Antitumor necrosis factor receptor antibody (anti-TNFR2 antibody) and its use

Abstract

The present disclosure provides various anti-TNFR2 antibodies (anti-tumor necrosis factor receptor 2 antibodies). The antibodies of the present disclosure are TNFR2 agonists. In certain cases, the antibodies of the present disclosure agonize TNFR2 in an Fc-independent manner. The antibodies of the present disclosure can modulate the proliferation and / or function of regulatory T cells and myeloid-derived suppressor cells. In certain cases, the antibodies of the present disclosure can be used to treat diseases such as GvHD and autoimmune diseases.

Description

[Technical Field]

[0001] (Cross-reference of related applications) This application claims priority under U.S. Provisional Patent Application No. 63 / 047, 490, filed 2 July 2020. The entire disclosure of the above application is incorporated herein by reference.

[0002] (Statement regarding sequence listings) This application includes a sequence listing submitted electronically in ASCII format, the entirety of which is incorporated herein by reference. The name of this ASCII copy, created on June 28, 2021, is "P-597451-PC_SL.txt", and its size is 573,679 bytes.

[0003] (Technical field) This disclosure generally relates to modified antibodies. In one or more embodiments, this disclosure relates to the manufacture and use of antibodies against tumor necrosis factor receptor 2 (TNFR2). [Background technology]

[0004] TNFα is an extremely multifaceted cytokine. It is produced not only by immune cells such as activated macrophages, T cells, and natural killer (NK) cells, but also by endothelial cells, microglia, cardiomyocytes, and fibroblasts. Once produced, TNFα is presented as membrane-bound mTNFα. Membrane-bound mTNFα is a 26 kDa transmembrane protein that, when cleaved by TNF-converting enzyme (TACE), forms soluble TNFα (sTNFα) released from the membrane.

[0005] TNFα interacts with two receptors: tumor necrosis factor receptor superfamily member 1A (TNFR1) and tumor necrosis factor receptor superfamily member 1B (TNFR2). The extracellular domains of these two TNF receptors share a common structure consisting of four cysteine-rich domains (CRDs) that are involved in binding to TNFα.

[0006] TNFR1 is constitutively expressed in virtually all nuclear cell types. TNFR1 has been reported to bind to and be activated by both sTNFα and mTNFα. One of the main functions of TNFR1 is to mediate TNF-induced apoptosis via the NF-κB pathway. Under certain circumstances, activation of the NF-κB pathway leads to the production of anti-apoptotic proteins and inflammatory cytokines such as IL-1 and IL-6.

[0007] TNFR2 is a recently discovered and characterized receptor that is primarily expressed in activated T cells, myeloid cells, and glial cells. TNFR2 is a 75 kDa transmembrane receptor with an extracellular domain composed of four cysteine-rich domains (CRDs), a single transmembrane domain, and a cytoplasmic domain that interacts with TNF receptor-related factor 2 (TRAF2). TRAF2 recruitment promotes a cascade of events leading to the activation of alternative NF-κB pathways. TNF2 can strongly bind to both sTNFα and mTNFα, and is primarily activated by mTNFα but not by sTNFα.

[0008] TNFR2 is CD4 + CD25 + Foxp3 + TNFR2 has been shown to be highly expressed on the surface of regulatory T cells (Tregs) and myeloid suppressor cells (MDSCs). Activation of TNFR2 in these cells promotes immunosuppressive activity and is crucial for the proliferation and survival of Tregs and MDSCs. In addition, TNFR2 is associated with CD8 + Effector T cells (Teff) play two roles. One role is CD8 during the initial immune response. + Another role is to mediate the activation signal to Teff, and CD8 + The goal is to terminate the immune response by transmitting apoptosis signals in Teff.

[0009] The crucial role of TNFR2 in regulating the immune system is reflected in several pathological conditions. In cancer, TNFR2 is highly expressed in invasive Tregs, MDSCs, and the tumor microenvironment (TME) of tumor cells. Activation of TNFR2 receptors in Tregs and MDSCs suppresses the immune system in the TME. In addition, in vitro blocking of TNFR2 in cancer cell lines has been shown to have a dose-dependent cytotoxic effect.

[0010] In autoimmune diseases, abnormalities in the TNFα signaling pathway have been reported in various autoimmune diseases, including rheumatoid arthritis (RA), Crohn's disease (CD), multiple sclerosis (MS), and type 1 diabetes. In many of these conditions, where TNFR1 is more dominant, blocking most of TNFα in its soluble form is sufficient to produce effective results. TNFα blockers such as infliximab and adalimumab have been successfully used in rheumatoid arthritis (RA), Crohn's disease (CD), and ulcerative colitis (UC). However, in diseases such as type 1 diabetes (T1D) and multiple sclerosis, the above approach has been shown to be particularly ineffective.

[0011] In certain cases, specific inhibition or activation of the TNFR2 pathway has been proven beneficial. For example, in mouse cancer models, specific inhibition of TNFR2 not only acts directly on cancer cells but also exhibits robust effects on immune system activation. TNFR2 is strongly involved as a driving force in several autoimmune diseases in which Tregs and MDCS play a major role. In experimental autoimmune encephalomyelitis (EAE) mouse models of multiple sclerosis (MS), deletion of the TNFR2 gene has been shown to have adverse effects. In graft-versus-host disease (GvHD) models, agonizing TNFR2 has been shown to reduce the severity of GvHD. Furthermore, TNFR2 activation is also involved in novel treatments for type 1 diabetes (T1D) with Bacillus calmette-Guérin (BCG).

[0012] Given that TNFR2 plays a crucial role in regulating immune responses in various pathological conditions, the development of improved targeting of TNFR2 with antagonists or agonists is needed as an improved therapeutic strategy for treating diseases such as cancer and autoimmune diseases. [Overview of the project] [Means for solving the problem]

[0013] This disclosure provides various anti-TNFR2 (tumor necrosis factor receptor 2) antibodies. In one embodiment, each of the anti-TNFR2 antibodies of this disclosure comprises a set of three complementarity-determining regions (CDRs) on the heavy chain (HCDR1, HCDR2, and HCDR3) and a set of three CDRs on the light chain (LCDR1, LCDR2, and LCDR3). In one embodiment, the sets of HCDR1, HCDR2, and HCDR3 comprise the amino acid sequence combinations shown in Table 7 below, and the corresponding sets of LCDR1, LCDR2, and LCDR3 on the light chain comprise the amino acid sequence combinations shown in Table 8 below. In another embodiment, the sets of HCDR1, HCDR2, and HCDR3 on the heavy chain and the corresponding sets of LCDR1, LCDR2, and LCDR3 on the light chain comprise the amino acid sequence combinations shown in Table 8 below.

[0014] [Table 7-1]

[0015] [Table 7-2]

[0016] [Table 7-3]

[0017] [Table 7-4]

[0018] Table 8-1

[0019] Table 8-2

[0020] Table 8-3

[0021] Table 9-1

[0022] Table 9-2

[0023] In one embodiment, each of the anti-TNFR2 antibodies of the present disclosure comprises a heavy chain variable region and a light chain variable region, the amino acid sequences of the heavy chain variable region and the light chain variable region being: SEQ ID NOs: 289 and 290, SEQ ID NOs: 3 and 4, SEQ ID NOs: 7 and 8, SEQ ID NOs: 11 and 12, SEQ ID NOs: 15 and 16, SEQ ID NOs: 19 and 20, SEQ ID NOs: 23 and 24, SEQ ID NOs: 27 and 28, SEQ ID NOs: 31 and 32, SEQ ID NOs: 35 and 36, SEQ ID NOs: 39 and 40, SEQ ID NOs: 43 and 44, SEQ ID NOs: 47 and 48, SEQ ID NOs: 51 and 52, SEQ ID NOs: 55 and 56, SEQ ID NOs: 59 and 60, sequence number Sequence numbers 63 and 64, Sequence numbers 67 and 68, Sequence numbers 71 and 72, Sequence numbers 75 and 76, Sequence numbers 79 and 80, Sequence numbers 83 and 84, Sequence numbers 87 and 88, Sequence numbers 91 and 92, Sequence numbers 95 and 96, Sequence numbers 99 and 100, Sequence numbers 103 and 104, Sequence numbers 107 and 108, Sequence numbers 111 and 112, Sequence numbers 115 and 116, Sequence numbers 119 and 120, Sequence numbers 123 and 124, Sequence numbers 127 and 128, Sequence numbers 131 and 132, Sequence numbers 135 and 136, Sequence numbers 139 and 140, Sequence number 143 and 144, SEQ ID NOs: 147 and 148, SEQ ID NOs: 151 and 152, SEQ ID NOs: 157 and 158, SEQ ID NOs: 163 and 164, SEQ ID NOs: 169 and 170, SEQ ID NOs: 175 and 176, SEQ ID NOs: 181 and 182, SEQ ID NOs: 193 and 194, SEQ ID NOs: 199 and 200, SEQ ID NOs: 205 and 206, SEQ ID NOs: 211 and 212, SEQ ID NOs: 217 and 218, SEQ ID NOs: 223 and 224, SEQ ID NOs: 229 and 230, SEQ ID NOs: 233 and 234, SEQ ID NOs: 237 and 238, SEQ ID NOs: 241 and 242, SEQ ID NOs: 245 and 246, SEQ ID NOs: 2 49 and 250, SEQ ID NOs: 253 and 254, 257 and 258, 261 and 262, 265 and 266, 269 and 270, 273 and 274, 277 and 278, 281 and 282, 285 and 286, 293 and 294, 297 and 298, 301 and 302, 305 and 306, 309 and 310, 313 and 314, 317 and 318, 321 and 322, 325 and 326, and,It has an amino sequence selected from the group consisting of sequence numbers 329 and 330.

