WO2022003693A1 - Anti-tumor necrosis factor receptor (tnfr2) antibodies and uses thereof - Google Patents

Abstract

The present disclosure describes a number of anti-TNFR2 (Tumor necrosis factor receptor 2) antibodies. These antibodies are tight binders to TNFR2 but are neither agonists nor antagonists. These antibodies can decrease the number of TNFR2+ regulatory T cells or cancer cells through antibody-dependent cellular cytotoxicity. In one embodiment, these antibodies can be used to treat diseases such as cancer or autoimmune diseases.

Description

ANTI-TUMOR NECROSIS FACTOR RECEPTOR (TNFR2) ANTIBODIES AND USES

THEREOF

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of United States Provisional Application Serial Number 63/047,514 filed July 2, 2020, which is hereby incorporated by reference in its entirety.

SEQUENCE LISTING STATEMENT

[0002] The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. The ASCII copy, created on June9, 2021, is named P-597452-PC-SQL-09JUN21_ST25 and is 25.5 kilobytes in size.

FIELD OF THE INVENTION

[0003] The present disclosure relates in general to engineered antibodies. In one embodiment, the present disclosure describes the making and uses of antibodies against Tumor necrosis factor receptor 2 (TNFR2).

BACKGROUND OF THE INVENTION

[0004] Tumor necrosis factor-alpha (TNFa) is an extremely pleiotropic cytokine. It is not only produced by immune cells which include activated macrophages, T cells, and natural killer (NK) cells, but can also be produced in endothelial cells, microglia, cardiac myocytes, and fibroblasts. Upon production, TNFa is presented as a membrane bound form (mTNFa), which is a 26 kDa transmembrane protein that can be cleaved by TNF-Converting Enzyme (TACE) to form a soluble form of TNFa (sTNFa) that is released from the membrane.

[0005] TNFa interacts with two receptors, Tumor Necrosis Factor Receptor Superfamily Member 1A (TNFR1) and Tumor Necrosis Factor Receptor Superfamily Member IB (TNFR2). The extracellular domains of the two TNF receptors share a common structure composed of four cysteine-rich domains (CRDs) that are responsible for binding TNFa.

[0006] TNFR1 is constitutively expressed on virtually all nuclear cell types. It has been reported to bind sTNFa and mTNFa and is activated by both. One of TNFR1 main function is to mediate TNF-induced apoptosis via the NF-kB pathway; however, in certain situations activation of NF- kB pathway is involved in production of anti-apoptotic proteins and pro-inflammatory cytokines such as IL-1 and IL-6.

[0007] TNFR2, which was discovered and characterized more recently, is mostly expressed on activated T cells, myeloid cells and glial cells. It is a 75 kDa trans-membrane receptor with an extracellular domain consisting of four cysteine rich domains (CRD), a single transmembrane domain and a cytoplasmic domain that interacts with TNF receptor-associated factor 2 (TRAF2). Recruitment of TRAF2 promotes a cascade of events resulting in activation of an alternative NF- kB pathway. Although TNFR2 can bind tightly to both sTNFa and mTNFa, it is mainly activated through mTNFa and not sTNFa.

[0008] It has been shown that TNFR2 is expressed in large numbers on the surface of CD4⁺CD25⁺Foxp3⁺ regulatory T cells (Tregs) and myeloid-derived suppressor cells (MDSCs). Activation of TNFR2 in these cells promotes their immunosuppressive activity, and is crucial for Tregs and MDSC proliferation and survival. Additionally, TNFR2 has a dual role on CD8⁺ effector T cells (Teffs), which on one hand mediates an activation signal to CD8⁺ Teffs during early immune response, while on the other hand signals for apoptosis in these cells to terminate the immune response.

[0009] The key role of TNFR2 in regulation of the immune system is reflected in several pathogenic conditions. In cancer, TNFR2 is highly expressed in the tumor microenvironment (TME), on infiltrating Tregs, MDSC and on the tumor cells themselves. Activation of the TNFR2 receptor on Tregs and MDSC results in suppression of the immune system in the TME. Additionally, blocking of TNFR2 on cancer cells lines in vitro has been shown to have a dose dependent killing effect.

[0010] In autoimmune diseases, abnormalities in TNFa signaling pathway have been reported in various autoimmune diseases including rheumatoid arthritis (RA), Crohn’s disease (CD), multiple sclerosis (MS) and Type I diabetes. In many of these pathological conditions where the TNFR1 is more dominant, blocking of TNFa mostly in its soluble form is sufficient to have an efficacious effect. TNFa blockers like Infliximab and Adalimumab have been applied successfully in rheumatoid arthritis (RA), Crohn’s disease (CD), and ulcerative colitis (UC). However, there are other conditions like type 1 diabetes (T1D) and multiple sclerosis where this approach doesn’t work well. [0011] It is clear that in certain cases specific inhibition or activation of the TNFR2 pathway may prove beneficial, for example, in murine cancer models specific inhibition of TNFR2 shows a robust effect both directly on cancer cells as well as on immune system activation. TNFR2 has been strongly implicated as a driving force in some autoimmune diseases where Tregs and MDCS play a major role. In EAE mice model of MS, deletion of TNFR2 gene has a deleterious effect. Agonizing TNFR2 in GvHD models has been shown to reduce severity of GvHD. TNFR2 activation has also been implicated with BCG novel treatment of T1D.

[0012] In view of the important roles of TNFR2 in the regulation of immune responses in various disease conditions, there is a need to develop improved targeting of TNFR2 by either antagonists or agonists as a strategy to treat diseases, for example cancer and autoimmune diseases.

SUMMARY OF THE INVENTION

[0013] The present disclosure provides a number of anti-TNFR2 (Tumor necrosis factor receptor 2) antibodies. In one aspect, each of the anti-TNFR2 antibodies comprises a set of three complementarity determining regions (CDRs) on a heavy chain (HCDR1, HCDR2, and HCDR3) and a set of three CDRs on a light chain (LCDR1, LCDR2, and LCDR3). In one aspect, the set of HCDR1, HCDR2, and HCDR3 comprises the amino acid sequences of SEQ ID NOs:5-7, and the set of LCDR1, LCDR2, and LCDR3 comprises the amino acid sequences of SEQ ID NOs:8-10. In another aspect, the set of HCDR1, HCDR2, and HCDR3 comprises the amino acid sequences of SEQ ID NOs: 17-19, and the set of LCDR1, LCDR2, and LCDR3 comprises the amino acid sequences of SEQ ID NOs:20-22.

[0014] In one aspect, each of the anti-TNFR2 antibodies comprises a heavy chain variable region and a light chain variable region, wherein the amino acid sequences for the heavy chain variable region and the light chain variable region can be one of the following pairs: SEQ ID NOs:3-4, or SEQ ID NOs:13-14.

[0015] In another aspect, each of the anti-TNFR2 antibodies comprises a heavy chain and a light chain, wherein the amino acid sequences for the heavy chain and the light chain can be one of the following pairs: SEQ ID NOs:23-24, or SEQ ID NOs:25-26.

[0016] In another aspect, the anti-TNFR2 antibodies are neither agonists nor antagonists of TNFR2.

[0017] In one aspect, the present disclosure provides a composition comprising a pharmaceutically acceptable carrier and an anti-TNFR2 antibody disclosed herein.

[0018] The present disclosure also provides polynucleotide sequences encoding the anti-TNFR2 antibodies disclosed herein, as well as vectors and host cells comprising such polynucleotide sequences.

[0019] In one aspect, the anti-TNFR2 antibodies disclosed herein can be used to decrease the number of regulatory T cells or cancer cells in a subject.

[0020] In one aspect, the anti-TNFR2 antibodies disclosed herein can be used to treat diseases such as cancer, autoimmune diseases, viral infection or bacterial infection.

[0021] These and other aspects of the anti-TNFR2 antibodies will be appreciated from the ensuing descriptions of the figures and detailed description of the anti-TNFR2 antibodies.

BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Some embodiments of the anti-TNFR2 antibodies and uses thereof are herein described, by way of example only, with reference to the accompanying drawings. With specific reference now to the drawings in detail, it is stressed that the particulars shown are by way of example and for purposes of illustrative discussion of embodiments of the anti-TNFR2 antibodies and uses thereof. In this regard, the description taken with the drawings makes apparent to those skilled in the art how embodiments of the anti-TNFR2 antibodies and uses thereof may be practiced.

[0023] Figures 1A and IB shows SEC analysis of clones 30.083 (Figure 1A) and 30.091 (Figure IB) in IgG format. The retention time of lOOug IgG run at 0.8ml/min on a Superdex® 200 10/300pg with PBS as mobile phase was monitored as absorbance at 280nm.

[0024] Figures 2A and 2B shows IgG binding specificity of clones 30.083 (Figure 2A) and 30.091(Figure 2B) to TNFR2. Wells were coated with 50ng/well IgGs and tested for binding to lOOnM TNFR1 or TNFR2.

[0025] Figures 3A and 3B shows ELISA EC50 binding of IgG 1 of clones 30.083 (Figure 3A) and 30.091 (Figure 3B) to TNFR2. [0026] Figure 4 shows HEK-TNFR2 cells express human TNFR2. lxlO⁶ cells were harvested and lysed with either Tris Lysis Buffer (TLB) or with RIPA lysis buffer. Protein concentration was determined by the Bradford method, and 22ug protein cell lysate was subjected to western blot, detecting TNFR2 and GAPDH as control. Left 2 lanes: Untransfected HEK-Blue™ Null cells, right 2 lanes: HEK-Blue™ Null cells transfected with pCDNA3.1 plasmid encoding human TNFR2.

