules suitable for conjugation to the antibodies described herein are known in the art and are described in detail in, for example, U.S. Patent Nos. 7,417,131 and 7,413,874, each of which is incorporated herein by reference.

[0251] Agonistic TNFR2 polypeptides that contain fluorescent molecules are particularly useful for monitoring the cell surface localization properties of antibodies and fragments thereof described herein.For example, cultured mammalian cells (e.g., Treg cells) can be exposed to agonistic TNFR2 polypeptides described herein that are covalently linked to fluorescent molecules, and then these cells can be analyzed using conventional fluorescence microscopy methods known in the art.Confocal fluorescence microscopy is a particularly powerful method for determining the cell surface localization of agonistic TNFR2 polypeptides.This is because individual planes of cells can be analyzed to distinguish antibodies or fragments thereof that are internalized in the cell, for example, by receptor-mediated endocytosis, from antibodies or fragments thereof that are bound to the outer surface of the cell membrane. Additionally, cells can be treated with an agonistic TNFR2 antibody conjugated to a fluorescent molecule that emits visible light at a particular wavelength (e.g., fluorescein, which fluoresces at about 535 nm) and an additional fluorescent molecule that fluoresces at a different wavelength known to localize to a particular site on the Treg cell surface (e.g., a molecule that localizes to CD25 and fluoresces at about 599 nm). The resulting emission pattern can be visualized by confocal fluorescence microscopy, and images from these two wavelengths can be merged to reveal information about the location of the agonistic TNFR2 antibody or its antigen-binding fragment on the Treg cell surface relative to other receptors.

[0252] Bioluminescent proteins can also be incorporated into the fusion protein for the purpose of detecting and visualizing agonistic TNFR2 polypeptides, such as single chain polypeptides, antibodies, or fragments thereof. Bioluminescent proteins, such as luciferase and aequorin, emit light as part of a chemical reaction with a substrate (e.g., luciferin or coelenterazine). Examples of bioluminescent proteins suitable for use as diagnostic sequences and their methods of use are described, for example, in U.S. Patent Nos. 5,292,658, 5,670,356, 6,171,809, and 7,183,092, each of which is incorporated herein by reference. Agonistic TNFR2 antibodies or fragments thereof labeled with bioluminescent proteins are useful tools for detecting the antibodies described herein following in vitro assays. For example, the presence of an agonistic TNFR2 antibody conjugated to a bioluminescent protein can be detected among a complex mixture of additional proteins by separating the components of the mixture using gel electrophoresis methods known in the art (e.g., native gel analysis) and then transferring the separated proteins to a membrane for Western blotting. Detection of an agonistic TNFR2 polypeptide in a mixture of other proteins can be achieved by treating the membrane with an appropriate luciferase substrate, followed by visualization of the mixture of proteins on a film using established protocols.

[0253] The polypeptides (e.g., single chain polypeptides, antibodies, antigen-binding fragments thereof, and constructs thereof) described herein can also be conjugated to a molecule containing a radioactive nuclide, and the antibodies or fragments thereof described herein can be detected by analyzing the radioactive emission pattern of the nuclide. Alternatively, the agonistic TNFR2 antibody or fragment thereof can be directly modified by incorporating a radioactive nuclide into the antibody during preparation of the protein. Methionine ( 35 S), nitrogen ( 15 N), or carbon ( 13Radioactive isotopes of C) can be incorporated into the antibodies or fragments thereof described herein, for example, by culturing bacteria in a medium supplemented with nutrients containing these isotopes. Optionally, tyrosine derivatives containing radioactive halogens can be incorporated into agonistic TNFR2 polypeptides, for example, by culturing bacterial cells in a medium supplemented with radiolabeled tyrosine. Tyrosine functionalized with radioactive halogens at the phenolic C2 position has been shown to be rapidly incorporated into growing polypeptide chains in vivo using endogenous translation enzymes (U.S. Patent No. 4,925,651, incorporated herein by reference). Halogens include fluorine, chlorine, bromine, iodine, and astatine. Additionally, agonistic TNFR2 polypeptides can be modified by functionalizing the polypeptides described herein with radioactive isotopes after isolation and purification from cell culture. Halogens represent a class of isotopes that can be readily incorporated into purified proteins by aromatic substitution at tyrosine or tryptophan, for example, by reaction of one or more of these residues with an electrophilic halogen species. Examples of radioactive halogen isotopes include: 18 F, 75 Br, 77 Br, 122 I, 123 I, 124 I, 125 I, 129 I, 131 I, or 211 Contains At.