[0024] In another embodiment, each of the anti-TNFR2 antibodies of the present disclosure comprises a heavy chain and a light chain, the heavy chain and light chain being sequence numbers 438 and 292, 291 and 292, 235 and 236, 239 and 240, 243 and 244, 247 and 248, 251 and 252, 255 and 256, 259 and 260, 263 and 264, 267 and 268, 271 and 272, 275 and 276, 279 and 280, 283 and 284, 287 and It has an amino sequence selected from the group consisting of 288, SEQ ID NOs: 295 and 296, SEQ ID NOs: 299 and 300, SEQ ID NOs: 303 and 304, SEQ ID NOs: 307 and 308, SEQ ID NOs: 311 and 312, SEQ ID NOs: 315 and 316, SEQ ID NOs: 319 and 320, SEQ ID NOs: 323 and 324, SEQ ID NOs: 327 and 328, SEQ ID NOs: 331 and 332, SEQ ID NOs: 432 and 260, SEQ ID NOs: 433 and 304, SEQ ID NOs: 434 and 308, SEQ ID NOs: 435 and 236, SEQ ID NOs: 436 and 244, SEQ ID NOs: 437 and 284, and SEQ ID NOs: 439 and 296.

[0025] In yet another embodiment, the Disclosure provides a composition comprising a pharmaceutically acceptable carrier and the anti-TNFR2 antibody of the Disclosure.

[0026] This disclosure also provides a polynucleotide sequence encoding the anti-TNFR2 antibody of this disclosure, as well as a vector and host cells comprising such a polynucleotide sequence.

[0027] In further embodiments, the anti-TNFR2 antibody of the present disclosure can be used to modulate the proliferation and / or function of regulatory T cells. In another embodiment, the anti-TNFR2 antibody of the present disclosure can be used to modulate the proliferation and / or function of myeloid-derived immunosuppressive cells.

[0028] In one or more additional embodiments, the anti-TNFR2 antibodies of the present disclosure can be used to treat diseases such as cancer, autoimmune diseases, GvHD, viral infections, or bacterial infections.

[0029] These embodiments and other embodiments of the anti-TNFR2 antibody of this disclosure will be understood from the following description of the drawings and the detailed description of the anti-TNFR2 antibody of this disclosure. The present invention provides, for example, the following items: (Item 1) An isolated anti-TNFR2 antibody (anti-tumor necrosis factor receptor 2 antibody) comprising a set of three CDRs (complementarity-determining regions) on the heavy chain, namely HCDR1, HCDR2, and HCDR3, and a set of three CDRs on the light chain, namely LCDR1, LCDR2, and LCDR3, (i) The set of HCDR1, HCDR2, and HCDR3 on the heavy chain and the corresponding set of LCDR1, LCDR2, and LCDR3 on the light chain contain the amino acid sequences shown in Table 9 below, or (ii) The set of HCDR1, HCDR2, and HCDR3 on the heavy chain contains the amino acid sequence shown in Table 7 below, and the corresponding set of LCDR1, LCDR2, and LCDR3 on the light chain contains the amino acid sequence shown in Table 8 below. Anti-TNFR2 antibody. (Item 2) The anti-TNFR2 antibody described in item 1, The antibody comprises a heavy chain variable region and a light chain variable region. The heavy chain variable region and the light chain variable region are sequence numbers 289 and 290, 3 and 4, 7 and 8, 11 and 12, 15 and 16, 19 and 20, 23 and 24, 27 and 28, 31 and 32, 35 and 36, 39 and 40, 43 and 44, 47 and 48, 51 and 52, 55 and 56, 59 and 60, 63 and 64, 67 and 68, 71 and 72, 75 and 76, and 79 and 80, SEQ ID NOs. 83 and 84, SEQ ID NOs. 87 and 88, SEQ ID NOs. 91 and 92, SEQ ID NOs. 95 and 96, SEQ ID NOs. 99 and 100, SEQ ID NOs. 103 and 104, SEQ ID NOs. 107 and 108, SEQ ID NOs. 111 and 112, SEQ ID NOs. 115 and 116, SEQ ID NOs. 119 and 120, SEQ ID NOs. 123 and 124, SEQ ID NOs. 127 and 128, SEQ ID NOs. 131 and 132, SEQ ID NOs. 135 and 136, SEQ ID NOs. 139 and 140, SEQ ID NOs. 143 and 144, SEQ ID NOs. 147 and 148, SEQ ID NOs. 151 and 152, SEQ ID NOs. 157 and 158, Column numbers 163 and 164, sequence numbers 169 and 170, sequence numbers 175 and 176, sequence numbers 181 and 182, sequence numbers 193 and 194, sequence numbers 199 and 200, sequence numbers 205 and 206, sequence numbers 211 and 212, sequence numbers 217 and 218, sequence numbers 223 and 224, sequence numbers 229 and 230, sequence numbers 233 and 234, sequence numbers 237 and 238, sequence numbers 241 and 242, sequence numbers 245 and 246, sequence numbers 249 and 250, sequence numbers 253 and 254, sequence numbers 257 and 258, sequence numbers 261 and 26 2. An anti-TNFR2 antibody having an amino sequence selected from the group consisting of SEQ ID NOs: 265 and 266, SEQ ID NOs: 269 and 270, SEQ ID NOs: 273 and 274, SEQ ID NOs: 277 and 278, SEQ ID NOs: 281 and 282, SEQ ID NOs: 285 and 286, SEQ ID NOs: 293 and 294, SEQ ID NOs: 297 and 298, SEQ ID NOs: 301 and 302, SEQ ID NOs: 305 and 306, SEQ ID NOs: 309 and 310, SEQ ID NOs: 313 and 314, SEQ ID NOs: 317 and 318, SEQ ID NOs: 321 and 322, SEQ ID NOs: 325 and 326, and SEQ ID NOs: 329 and 330. (Item 3) The anti-TNFR2 antibody described in item 1, The antibody in question contains a heavy chain and a light chain. The heavy chain and the light chain are sequence numbers 438 and 292, 291 and 292, 235 and 236, 239 and 240, 243 and 244, 247 and 248, 251 and 252, 255 and 256, 259 and 260, 263 and 264, 267 and 268, 271 and 272, 275 and 276, 279 and 280, 283 and 284, 287 and 288, 295 and 296, sequence number An anti-TNFR2 antibody having an amino sequence selected from the group consisting of 299 and 300, SEQ ID NOs: 303 and 304, SEQ ID NOs: 307 and 308, SEQ ID NOs: 311 and 312, SEQ ID NOs: 315 and 316, SEQ ID NOs: 319 and 320, SEQ ID NOs: 323 and 324, SEQ ID NOs: 327 and 328, SEQ ID NOs: 331 and 332, SEQ ID NOs: 432 and 260, SEQ ID NOs: 433 and 304, SEQ ID NOs: 434 and 308, SEQ ID NOs: 435 and 236, SEQ ID NOs: 436 and 244, SEQ ID NOs: 437 and 284, and SEQ ID NOs: 439 and 296. (Item 4) An anti-TNFR2 antibody described in any of items 1 to 3, The antibodies in question are IgG, Fv, scFv, Fab, and F(ab'). 2 Anti-TNFR2 antibodies, which are mini-bodies, dia-bodies, tri-bodies, nano-bodies, bispecific antibodies, or single-domain antibodies. (Item 5) The anti-TNFR2 antibody described in item 4, The IgG is an anti-TNFR2 antibody, which is IgG1, IgG2, IgG3, or IgG4. (Item 6) The anti-TNFR2 antibody described in item 1, This antibody is an anti-TNFR2 antibody that agonizes TNFR2. (Item 7) The anti-TNFR2 antibody described in item 6, This antibody is an anti-TNFR2 antibody that agonizes TNFR2 in an Fc-independent manner. (Item 8) The anti-TNFR2 antibody described in item 1, This antibody has an EC of approximately 0.05 to 100 nM. 50 Anti-TNFR2 antibody with binding measurement value. (Item 9) The anti-TNFR2 antibody described in item 1, This antibody has an EC of approximately 0.05 to 100 nM. 50 Anti-TNFR2 antibody with functional agonism measurement capabilities. (Item 10) The anti-TNFR2 antibody described in item 1, An anti-TNFR2 antibody that activates TNFR2-mediated signaling at at least 80% of the level compared to signaling activated by TNF. (Item 11) The anti-TNFR2 antibody described in item 1, An anti-TNFR2 antibody that activates TNFR2-mediated signaling at at least 95% of the level compared to signaling activated by TNF. (Item 12) An anti-TNFR2 antibody described in any of items 1-3, A composition comprising a pharmaceutically acceptable carrier. (Item 13) An isolated polynucleotide sequence encoding an anti-TNFR2 antibody as described in any of items 1-3. (Item 14) A vector containing the polynucleotide sequence described in item 13. (Item 15) Host cells containing the vector described in item 14. (Item 16) A method for regulating the activity or function of regulatory T cells in a subject, A method comprising the step of administering to a subject a composition containing an effective amount of an anti-TNFR2 antibody described in any of items 1 to 3. (Item 17) The method described in item 16, The regulatory T cells are CD4 + CD25 + Foxp3 + The method. (Item 18) The method described in item 16, A method for stimulating the proliferation of the aforementioned regulatory T cells. (Item 19) The method described in item 16, A method for activating the aforementioned regulatory T cells. (Item 20) The method described in item 16, A method for regulating the immune response mediated by the regulatory T cells. (Item 21) A method for regulating the activity or function of myeloid-derived immunosuppressive cells (MDSCs) in a subject, A method comprising the step of administering to a subject a composition containing an effective amount of an anti-TNFR2 antibody described in any of items 1 to 3. (Item 22) The method described in item 21, A method for stimulating the proliferation of myeloid-derived immunosuppressive cells (MDSCs). (Item 23) The method described in item 21, A method for activating the aforementioned myeloid-derived immunosuppressive cells (MDSCs). (Item 24) A method for treating a disease in the subject, A method comprising the step of administering to a subject a composition containing an effective amount of an anti-TNFR2 antibody described in any of items 1 to 3. (Item 25) The method described in item 24, The disease is cancer, autoimmune disease, GvHD, viral infection, or bacterial infection, by means of. [Brief explanation of the drawing]