[0027] Figures 5A-5C show identification of TNFa responsive clone. Figure 5A shows a schematic representation of the selection process. Briefly, a parental plate of single clones was replicated and TNFa-Fc was added to the replica plate, and secreted embryonic alkaline phosphatase (SEAP) activity was measured in both plates using QB reagent. Cells that had SEAP signal only as a response to TNFa addition were selected. Figure 5B shows a photo of SEAP activity colors of selected clones with or without TNFa. Figure 5C shows quantification of colorimetric reaction of QB at OD 655 of selected clones.

[0028] Figures 6A-6B show TNFR2-TNF dependent activation of the NFKB pathway. Figure 6A shows dose response of clone G6 to TNFa-Fc (circles). I.C is isotype control antibody (square). Figure 6B shows activation of TNFa is inhibited by the addition of soluble TNFR2-Fc. OD 620 values are presented.

[0029] Figures 7A and 7B shows EC50 of functional agonism of TNFR2 in HEK293-NFKB reporter cell line. Clone 30.083 (Figure 7A) and Clone 30.091 (Figure 7B)

[0030] Figures 8A and 8B shows the effects of TNFa on antibody-dependent TNFR2 activation. HEK-TNFR2 reporter cells were incubated with 200nM soluble anti-TNFR2 antibodies for one hour. Subsequently, 0.05nM to lOOnM TNFa was added. Clone 30.083 (Figure 8A) and Clone 30.091 (Figure 8B)

DETAILED DESCRIPTION OF THE INVENTION

[0031] The present disclosure presents a panel of high affinity anti-TNFR2 antibodies that are neither agonist nor antagonist of TNFR2. These antibodies bind to the TNF receptor without affecting signaling through the receptor. These antibodies can decrease the number of TNFR2⁺ regulatory T cells or cancer cells through antibody-dependent cellular cytotoxicity. Accordingly, these antibodies could indirectly modulate immune response through elimination of regulatory T cells or cancer cells.

[0032] As used herein, the terms “comprise”, "comprises", "comprising", "includes", "including", “having” and their conjugates mean "including but not limited to".

[0033] As used herein, the singular form "a", "an" and "the" include plural references unless the context clearly dictates otherwise. For example, the term "an antibody" or "at least one antibody" may include a plurality of antibodies.

[0034] Throughout this application, various embodiments of the present disclosure may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the anti-TNFR2 antibodies and uses thereof. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.

[0035] Whenever a numerical range is indicated herein, it is meant to include any cited numeral (fractional or integral) within the indicated range. The phrases “ranging/ranges between” a first indicate number and a second indicate number and “ranging/ranges from” a first indicate number “to” a second indicate number are used herein interchangeably and are meant to include the first and second indicated numbers and all the fractional and integral numerals therebetween.

[0036] When values are expressed as approximations, by use of the antecedent "about," it is understood that the particular value forms another embodiment. All ranges are inclusive and combinable. In one embodiment, the term “about” refers to a deviance of between 0.1-5% from the indicated number or range of numbers. In another embodiment, the term “about” refers to a deviance of between 1-10% from the indicated number or range of numbers. In another embodiment, the term “about” refers to a deviance of up to 20% from the indicated number or range of numbers. In one embodiment, the term “about” refers to a deviance of ± 10% from the indicated number or range of numbers. In another embodiment, the term “about” refers to a deviance of ± 5% from the indicated number or range of numbers. [0037] Unless otherwise defined, all technical and/or scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the anti-TNFR2 antibodies and uses thereof pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the anti-TNFR2 antibodies and uses thereof, methods and/or materials are described below. In case of conflict, the patent specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and are not intended to be necessarily limiting. Each literature reference or other citation referred to herein is incorporated herein by reference in its entirety.

[0038] In the description presented herein, each of the steps of making and using the anti-TNFR2 antibodies and variations thereof are described. This description is not intended to be limiting and changes in the components, sequence of steps, and other variations would be understood to be within the scope of the present anti-TNFR2 antibodies and uses thereof.

[0039] It is appreciated that certain features of the anti-TNFR2 antibodies and uses thereof , which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the anti-TNFR2 antibodies and uses thereof, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination or as suitable in any other described embodiment of the anti-TNFR2 antibodies and uses thereof. Certain features described in the context of various embodiments are not to be considered essential features of those embodiments, unless the embodiment is inoperative without those elements.

[0040] As used herein, the term “antibody” may be used interchangeably with the term “immunoglobulin”, having all the same qualities and meanings. An antibody binding domain or an antigen binding site can be a fragment of an antibody or a genetically engineered product of one or more fragments of the antibody, which fragment is involved in specifically binding with a target antigen. By "specifically binding" is meant that the binding is selective for the antigen of interest and can be discriminated from unwanted or nonspecific interactions. For example, an antibody is said to specifically bind a TNFR2 epitope when the equilibrium dissociation constant is < 10⁵, 10⁶, or 10⁷ M. In some embodiments, the equilibrium dissociation constant may be < 10⁸ M or 10⁹ M. In some further embodiments, the equilibrium dissociation constant may be < 10 ¹⁰ M, 10 ¹¹ M, or 10 ¹²M. In some embodiments, the equilibrium dissociation constant may be in the range of < 10⁵ M to 10 ¹²M.

[0041] Half maximal effective concentration (EC50) refers to the concentration of a drug, antibody or toxicant which induces a response halfway between the baseline and maximum responses after a specified exposure time. In some embodiments, the response comprises a binding affinity. In some embodiments, the response comprises a functional response for example an agonistic or antagonistic response. A skilled artisan would appreciate that as used herein in certain embodiments, the EC50 measurement of an anti-TNFR2 antibody disclosed herein provides a measure of a half-maximal binding of the anti-TNFR2 antibody to the TNFR2 antigen (EC50 binding). Measure of EC50 binding affinity comprises measuring the binding the an anti-TNFR2 antibody described herein to the TNFR2 antigen, as exemplified in Table 3 in the Examples. The skilled artisan would appreciate that as used herein in certain embodiments, the EC50 measurement of an anti-TNFR2 antibody disclosed herein provides a measure of a half-maximal effective concentration of the anti-TNFR2 antibody to induce an agonist response (EC50 functional agonism). Measure of EC50 functional agonism comprises measuring the effects of the anti-TNFR2 antibodies described herein on cellular signaling of TNFR2, wherein as shown in Figure 7 the anti-TNFR2 antibodies disclosed herein do not exhibit functional agonism activity.

[0042] In certain embodiments, an anti-TNFR2 antibody disclosed herein is neither an agonist nor an antagonist for the TNFR2 receptor. In certain embodiments, an anti-TNFR2 antibody disclosed herein comprises a tight binder for the TNFR2 receptor but is neither an agonist nor an antagonist.

[0043] In some embodiments, EC50 comprises the concentration of antibody required to obtain a 50% agonist response that would be observed upon binding of TNFa. In certain embodiments, a measure of EC50 is commonly used as a measure of a drug's potency and may in some embodiments, reflect the binding of the antibody to the receptor. In some embodiments, anti-TNFR2 antibodies having nanomolar EC50 binding concentration measurements comprise tight binding anti-TNFR2 antibodies. In certain embodiments, an anti-TNFR2 antibody disclosed herein comprises a tight binder to the TNFR2 receptor. In certain embodiments, an anti-TNFR2 antibody disclosed herein lacks agonist or antagonist activity for the TNFR2 receptor. In certain embodiments, an anti-TNFR2 antibody disclosed herein comprises a tight binding antibody that lack TNFR2 agonist or antagonist activity.

[0044] In some embodiments, the binding EC50 of an anti-TNFR2 antibody is in the nanomolar range. In some embodiments, the binding EC50 of an anti-TNFR2 antibody comprises a range of about 0.05- 100 nM. In some embodiments, the binding EC50 of an anti-TNFR2 antibody comprises a range of about 0.05-50 nM. In some embodiments, the binding EC50 of an anti-TNFR2 antibody comprises a range of about 0.05-20 nM. In some embodiments, the binding EC50 of an anti-TNFR2 antibody comprises a range of about 0.05-10 nM. In some embodiments, the binding EC50 of an anti-TNFR2 antibody comprises a range of about 0.1-100 nM. In some embodiments, the binding EC50 of an anti- TNFR2 antibody comprises a range of about 0.1-50 nM. In some embodiments, the binding EC50 of an anti-TNFR2 antibody comprises a range of about 0.1-20 nM. In some embodiments, the binding EC50 of an anti-TNFR2 antibody comprises a range of about 0.1-10 nM. In some embodiments, the binding EC50 of an anti-TNFR2 antibody comprises a range of about 1-100 nM. In some embodiments, the binding EC50 of an anti-TNFR2 antibody comprises a range of about 1-20 nM. In some embodiments, the binding EC50 of an anti-TNFR2 antibody comprises a range of about 20-40 nM. In some embodiments, the binding EC50 of an anti-TNFR2 antibody comprises a range of about 40-60 nM. In some embodiments, the binding EC50 of an anti-TNFR2 antibody comprises a range of about 60-80 nM. In some embodiments, the binding EC50 of an anti-TNFR2 antibody comprises a range of about 80-100 nM. In some embodiments, the binding EC50 of an anti-TNFR2 antibody comprises a range of about 1-40 nM. In some embodiments, the binding EC50 of an anti-TNFR2 antibody comprises a range of about 1-60 nM. In some embodiments, the binding EC50 of an anti- TNFR2 antibody comprises a range of about 1-80 nM. In some embodiments, the binding EC50 of an anti-TNFR2 antibody comprises a range of about 1-50 nM. In some embodiments, the binding EC50 of an anti-TNFR2 antibody comprises a range of about 0.05-5 nM. In some embodiments, the binding EC50 of an anti-TNFR2 antibody comprises a range of about 0.1-5 nM. In some embodiments, the binding EC50 of an anti-TNFR2 antibody comprises a range of about 0.05-20 nM.