[0254] Another alternative strategy for the incorporation of radioisotopes is to covalently bind a chelating group to an agonistic TNFR2 polypeptide, such as a single chain polypeptide, an antibody, a fragment thereof, or a construct. The chelating group can be covalently bound to an agonistic TNFR2 antibody or a fragment thereof by binding to a reactive functional group such as a thiol, an amino group, an alcohol, or a carboxylic acid. The chelating group can then be modified to: 125 I, 67 Ga, 111 In, 99 Tc, 169 Yb, 186Re, 123 I, 124 I, 125 I, 131 I, 99m Tc, 111 In, 64 Cu, 67 Cu, 186 Re, 188 Re, 177 Lu, 90 Y, 77 As, 72 As, 86 Y, 89 Zr, 211 At, 212 Bi, 213 Bi, or 225 It can include any of a variety of metallic radioisotopes, including, but not limited to, radionuclides such as Ac.

[0255] In some embodiments, the polypeptides described herein (e.g., single chain polypeptides, antibodies, fragments thereof, or constructs thereof) are administered by injecting Gd 3+ , Fe 3+ , Mn 3+ , or Cr 2+It may be desirable to covalently bind a chelating group capable of binding metal ions from heavy elements such as Zn, Fe, Fe(II), Fe(III), Co(II), Ni(II), Co(II), Ni(II), Co(II), Pr(III), Neodymium(III), Samarium(III), Gadolinium(III), Terbium(III), Dysprosium(III), Holmium(III), Erbium(III), and Ytterbium(III) to a mammalian subject (e.g., a human patient) to monitor the distribution of the antibody after administration. This can be accomplished by administering the antibody to a patient by any of the routes of administration described herein, such as intravenously, and then analyzing the location of the administered antibody by recording an MRI of the patient according to established protocols.

[0256] Agonistic TNFR2 polypeptides (e.g., single-chain peptides, antibodies, antigen-binding fragments thereof, and constructs thereof) can be additionally conjugated to other molecules to improve the solubility and stability of the protein in aqueous solution. Examples of such molecules include, among others, PEG, PSA, bovine serum albumin (BSA), and human serum albumin (HSA). For example, agonistic TNFR2 polypeptides can be conjugated to carbohydrate moieties to avoid detection of the antibody or fragments thereof by the immune system of the patient undergoing treatment. This process of hypergly...

Claims

1. 1. An antibody or antigen-binding fragment thereof that specifically binds to human tumor necrosis factor receptor 2 (TNFR2) at an epitope defined by one or more amino acids in cysteine-rich domain (CRD) 1 (CRD1), CRD2, and / or CRD3, and does not specifically bind to human TNFR2 at an epitope defined by one or more amino acids in CRD4, The antibody or antigen-binding fragment thereof has the following CDRs: (i) a complementarity determining region heavy chain (CDR-H) 1 having the amino acid sequence GYTFTDYNI (SEQ ID NO: 3) or GYTFTDYNL (SEQ ID NO: 4); (ii) CDR-H2 having the amino acid sequence INPNYDST (SEQ ID NO: 10); (iii) CDR-H3 having the amino acid sequence CARGNSWYFDV (SEQ ID NO:11); (iv) a complementarity determining region light chain (CDR-L) 1 having the amino acid sequence SSVRY (SEQ ID NO: 12); (v) a CDR-L2 having the amino acid sequence LTS; and (vi) CDR-L3 having the amino acid sequence CQQWSSNPLT (SEQ ID NO: 13) Including, and, The antibody or antigen-binding fragment thereof (a) a human IgG1 or IgG2 heavy chain constant 1 (CH1) domain comprising a deletion or substitution at cysteine ​​residue 127; (b) comprising antigen-binding sites that are separated from each other by a distance of less than about 133 Å; and / or (c) TJDJSJJJX 1 YX 2 X 3 LJX 4 LJS (SEQ ID NO:5), or an amino acid sequence having 10 or more of the residues of SEQ ID NO:5, wherein each J is independently a naturally occurring amino acid; 1 is independently A, V, or F; each X 2 is independently M or I; each X 3 is independently E or Q; and each X 4 is independently S or R; The antibody or antigen-binding fragment thereof.