[0030] In this specification, several embodiments of the anti-TNFR2 antibody and its use are described merely as examples with reference to the accompanying drawings. A detailed reference to the drawings hereby emphasizes that the details shown are illustrative and are for illustrative purposes only, to illustrate the anti-TNFR2 antibody and its uses. In this regard, the description made with reference to the drawings will make it clear to those skilled in the art how the anti-TNFR2 antibody and its uses can be carried out.

[0031] [Figure 1A] Figures 1A to 1D show the yeast surface display (YSD) EC50 (binding) to TNFR2-His of selected clones. Markers in Figure 1A: CID_327 (circle), CID_329 (square), CID_330 (upward triangle), CID_326 (downward triangle), CID_325 (diamond), CID_324 (star), CID_323 (asterisk), CID_328 (ring). [Figure 1B] Figure 1B shows the results for CID_251. [Figure 1C] Figure 1C shows the results for CID_436. [Figure 1D] Figure 1D shows the results for CID_437. [Figure 2A]Figures 2A to 2R show the results of size exclusion chromatography (SEC) analysis of IgG clones. The retention time of each IgG was monitored at a wavelength of 280 nm. Samples were analyzed under the following conditions: A 100 mg IgG sample was eluted at a rate of 0.8 ml / min using a Superdex® 200 10 / 300 pg column with PBS as the mobile phase (Figures 2A to 2F). The axes in Figures 2A to 2F represent Abs. 280 nm versus volume (ml). A 12 mg IgG sample was eluted at a rate of 0.5 ml / min using a BioResolve SEC mAb column with PBS as the mobile phase (Figures 2G to 2R). The axes in Figures 2G to 2R represent absorbance units (AU) versus minutes. Figure 2A shows the analysis results for IgG clone 30.086. [Figure 2B] Figure 2B shows the analysis results for IgG clone 30.095. [Figure 2C] Figure 2C shows the analysis results for IgG clone 30.116. [Figure 2D] Figure 2D shows the analysis results for IgG clone 30.111. [Figure 2E] Figure 2E shows the analysis results for IgG clone 30.119. [Figure 2F] Figure 2F shows the analysis results for IgG clone 30.123. [Figure 2G] Figure 2G shows the analysis results for IgG clone 30.204. [Figure 2H] Figure 2H shows the analysis results for IgG clone 30.116. [Figure 2I] Figure 2I shows the analysis results for IgG clone 30.202. [Figure 2J] Figure 2J shows the analysis results for IgG clone 30.202. [Figure 2K] Figure 2K shows the analysis results for IgG clone 30.203. [Figure 2L] Figure 2L shows the analysis results for IgG clone 30.115. [Figure 2M] Figure 2M shows the analysis results for IgG clone 30,200. [Figure 2N]Figure 2N shows the analysis results for IgG clone 30.201. [Figure 2O] Figure 20 shows the analysis results for IgG clone 30.114. [Figure 2P] Figure 2P shows the analysis results for IgG clone 30.117. [Figure 2Q] Figure 2Q shows the analysis results for IgG clone 30.122. [Figure 2R] Figure 2R shows the analysis results for IgG clone 30.118. [Figure 3A] Figures 3A and 3B show the IgG binding specificity of selected clones to TNFR2 (Figure 3A shows data for clones 30.092, 30.085, and 30.089, and Figure 3B shows data for clones 30.086, 30.116, 30.093, 30.117, 30.094, 30.118, 30.095, 30.119, 30.109, 30.111, 30.113, and 30.114). Wells were coated with 50 ng / well of IgG and tested for binding to 100 nM TNFR1 or TNFR2 (Figure 3A). [Figure 3B] NC is a negative control, and human anti-IL-2IgG1 (50 ng / well) does not bind to either TNFR1 or TNFR2. LALA_N.C is a negative control, and human anti-IL-2IgG1 (50 ng / well) with an LALA mutation in the Fc region does not bind to either TNFR1 or TNFR2. PC is a positive control for TNFR1 and TNFR2, and the wells were coated with TNFα-Fc (50 ng / well) to test the binding of 100 nM TNFR2 or TNFR1 to TNFα (Figure 3B). [Figure 4A] Figures 4A to 4F show ELISA EC50 binding of selected IgG1 to TNFR2. Figure 4A: IgG clones 30.080 (circle), 30.081 (square), and 30.084 (downward triangle). [Figure 4B] Figure 4B: IgG clones 30.085 (circle), 30.087 (upward-pointing triangle), 30.088 (downward-pointing triangle), 30.089 (rhombus), and 30.032 (quadrilateral). [Figure 4C] Figure 4C: IgG clones 30.092 (square), 30.046 (circle). [Figure 4D] Figure 4D: IgG 30.086 (square), 30.116 (triangle), isotype control antibody (IC; circle). [Figure 4E] Figure 4E: IgG clones 30.093 (square), 30.117 (circle), 30.094 (upward-pointing triangle), 30.118 (small hexagon), 30.095 (asterisk), 30.119 (large hexagon). [Figure 4F] Figure 4F: IgG clones 30.109 (circle), 30.111 (square), 30.113 (upward-pointing triangle), 30.114 (downward-pointing triangle). [Figure 5] Figure 5 shows the expression of human TNFR2 by HEK-TNFR2 cells. 1 × 10⁶ cells were collected and lysed in Tris lysis buffer (TLB) or RIPA lysis buffer. Protein concentration was measured by the Bradford method, and 22 ug of protein cell lysates were subjected to Western blotting to detect TNFR2 and GAPDH as a control. The two lanes on the left are untransfected HEK-Blue(TM) Null cells, and the two lanes on the right are HEK-Blue(TM) Null cells transfected with the pCDNA3.1 plasmid encoding human TNFR2. [Figure 6A] Figures 6A–6C show the identification of TNFα-reactive clones. Figure 6A shows a schematic diagram of the selection process. Briefly, a parent plate of a single clone was replicated, and TNFα-Fc was added to the replicated plate. Soluble embryonic alkaline phosphatase (SEAP) activity was then measured in both plates using QB reagent. Cells that responded only to TNFα addition and showed a SEAP signal were selected. [Figure 6B] Figure 6B shows photographs of the SEAP activity color of selected clones with and without TNFα addition. [Figure 6C] Figure 6C shows the quantification of the colorimetric reaction of the QB reagent at OD655 of the selected clones. [Figure 7A] Figures 7A and 7B show the activation of the TNFR2-TNF-dependent NFκB pathway. Figure 7A shows the dose-response of clone G6 to TNFα-Fc (circle). IC is the isotype control antibody (upward triangle). OD620 values ​​are shown. [Figure 7B] Figure 7B shows that the activation of TNFα (circle) is inhibited by the addition of soluble TNFR2-Fc (asterisk). The OD620 value is shown. [Figure 8A] Figures 8A–8G show the EC50 of functional TNFR2 agonists with the indicated antibodies, incubated with the HEK293-TNFR2 cell line, which harbors soluble embryonic alkaline phosphatase (SEAP) under the control of an NFκB-regulated promoter. IC is the isotype control antibody for IgG, and NC is the isotype control antibody for IgG-LALA. Figure 8A: IgG clones 30.032 (circle), 30.085 (square), 30.087 (downward triangle), 30.088 (diamond). [Figure 8B] Figure 8B: IgG clones 30.046 (circle), 30.092 (square), 30.093 (upward-pointing triangle), 30.094 (downward-pointing triangle). [Figure 8C] Figure 8C: IC (circle), IgG clones 30.109 (square), 30.111 (upward-pointing triangle), 30.113 (downward-pointing triangle), 30.114 (rhombus). [Figure 8D] Figure 8D: IC (circle), IgG clones 30.086 (square), 30.116 (upward-pointing triangle), 30.117 (downward-pointing triangle), 30.118 (rhombus), 30.119 (hexagon). [Figure 8E] Figure 8E: IgG clones 30.200 (square), 30.201 (circle), 30.122 (upward-pointing triangle), NC (downward-pointing triangle). [Figure 8F] Figure 8F: IgG clones 30.115 (square), 30.202 (circle), 30.203 (upward-pointing triangle), 30.204 (rhombus), NC (downward-pointing triangle). [Figure 8G]Figure 8G: IgG clone 30.123 (square), NC negative control (downward triangle). [Figure 9A] Figures 9A to 9C show that antibody-dependent activation of the NFκB regulatory promoter is specific to TNFR2, and TNFR1 is not agonized by the TNFR2-specific antibody. Representative flow cytometry labeling of HEK-TNFR2 and HEK-TNFR1 cell lines shows that only HEK-TNFR2 is labeled by the 30,116-anti-TNFR2 antibody (Figure 9A). [Figure 9B] As shown in Figures 9B to 9C, 1 mM antibody clones 30.113, 30.114, 30.115, 30.116, 30.117, 30.118, 30.200, 30.201, 30.116, 30.119, 30.123, 30.202, 30.203, 30.204, or IgG-LALA isotype control NC17069 did not activate the TNFR1-dependent NFκB pathway, while 11 nM human TNFα did activate the TNFR1-dependent NFκB pathway. [Figure 9C] As shown in Figures 9B to 9C, 1 mM antibody clones 30.113, 30.114, 30.115, 30.116, 30.117, 30.118, 30.200, 30.201, 30.116, 30.119, 30.123, 30.202, 30.203, 30.204, or IgG-LALA isotype control NC17069 did not activate the TNFR1-dependent NFκB pathway, while 11 nM human TNFα did activate the TNFR1-dependent NFκB pathway. [Figure 10A] Figures 10A–10D show the effect of TNFα on antibody-dependent TNFR2 activation of selected clones. HEK-TNFR2 reporter cells were incubated with 200 nM soluble anti-TNFR2 antibody (square) for 1 hour, followed by the addition of 0.05 nM–100 nM TNFα (circle). Isotype control antibody (IC; upward triangle). [Figure 10B]Figures 10A–10D show the effect of TNFα on antibody-dependent TNFR2 activation of selected clones. HEK-TNFR2 reporter cells were incubated with 200 nM soluble anti-TNFR2 antibody (square) for 1 hour, followed by the addition of 0.05 nM–100 nM TNFα (circle). Isotype control antibody (IC; upward triangle). [Figure 10C] Figures 10A–10D show the effect of TNFα on antibody-dependent TNFR2 activation of selected clones. HEK-TNFR2 reporter cells were incubated with 200 nM soluble anti-TNFR2 antibody (square) for 1 hour, followed by the addition of 0.05 nM–100 nM TNFα (circle). Isotype control antibody (IC; upward triangle). [Figure 10D] Figures 10A–10D show the effect of TNFα on antibody-dependent TNFR2 activation of selected clones. HEK-TNFR2 reporter cells were incubated with 200 nM soluble anti-TNFR2 antibody (square) for 1 hour, followed by the addition of 0.05 nM–100 nM TNFα (circle). Isotype control antibody (IC; upward triangle). [Modes for carrying out the invention]