[0045] In some embodiments, the binding EC50 of an anti-TNFR2 antibody comprises a range of about 0.05-5 nM. In some embodiments, the binding EC50 of an anti-TNFR2 antibody comprises a range of about 0.1-5 nM. In some embodiments, the binding EC50 of an anti-TNFR2 antibody comprises a range of about 1-5 nM. In some embodiments, the binding EC50 of an anti-TNFR2 antibody comprises a range of about 0.05-10 nM. In some embodiments, the binding EC50 of an anti- TNFR2 antibody comprises a range of about 0.1-10 nM. In some embodiments, the binding EC50 of an anti-TNFR2 antibody comprises a range of about 1-10 nM. In some embodiments, the binding EC50 of an anti-TNFR2 antibody comprises a range of about 5-10 nM. In some embodiments, the binding EC50 of an anti-TNFR2 antibody comprises a range of about 0.05-15 nM. In some embodiments, the binding EC50 of an anti-TNFR2 antibody comprises a range of about 0.01-15 nM. In some embodiments, the binding EC50 of an anti-TNFR2 antibody comprises a range of about 1-15 nM.

[0046] As used herein, the term “antibody” encompasses an antibody fragment or fragments that retain binding specificity including, but not limited to, IgG, heavy chain variable region (VH), light chain variable region (VL), Fab fragments, F(ab')2 fragments, scFv fragments, Fv fragments, a nanobody, minibodies, diabodies, triabodies, tetrabodies, and single domain antibodies (see, e.g., Hudson and Souriau, Nature Med. 9: 129-134 (2003)). Also encompassed are humanized, primatized, and chimeric antibodies as these terms are generally understood in the art.

[0047] As used herein, the term “heavy chain variable region” may be used interchangeably with the term “VH domain” or the term “VH”, having all the same meanings and qualities. As used herein, the term “light chain variable region” may be used interchangeably with the term “VF domain” or the term “VF”, having all the same meanings and qualities. A skilled artisan would recognize that a “heavy chain variable region” or “VH” with regard to an antibody encompasses the fragment of the heavy chain that contains three complementarity determining regions (CDRs) interposed between flanking stretches known as framework regions. The framework regions are more highly conserved than the CDRs, and form a scaffold to support the CDRs. Similarly, a skilled artisan would also recognize that a “light chain variable region” or “VF” with regard to an antibody encompasses the fragment of the light chain that contains three CDRs interposed between framework regions.

[0048] As used herein, the term “complementarity determining region” or “CDR” refers to the hypervariable region(s) of a heavy or light chain variable region. Proceeding from the N-terminus, each of a heavy or light chain polypeptide has three CDRs denoted as “CDR1,” “CDR2,” and “CDR3”. Crystallographic analysis of a number of antigen-antibody complexes has demonstrated that the amino acid residues of CDRs form extensive contact with a bound antigen, wherein the most extensive antigen contact is with the heavy chain CDR3. Thus, the CDR regions are primarily responsible for the specificity of an antigen-binding site. In one embodiment, an antigen-binding site includes six CDRs, comprising the CDRs from each of a heavy and a light chain variable region.

[0049] As used herein, the term “framework region” or “FR” refers to the four flanking amino acid sequences which frame the CDRs of a heavy or light chain variable region. Some FR residues may contact bound antigen; however, FR residues are primarily responsible for folding the variable region into the antigen-binding site. In some embodiments, the FR residues responsible for folding the variable regions comprise residues directly adjacent to the CDRs. Within FRs, certain amino residues and certain structural features are very highly conserved. In this regard, all variable region sequences contain an internal disulfide loop of around 90 amino acid residues. When a variable region folds into an antigen binding site, the CDRs are displayed as projecting loop motifs that form an antigen binding surface. It is generally recognized that there are conserved structural regions of FR that influence the folded shape of the CDR loops into certain “canonical” structures regardless of the precise CDR amino acid sequence. Furthermore, certain FR residues are known to participate in non- covalent interdomain contacts which stabilize the interaction of the antibody heavy and light chains.

[0050] Wu and Rabat (Wu and Rabat, An analysis of the sequences of the variable regions of bence jones proteins and myeloma light chains and their implications for antibody complementarity. Journal of Experimental Medicine, 132, 2, 8 (1970); Rabat et al., Sequence of proteins of immunological interest. Bethesda: National Institute of Health; 1983. 323 (1983)) pioneered the alignment of antibody peptide sequences, and their contributions in this area were several-fold: Firstly, through study of sequence similarities between variable domains, they identified correspondent residues that to a greater or lesser extent were homologous across all antibodies in all vertebrate species, inasmuch as they adopted similar three-dimensional structure, played similar functional roles, interacted similarly with neighboring residues, and existed in similar chemical environments. Secondly, they devised a peptide sequence numbering system in which homologous immunoglobulin residues were assigned the same position number. One skilled in the art can unambiguously assign to any variable domain sequence what is now commonly called Rabat numbering without reliance on any experimental data beyond the sequence itself. Thirdly, Rabat and Wu calculated variability for each Rabat-numbered sequence position, which is the finding of few or many possible amino acids when variable domain sequences are aligned. They identified three contiguous regions of high variability embedded within four less variable contiguous regions. Rabat and Wu formally demarcated residues constituting these variable tracts, and designated these “complementarity determining regions” (CDRs), referring to chemical complementarity between antibody and antigen. A role in three- dimensional folding of the variable domain, but not in antigen recognition, was ascribed to the remaining less-variable regions, which are now termed “framework regions”. Fourth, Rabat and Wu established a public database of antibody peptide and nucleic acid sequences, which continues to be maintained and is well known to those skilled in the art. [0051] Chothia and coworkers (Cyrus Chothia, Arthur M. Lesk. Canonical structures for the hypervariable regions of immunoglobulins. Journal of Molecular Biology, 196, 4, 8 (1987)) found that certain sub portions within Kabat CDRs adopt nearly identical peptide backbone conformations, despite having great diversity at the level of amino acid sequence. These sub portions were designated as LI, L2 and L3 or HI, H2 and H3, where the “L” and the “H” designates the light chain and the heavy chains regions, respectively. These regions may be referred to as Chothia CDRs, which have boundaries that overlap with Kabat CDRs.

[0052] More recent studies have shown that virtually all antibody binding residues fall within regions of structural consensus. (Kunik, V. et al., PloS Computational Biology 8(2):el002388 (February 2012)). In some embodiments, these regions are referred to as antibody binding regions. It was shown that these regions can be identified from the antibody sequence as well. "Paratome", an implementation of a structural approach for the identification of structural consensus in antibodies, was used for this purpose. (Ofran, Y. et al., J. Immunol. 757:6230-6235 (2008)). While residues identified by Paratome cover virtually all the antibody binding sites, the CDRs (as identified by the commonly used CDR identification tools) miss significant portions of them. Antibody binding residues which were identified by Paratome but were not identified by any of the common CDR identification methods are referred to as Paratome-unique residues. Similarly, antibody binding residues that are identified by any of the common CDR identification methods but are not identified by Paratome are referred to as CDR-unique residues. Paratome-unique residues make crucial energetic contributions to antibody-antigen interactions, while CDRs-unique residues have a rather minor contribution. These results allow for better identification of antigen binding sites.

[0053] IMGT® is the international ImMunoGeneTics information system®, (See, Nucleic Acids Res. 2015 Jan; 43 (Database issue) :D413 -22. doi: 10.1093/nar/gkul056. Epub 2014 Nov 5 Free article. PMID: 25378316 LIGM:441 and Dev Comp Immunol. 2003 Jan;27(l):55-77). IMGT is a unique numbering system for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains (Lefranc et al., Dev Comp Immunol. 27: 55-77 (2003)). IMGT® presents a uniform numbering system for these IG and TcR variable domain sequences, based on aligning 5 or more IG and TcR variable region sequences, taking into account and combining the Kabat definition of FRs and CDRs, structural data, and Chothia's characterization of the hypervariable loops. IMGT is considered well known in the art as a universal numbering scheme for antibodies. [0054] In describing variant amino acid positions present in the VH and VL domains, in some embodiments the IMGT numbering is used. In describing variant amino acid positions present in the VH and VL domains, in some embodiments the Paratome numbering is used. In describing variant amino acid positions present in the VH and VL domains, in some embodiments the Kabat numbering is used. In describing variant amino acid positions present in the VH and VL domains, in some embodiments the Clothia numbering is used.