2. (a) the antibody or antigen-binding fragment thereof comprises a human IgG1 or IgG2 CH1 domain comprising a deletion or substitution at cysteine ​​residue 127; (b) the CH1 domain comprises a C127S mutation; (c) the IgG1 CH1 domain has an amino acid sequence at least 85% identical to the amino acid sequence of SEQ ID NO:6 or the amino acid sequence of SEQ ID NO:7; (d) the IgG2 CH1 Domain has an amino acid sequence at least 85% identical to the amino acid sequence of SEQ ID NO:8 or the amino acid sequence of SEQ ID NO:9; (e) the antibody or antigen-binding fragment thereof has an IgG3 or IgG4 isotype; (f) the antibody or antigen-binding fragment thereof comprises at least two antigen-binding sites separated by a distance less than about 133 Å; (g) the antigen binding sites are separated by a distance of about 90 Å to about 132 Å; (h) the antibody or antigen-binding fragment thereof comprises a non-native constant region; (i) the antibody or antigen-binding fragment thereof is 1 YX 2 X 3 LJX 4 LJS (SEQ ID NO:5), or an amino acid sequence having 10 or more of the residues of SEQ ID NO:5, wherein each J is independently a naturally occurring amino acid; 1 is independently A, V, or F; each X 2 is independently M or I; each X 3 is independently E or Q; and each X 4 is independently S or R; (j) the antibody or antigen-binding fragment thereof (i) TVDKSSSTAYMELRSLTS (SEQ ID NO: 16), or an amino acid sequence having up to two conservative amino acid substitutions thereto; (ii) TADTSSNTAYIQLSSLTS (SEQ ID NO: 17), or an amino acid sequence having up to two conservative amino acid substitutions thereto; (iii) TADTSDTAYMELSSLRS (SEQ ID NO: 18), or an amino acid sequence having up to two conservative amino acid substitutions thereto; (iv) TRDTSISTAYMELSRLTS (SEQ ID NO: 19), or an amino acid sequence having up to two conservative amino acid substitutions thereto; (v) TFYMELSSLRS (SEQ ID NO: 20), or an amino acid sequence having up to two conservative amino acid substitutions thereto; (vi) TRDTSISTAYMELNRLTS (SEQ ID NO: 21), or an amino acid sequence having up to two conservative amino acid substitutions thereto; (vii) TRDTSTNTVYMELTSLRS (SEQ ID NO: 22), or an amino acid sequence having up to two conservative amino acid substitutions thereto; and (ix) TADTSTDRAYMELSSLRS (SEQ ID NO: 23), or an amino acid sequence having up to two conservative amino acid substitutions thereto; a framework region having an amino acid sequence selected from the group consisting of: (k) the framework regions are adjacent to CDR-H2 in the antibody or antigen-binding fragment thereof; (l) the framework region is adjacent to CDR-H3 in the antibody or antigen-binding fragment thereof; and / or (m) the framework regions are located between CDR-H2 and CDR-H3; The antibody or antigen-binding fragment thereof according to claim 1.

3. (a) the antibody or antigen-binding fragment thereof has an IgG3 isotype; (b) the antibody or antigen-binding fragment thereof specifically binds to human TNFR2 at an epitope defined by one or more amino acids within CRD1; (c) the antibody or antigen-binding fragment thereof specifically binds to human TNFR2 at an epitope defined by one or more amino acids within CRD2; (d) the antibody or antigen-binding fragment thereof specifically binds to human TNFR2 at an epitope defined by one or more amino acids within CRD3; (e) the antibody or antigen-binding fragment thereof comprises a heavy chain variable domain (V) having an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO:

14. H ) (f) the antibody or antigen-binding fragment thereof comprises a heavy chain variable domain (V) having an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO:

15. H ) and / or (g) the non-native constant region is a human constant region; The antibody or antigen-binding fragment thereof according to claim 1 or 2.

4. (a) the epitope is defined by one or more of amino acid residues 56-60 (KCSPG) of SEQ ID NO:1; (b) the antibody or antigen-binding fragment thereof comprises a heavy chain variable domain (V H ) and / or (c) the antibody or antigen-binding fragment thereof comprises a heavy chain variable domain (V H ) including; The antibody or antigen-binding fragment thereof according to claim 3.

5. the antibody or antigen-binding fragment thereof (a) amino acids 56-60 of SEQ ID NO:1 (KCSPG); (b) amino acids 101-107 of SEQ ID NO:1 (CSSDQVET); (c) amino acids 115-142 of SEQ ID NO:1 (NRICTCRPGWYCALSKQEGCRLCAPLRK); (d) amino acids 26-45 of SEQ ID NO:1 (QTAQMCCSKCSPGQHAKVFC); (e) amino acids 90-109 of SEQ ID NO:1 (REQNRICTCRPGWYCALSKQ); (f) amino acids 98-117 of SEQ ID NO:1 (CRPGWYCALSKQEGCRLCAP); (g) amino acids 106-125 of SEQ ID NO:1 (LSKQEGCRLCAPLRKCRPGF); and / or (h) amino acids 108-127 of SEQ ID NO:1 (KQEGCRLCAPLRKCRPGFGV); The antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, which specifically binds to TNFR2 at an epitope within said TNFR2.

6. the antibody or antigen-binding fragment thereof (a) activates TNFR2 signaling; (b) a K of no more than about 10 nM D binds to TNFR2 at (c) binds to TNFR2 and has a specific binding affinity of at least about 10 4 M -1 s -1 No. on to form an antibody-antigen complex; (d) binding to TNFR2 to form an antibody-antigen complex, and said complex is about 10 -3 s -1 Not exceeding k off Dissociates at; (e) promoting the expansion of T regulatory (Treg) cells; (f) directly killing or promoting the death of CD8+ T cells; (g) promoting an increase in the level of one or more mRNA molecules encoding a protein selected from the group consisting of cIAP2, TRAF2, Etk, VEGFR2, PI3K, Akt, proteins involved in the angiogenesis pathway, IKK complex, RIP, NIK, MAP3K, proteins involved in the NFkB pathway, NIK, JNK, AP-1, MEK (e.g., MEK1, MEK7), MKK3, NEMO, IL2R, Foxp3, IL2, TNF, lymphotoxin, lymphotoxin alpha, and lymphotoxin beta; (h) promoting an increase in the level of one or more proteins selected from the group consisting of cIAP2, TRAF2, Etk, VEGFR2, PI3K, Akt, proteins involved in the angiogenesis pathway, IKK complex, RIP, NIK, MAP3K, proteins involved in the NFkB pathway, NIK, JNK, AP-1, MEK (e.g., MEK1, MEK7), MKK3, NEMO, IL2R, Foxp3, IL2, TNF, lymphotoxin, lymphotoxin alpha, and lymphotoxin beta; and / or (i) promoting an increase in the level of itaconic acid upon administration to a subject; An antibody or antigen-binding fragment thereof according to any one of claims 1 to 5.