[0032] This disclosure provides a panel of high-affinity antibody agonists capable of agonizing the TNFR2 receptor in an Fc-independent manner, which could potentially be used to treat patients with medical conditions requiring (or benefiting from) activation of the TNFR2 pathway. As used herein, “agonist antibody” of the TNFR2 receptor means an antibody capable of essentially mimicking the activity of a natural ligand. Agonist activity can result in antibody-mediated agonism by the antibody binding to the TNF receptor in a manner that mimics the binding of a physiological ligand. As used herein, the terms “natural ligand” and “common ligand” have the exact same meaning and properties and are used interchangeably.

[0033] In some embodiments, the agonist antibodies of this disclosure mimic the activity of a native ligand. In some embodiments, the agonist antibodies of this disclosure completely replace the activity of a conventional ligand. In some embodiments, the agonist antibodies of this disclosure specifically activate the TNFR2 receptor. In some embodiments, the agonist antibodies of this disclosure completely replace the activity of a conventional ligand. In some embodiments, the agonist antibodies of this disclosure replace the activity of a conventional ligand and specifically activate the TNFR2 receptor.

[0034] In some embodiments, 100% of the activity of the normal ligand is substituted compared to the activity in the presence of the natural ligand. In some embodiments, 50-100% of the activity of the normal ligand is substituted compared to the activity in the presence of the natural ligand. In some embodiments, 75-100% of the activity of the normal ligand is substituted compared to the activity in the presence of the natural ligand.

[0035] As used herein, the terms "comprises, comprising, includes, including" or "having" and their conjugations mean "including, but not limited to, ~".

[0036] As used herein, the singular forms “a,” “an,” and “the” also include the plural form of the object they refer to, unless the context clearly indicates otherwise. For example, the term “one antibody” or “at least one antibody” may include multiple antibodies, including any combination thereof.

[0037] Throughout this application, various embodiments of the invention may be described in range form. It should be understood that the use of range form is solely for convenience and brevity and should not be interpreted as an inflexible limitation on the scope of the invention. Therefore, a range form should be considered to specifically disclose not only the individual numerical values ​​within that range, but also all possible partial ranges within that range. For example, a range description of 1 to 6 should be considered to specifically disclose the partial ranges of 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, ..., as well as the individual numerical values ​​within that range (e.g., 1, 2, 3, 4, 5, and 6). This applies regardless of the width of the range.

[0038] In this specification, when a numerical range is specified, it always means that all numbers (fractions or integers) within the specified numerical range are included. The expressions "range between" the first and second specified numbers, and "range from" the first specified number to the second specified number, are used interchangeably in this specification and mean that the first and second specified numbers, as well as all fractions and integers between them, are included.

[0039] When a value is expressed as an approximation, the use of "approximately" will be understood to mean that the particular value forms another embodiment. All ranges are inclusive and can be combined. In one embodiment, the term "approximately" refers to a deviation of 0.1 to 5% from the given number or range. In another embodiment, the term "approximately" refers to a deviation of 1 to 10% from the given number or range. In yet another embodiment, the term "approximately" refers to a deviation of up to 20% from the given number or range. In one embodiment, the term "approximately" refers to a deviation of ±10% from the given number or range. In yet another embodiment, the term "approximately" refers to a deviation of ±5% from the given number or range.

[0040] Unless otherwise defined, all technical and / or scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which the present invention pertains. Methods and materials that may be used to carry out or test embodiments of the anti-TNFR2 antibody and its uses according to the present invention are described below, but similar or equivalent methods and materials as those described herein may also be used. In the event of any conflict, including definitions, the foregoing shall prevail. In addition, materials, methods and examples are for illustrative purposes only and are not intended to be limiting. Each of the documents or other publications referenced herein is incorporated herein by reference in their entirety.

[0041] The descriptions presented herein describe the steps for the manufacture and use of anti-TNFR2 antibodies and their variations. This description is not intended to be limiting, and it should be understood that changes in components, the order of steps, and other variations are within the scope of the anti-TNFR2 antibodies, their manufacturing methods, and their uses as described herein.

[0042] For clarity, it should be understood that certain features of anti-TNFR2 antibodies and their uses described in the context of separate embodiments may be provided in combination in a single embodiment. Conversely, for brevity, various features of anti-TNFR2 antibodies and their uses described in the context of a single embodiment may be provided separately or in any suitable subcombination in any other embodiment of anti-TNFR2 antibodies and their uses. Certain features described in the context of various embodiments should not be considered essential features of those embodiments unless the embodiment is inoperable without those elements.