[0055] A skilled artisan would appreciate that a definition of CDR regions may encompass any common definition known in the art, for example but not limited to the definition according to the approaches utilized by IMGT^®, KABAT, or Chothia and Paratome.

[0056] An antibody may exist in various forms or having various domains including, without limitation, a complementarity determining region (CDR), a variable region (Fv), a VH domain, a VL domain, a single chain variable region (scFv), and a Fab fragment.

[0057] A person of ordinary skill in the art would appreciate that a scFv is a fusion polypeptide comprising the variable heavy chain (VH) and variable light chain (VL) regions of an immunoglobulin, connected by a short linker peptide. The linker may have, for example, 10 to about 25 amino acids.

[0058] A skilled artisan would also appreciate that the term “Fab” with regard to an antibody generally encompasses that portion of the antibody consisting of a single light chain (both variable and constant regions) bound to the variable region and first constant region of a single heavy chain by a disulfide bond, whereas F(ab')2 comprises a fragment of a heavy chain comprising a VH domain and a light chain comprising a VL domain.

[0059] In some embodiments, an antibody encompasses whole antibody molecules, including monoclonal and polyclonal antibodies. In some embodiments, an antibody encompasses an antibody fragment or fragments that retain binding specificity including, but not limited to, variable heavy chain (VH) fragments, variable light chain (VL) fragments, Fab fragments, F(ab')2 fragments, scFv fragments, Fv fragments, minibodies, diabodies, triabodies, and tetrabodies.

[0060] In one embodiment, the anti-TNFR2 antibodies disclosed herein can be incorporated as part of a bispecific antibody. As it is generally known in the art, bispecific antibody is a recombinant protein that includes antigen-binding fragments of two different monoclonal antibodies, and is thereby capable of binding two different antigens. In some embodiments, bispecific antibodies are used for cancer immunotherapy by simultaneously targeting, for example, both CTLs (such as a CTL receptor component such as CD3) or effector natural killer (NK) cells, and a tumor antigen. Similarly, a multi specific antibody is a recombinant protein that includes antigen-binding fragments of at least two different monoclonal antibodies, such as two, three or four different monoclonal antibodies.

Anti-TNFR2 Antibodies

[0061] The present disclosure provides a number of anti-TNFR2 (Tumor necrosis factor receptor 2) antibodies. In one embodiment, each of the anti-TNFR2 antibodies comprises a set of three complementarity determining regions (CDRs) on a heavy chain (HCDR1, HCDR2, and HCDR3) and a set of three CDRs on a light chain (LCDR1, LCDR2, and LCDR3). In one embodiment, the set of HCDR1, HCDR2, and HCDR3 comprises the amino acid sequences of SEQ ID NOs:5-7, and the set of LCDR1, LCDR2, and LCDR3 comprises the amino acid sequences of SEQ ID NOs:8-10. In another embodiment, the set of HCDR1, HCDR2, and HCDR3 comprises the amino acid sequences of SEQ ID NOs: 17-19, and the set of LCDR1, LCDR2, and LCDR3 comprises the amino acid sequences of SEQ ID NOs:20-22. In another embodiment, the anti-TNFR2 antibodies comprises heavy chain and light chain CDR sequences that are at least 80% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, or 99%) identical to the amino acid sequences set forth above, for example but not limited to identity as determined using BlastP software of the National Center of Biotechnology Information (NCBI) using default parameters.

[0062] A skilled artisan would appreciate that percent identity (% identity) provides a number that describes how similar the query sequence is to the target sequence (i.e., how many amino acids in each sequence are identical). The higher the percent identity is, the more significant the match.

[0063] When used in relation to polypeptide (or protein) sequences, the term “identity” refers to the degree of identity between two or more polypeptide (or protein) sequences or fragments thereof. Typically, the degree of similarity between two or more polypeptide (or protein) sequences refers to the degree of similarity of the composition, order, or arrangement of two or more amino acids of the two or more polypeptides (or proteins).

[0064] In one embodiment, each of the anti-TNFR2 antibodies presented herein comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the amino acid sequences for the heavy chain variable region and the light chain variable region can be one of the following pairs: SEQ ID NOs:3-4, or SEQ ID NOs: 13-14. In some embodiments, an anti-TNFR2 antibody disclosed herein comprises a heavy chain variable region (VH) and a light chain variable region (VL), as set forth in SEQ ID NOs:3-4, respectively. In some embodiments, an anti-TNFR2 antibody disclosed herein comprises a heavy chain variable region (VH) and a light chain variable region (VL), as set forth in SEQ ID NOs: 13-14, respectively. In another embodiment, the anti- TNFR2 antibodies comprise VH and VL sequences that are at least 80% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, or 99%) identical to the amino acid sequences set forth above, for example but not limited to identity as determined using BlastP software of the National Center of Biotechnology Information (NCBI) using default parameters.

[0065] In one embodiment, in view of the sequences for the heavy chain variable regions and light chain variable regions disclosed herein, one of ordinary skill in the art would readily employ standard techniques known in the art to construct an anti-TNFR2 scFv.

[0066] In certain embodiments, the present disclosure provides polypeptides comprising the VH and VL domains which could be dimerized under suitable conditions. For example, the VH and VL domains may be combined in a suitable buffer and dimerized through appropriate interactions such as hydrophobic interactions. In another embodiment, the VH and VL domains may be combined in a suitable buffer containing an enzyme and/or a cofactor which can promote dimerization of the VH and VL domains. In another embodiment, the VH and VL domains may be combined in a suitable vehicle that allows them to react with each other in the presence of a suitable reagent and/or catalyst.

[0067] In certain embodiments, the VH and VL domains may be contained within longer polypeptide sequences that may include for example, but not limited to, constant regions, hinge regions, linker regions, Fc regions, or disulfide binding regions, or any combination thereof. A constant domain is an immunoglobulin fold unit of the constant part of an immunoglobulin molecule, also referred to as a domain of the constant region (e.g. CHI, CH2, CH3, CH4, Ck, Cl). In some embodiments, the longer polypeptides may comprise multiple copies of one or both of the VH and VL domains generated according to the method disclosed herein; for example, when the polypeptides generated herein are used to forms a diabody or a triabody.

[0068] In another embodiment, each of the anti-TNFR2 antibodies presented herein comprises a heavy chain and a light chain, wherein the amino acid sequences for the heavy chain and the light chain can be one of the following pairs: SEQ ID NOs:23-24, or SEQ ID NOs:25-26. In some embodiments, an anti-TNFR2 antibody disclosed herein comprises a heavy chain and a light chain as set forth in SEQ ID NOs:23-24, respectively. In some embodiments, an anti-TNFR2 antibody disclosed herein comprises a heavy chain and a light chain as set forth in SEQ ID NOs:25-26, respectively. In another embodiment, the anti-TNFR2 antibodies comprises heavy chains and light chains amino acid sequences that are at least 80% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, or 99%) identical to the amino acid sequences set forth above, for example but not limited to identity as determined using BlastP software of the National Center of Biotechnology Information (NCBI) using default parameters.

[0069] In one embodiment, the anti-TNFR2 antibody presented herein can be an IgG, a Fv, a scFv, a Fab, a F(ab')2, a minibody, a diabody, a triabody, a nanobody, a bispecific antibody, or a single domain antibody. For example, the anti-TNFR2 antibody can be IgG such as IgGl, IgG2, IgG3, or IgG4. In some embodiments, the anti-TNFR2 antibody comprises an IgGl. In some embodiments, the anti-TNFR2 antibody comprises an IgG2. In some embodiments, the anti- TNFR2 antibody comprises an IgG3. In some embodiments, the anti-TNFR2 antibody comprises an IgG4.

[0070] In one embodiment, the present disclosure provides antibodies that bind with high affinity to TNFR2. In one embodiment, binding affinity is calculated by a modification of the Scatchard method as described by Frankel et al. (Mol. Immunol., 16: 101-106, 1979). In another embodiment, binding affinity is measured by an antigen/antibody dissociation rate. In another embodiment, binding affinity is measured by a competition radioimmunoassay. In another embodiment, binding affinity is measured by ELISA. In another embodiment, antibody affinity is measured by flow cytometry.

[0071] In one embodiment, the present disclosure also provides isolated polynucleotide sequences encoding the heavy chain and light chain CDRs as described herein. In another embodiment, the present disclosure also provides a vector comprising such polynucleotide sequences. In view of the amino acid sequences disclosed herein, one of ordinary skill in the art would readily construct a vector or plasmid to encode for the amino acid sequences. In another embodiment, the present disclosure also provides a host cell comprising the vector provided herein. Depending on the uses and experimental conditions, one of skill in the art would readily employ a suitable host cell to carry and/or express the above-mentioned polynucleotide sequences. [0072] In one embodiment, the present disclosure also provides isolated polynucleotide sequence encoding the heavy chain and light chain variable regions as described herein. In another embodiment, the present disclosure also provides a vector comprising such polynucleotide sequences. In view of the amino acid sequences disclosed herein, one of ordinary skill in the art would readily construct a vector or plasmid to encode for the amino acid sequences. In another embodiment, the present disclosure also provides a host cell comprising the vector provided herein. Depending on the uses and experimental conditions, one of skill in the art would readily employ a suitable host cell to carry and/or express the above-mentioned polynucleotide sequences.