7. (a) the antibody or antigen-binding fragment thereof has a K of no more than about 1 nM D binds to TNFR2 at (b) the antibody or antigen-binding fragment thereof binds to TNFR2 and has a cytotoxicity of at least about 10 5 M -1 s -1 No. on to form an antibody-antigen complex; (c) the antibody or antigen-binding fragment thereof is about 10 -4 s -1 Not exceeding k off dissociates from TNFR2 with; (d) the Treg cells are Hi and CD45RA Low and / or (e) the subject is a human subject; The antibody or antigen-binding fragment thereof according to claim 6.

8. the antibody or antigen-binding fragment thereof (a) a monoclonal antibody or antigen-binding fragment thereof, a polyclonal antibody or antigen-binding fragment thereof, a human antibody or antigen-binding fragment thereof, a humanized antibody or antigen-binding fragment thereof, a primatized antibody or antigen-binding fragment thereof, a bispecific antibody or antigen-binding fragment thereof, a multispecific antibody or antigen-binding fragment thereof, a dual variable immunoglobulin domain, a monovalent antibody or antigen-binding fragment thereof, a chimeric antibody or antigen-binding fragment thereof, a single chain Fv molecule (scFv), a diabody, a triabody, an antibody-like protein scaffold, an Fv fragment, a Fab fragment, an F(ab') 2 molecule, and tandem scFv (taFv); or (b) is a single chain polypeptide; An antibody or antigen-binding fragment thereof according to any one of claims 1 to 7.

9. The antibody or antigen-binding fragment thereof of claim 8 , wherein the antibody or antigen-binding fragment thereof is a humanized antibody or antigen-binding fragment thereof, or a chimeric antibody or antigen-binding fragment thereof.

10. A construct comprising a first polypeptide domain and a second polypeptide domain, wherein the first polypeptide domain and the second polypeptide domain each independently comprise a single chain polypeptide according to claim 8.

11. (a) the construct comprises a human Fc domain; (b) the construct lacks a mouse Fc domain; and / or (c) the first polypeptide domain and the second polypeptide domain are linked by a covalent linker; The construct of claim 10.

12. The construct of claim 11 , wherein the covalent linker comprises an amide bond or a disulfide bond.

13. A polynucleotide encoding the antibody or antigen-binding fragment thereof described in any one of claims 1 to 9.

14. A polynucleotide encoding the construct described in any one of claims 10 to 12.

15. A vector comprising the polynucleotide described in claim 13 or 14.

16. The vector of claim 15 , wherein the vector is an expression vector or a viral vector.

17. The vector of claim 16 , wherein the expression vector is a eukaryotic expression vector.

18. 17. The vector of claim 16, wherein the viral vector is selected from the group consisting of adenovirus (Ad), retrovirus, poxvirus, adeno-associated virus, baculovirus, herpes simplex virus, and vaccinia virus.

19. 19. The vector of claim 18, wherein the adenovirus is a serotype 2, 5, 11, 12, 24, 26, 34, 35, 40, 48, 49, 50, 52, or Pan 9 adenovirus, or a human, chimpanzee, or rhesus adenovirus.

20. The vector of claim 18, wherein the retrovirus is a gamma-retrovirus or a lentivirus.

21. 19. The vector of claim 18, wherein the vaccinia virus is Modified Vaccinia Ankara (MVA).

22. An isolated host cell comprising a vector described in any one of claims 15 to 21.

23. The host cell of claim 22 , wherein the host cell is a prokaryotic cell.

24. The host cell of claim 22 , wherein the host cell is a eukaryotic cell.

25. The host cell of claim 24 , wherein the eukaryotic cell is a mammalian cell.

26. 26. The host cell of claim 25, wherein the mammalian cell is a CHO cell or a HEK cell.

27. A pharmaceutical composition comprising an antibody or antigen-binding fragment thereof described in any one of claims 1 to 9, wherein at least 50% of the antibody or antigen-binding fragment thereof in the pharmaceutical composition is present as a single disulfide bond isoform.