[0043] As used herein, the term “antibody” has the exact same properties and meaning as the term “immunoglobulin” and is used interchangeably. The antibody-binding domain or antigen-binding site may be a fragment of an antibody, or a genetically modified product of one or more fragments of an antibody, that is involved in specific binding to a target antigen. “Specific binding” means that it binds selectively to the antigen of interest and can be distinguished from unwanted or nonspecific interactions. For example, if the equilibrium dissociation constant is 10 -5 M or less, 10 -6 M or less, or 10 -7 When the M value is less than or equal to M, the antibody is said to bind specifically to the IL-2 epitope. In some embodiments, the equilibrium dissociation constant is 10 -8 M or less or 10 -9 It may be less than or equal to M. In some further embodiments, the equilibrium dissociation constant is 10 -10 M or less, 10 -11 M or less, or 10 -12 It may be less than or equal to M. In some embodiments, the equilibrium dissociation constant is 10 -5 M~10 -12 It may be within the range of M or less.

[0044] 50% effective concentration (EC 50 ) refers to the concentration at which a drug, antibody, or toxin exhibits 50% of the maximum response from baseline after a specified exposure time. In some embodiments, the reaction includes binding affinity. In some embodiments, the reaction includes a functional reaction, such as an agonist reaction. Those skilled in the art will know the EC of the anti-TNFR2 antibody of this disclosure when used in a particular embodiment. 50 The measurement is the maximum half-amount binding of anti-TNFR2 antibody to the TNFR2 antigen (EC2). 50 It will be understood that this provides measurement of bonding. 50 The measurement of binding affinity involves measuring the binding of the anti-TNFR2 antibody of this disclosure to the TNFR2 antigen, as illustrated in Tables 2 and 5 of the Examples. Those skilled in the art will know that when used in a particular embodiment, the EC of the anti-TNFR2 antibody of this disclosure 50 The measurement is performed using an agonist reaction (EC). 50It will be understood that this provides the measurement of the 50% effective concentration of an anti-TNFR2 antibody to induce functional agonism. 50 Measurement of functional agonism includes measuring the effect of the anti-TNFR2 antibody of this disclosure on cellular signaling of TNFR2, as illustrated in Table 6 of the Examples.

[0045] In some embodiments, EC 50 This includes the concentration of antibody necessary to obtain the 50% agonist reaction that will be observed upon binding of TNFα. In certain embodiments, EC 50 The measurement of EC is commonly used as a measure of drug potency and, in some embodiments, can reflect the binding of antibodies to receptors. In some embodiments, the EC of nanomoles 50 Anti-TNFR2 antibodies with binding concentration measurements include anti-TNFR2 antibodies that bind strongly. In some embodiments, nanomolar EC 50 Anti-TNFR2 antibodies having functional agonism concentration measurement include functionally effective agonist antibodies. In certain embodiments, the anti-TNFR2 antibodies of this disclosure include strong binding to the TNFR2 receptor. In certain embodiments, the anti-TNFR2 antibodies of this disclosure include an agonist of the TNFR2 receptor. In certain embodiments, the anti-TNFR2 antibodies of this disclosure include a strongly binding agonist to the TNFR2 receptor.

[0046] In some embodiments, the conjugation of anti-TNFR2 antibody EC 50 This is in the range of nM (nanomoles). In some embodiments, the conjugated EC of the anti-TNFR2 antibody 50 The EC2 concentration is in the range of approximately 0.05 to 100 nM. In some embodiments, the conjugated EC2 concentration of the anti-TNFR2 antibody is used. 50 The EC2 is in the range of approximately 0.05 to 50 nM. In some embodiments, the conjugated EC2 of the anti-TNFR2 antibody is used. 50 The EC2 is in the range of approximately 0.05 to 20 nM. In some embodiments, the conjugated EC2 of the anti-TNFR2 antibody is used. 50 The EC2 concentration is in the range of approximately 0.05 to 10 nM. In some embodiments, the conjugated EC2 concentration of the anti-TNFR2 antibody is used. 50The EC2 concentration is in the range of approximately 0.1 to 100 nM. In some embodiments, the conjugated EC2 concentration of the anti-TNFR2 antibody is used. 50 The EC2 is in the range of approximately 0.1 to 50 nM. In some embodiments, the conjugated EC2 of the anti-TNFR2 antibody is used. 50 The EC2 is in the range of approximately 0.1 to 20 nM. In some embodiments, the conjugated EC2 of the anti-TNFR2 antibody is used. 50 The EC2 concentration is in the range of approximately 0.1 to 10 nM. In some embodiments, the conjugated EC2 concentration of the anti-TNFR2 antibody is used. 50 The EC2 is in the range of approximately 1 to 100 nM. In some embodiments, the conjugated EC2 of the anti-TNFR2 antibody is used. 50 The EC2 is in the range of approximately 1 to 20 nM. In some embodiments, the conjugated EC2 of the anti-TNFR2 antibody is used. 50 The EC2 concentration is in the range of approximately 20-40 nM. In some embodiments, the conjugated EC2 concentration of the anti-TNFR2 antibody is used. 50 The EC2 is in the range of approximately 40-60 nM. In some embodiments, the conjugated EC2 of the anti-TNFR2 antibody is used. 50 The EC2 concentration is in the range of approximately 60-80 nM. In some embodiments, the conjugated EC2 concentration of the anti-TNFR2 antibody is used. 50 The EC2 is in the range of approximately 80-100 nM. In some embodiments, the conjugated EC2 of the anti-TNFR2 antibody is used. 50 The EC2 is in the range of approximately 1 to 40 nM. In some embodiments, the conjugated EC2 of the anti-TNFR2 antibody is used. 50 The EC2 concentration is in the range of approximately 1 to 60 nM. In some embodiments, the conjugated EC2 concentration of the anti-TNFR2 antibody is used. 50 The EC2 is in the range of approximately 1 to 80 nM. In some embodiments, the conjugated EC2 of the anti-TNFR2 antibody is used. 50 The EC2 is in the range of approximately 1 to 50 nM. In some embodiments, the conjugated EC2 of the anti-TNFR2 antibody is used. 50 The EC2 is in the range of approximately 0.05 to 5 nM. In some embodiments, the conjugated EC2 of the anti-TNFR2 antibody is used. 50 The EC2 concentration is in the range of approximately 0.1 to 5 nM. In some embodiments, the conjugated EC2 concentration of the anti-TNFR2 antibody is used. 50 This range is approximately 0.05 to 20 nM.

[0047] In some embodiments, the conjugation of anti-TNFR2 antibody EC 50The EC2 is in the range of approximately 0.05 to 5 nM. In some embodiments, the conjugated EC2 of the anti-TNFR2 antibody is used. 50 The EC2 concentration is in the range of approximately 0.1 to 5 nM. In some embodiments, the conjugated EC2 concentration of the anti-TNFR2 antibody is used. 50 The EC2 is in the range of approximately 1 to 5 nM. In some embodiments, the conjugated EC2 of the anti-TNFR2 antibody is used. 50 The EC2 concentration is in the range of approximately 0.05 to 10 nM. In some embodiments, the conjugated EC2 concentration of the anti-TNFR2 antibody is used. 50 The EC2 concentration is in the range of approximately 0.1 to 10 nM. In some embodiments, the conjugated EC2 concentration of the anti-TNFR2 antibody is used. 50 The EC2 is in the range of approximately 1 to 10 nM. In some embodiments, the conjugated EC2 of the anti-TNFR2 antibody is used. 50 The EC2 is in the range of approximately 5-10 nM. In some embodiments, the conjugated EC2 of the anti-TNFR2 antibody is used. 50 The EC2 is in the range of approximately 0.05 to 15 nM. In some embodiments, the conjugated EC2 of the anti-TNFR2 antibody is used. 50 The EC2 concentration is in the range of approximately 0.01 to 15 nM. In some embodiments, the conjugated EC2 concentration of the anti-TNFR2 antibody is used. 50 This range is approximately 1 to 15 nM.