[0073] In one embodiment, the present disclosure also provides isolated polynucleotide sequences encoding the heavy chains and light chains as described herein. In another embodiment, the present disclosure also provides a vector comprising such polynucleotide sequences. In view of the amino acid sequences disclosed herein, one of ordinary skill in the art would readily construct a vector or plasmid to encode for the amino acid sequences. In another embodiment, the present disclosure also provides a host cell comprising the vector provided herein. Depending on the uses and experimental conditions, one of skill in the art would readily employ a suitable host cell to carry and/or express the above-mentioned polynucleotide sequences.

Compositions of Use

[0074] In one embodiment, the present disclosure also provides a composition comprising the anti- TNFR2 antibody disclosed herein and a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers of use are well-known in the art. For example, Remington's Pharmaceutical Sciences, by E.W. Martin, Mack Publishing Co., Easton, PA, 15th Edition, 1975, describes compositions and formulations suitable for pharmaceutical delivery of the antibodies disclosed herein. In one embodiment, the composition comprises anti-TNFR2 antibodies that comprise a set of three complementarity determining regions (CDRs) on a heavy chain (HCDR1, HCDR2, and HCDR3) and a set of three CDRs on a light chain (LCDR1, LCDR2, and LCDR3). In one embodiment, the set of HCDR1, HCDR2, and HCDR3 comprises the amino acid sequences of SEQ ID NOs:5-7, and the set of LCDR1, LCDR2, and LCDR3 comprises the amino acid sequences of SEQ ID NOs:8-10. In another embodiment, the set of HCDR1, HCDR2, and HCDR3 comprises the amino acid sequences of SEQ ID NOs: 17-19, and the set of LCDR1, LCDR2, and LCDR3 comprises the amino acid sequences of SEQ ID NOs:20-22. In another embodiment, the anti-TNFR2 antibodies comprises heavy chain and light chain CDR sequences that are at least 80% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, or 99%) identical to the amino acid sequences set forth above, for example but not limited to identity as determined using BlastP software of the National Center of Biotechnology Information (NCBI) using default parameters.

[0075] In another embodiment, the composition comprises anti-TNFR2 antibodies having one of the following pairs of heavy chain variable region and light chain variable region: SEQ ID NOs:3- 4, or SEQ ID NOs: 13-14. In some embodiments, the composition comprises anti-TNFR2 antibodies comprising a heavy chain variable region and a light chain variable region as set forth in SEQ ID NOs:3-4, respectively. In some embodiments, the composition comprises anti-TNFR2 antibodies comprising a heavy chain variable region and a light chain variable region as set forth in SEQ ID NOs: 13-14, respectively. In another embodiment, the anti-TNFR2 antibodies comprise VH and VL sequences that are at least 80% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, or 99%) identical to the amino acid sequences set forth above, for example but not limited to identity as determined using BlastP software of the National Center of Biotechnology Information (NCBI) using default parameters.

[0076] In yet another embodiment, the composition comprises anti-TNFR2 antibodies having one of the following pairs of heavy chain and light chain: SEQ ID NOs:23-24, or SEQ ID NOs:25-26. In another embodiment, the anti-TNFR2 antibodies comprises heavy chains and light chains amino acid sequences that are at least 80% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, or 99%) identical to the amino acid sequences set forth above, for example but not limited to identity as determined using BlastP software of the National Center of Biotechnology Information (NCBI) using default parameters.

[0077] In one embodiment, the antibodies disclosed herein can be in the form of a conjugate. As used herein, a "conjugate" is an antibody or antibody fragment (such as an antigen-binding fragment) covalently linked to an effector molecule or a second protein (such as a second antibody). The effector molecule can be, for example, a drug, toxin, therapeutic agent, detectable label, protein, nucleic acid, lipid, nanoparticle, carbohydrate or recombinant virus. An antibody conjugate can also be referred to as an "immunoconjugate." When the conjugate comprises an antibody linked to a drug (e.g., a cytotoxic agent), the conjugate can be referred to as an "antibody- drug conjugate". Other antibody conjugates include, for example, multi- specific (such as bispecific or trispecific) antibodies and chimeric antigen receptors (CARs). [0078] A composition comprising the anti-TNFR2 antibody or an antigen-binding fragment thereof can be administered to a subject (e.g. a human or an animal) alone, or in combination with a carrier, i.e., a pharmaceutically acceptable carrier. By pharmaceutically acceptable is meant a material that is not biologically or otherwise undesirable, i.e., the material can be administered to a subject without causing any undesirable biological effects or interacting in a deleterious manner with any of the other components of the pharmaceutical composition in which it is contained. As would be well-known to one of ordinary skill in the art, the carrier is selected to minimize any degradation of the polypeptides disclosed herein and to minimize any adverse side effects in the subject. The pharmaceutical compositions may be prepared by methodology well known in the pharmaceutical art.

[0079] The pharmaceutical compositions comprising the antibodies or antigen-binding fragments thereof disclosed herein can be administered (e.g., to a mammal, a cell, or a tissue) in any suitable manner depending on whether local or systemic treatment is desired. For example, the composition can be administered topically (e.g. ophthalmically, vaginally, rectally, intranasally, transdermally, and the like), orally, by inhalation, or parenterally (including by intravenous drip or subcutaneous, intracavity, intraperitoneal, intradermal, or intramuscular injection). Topical intranasal administration refers to delivery of the compositions into the nose and nasal passages through one or both of the nares. The composition can be delivered by a spraying mechanism or droplet mechanism, or through aerosolization. Alternatively, administration can be intratumoral, e.g. local or intravenous injection.

[0080] If the composition is to be administered parenterally, the administration is generally by injection. Injectables can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for suspension in liquid prior to injection, or as emulsions. Additionally, parenteral administration can involve preparation of a slow-release or sustained- release system so as to maintain a constant dosage.

Methods of Use

[0081] In one embodiment, the anti-TNFR2 antibodies disclosed herein can be used to decrease the number of regulatory T cells or cancer cells in a subject.

[0082] Regulatory T cells (Tregs) represent a heterogeneous class of T cells that can be distinguished based on their unique surface protein presentation. The most studied Tregs include CD4⁺, CD25⁺, FoxP3⁺T-reg cells and CD17⁺Tregs. It has been shown that certain classes of Tregs inhibit 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. Moreover, it has been shown that CD4⁺, CD25⁺, FoxP3⁺ Tregs are also present in B cell-rich areas and are capable of directly suppressing immunoglobulin production independent of their ability to attenuate TH2-cell activity.

[0083] In another embodiment, the anti-TNFR2 antibodies disclosed herein can be used to treat diseases such as cancer, autoimmune diseases, viral infection or bacterial infection.

[0084] As used herein, the term "method" refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of the chemical, pharmacological, biological, biochemical and medical arts.

[0085] As used herein, the terms “treat”, “treatment”, or “therapy” (as well as different forms thereof) refer to therapeutic treatment, including prophylactic or preventative measures, wherein the object is to prevent or slow down (lessen) an undesired physiological change associated with a disease or condition. Beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of the extent of a disease or condition, stabilization of a disease or condition (i.e., where the disease or condition does not worsen), delay or slowing of the progression of a disease or condition, amelioration or palliation of the disease or condition, and remission (whether partial or total) of the disease or condition, whether detectable or undetectable. Those in need of treatment include those already with the disease or condition as well as those prone to having the disease or condition or those in which the disease or condition is to be prevented.

[0086] The terms "subject," "individual," and "patient" are used interchangeably herein, and refer to human or non-human animals to whom treatment with a composition or formulation in accordance with the present anti-TNFR2 antibodies is provided. The terms "non-human animals" and "non-human mammals" are used interchangeably herein and include all vertebrates, e.g., mammals, such as non-human primates (e.g. higher primates), sheep, dog, rodent (e.g. mouse or rat), guinea pig, goat, pig, cat, rabbits, cows, horses, or non-mammals such as reptiles, amphibians, chickens, and turkeys. The compositions described herein can be used to treat any suitable mammal, including primates, such as monkeys and humans, horses, cows, cats, dogs, rabbits, and rodents such as rats and mice. In one embodiment, the mammal to be treated is human. The human can be any human of any age. In one embodiment, the human is an adult. In another embodiment, the human is a child. The human can be male, female, pregnant, middle-aged, adolescent, or elderly.

[0087] Pharmaceutical compositions suitable for use in the methods disclosed herein include compositions wherein the active ingredients are contained in an amount effective to achieve the intended purpose. In one embodiment, a therapeutically effective amount means an amount of active ingredients effective to prevent, alleviate or ameliorate symptoms of disease or prolong the survival of the subject being treated. Determination of a therapeutically effective amount is well within the capability of those skilled in the art.

[0088] In one embodiment, the present disclosure provides a method of decreasing the number of regulatory T cells or cancer cells in a subject. In one embodiment, the method comprises the step of administering to the subject a composition comprising an effective amount of the anti-TNFR2 antibody disclosed herein. In one embodiment, the composition comprises anti-TNFR2 antibodies having the heavy chain and light chain CDR sequences as described herein. In another embodiment, the composition comprises anti-TNFR2 antibodies having the VH and VL sequences as described herein. In yet another embodiment, the composition comprises anti-TNFR2 antibodies having the heavy chain and light chain sequences as described herein. In one embodiment, the regulatory T cells are CD4+CD25+Foxp3+.