28. (a) at least 75% of the antibody or antigen-binding fragment thereof in the pharmaceutical composition is present in a single disulfide-bonded isoform; (b) the antibody or antigen-binding fragment thereof gives rise to a single detectable band upon gel electrophoretic analysis performed under non-reducing conditions; (c) the single disulfide bond isoform is IgG2-B; (d) the antibody or antigen-binding fragment thereof is present in the pharmaceutical composition in an amount from about 0.001 mg / ml to about 100 mg / ml; and / or (e) the pharmaceutical composition further comprises an additional therapeutic agent; 28. The pharmaceutical composition of claim 27.

29. A pharmaceutical composition comprising an antibody or antigen-binding fragment thereof described in any one of claims 1 to 9, a construct described in any one of claims 10 to 12, a polynucleotide described in claim 13 or 14, a vector described in any one of claims 15 to 21, or a host cell described in any one of claims 22 and 24 to 26, and a pharma- ceutically acceptable carrier or excipient.

30. 30. The pharmaceutical composition of claim 28, wherein the additional therapeutic agent is an immunotherapeutic agent.

31. The immunotherapeutic agent is (a) agonistic anti-CTLA-4 agents, agonistic anti-PD-1 agents, agonistic anti-PD-L1 agents, agonistic anti-PD-L2 agents, agonistic anti-CD27 agents, agonistic anti-CD30 agents, agonistic anti-CD40 agents, agonistic anti-4-1BB agents, agonistic anti-GITR agents, agonistic anti-OX40 agents, agonistic anti-TRAILR1 agents, agonistic anti-TRAILR2 agents, agonistic anti-TWEAK agents , agonistic anti-TWEAKR agents, agonistic anti-cell surface lymphocyte protein agents, agonistic anti-BRAF agents, agonistic anti-MEK agents, agonistic anti-CD33 agents, agonistic anti-CD20 agents, agonistic anti-HLA-DR agents, agonistic anti-HLA class I agents, agonistic anti-CD52 agents, agonistic anti-A33 agents, agonistic anti-GD3 agents, agonistic anti-PSMA agents, agonistic anti-Ceacan 1 agents, agonistic anti-Galedin 9 agents, agonistic anti-HVEM agents, agonistic anti-VISTA agents, agonistic anti-B7 agents H4 agents, agonistic anti-HHLA2 agents, agonistic anti-CD155 agents, agonistic anti-CD80 agents, agonistic anti-BTLA agents, agonistic anti-CD160 agents, agonistic anti-CD28 agents, agonistic anti-CD226 agents, agonistic anti-CEACAM1 agents, agonistic anti-TIM3 agents, agonistic anti-TIGIT agents, agonistic anti-CD96 agents, agonistic anti-CD70 agents, agonistic anti-CD27 agents, agonistic anti-LIGHT agents, agonistic anti-CD137 agents, agonistic anti-DR4 agents, agonistic anti-CR5 agents, agonists or an agonistic anti-TNFRS agent, an agonistic anti-TNFR1 agent, an agonistic anti-FAS agent, an agonistic anti-CD95 agent, an agonistic anti-TRAIL agent, an agonistic anti-DR6 agent, an agonistic anti-EDAR agent, an agonistic anti-NGFR agent, an agonistic anti-OPG agent, an agonistic anti-RANKL agent, an agonistic anti-LTβ receptor agent, an agonistic anti-BCMA agent, an agonistic anti-TACI agent, an agonistic anti-BAFFR agent, an agonistic anti-EDAR2 agent, an agonistic anti-TROY agent, and an agonistic anti-RELT agent; or (b) an agonistic anti-CTLA-4 antibody or antigen-binding fragment thereof, an agonistic anti-PD-1 antibody or antigen-binding fragment thereof, an agonistic anti-PD-L1 antibody or antigen-binding fragment thereof, an agonistic anti-PD-L2 antibody or antigen-binding fragment thereof, an agonistic anti-CD27 antibody or antigen-binding fragment thereof, an agonistic anti-CD30 antibody or antigen-binding fragment thereof, an agonistic anti-CD40 antibody or antigen-binding fragment thereof, an agonistic anti-4-1BB antibody or antigen-binding fragment thereof, an agonistic anti-GITR antibody or antigen-binding fragment thereof, an agonistic anti-OX40 antibody or antigen-binding fragment thereof, an agonistic anti-TRAILR1 antibody or antigen-binding fragment thereof, an agonistic anti-TRAILR2 antibody or antigen-binding fragment thereof, an agonistic anti-TWEAK antibody or antigen-binding fragment, agonistic anti-TWEAKR antibody or antigen-binding fragment thereof, agonistic anti-cell surface lymphocyte protein antibody or antigen-binding fragment thereof, agonistic anti-BRAF antibody or antigen-binding fragment thereof, agonistic anti-MEK antibody or antigen-binding fragment thereof, agonistic anti-CD33 antibody or antigen-binding fragment thereof, agonistic anti-CD20 antibody or antigen-binding fragment thereof, agonistic anti-HLA-DR antibody or antigen-binding fragment thereof, agonistic anti-HLA class I antibody or antigen-binding fragment thereof, agonistic anti-CD52 antibody or antigen-binding fragment thereof, agonistic anti-A33 antibody or antigen-binding fragment thereof, agonistic anti-GD3 antibody or antigen-binding fragment thereof, agonistic anti-PSMA antibody or antigen-binding fragment thereof, agonistic anti-Ceacan 1 antibody or antigen-binding fragment thereof, agonistic anti-Galedin 9 antibody or antigen-binding fragment thereof, agonistic anti-HVEM antibody or antigen-binding fragment thereof, agonistic anti-VISTA antibody or antigen-binding fragment thereof, agonistic anti-B7H4 antibody or antigen-binding fragment thereof, agonistic anti-HHLA2 antibody or antigen-binding fragment thereof, agonistic anti-CD155 antibody or antigen-binding fragment thereof, agonistic anti-CD80 antibody or antigen-binding fragment thereof, agonistic anti-BTLA antibody or antigen-binding fragment thereof, agonistic anti-CD160 antibody or antigen-binding fragment thereof, agonistic anti-CD28 antibody or antigen-binding fragment thereof, agonistic anti-CD226 antibody or antigen-binding fragment thereof, agonistic anti-CEACAM1 antibody or antigen-binding fragment thereof, agonistic anti-TIM3 antibody or antigen-binding fragment thereof, agonistic anti-TIGIT antibody or antigen-binding fragment thereof, agonistic anti-CD96 antibody or antigen-binding fragment thereof, agonistic anti-CD70 antibody or antigen-binding fragment thereof, agonistic anti-CD27 antibody or antigen-binding fragment thereof, agonistic anti-LIGHT antibody or antigen-binding fragment thereof, agonistic anti-CD137 antibody or antigen-binding fragment thereof, agonistic anti-DR4 antibody or antigen-binding fragment thereof, agonistic anti-CR5 antibody or antigen-binding fragment thereof fragment thereof, an agonistic anti-TNFRS antibody or antigen-binding fragment thereof, an agonistic anti-TNFR1 antibody or antigen-binding fragment thereof, an agonistic anti-FAS antibody or antigen-binding fragment thereof, an agonistic anti-CD95 antibody or antigen-binding fragment thereof, an agonistic anti-TRAIL antibody or antigen-binding fragment thereof, an agonistic anti-DR6 antibody or antigen-binding fragment thereof, an agonistic anti-EDAR antibody or antigen-binding fragment thereof, an agonistic anti-NGFR antibody or antigen-binding fragment thereof, an agonistic anti-OPG antibody or antigen-binding fragment thereof an agonistic anti-RANKL antibody or antigen-binding fragment thereof, an agonistic anti-LTβ receptor antibody or antigen-binding fragment thereof, an agonistic anti-BCMA antibody or antigen-binding fragment thereof, an agonistic anti-TACI antibody or antigen-binding fragment thereof, an agonistic anti-BAFFR antibody or antigen-binding fragment thereof, an agonistic anti-EDAR2 antibody or antigen-binding fragment thereof, an agonistic anti-TROY antibody or antigen-binding fragment thereof, and an agonistic anti-RELT antibody or antigen-binding fragment thereof; 31. The pharmaceutical composition of claim 30.