[0048] In some embodiments, EC measures functional agonism. 50 In this specification, all functional ECs having the same properties are defined as 50 This is called the functional EC of anti-TNFR2 antibodies. 50 This is in the range of nM (nanomoles). In some embodiments, the functional EC of the anti-TNFR2 antibody 50 The range is approximately 0.05 to 100 nM. In some embodiments, the functional EC of the anti-TNFR2 antibody 50 The range is approximately 0.05 to 50 nM. In some embodiments, the functional EC of the anti-TNFR2 antibody 50 The range is approximately 0.05 to 20 nM. In some embodiments, the functional EC of the anti-TNFR2 antibody 50 The range is approximately 0.05 to 10 nM. In some embodiments, the functional EC of the anti-TNFR2 antibody 50The range is approximately 0.1 to 100 nM. In some embodiments, the functional EC of the anti-TNFR2 antibody 50 The range is approximately 0.1 to 50 nM. In some embodiments, the functional EC of the anti-TNFR2 antibody 50 The range is approximately 0.1 to 20 nM. In some embodiments, the functional EC of the anti-TNFR2 antibody 50 The range is approximately 0.1 to 10 nM. In some embodiments, the functional EC of the anti-TNFR2 antibody 50 The range is approximately 1 to 100 nM. In some embodiments, the functional EC of the anti-TNFR2 antibody 50 The range is approximately 1 to 20 nM. In some embodiments, the functional EC of the anti-TNFR2 antibody 50 The functional EC of the anti-TNFR2 antibody is in the range of approximately 20-40 nM. In some embodiments, the functional EC of the anti-TNFR2 antibody 50 The functional EC of the anti-TNFR2 antibody is in the range of approximately 40-60 nM. In some embodiments, the functional EC of the anti-TNFR2 antibody 50 The functional EC of the anti-TNFR2 antibody is in the range of approximately 60-80 nM. In some embodiments, the functional EC of the anti-TNFR2 antibody 50 The functional EC of the anti-TNFR2 antibody is in the range of approximately 80-100 nM. In some embodiments, the functional EC of the anti-TNFR2 antibody is 50 The range is approximately 1 to 40 nM. In some embodiments, the functional EC of the anti-TNFR2 antibody 50 The range is approximately 1 to 60 nM. In some embodiments, the functional EC of the anti-TNFR2 antibody 50 The range is approximately 1 to 80 nM. In some embodiments, the functional EC of the anti-TNFR2 antibody 50 The range is approximately 1 to 50 nM. In some embodiments, the functional EC of the anti-TNFR2 antibody 50 The range is approximately 0.05 to 5 nM. In some embodiments, the functional EC of the anti-TNFR2 antibody 50 The range is approximately 0.1 to 5 nM. In some embodiments, the functional EC of the anti-TNFR2 antibody 50 This range is approximately 0.05 to 20 nM.

[0049] In some embodiments, functional EC of anti-TNFR2 antibody 50is in the range of about 0.05 to 5 nM. In some embodiments, the functional EC of the anti-TNFR2 antibody 50 is in the range of about 0.1 to 5 nM. In some embodiments, the functional EC of the anti-TNFR2 antibody 50 is in the range of about 1 to 5 nM. In some embodiments, the functional EC of the anti-TNFR2 antibody 50 is in the range of about 0.05 to 10 nM. In some embodiments, the functional EC of the anti-TNFR2 antibody 50 is in the range of about 0.1 to 10 nM. In some embodiments, the functional EC of the anti-TNFR2 antibody 50 is in the range of about 1 to 10 nM. In some embodiments, the functional EC of the anti-TNFR2 antibody 50 is in the range of about 5 to 10 nM. In some embodiments, the functional EC of the anti-TNFR2 antibody 50 is in the range of about 0.05 to 15 nM. In some embodiments, the functional EC of the anti-TNFR2 antibody 50 is in the range of about 0.01 to 15 nM. In some embodiments, the functional EC of the anti-TNFR2 antibody 50 is in the range of about 1 to 15 nM.

[0050] As used herein, the term "antibody" encompasses antibody fragments that retain binding specificity, including but not limited to, for example, IgG, heavy chain variable region (VH), light chain variable region (VL), Fab fragments, F(ab')2 fragments, scFv fragments, Fv fragments, nanobodies, minibodies, diabodies, triabodies, tetrabodies, and single domain antibodies (see, e.g., Hudson and Souriau, Nature Med. 9: 129-134 (2003)). As also generally understood in the art, these terms also encompass humanized antibodies, primatized antibodies, and chimeric antibodies.

[0051] As used herein, the term “heavy chain variable region (VH)” has the exact same meaning and properties as the terms “VH domain” or “VH” and is used interchangeably. As used herein, the term “light chain variable region (VL)” has the exact same meaning and properties as the terms “VL domain” or “VL” and is used interchangeably. Those skilled in the art will recognize that the “heavy chain variable region (VH)” or “VH” in relation to an antibody encompasses a fragment of the heavy chain containing three complementarity-determining regions (CDRs) interposed between adjacent stretches known as framework regions. Framework regions are more conserved than complementarity-determining regions (CDRs) and form a scaffold for supporting the complementarity-determining regions (CDRs). Similarly, those skilled in the art will also recognize that the “light chain variable region (VL)” or “VL” in relation to an antibody also encompasses a fragment of the light chain containing three complementarity-determining regions (CDRs) interposed between framework regions.

[0052] As used herein, the term “complementarity-determining region,” or “CDR,” refers to the hypervariable region of the heavy chain variable region (VH) or the light chain variable region (VL). Starting from the N-terminus, each heavy chain or light chain polypeptide has three complementarity-determining regions (CDRs), denoted as “CDR1,” “CDR2,” and “CDR3.” Crystal structure analysis of various antigen-antibody complexes has revealed that amino acid residues in the complementarity-determining regions (CDRs) form extensive contact with the bound antigen, with the most extensive antigen contact occurring with CDR3 of the heavy chain. Thus, the complementarity-determining regions (CDRs) are the primary factor in the specificity of the antigen-binding site. In one embodiment, the antigen-binding site includes six complementarity-determining regions (CDRs), including those from the heavy chain variable region (VH) and the light chain variable region (VL).

[0053] As used herein, the term “framework region,” or “FR,” refers to four adjacent amino acid sequences that form the complementarity-determining region (CDR) of a heavy chain variable region (VH) or light chain variable region (VL). While some FR residues contact the bound antigen, the FR residues primarily play a role in folding the variable region into the antigen-binding site. In some embodiments, the FR residues responsible for folding the variable region include residues directly adjacent to the complementarity-determining region (CDR). Within the framework region (FR), specific amino residues and specific structural features are highly conserved. In this regard, all variable region sequences contain an internal disulfide loop of approximately 90 amino acid residues. When the variable region is folded into the antigen-binding site, the complementarity-determining region (CDR) is represented as a protruding loop motif that forms the antigen-binding surface. It is generally recognized that, regardless of the exact amino acid sequence of the complementarity-determining region (CDR), there are conserved structural regions of the framework region (FR) that give the folded shape of the complementarity-determining region (CDR) loop a specific "standard" structure. Furthermore, certain FR residues are known to be involved in non-covalent interdomain contacts that stabilize the interaction between the heavy and light chains of antibodies.

[0054] Wu and Kabat were pioneers in antibody peptide sequence alignment, and their contributions to this field were immense ("Wu and Kabat, "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); Kabat et al., "Sequence of proteins of immunological interest", Bethesda: National Institutes of Health; 323 (1983)"). Firstly, by examining the sequence similarities between variable domains, they identified corresponding residues that exhibit more or less homology in all antibodies of all vertebrate species, provided they adopt similar three-dimensional structures, perform similar functional roles, interact similarly with adjacent residues, and exist in similar chemical environments. Secondly, they devised a peptide sequence numbering system that assigns the same positional number to homologous immunoglobulin residues. Those skilled in the art can clearly assign a number now commonly known as Kabat numbering to any variable domain sequence, without relying on experimental data other than the sequence itself. Thirdly, Kabat and Wu calculated variability for each Kabat-numbered sequence position to determine whether there were fewer or more amino acids when the variable domain sequences were aligned. They found that there were three consecutive regions with high variability within four consecutive regions with low variability. Kabat and Wu formally defined the residues that make up these variable regions and named them "complementarity-determining regions" (CDRs) after the chemical complementarity between antibodies and antigens. Variable regions are thought to be involved in three-dimensional folding, although they are not involved in antigen recognition, and are now called "framework regions."Fourth, Kabat and Wu established a public database of antibody peptide and nucleic acid sequences, which is still maintained and well known to those skilled in the art.

[0055] Chothia and his collaborators discovered that certain subregions within the complementarity-determining region (CDR) of Kabat have nearly identical peptide skeletal structures despite significant diversity at the amino acid sequence level ("Cyrus Chothia, Arthur M. Lesk (1987), Journal of Molecular Biology. 196(4): 901-917"). These subregions were named L1, L2, and L3, or H1, H2, and H3, where "L" and "H" represent the light chain and heavy chain regions, respectively. These regions are also known as Chothia's complementarity-determining region (CDR), which has a boundary that overlaps with that of Kabat's CDR.

[0056] Recent studies have revealed that virtually all antibody-binding residues are contained within a structural consensus region ("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 has been shown that these regions can also be identified from antibody sequences. For this purpose, "Paratome," an implementation of a structural approach for identifying the structural consensus of antibodies, was used ("Ofran, Y. et al., J. Immunol. 757:6230-6235 (2008)"). While residues identified by Paratome encompass virtually all antibody-binding sites, complementarity-determining regions (CDRs) (identified by commonly used CDR identification tools) miss a significant portion of them. Antibody-binding residues identified by Paratome but not by common CDR identification methods are called Paratome-unique residues. Similarly, antibody-binding residues that are identified by general complementarity-determining region (CDR) identification methods but not by paratome are called CDR-unique residues. The energetic contribution to antibody-antigen interaction is large for paratome-unique residues but considerably smaller for CDR-unique residues. These results make the identification of antigen-binding sites more reliable.