[0089] In one embodiment, the anti-TNFR2 antibodies disclosed herein could decreases the number of regulatory T cells by antibody-dependent cellular cytotoxicity (ADCC). As used herein, “antibody-dependent cellular cytotoxicity”, or “antibody-dependent cell-mediated cytotoxicity”, refers to a type of immune reaction in which a target cell or microbe is coated with antibodies and killed by certain types of white blood cells. The white blood cells bind to the antibodies and release substances that kill the target cells or microbes.

[0090] In one embodiment, the anti-TNFR2 antibodies disclosed herein could promote ADCC of human TNFR2⁺ CD4⁺ T regulatory cells in a dose- and time-dependent manner. In another embodiment, the present anti-TNFR2 antibodies are capable of efficiently inducing ADCC of CD4⁺/TNFR2⁺ T regulatory cells in part due to the IgGl isotype of the antibody, which possesses high affinity for Fey (gamma) receptors on NK cells, a common cell type that mediates ADCC.

[0091] In another embodiment, the present anti-TNFR2 antibodies further comprise a cytotoxic agent to mediate cellular cytotoxicity. A number of cytotoxic agents are well-known and generally used in the art. Examples of cytotoxic agents include, but are not limited to, chemotherapeutic agent, toxin (e.g. an enzymatically active toxin of bacterial, fungal, plant or animal origin, or fragments thereof), or radioactive isotope (i.e., a radioconjugate).

[0092] In addition to the ability of decreasing the number of regulatory T cells in a subject as described above, the anti-TNFR2 antibodies presented herein can similarly be used to target TNFR2⁺ tumor cells and confer cytotoxic effects on such tumor cells via ADCC.

[0093] In one embodiment, the present disclosure also provides uses of a composition comprising anti-TNFR2 antibodies for decreasing the number of regulatory T cells or tumor cells in a subject. In one embodiment, the composition comprises anti-TNFR2 antibodies having the heavy chain and light chain CDR sequences as described herein. In another embodiment, the composition comprises anti-TNFR2 antibodies having the VH and VL sequences as described herein. In yet another embodiment, the composition comprises anti-TNFR2 antibodies having the heavy chain and light chain sequences as described herein.

[0094] In another embodiment, the present disclosure provides a method of treating a disease in a subject, comprising the step of administering to the subject a composition comprising an effective amount of the anti-TNFR2 antibody disclosed herein. In one embodiment, the composition comprises anti-TNFR2 antibodies having the heavy chain and light chain CDR sequences as described herein. In another embodiment, the composition comprises anti-TNFR2 antibodies having the VH and VL sequences as described herein. In yet another embodiment, the composition comprises anti-TNFR2 antibodies having the heavy chain and light chain sequences as described herein.

[0095] In one embodiment, the present disclosure also provides uses of a composition comprising anti-TNFR2 antibodies for treating a disease in a subject. In one embodiment, the composition comprises anti-TNFR2 antibodies having the heavy chain and light chain CDR sequences as described herein. In another embodiment, the composition comprises anti-TNFR2 antibodies having the VH and VL sequences as described herein. In yet another embodiment, the composition comprises anti-TNFR2 antibodies having the heavy chain and light chain sequences as described herein.

[0096] In one embodiment, the exact amount of the present polypeptides or compositions thereof required to elicit the desired effects will vary from subject to subject, depending on the species, age, gender, weight, and general condition of the subject, the particular polypeptides, the route of administration, and whether other drugs are included in the regimen. Thus, it is not possible to specify an exact amount for every composition. However, an appropriate amount can be determined by one of ordinary skill in the art using routine experimentation. Dosages can vary, and the polypeptides can be administered in one or more (e.g., two or more, three or more, four or more, or five or more) doses daily, for one or more days. Guidance in selecting appropriate doses for antibodies can be readily found in the literature.

[0097] In one embodiment, the disease can be viral infection, bacterial infection, cancer, autoimmune disease or immune disorder. In one embodiment, the disease can be upper respiratory viral infections, early stage lung infections, or late stage lung infections. A number of diseases and cancer are known to be caused by viruses. Examples of disease-causing viruses include, but are not limited to, norovirus; rotavirus; hepatitis virus A, B, C, D, or E; rabies virus, West Nile virus, enterovirus, echovirus, coxsackievirus, herpes simplex virus (HSV), HSV-2, varicella-zoster virus, mosquito-borne viruses, arbovirus, St. Louis encephalitis virus, California encephalitis virus, lymphocytic choriomeningitis virus, human immunodeficiency virus (HIV), poliovirus, zika virus, rubella virus, cytomegalovirus, human papillomavirus (HPV), enterovirus D68, severe acute respiratory syndrome (SARS) coronavirus, Middle East respiratory syndrome coronavirus, SARS coronavirus 2, Epstein-Barr virus, influenza virus, respiratory syncytial virus, polyoma viruses (such as JC virus, BK virus), Ebola virus, Dengue virus, or any combination thereof.

[0098] In another embodiment, the disease is a cancer that can be, but is not limited to, carcinoma, sarcoma, lymphoma, leukemia, germ cell tumor, blastoma, chondrosarcoma, Ewing's sarcoma, malignant fibrous histiocytoma of bone, osteosarcoma, rhabdomyosarcoma, heart cancer, brain cancer, astrocytoma, glioma, medulloblastoma, neuroblastoma, breast cancer, medullary carcinoma, adrenocortical carcinoma, thyroid cancer, Merkel cell carcinoma, eye cancer, gastrointestinal cancer, colon cancer, gallbladder cancer, gastric (stomach) cancer, gastrointestinal carcinoid tumor, hepatocellular cancer, pancreatic cancer, rectal cancer, bladder cancer, cervical cancer, endometrial cancer, ovarian cancer, renal cell carcinoma, prostate cancer, testicular cancer, urethral cancer, uterine sarcoma, vaginal cancer, head cancer, neck cancer, nasopharyngeal carcinoma, hematopoetic cancer, Non-hodgkin lymphoma, skin cancer, basal-cell carcinoma, melanoma, small cell lung cancer, non-small cell lung cancer, or any combination thereof.

[0099] In another embodiment, the disease is an autoimmune disease that can be, but is not limited to, achalasia, amyloidosis, ankylosing spondylitis, anti-gbm/anti-tbm nephritis, antiphospholipid syndrome, arthritis, autoimmune angioedema, autoimmune encephalomyelitis, autoimmune hepatitis, autoimmune myocarditis, autoimmune oophoritis, autoimmune orchitis, autoimmune pancreatitis, autoimmune retinopathy, autoimmune urticaria, Behcet’s disease, celiac disease, chagas disease, chronic inflammatory demyelinating polyneuropathy, Cogan’s syndrome, congenital heart block, Crohn’s disease, dermatitis, dermatomyositis, discoid lupus, Dressier’ s syndrome, endometriosis, fibromyalgia, fibrosing alveolitis, granulomatosis with polyangiitis, Graves’ disease, Guillain-Barre syndrome, herpes gestationis, immune thrombocytopenic purpura, interstitial cystitis, juvenile arthritis, juvenile diabetes (type 1 diabetes), juvenile myositis, Kawasaki disease, Lambert-Eaton syndrome, lichen planus, lupus, Lyme disease, multiple sclerosis, myasthenia gravis, myositis, neonatal lupus, neutropenia, palindromic rheumatism, peripheral neuropathy, polyarteritis nodosa, polymyalgia rheumatica, polymyositis, postmyocardial infarction syndrome, postpericardiotomy syndrome, primary biliary cirrhosis, primary sclerosing cholangitis, progesterone dermatitis, psoriasis, psoriatic arthritis, reactive arthritis, retroperitoneal fibrosis, rheumatic fever, rheumatoid arthritis, sarcoidosis, Schmidt syndrome, scleritis, scleroderma, Sjogren’s syndrome, thrombocytopenic purpura, type 1 diabetes, ulcerative colitis, uveitis, vasculitis, and vitiligo.

[0100] The TNFR2 antibodies (or antigen-binding fragments thereof) described herein may also be admixed, conjugated, or administered with, or administered separately from, another agent that regulates proliferation of regulatory T cells or cancer cells.

[0101] Additionally or alternatively, the TNFR2 antibodies (or antigen-binding fragments thereof) described herein may be admixed, conjugated, or administered with, or administered separately from, an immunotherapy agent. Exemplary immunotherapy agents useful in conjunction with the compositions and methods presented herein include, but are not limited to, an anti-CTLA-4 agent, an anti-PD-1 agent, an anti-PD-Ll agent, an anti-PD-L2 agent, a TNF-a cross-linking agent, a TRAIL cross-linking agent, an agent targeting CD27, an agent targeting CD30, an agent targeting CD40, an agent targeting 4-1BB, an agent targeting GITR, an agent targeting 0X40, an agent targeting TRAILR1, or an agent targeting TRAILR2.

[0102] In another embodiment, the present disclosure provides a method of using a polynucleotide to treat a disease or condition as described above, wherein the polynucleotide encodes an anti- TNFR2 antibody as described herein.

[0103] Various embodiments and aspects of the present anti-TNFR2 antibodies as delineated hereinabove and as claimed in the claims section below find experimental support in the following examples.