32. A method for producing an antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, the method comprising expressing a polynucleotide encoding the antibody or antigen-binding fragment thereof in a host cell, and recovering the antibody or antigen-binding fragment thereof from host cell culture medium.

33. A method for producing a construct according to any one of claims 10 to 12, comprising expressing a polynucleotide encoding the construct in a host cell and recovering the construct from host cell culture medium.

34. An antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, a construct according to any one of claims 10 to 12, a polynucleotide according to claim 13 or 14, a vector according to any one of claims 15 to 21, a host cell according to any one of claims 22 and 24 to 26, or a pharmaceutical composition according to any one of claims 27 to 29 for use in inhibiting an immune response mediated by B cells or CD8+ T cells in a human subject.

35. An antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, a construct according to any one of claims 10 to 12, a polynucleotide according to claim 13 or 14, a vector according to any one of claims 15 to 21, a host cell according to any one of claims 22 and 24 to 26, or a pharmaceutical composition according to any one of claims 27 to 29 for use in the treatment of an immunological disease in a human subject.

36. The antibody or antigen-binding fragment thereof, construct, polynucleotide, vector, host cell or pharmaceutical composition of claim 35, wherein the subject is in need of tissue or organ regeneration.

37. 37. The antibody or antigen-binding fragment thereof, construct, polynucleotide, vector, host cell or pharmaceutical composition of claim 36, wherein the tissue or organ is selected from the group consisting of pancreas, salivary gland, pituitary gland, kidney, heart, lung, hematopoietic system, cranial nerves, heart, aorta, olfactory gland, ear, nerves, head structures, eye, thymus, tongue, bone, liver, small intestine, large intestine, gut, lung, brain, skin, peripheral nervous system, central nervous system, spinal cord, breast, embryonic structure, embryo, and testis.