[0057] IMGT® is an international ImMunoGeneTics® information system (see "Nucleic Acids Res. 2015 Jan; 43 (Database issue):D413-22. doi: 10.1093 / nar / gku1056. Epub 2014 Nov 5 Free article. PMID: 25378316 LIGM:441 and Dev Comp Immunol. 2003 Jan;27(1):55-77"). IMGT is a unique numbering system for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains (see "Lefranc et al., Dev Comp Immunol. 27: 55-77 (2003)"). IMGT® presents a unified numbering system for IG and TcR variable domain sequences, based on the alignment of five or more IG and TcR variable region sequences, and combining Kabat's definitions and structural data of FR and complementarity-determining regions (CDRs), as well as Chothia's characterization of hypervariable loops. IMGT® is considered to be well-known in the art as a universal numbering scheme for antibodies.

[0058] In describing the positions of mutant amino acids in the heavy chain variable region (VH) and light chain variable region (VL) domains, in some embodiments, any common CDR definition, but not limited to, the methods used by IMGT®, KABAT, Clothia, or Paratome, is used to describe the CDR region.

[0059] Antibodies exist in various forms and have various domains, including, but are not limited to, complementarity-determining regions (CDRs), variable regions (Fvs), heavy chain variable region (VH) domains, light chain variable region (VL) domains, single chain variable regions (scFvs), and Fab fragments.

[0060] Those skilled in the art will understand that scFv is a fusion polypeptide comprising a heavy chain variable region (VH) and a light chain variable region (VL) of an immunoglobulin, linked by a short linker peptide. The linker may have, for example, 10 to about 25 amino acids.

[0061] Furthermore, those skilled in the art will understand that the term "Fab" in relation to antibodies generally encompasses a portion of the antibody consisting of a single light chain (both variable and constant regions) bonded by disulfide bonds to a variable region and a first constant region of a single heavy chain, while the term F(ab')2 includes a fragment consisting of a heavy chain having a heavy chain variable region (VH) and a light chain having a light chain variable region (VL) domain.

[0062] In some embodiments, the antibody encompasses the entire antibody molecule, including monoclonal and polyclonal antibodies. In some embodiments, the antibody encompasses, but is not limited to, one or more antibody fragments that maintain binding specificity, such as heavy chain variable region (VH) fragments, light chain variable region (VL) fragments, Fab fragments, F(ab')2 fragments, scFv fragments, Fv fragments, minibodies, diabodies, triabodies, and tetrabodies.

[0063] In one embodiment, the anti-TNFR2 antibody of this disclosure can be incorporated as part of a bispecific antibody. As is commonly known in the art, a bispecific antibody is a recombinant protein comprising antigen-binding fragments of two different monoclonal antibodies, thereby capable of binding to two different antigens. In some embodiments, bispecific antibodies are used in cancer immunotherapy by simultaneously targeting, for example, CTLs (e.g., CTL receptor components such as CD3) or effector natural killer (NK) cells and tumor antigens. Similarly, a multispecific antibody is a recombinant protein comprising antigen-binding fragments of at least two different monoclonal antibodies, such as two, three, or four different monoclonal antibodies.

[0064] Anti-TNFR2 antibody

[0065] This disclosure provides various anti-TNFR2 (tumor necrosis factor receptor 2) antibodies. In one embodiment, each anti-TNFR2 antibody comprises a set of three complementarity-determining regions (CDRs) on the heavy chain (HCDR1, HCDR2, and HCDR3) and a set of three CDRs on the light chain (LCDR1, LCDR2, and LCDR3). In one embodiment, the set of HCDR1, HCDR2, and HCDR3 on the heavy chain comprises the amino acid sequence combination shown in Table 7 below, and the corresponding set of LCDR1, LCDR2, and LCDR3 on the light chain comprises the amino acid sequence combination shown in Table 8 below. For illustrative purposes, taking clone CID251 as an example (see Table 7), the HCDR1, HCDR2, and HCDR3 sets of this antibody contain the amino acid sequences of SEQ ID NOs. 185, 190, and 347, respectively (see Table 7), while the LCDR1, LCDR2, and LCDR3 sets of the same antibody contain the amino acid sequences of SEQ ID NOs. 359, 372, and 378, respectively (see Table 8).