EXAMPLES EXAMPLE 1

Generation of Anti-TNFR2 Antibodies EXPERIMENTAL PROCEDURES Library Generation Library Construction

[0104] Libraries were constructed based on three template antibodies (PDB: 215 Y, 4IOI and 3E8U) by overlapping extension PCR with degenerate oligonucleotides. PCR used to introduce diversity was done using Phusion™ high fidelity DNA polymerase (New England Biolabs USA, Cat: M0530) according to manufacturer instructions in a 3-step reaction (98°C for 30 sec, 65°C for 20 sec, 72°C for 30 sec, 30 cycles). The PCR products were gel purified by gel purification kit and assembled in equimolar ratios in a 3-step PCR reaction, as above, but in the absence of primers. The assembled PCR product was reused as template for PCR amplifying the full scFv library, as above, using forward and reverse primers adding the yeast surface display (YSD) expression vector homology sequences at the 5’ and 3’ to the scFv library to efficiently perform homologous recombination in yeast cells.

[0105] scFv libraries were constructed with three repeats of flexible linkers of Gly-Gly-Gly-Gly- Ser (SEQ ID NO:27) between the VH and VL.

[0106] Fab display libraries were constructed in a similar fashion to the scFv libraries with the following modifications: the VL and VH were constructed separately and were cloned under two promoters. The VH was cloned in-frame under the Gal 10 promoter, between the aga2 gene and the constant heavy chain domain 1 (CHI). The VL was cloned in-frame under the Gall promoter between a signal peptide and a constant light domain (CL). The Fab fragments were combined using PCR into one fragment and cloned into the pFAB 1 expression vector in a similar fashion to the scFv libraries.

Library Transformation

[0107] Library transformation was carried out as published (Benatuil el al., An improved yeast transformation method for the generation of very large human antibody libraries. Protein Eng. Des. Sel. 23, 155-159 (2010)). Four hundred pi of a yeast suspension (EBY 100, ATCC, USA) per 0.2cm cuvette (cell projects) was electroporated (BioRad, USA, GenePulser) with 4pg linearized vector (pCTcon3 or pFAB 1) and 12pg DNA insert (either scFv fragment or assemble Fab) in a 1 :3 vector to insert ratio (Chao, G. et al, Isolating and engineering human antibodies using yeast surface display. Nat. Protoc. 1, 755-768 (2006)). The average number of transformants in a library was determined to be ~ IX 10^s by serial dilutions of transformed cells.

Library Screening

Screening and Selection Using Yeast Surface Display

[0108] Yeast display libraries were grown in a SDCAA selective medium and induced for expression with 2% w/v galactose at 30°C overnight according to established protocols (Chao, G. et al, (2006)). Briefly, the library was incubated with 1000 to O.lnM of recombinant human TNFR2 with 6xhis tag or TNFR2-Fc fusion (Reprokine, Israel) in PBS 0.1% BSA for 1 hour, then washed three times with PBS 0.1% BSA and labeled with either: mouse anti c-Myc FITC (Miltenyi Biotec, cat #130-116-485), mouse anti c-Myc (Santa Cruze, USA cat# sc-40), and also fluorescently labeled goat anti-mouse IgG-FITC (Sigma- Aldrich, cat # F4143- lml), monoclonal anti His APC (Miltenyi Biotec, Germany cat 0020130-119-782), or anti Fc APC (Jackson ImmunoResearch, USA. cat. 109-135-098 ).

[0109] When it is necessary to avoid non-specific binding, the fluorescent-labeled antibodies were alternated with rabbit anti c-Myc (Abeam, cat# ab9106), goat anti-rabbit APC (Abeam, Cat# ab!30805), and anti-His Alexa488 (Qiagen, cat# 20-35310). [0110] Post labeling the library was selected on MACS beads, until the library size was reduced to lxlO⁶ and then sorted on BioRad S3e or BD ARIA III Fluorescence Activated Cell Sorter (FACS) for high affinity binders of recombinant human TNFR2. Clones isolated from the final sort were sequenced by extraction of plasmid DNA from the yeast clones using a Zymoprep kit (Zymo Research, USA) and the DNA was sequenced.

[0111] When applicable yeast displaying Fab were labeled and selected under the same conditions mentioned for scFv selection with the addition of anti-FLAG-PE (Miltenyi Biotec, cat #130-101- 576) for the detection of Fab light chain display. k_off Selection

[0112] To select for binders with improved off rate, the yeast were incubated for 30min with lOnM to InM TNFR2-His. Subsequently the yeast were washed three times with 1ml PBS 0.1% BSA and incubated for 4h, 6h, and 24h with lOOnM TNFR2-Fc. Alternatively, post wash the cells were diluted 10 fold in PBS 0.1% BSA and incubated for the indicated time points. The yeast were then washed three times and labeled with anti-Myc-FITC (Santa Cruze, USA, Cat# 9E10) and monoclonal anti-His/APC (Miltenyi Biotec, Germany cat 0020130-119-782), and sorted on Se3 FACS as described above.

[0113] Furthermore, two rounds of k_off selections were done at the most suitable time point in which approximately 50%-70% of initial binding was lost.

IgG Production

Reformatting

[0114] Selected scFv and Fab clones were reformatted to human IgGl format. The sequences of the light chain (LC) and heavy chain (HC) variable regions were optimized to mammalian codon usage and ordered as genblocks (GB) from IDT (Integrated DNA Technologies. Coralville, Iowa USA). The GB were cloned using standard cloning techniques into pSF-CMV-HuIgGl_HC (HC plasmid) and pSF-CMV-HuLambda_LC (LC plasmid) (Oxford genetics, Oxford UK).

IgG Expression

[0115] Expi-CHO cells (Thermo Fisher Scientific, USA) were transfected with LC and HC plasmids at a ratio of 2:1 and expression was done according to the manufacturer's instructions. Briefly: 50ml Expi-CHO cells were cultured at 37°C, 120rpm, CO2 8% to a density of 6xl0⁶ cells/ml. Then, 50mg of expression heavy chain and light chain plasmids at a ratio of 1:2 were transfected into the CHO cells. Post transfections, a booster and feed was added to the culture, and growth conditions were changed to 32°C, 120rpm, 5% CO2. The cells were harvested 10 days after transfection. The IgGs were purified from the supernatant using proteinA beads (Tosoh Bioscience GmbH, Germany), followed by size exclusion chromatography (SEC) purification on superdex 200 10/300 increase column, with PBS as mobile phase (GE healthcare, USA).

Size Exclusion Chromatography

[0116] To analyze and purify the IgGs, samples were loaded on a Superdex® 200 10/300 increase column (GE healthcare, USA) at a flow rate of 0.8ml/min on a GE AKTA Explorer chromatography system (GE healthcare, USA). Monitoring of antibody retention time was done at 280nm.

Ligand Binding ELISA

[0117] IgG binding affinity to TNFR2 was examined by ELISA. 96 well plates (Greiner Bio-One high binding) were coated with (50ng/well) of the analyzed antibody and incubated overnight at 4°C. The plates were then washed three times with 300m1 PBS buffer containing 0.05% Tween 20 (PBS-T), blocked with 300m PBS-T supplemented with 1% to 2% BSA, and incubated for 1 hour at room temperature. Antibodies-coated plates were washed three times with 300m1 PBS-T and incubated with serial dilutions of test ligand hTNFR2-His (Reprokine, Israel) in a final volume of 50ul for 1-2 hours. Plates were then washed three times with 300m1 PBS-T and incubated with 50m1 anti-HIS-HRP (Santa Cruz Biotechnology, USA, SC-8036HRP) conjugate that was diluted 1:250 in PBS. After an additional wash step of six washes, the reaction was developed with 50ul tetramethylbenzidine (TMB) reagent (Southern biotech, USA), and stopped with 50ul 0.5N H2SO4. Detection was done on a Synergy LX BioTek (BioTek, USA) plate reader with an absorbance filter set to 450nM. The binding affinity was determined by fitting the data to a specific binding non linear regression model on Prisma 8 GraphPad software.

[0118] TNFR2/TNFR1 specificity ELISA was done in a similar manner to the EC₅₀ ELISA assay (binding EC₅o)but with a concentration of lOOnM for both TNFR1 and TNFR2.

RESULTS

Library Design [0119] In order to generate an antibody that binds TNFR2, a “re-epitoping” approach was applied to an existing antibody. The re-epitoping process allows for the introduction of new specificity to an existing antibody, and can allow for choosing a known antibody with favorable biophysical and biochemical properties as a template. Therefore, the re-epitoped antibody can have both a new specificity and desirable developability profile. The computational process of re-epitoping requires two steps: (i) using any computational analysis that identifies putative complementarity between existing antibody and a new epitope, and (ii) application of any computational analysis or tool that can suggest introduction of specific mutations predicted to enhance antibody binding to the new, desired epitope. Examples of such computational processes are presented in US20180068055, and Nimrod et al., Cell Rep. 25(8):2121-2131 (2018). In one embodiment, three libraries were designed using the sequences of the variable domains of re-epitoping template antibodies 2I5Y, 4IOI and 3E8U, which are predicted to be good candidates for introducing new specificity towards TNFR2. The sequences of some of these templates were further modified in later generation libraries to account for better developability or for humanization. The libraries for re-epitoping were constructed and introduced to yeast as described above.