38. The antibody or antigen-binding fragment thereof, construct, polynucleotide, vector, host cell or pharmaceutical composition according to any one of claims 35 to 37, wherein the immunological disease is selected from the group consisting of autoimmune diseases, neurological conditions, allergies, asthma, macular degeneration, muscle atrophy, diseases associated with miscarriage, atherosclerosis, bone loss, musculoskeletal diseases, obesity, graft-versus-host disease, and allograft rejection.

39. (a) The autoimmune disease is selected from the group consisting of type I diabetes mellitus, alopecia areata, ankylosing spondylitis, antiphospholipid syndrome, autoimmune Addison's disease, autoimmune hemolytic anemia, autoimmune hepatitis, Behcet's disease, bullous pemphigoid, cardiomyopathy, celiac sprue dermatitis, chronic fatigue and immune dysfunction syndrome (CFIDS), chronic inflammatory demyelinating polyneuropathy, Churg-Strauss syndrome, cicatricial pemphigoid, CREST syndrome, cold agglutinin disease, Crohn's disease, essential mixed cryoglobulinemia, fibromyalgia-fibromyositis, Graves' disease, Guillain-Barré, Hashimoto's thyroiditis, hypothyroidism, idiopathic pulmonary fibrosis, and idiopathic thrombocytopenic purpura. ITP), IgA nephropathy, juvenile arthritis, lichen planus, lupus, Meniere's disease, mixed connective tissue disease, multiple sclerosis, myasthenia gravis, pemphigus vulgaris, pernicious anemia, polyarteritis nodosa, polychondritis, polyglandular syndrome, polymyalgia rheumatica, polymyositis and dermatomyositis, primary agammaglobulinemia, primary biliary cirrhosis, psoriasis, Raynaud's phenomenon, Reiter's syndrome, rheumatic fever, rheumatoid arthritis, sarcoidosis, scleroderma, Sjogren's syndrome, stiff-body syndrome, Takayasu's arteritis, temporal arteritis / giant cell arteritis, ulcerative colitis, uveitis, vasculitis, vitiligo, and Wegener's granulomatosis; (b) the neurological condition is selected from the group consisting of brain tumors, brain metastases, spinal cord injury, schizophrenia, epilepsy, amyotrophic lateral sclerosis (ALS), Parkinson's disease, Alzheimer's disease, Huntington's disease, and stroke; (c) the allergy is selected from the group consisting of food allergies, seasonal allergies, pet allergies, hives, hay fever, allergic conjunctivitis, poison ivy allergies, oak allergies, mold allergies, drug allergies, dust allergies, cosmetic allergies, and chemical allergies; (d) the allograft rejection is selected from the group consisting of skin graft rejection, bone graft rejection, vascularized tissue graft rejection, ligament graft rejection, and organ graft rejection; or (e) the graft-versus-host disease arises from a bone marrow transplant or from one or more blood cells selected from the group consisting of hematopoietic stem cells, common myeloid progenitor cells, common lymphoid progenitor cells, megakaryocytes, monocytes, basophils, eosinophils, neutrophils, macrophages, T cells, B cells, natural killer cells, and dendritic cells; 39. The antibody or antigen-binding fragment thereof, construct, polynucleotide, vector, host cell or pharmaceutical composition of claim 38.

40. (a) the ligament graft rejection is selected from the group consisting of cricothyroid ligament graft rejection, periodontal ligament graft rejection, crystalline suspensory ligament graft rejection, palmar radiocarpal ligament graft rejection, dorsal radiocarpal ligament graft rejection, medial collateral ligament graft rejection, lateral collateral ligament graft rejection, mammary suspensory ligament graft rejection, anterior sacroiliac ligament graft rejection, posterior sacroiliac ligament graft rejection, sacrotuberous ligament graft rejection, sacrospinous ligament graft rejection, inferior pubic ligament graft rejection, superior pubic ligament graft rejection, anterior cruciate ligament graft rejection, lateral collateral ligament graft rejection, posterior cruciate ligament graft rejection, medial collateral ligament graft rejection, cranial cruciate ligament graft rejection, caudal cruciate ligament graft rejection, and patellar ligament graft rejection; or (b) the organ transplant rejection is selected from the group consisting of heart transplant rejection, lung transplant rejection, kidney transplant rejection, liver transplant rejection, pancreas transplant rejection, intestinal transplant rejection, and thymus transplant rejection; 40. The antibody or antigen-binding fragment thereof, construct, polynucleotide, vector, host cell or pharmaceutical composition of claim 39.

41. An antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, a construct according to any one of claims 10 to 12, a polynucleotide according to claim 13 or 14, a vector according to any one of claims 15 to 21, a host cell according to any one of claims 22 and 24 to 26, or a pharmaceutical composition according to any one of claims 27 to 29 for use in the treatment of an inflammatory disease in a human subject.

42. The inflammatory disease is (a) acute or chronic inflammation; and / or (b) selected from the group consisting of osteoarthritis, fibrotic lung disease, and cardiac inflammation; 42. An antibody or antigen-binding fragment thereof, construct, polynucleotide, vector, host cell or pharmaceutical composition according to claim 41.

43. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, the construct according to any one of claims 10 to 12, the polynucleotide according to claim 13 or 14, the vector according to any one of claims 15 to 21, the host cell according to any one of claims 22 and 24 to 26, or the pharmaceutical composition according to any one of claims 27 to 29 for use in the regeneration of TNFR2+ tissue in a human subject.

44. The antibody or antigen-binding fragment thereof, construct, polynucleotide, vector, host cell or pharmaceutical composition of claim 43, wherein the TNFR2+ tissue is selected from the group consisting of pancreas, salivary gland, pituitary gland, kidney, heart, lung, hematopoietic system, cranial nerves, heart, aorta, olfactory gland, ear, nerves, eye, thymus, tongue, bone, liver, small intestine, large intestine, gastrointestinal, lung, brain, skin, peripheral nervous system, central nervous system, spinal cord, breast, embryonic structure, embryo, and testis tissue.

45. The antibody or antigen-binding fragment thereof, construct, polynucleotide, vector, host cell or pharmaceutical composition according to any one of claims 34 to 44, wherein the antibody or antigen-binding fragment thereof is for administration to a human in combination with an immunotherapeutic agent.

46. The immunotherapeutic agent is an agonistic anti-CTLA-4 agent, an agonistic anti-PD-1 agent, an agonistic anti-PD-L1 agent, an agonistic anti-PD-L2 agent, an agonistic anti-CD27 agent, an agonistic anti-CD30 agent, an agonistic anti-CD40 agent, an agonistic anti-4-1BB agent, an agonistic anti-GITR agent, an agonistic anti-OX40 agent, an agonistic anti-TRAILR1 agent, an agonistic anti-TRAILR2 agent, an agonistic anti-TWE agent, AK agents, agonistic anti-TWEAKR agents, agonistic anti-cell surface lymphocyte protein agents, agonistic anti-BRAF agents, agonistic anti-MEK agents, agonistic anti-CD33 agents, agonistic anti-CD20 agents, agonistic anti-HLA-DR agents, agonistic anti-HLA class I agents, agonistic anti-CD52 agents, agonistic anti-A33 agents, agonistic anti-GD3 agents, agonistic anti-PSMA agents, agonistic anti-Ceacan 1 agents, agonistic anti-Galedin 9 agents, agonistic anti-HVEM agents, agonistic anti-VISTA agents, agonistic anti-B7 H4 agents, agonistic anti-HHLA2 agents, agonistic anti-CD155 agents, agonistic anti-CD80 agents, agonistic anti-BTLA agents, agonistic anti-CD160 agents, agonistic anti-CD28 agents, agonistic anti-CD226 agents, agonistic anti-CEACAM1 agents, agonistic anti-TIM3 agents, agonistic anti-TIGIT agents, agonistic anti-CD96 agents, agonistic anti-CD70 agents, agonistic anti-CD27 agents, agonistic anti-LIGHT agents, agonistic anti-CD137 agents, agonistic anti-DR4 agents, agonistic anti-CR5 agents, agonistic anti-TNFRS agents, agonistic anti-TNFR1 agents, agonists 46. ​​The antibody or antigen-binding fragment thereof, construct, polynucleotide, vector, host cell or pharmaceutical composition of claim 45, wherein the antibody or antigen-binding fragment thereof is selected from the group consisting of an agonistic anti-FAS agent, an agonistic anti-CD95 agent, an agonistic anti-TRAIL agent, an agonistic anti-DR6 agent, an agonistic anti-EDAR agent, an agonistic anti-NGFR agent, an agonistic anti-OPG agent, an agonistic anti-RANKL agent, an agonistic anti-LTβ receptor agent, an agonistic anti-BCMA agent, an agonistic anti-TACI agent, an agonistic anti-BAFFR agent, an agonistic anti-EDAR2 agent, an agonistic anti-TROY agent, and an agonistic anti-RELT agent.

47. The antibody or antigen-binding fragment thereof, construct, polynucleotide, vector, host cell or pharmaceutical composition according to any one of claims 34 to 46, wherein the antibody or antigen-binding fragment thereof is for administration to a human in combination with an additional agent selected from the group consisting of TNFα, an agonistic TNFα mutein, and Bacillus Calmette-Guerin (BCG).

48. The antibody or antigen-binding fragment thereof, construct, polynucleotide, vector, host cell or pharmaceutical composition according to any one of claims 34 to 47, wherein the antibody or antigen-binding fragment thereof that specifically binds to TNFR2 is for administration to a human in an amount of about 0.001 mg / kg to about 100 mg / kg.

49. A kit comprising an agent selected from the group consisting of an antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, a construct according to any one of claims 10 to 12, a polynucleotide according to claim 13 or 14, a vector according to any one of claims 15 to 21, a host cell according to any one of claims 22 and 24 to 26, or a pharmaceutical composition according to any one of claims 27 to 29.

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