YSD Screen for TNFR2 Binders

[0120] Following introduction of variations, the libraries were screened in a yeast surface display (YSD) format to identify clones that specifically bind to TNFR2. Initially, the libraries underwent MACS (magnetic beads) selection followed by FCAS selection. Clones from all three re-epitoping templates showed relative binding.

[0121] To further enhance affinity, affinity maturation libraries were constructed in a similar fashion to the construction of libraries described above. The affinity maturation libraries were passed through regular and specific k_0ff improvement selection as described above. Best binders were gated, yeast clones were isolated and sequenced. These clones are listed in Table 1.

TABLE 1

Sequences of TNFR2 Binders in scFv, Fab and IgG Format

Expression in IgG Format

[0122] To test these antibodies' tendency to aggregate, the IgGs went through Size Exclusion Chromatography (SEC) on a Superdex 10/300 increase column using PBS as a mobile phase as described above. Examples of SEC analysis of selected antibodies are shown in Figure 1 and a summary of the complete SEC analysis of the clones is presented in Table 2. These results indicate that IgGs 30.083 and 30.091 were eluted from protein A mostly as non-aggregated species, and migrated on a SEC column as a typically folded IgGl.

TABLE 2 SEC Profile of TNFR2 Binding Clones in IgG Format

Specificity ELISA

[0123] The extracellular domains of TNFR2 and TNFR1 share 27% homology. To test specificity to TNFR2, the antibodies were analyzed by an ELISA assay against TNFR1 and TNFR2 as described herein. Briefly, antibodies were coated directly on the ELISA plate wells, the wells were blocked, and lOOnM of TNFRl-His-Fc or TNFR2-His-Fc were added to the wells, washed and detected using anti His-HRP. As can be seen in Figure 2, the IgG 30.083 and 30.091 showed specific binding to TNFR2 and did not bind TNFR1.

Binding Affinity of IgGs Towards TNFR2 IgG ELISA EC 50

[0124] To test binding affinity of clones reformatted as IgGl to TNFR2, an ELISA binding EC50 experiment was conducted for each antibody. As can be seen in Figure 3, the 30.083 and 30.091 antibodies bind soluble TNFR2-His at an EC50 range of 23.4nM to 8.6nM respectively, indicating that these antibodies are tight binders. Table 3 lists the TNFR2 EC50 values for the indicated antibodies, the values are an average of at least two biological repeats.

TABLE 3

TNFR2 EC50 Binding Value for Indicated Antibodies

Establishment of TNFR2-Dependent NFKB Cell Based Assay

[0125] To characterize whether the antibodies affect the TNFR2 cellular signaling functionally, and whether they have an agonistic, antagonistic or no effect on the TNFR2, a reporter cell line was created. HEK-Blue™ Null cells were purchased from InvivoGen (Toulouse France). These cells contain TNFR1 null mutation and a plasmid encoding soluble embryonic alkaline phosphatase (SEAP) under the control IFN-b minimal promoter fused to five NF-KB and AP-1 binding sites. A pCDNA3.1 plasmid encoding human TNFR2 (residues 1-461) under the CMV promoter was transfected into HEK-Blue™ Null cells. The cells were kept for 14 days under the selection of 50ug/ml hygromycin B. After the selection period, TNFR2 expression was validated by Western blot analysis (Figure 4).

[0126] Subsequently, the cells were diluted to a limiting dilution of 0.5 cells per well in 96 well plate and propagated in a growth medium containing DMEM supplemented with 10% FBS, L- Glutamin, pen/strep and 50ug/ml hygromycin B. To identify TNFa reactive clones, a replica plate was made of the single clones and tested for TNFa dependent activation of NFKB using Quanti- Blue (QB, InvivoGen) substrate as instructed by the manufacturer (Figures 5A-5C). [0127] Figures 6A-6B show the results of TNFa-dependent NFKB response of a specific clone, with dynamic range of 20pM to lOOOpM. The activation was inhibited in a dose dependent manner by soluble TNFR2 (Figure 6B), but was not affected by isotype control antibody (Figure 6A). Clone G6 was selected for evaluating the target antibodies for agonizing or antagonizing the TNFR2 receptor.

Functional Testing of The IgGs In HEK-TNFR2 Reporter Call Line

[0128] To test the effects of the anti-TNFR2 antibodies on cellular signaling of TNFR2, IgGs were incubated overnight with the HEK-TNFR2 reporter call line up to a concentration of 600nM and tested for antibody dependent NFkB activation as detailed herein.

[0129] Figure 7 shows that IgG 30.083 and 30.091 do not activate the TNFR2-dependent NFKB pathway, demonstrating that these antibodies are high affinity binders but functionally cannot agonize the receptor even at antibody concentration as high as lOOnM.

[0130] To examine the effects of TNFa on antibody dependent NFKB activation, the HEK-TNFR2 cells were incubated with 200nM anti-TNFR2 antibodies and TNFa was added up to a concentration of lOOnM. As shown in Figure 8, IgGs 30.083 and 30.091 did not affect TNFa- dependent NFKB activation, indicating that these antibodies do not antagonize the TNFR2 receptor, and likely bind the TNFR2 receptor at an epitope that is not blocking the TNFa binding site.

[0131] Taking together, the results shown above demonstrate that the antibodies presented in this disclosure bind tightly to TNFR2 and are expressed in a folded form. However, these antibodies functionally are neither agonist nor antagonist, and they bind the TNF receptor without effecting its signaling.

[0132] While certain features of the anti-TNFR2 antibodies have been illustrated and described herein, many modifications, substitutions, changes, and equivalents will now occur to those of ordinary skill in the art. It is, therefore, to be understood that the appended claims are intended to cover all such modifications and changes as fall within the true spirit of the anti-TNFR2 antibodies.

Claims

CLAIMS What is claimed is:

1. An isolated anti-TNFR2 (Tumor necrosis factor receptor 2) antibody comprising a set of three complementarity determining regions (CDRs) on a heavy chain (HCDR1, HCDR2, and HCDR3) and a set of three CDRs on a light chain (LCDR1, LCDR2, and LCDR3), wherein

(i) the set of HCDR1, HCDR2, and HCDR3 comprises the amino acid sequences of SEQ ID NOs:5-7, and the set of LCDR1, LCDR2, and LCDR3 comprises the amino acid sequences of SEQ ID NOs:8-10; or

(ii) the set of HCDR1, HCDR2, and HCDR3 comprises the amino acid sequences of SEQ ID NOs: 17-19, and the set of LCDR1, LCDR2, and LCDR3 comprises the amino acid sequences of SEQ ID NOs:20-22.

2. The anti-TNFR2 antibody of claim 1, wherein the antibody comprises a heavy chain variable region and a light chain variable region, said heavy chain variable region and light chain variable region comprise the amino acid sequences of SEQ ID NOs:3-4, or SEQ ID NOs:13-14.

3. The anti-TNFR2 antibody of claim 1, wherein the antibody comprises a heavy chain and a light chain, said heavy chain and a light chain comprise the amino acid sequences of SEQ ID NOs: 25-26, or SEQ ID NOs: 23-24.

4. The anti-TNFR2 antibody of any one of claims 1-3, wherein the antibody is an IgG, a Fv, a scFv, a Fab, a F(ab')2, a minibody, a diabody, a triabody, a nanobody, a bispecific antibody, or a single domain antibody.

5. The anti-TNFR2 antibody of claim 4, wherein said IgG is IgGl, IgG2, IgG3, or IgG4.

6. The anti-TNFR2 antibody of claim 1, wherein the antibody has an EC50 binding measurement of about 0.05-100 nM.

7. The anti-TNFR2 antibody of claim 1, wherein the antibody is neither an agonist nor an antagonist of TNFR2.

8. A composition comprising the anti-TNFR2 antibody of any of claims 1-3, and a pharmaceutically acceptable carrier.

9. An isolated polynucleotide sequence encoding the anti-TNFR2 antibody of any one of claims 1-3.

10. A vector comprising the polynucleotide sequence of claim 9.

11. A host cell comprising the vector of claim 10.

12. A method of decreasing the number of regulatory T cells in a subject, comprising the step of administering to the subject a composition comprising an effective amount of the anti- TNFR2 antibody of any one of claims 1-3.

13. The method of claim 12, wherein the regulatory T cells are CD4⁺CD25⁺Foxp3⁺.

14. The method of claim 12, wherein the anti-TNFR2 antibody decreases the number of regulatory T cells by antibody-dependent cellular cytotoxicity.

15. A method of decreasing the number cancer cells in a subject, comprising the step of administering to the subject a composition comprising an effective amount of the anti- TNFR2 antibody of any of claims 1-3.

16. The method of claim 14, wherein the anti-TNFR2 antibody decreases the number of cancer cells by antibody-dependent cellular cytotoxicity.

17. The method of claim 14, wherein the anti-TNFR2 antibody comprises a cytotoxic agent.

18. A method of treating a disease in a subject, comprising the step of administering to the subject a composition comprising an effective amount of the anti-TNFR2 antibody of any of claims 1-3.

19. The method of claim 18, wherein the disease is a cancer, an autoimmune disease, a viral infection, or a bacterial infection.

20. The method of claim 18, wherein the antibody is neither an agonist nor an antagonist of TNFR2.