[0066] In some embodiments, the sets of HCDR1, HCDR2, and HCDR3 include the amino acid sequences described in SEQ ID NOs. 185, 190, and 347, respectively, and the sets of LCDR1, LCDR2, and LCDR3 of the same antibody include the amino acid sequences described in SEQ ID NOs. 359, 372, and 378, respectively. In some embodiments, the sets of HCDR1, HCDR2, and HCDR3 include the amino acid sequences described in SEQ ID NOs. 185, 334, and 347, respectively, and the sets of LCDR1, LCDR2, and LCDR3 of the same antibody include the amino acid sequences described in SEQ ID NOs. 360, 372, and 379, respectively. In some embodiments, the sets of HCDR1, HCDR2, and HCDR3 include the amino acid sequences described in SEQ ID NOs. 185, 190, and 347, respectively, and the sets of LCDR1, LCDR2, and LCDR3 of the same antibody include the amino acid sequences described in SEQ ID NOs. 359, 372, and 380, respectively. In some embodiments, the sets of HCDR1, HCDR2, and HCDR3 include the amino acid sequences described in SEQ ID NOs. 185, 190, and 347, respectively, and the sets of LCDR1, LCDR2, and LCDR3 of the same antibody include the amino acid sequences described in SEQ ID NOs. 359, 372, and 381, respectively. In some embodiments, the sets of HCDR1, HCDR2, and HCDR3 include the amino acid sequences described in SEQ ID NOs. 185, 190, and 347, respectively, and the sets of LCDR1, LCDR2, and LCDR3 of the same antibody include the amino acid sequences described in SEQ ID NOs. 361, 372, and 381, respectively. In some embodiments, the sets of HCDR1, HCDR2, and HCDR3 include the amino acid sequences described in SEQ ID NOs. 185, 190, and 347, respectively, and the sets of LCDR1, LCDR2, and LCDR3 of the same antibody include the amino acid sequences described in SEQ ID NOs. 362, 372, and 382, ​​respectively. In some embodiments, the sets of HCDR1, HCDR2, and HCDR3 include the amino acid sequences described in SEQ ID NOs: 185, 335, and 347, respectively, and the sets of LCDR1, LCDR2, and LCDR3 of the same antibody include the amino acid sequences described in SEQ ID NOs: 363, 372, and 383, respectively.In some embodiments, the sets of HCDR1, HCDR2, and HCDR3 include the amino acid sequences described in SEQ ID NOs. 185, 190, and 347, respectively, and the sets of LCDR1, LCDR2, and LCDR3 of the same antibody include the amino acid sequences described in SEQ ID NOs. 364, 372, and 384, respectively. In some embodiments, the sets of HCDR1, HCDR2, and HCDR3 include the amino acid sequences described in SEQ ID NOs. 185, 336, and 348, respectively, and the sets of LCDR1, LCDR2, and LCDR3 of the same antibody include the amino acid sequences described in SEQ ID NOs. 365, 372, and 378, respectively. In some embodiments, the sets of HCDR1, HCDR2, and HCDR3 include the amino acid sequences described in SEQ ID NOs. 185, 337, and 347, respectively, and the sets of LCDR1, LCDR2, and LCDR3 of the same antibody include the amino acid sequences described in SEQ ID NOs. 359, 373, and 385, respectively. In some embodiments, the sets of HCDR1, HCDR2, and HCDR3 include the amino acid sequences described in SEQ ID NOs. 185, 338, and 348, respectively, and the sets of LCDR1, LCDR2, and LCDR3 of the same antibody include the amino acid sequences described in SEQ ID NOs. 359, 372, and 386, respectively. In some embodiments, the sets of HCDR1, HCDR2, and HCDR3 include the amino acid sequences described in SEQ ID NOs. 185, 339, and 349, respectively, and the sets of LCDR1, LCDR2, and LCDR3 of the same antibody include the amino acid sequences described in SEQ ID NOs. 359, 372, and 378, respectively. In some embodiments, the sets of HCDR1, HCDR2, and HCDR3 include the amino acid sequences described in SEQ ID NOs. 185, 190, and 350, respectively, and the sets of LCDR1, LCDR2, and LCDR3 of the same antibody include the amino acid sequences described in SEQ ID NOs. 366, 372, and 387, respectively. In some embodiments, the sets of HCDR1, HCDR2, and HCDR3 include the amino acid sequences described in SEQ ID NOs: 185, 190, and 351, respectively, and the sets of LCDR1, LCDR2, and LCDR3 of the same antibody include the amino acid sequences described in SEQ ID NOs: 359, 372, and 378, respectively.In some embodiments, the sets of HCDR1, HCDR2, and HCDR3 include the amino acid sequences described in SEQ ID NOs. 185, 334, and 347, respectively, and the sets of LCDR1, LCDR2, and LCDR3 of the same antibody include the amino acid sequences described in SEQ ID NOs. 360, 372, and 388, respectively. In some embodiments, the sets of HCDR1, HCDR2, and HCDR3 include the amino acid sequences described in SEQ ID NOs. 185, 334, and 352, respectively, and the sets of LCDR1, LCDR2, and LCDR3 of the same antibody include the amino acid sequences described in SEQ ID NOs. 359, 372, and 378, respectively. In some embodiments, the sets of HCDR1, HCDR2, and HCDR3 include the amino acid sequences described in SEQ ID NOs. 185, 190, and 352, respectively, and the sets of LCDR1, LCDR2, and LCDR3 of the same antibody include the amino acid sequences described in SEQ ID NOs. 359, 372, and 389, respectively. In some embodiments, the sets of HCDR1, HCDR2, and HCDR3 include the amino acid sequences described in SEQ ID NOs. 185, 334, and 347, respectively, and the sets of LCDR1, LCDR2, and LCDR3 of the same antibody include the amino acid sequences described in SEQ ID NOs. 359, 372, and 378, respectively. In some embodiments, the sets of HCDR1, HCDR2, and HCDR3 include the amino acid sequences described in SEQ ID NOs. 185, 190, and 347, respectively, and the sets of LCDR1, LCDR2, and LCDR3 of the same antibody include the amino acid sequences described in SEQ ID NOs. 365, 372, and 378, respectively. In some embodiments, the sets of HCDR1, HCDR2, and HCDR3 include the amino acid sequences described in SEQ ID NOs. 185, 336, and 352, respectively, and the sets of LCDR1, LCDR2, and LCDR3 of the same antibody include the amino acid sequences described in SEQ ID NOs. 365, 372, and 390, respectively. In some embodiments, the sets of HCDR1, HCDR2, and HCDR3 include the amino acid sequences described in SEQ ID NOs: 185, 336, and 352, respectively, and the sets of LCDR1, LCDR2, and LCDR3 of the same antibody include the amino acid sequences described in SEQ ID NOs: 365, 374, and 391, respectively.In some embodiments, the sets of HCDR1, HCDR2, and HCDR3 include the amino acid sequences described in SEQ ID NOs: 186, 340, and 353, respectively, and the sets of LCDR1, LCDR2, and LCDR3 of the same antibody include the amino acid sequences described in SEQ ID NOs: 367, 375, and 392, respectively. In some embodiments, the sets of HCDR1, HCDR2, and HCDR3 include the amino acid sequences described in SEQ ID NOs: 186, 341, and 354, respectively, and the sets of LCDR1, LCDR2, and LCDR3 of the same antibody include the amino acid sequences described in SEQ ID NOs: 367, 375, and 392, respectively. In some embodiments, the sets of HCDR1, HCDR2, and HCDR3 include the amino acid sequences described in SEQ ID NOs: 186, 341, and 354, respectively, and the sets of LCDR1, LCDR2, and LCDR3 of the same antibody include the amino acid sequences described in SEQ ID NOs: 368, 375, and 392, respectively. In some embodiments, the sets of HCDR1, HCDR2, and HCDR3 include the amino acid sequences described in SEQ ID NOs: 186, 342, and 354, respectively, and the sets of LCDR1, LCDR2, and LCDR3 of the same antibody include the amino acid sequences described in SEQ ID NOs: 367, 375, and 392, respectively. In some embodiments, the sets of HCDR1, HCDR2, and HCDR3 include the amino acid sequences described in SEQ ID NOs: 186, 343, and 354, respectively, and the sets of LCDR1, LCDR2, and LCDR3 of the same antibody include the amino acid sequences described in SEQ ID NOs: 369, 375, and 393, respectively. In some embodiments, the sets of HCDR1, HCDR2, and HCDR3 include the amino acid sequences described in SEQ ID NOs: 186, 343, and 355, respectively, and the sets of LCDR1, LCDR2, and LCDR3 of the same antibody include the amino acid sequences described in SEQ ID NOs: 367, 375, and 392, respectively. In some embodiments, the sets of HCDR1, HCDR2, and HCDR3 include the amino acid sequences described in SEQ ID NOs: 186, 341, and 354, respectively, and the sets of LCDR1, LCDR2, and LCDR3 of the same antibody include the amino acid sequences described in SEQ ID NOs: 367, 375, and 392, respectively.In some embodiments, the sets of HCDR1, HCDR2, and HCDR3 include the amino acid sequences described in SEQ ID NOs: 186, 341, and 355, respectively, and the sets of LCDR1, LCDR2, and LCDR3 of the same antibody include the amino acid sequences described in SEQ ID NOs: 367, 376, and 392, respectively. In some embodiments, the sets of HCDR1, HCDR2, and HCDR3 include the amino acid sequences described in SEQ ID NOs: 186, 344, and 354, respectively, and the sets of LCDR1, LCDR2, and LCDR3 of the same antibody include the amino acid sequences described in SEQ ID NOs: 370, 375, and 392, respectively. In some embodiments, the sets of HCDR1, HCDR2, and HCDR3 include the amino acid sequences described in SEQ ID NOs: 186, 341, and 354, respectively, and the sets of LCDR1, LCDR2, and LCDR3 of the same antibody include the amino acid sequences described in SEQ ID NOs: 367, 375, and 394, respectively. In some embodiments, the sets of HCDR1, HCDR2, and HCDR3 include the amino acid sequences described in SEQ ID NOs: 186, 344, and 354, respectively, and the sets of LCDR1, LCDR2, and LCDR3 of the same antibody include the amino acid sequences described in SEQ ID NOs: 367, 375, and 392, respectively. In some embodiments, the sets of HCDR1, HCDR2, and HCDR3 include the amino acid sequences described in SEQ ID NOs: 186, 341, and 359, respectively, and the sets of LCDR1, LCDR2, and LCDR3 of the same antibody include the amino acid sequences described in SEQ ID NOs: 367, 375, and 393, respectively. In some embodiments, the sets of HCDR1, HCDR2, and HCDR3 include the amino acid sequences described in SEQ ID NOs: 187, 345, and 356, respectively, and the sets of LCDR1, LCDR2, and LCDR3 of the same antibody include the amino acid sequences described in SEQ ID NOs: 371, 377, and 395, respectively. In some embodiments, the sets of HCDR1, HCDR2, and HCDR3 include the amino acid sequences described in SEQ ID NOs: 188, 345, and 357, respectively, and the sets of LCDR1, LCDR2, and LCDR3 of the same antibody include the amino acid sequences described in SEQ ID NOs: 371, 377, and 396, respectively.In some embodiments, the sets of HCDR1, HCDR2, and HCDR3 include the amino acid sequences described in SEQ ID NOs: 189, 346, and 358, respectively, and the sets of LCDR1, LCDR2, and LCDR3 of the same antibody include the amino acid sequences described in SEQ ID NOs: 371, 377, and 397, respectively.

[0067] In another embodiment, the anti-TNFR2 antibody comprises a heavy chain CDR sequence and a light chain CDR sequence that are at least 80% (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99% or more) identical to the amino acid sequences described above, not limited to the identity determined using, for example, the BlastP software of the National Center for Biotechnology Information (NCBI) with default parameters.

[0068] Those skilled in the art will understand that the identity percentage (identity %) provides a numerical value indicating how similar a query sequence is to a target sequence (i.e., how many amino acids in each sequence are identical). A higher identity percentage indicates a greater degree of match.

[0069] When used in reference 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. Generally, similarity between two or more polypeptide (or protein) sequences refers to the similarity of the composition, order, or sequence of two or more amino acids of the two or more polypeptides (or proteins).

[0070] In some further embodiments, the sets of HCDR1, HCDR2, and HCDR3 on the heavy chain and the corresponding sets of LCDR1, LCDR2, and LCDR3 on the light chain contain the amino acid sequences shown in Table 9 below. For illustrative purposes, taking clone CID_264 as an example (see Table 9), the sets of HCDR1, HCDR2, and HCDR3 in this antibody contain the amino acid sequences of SEQ ID NO: 402, SEQ ID NO: 345, and SEQ ID NO: 413, respectively (see Table 9), and the sets of LCDR1, LCDR2, and LCDR3 in the same antibody contain the amino acid sequences of SEQ ID NO: 371, SEQ ID NO: 418, and SEQ ID NO: 427, respectively (see Table 9).

[0071] In some embodiments, the sets of HCDR1, HCDR2, and HCDR3 include the amino acid sequences described in SEQ ID NOs. 402, 345, and 413, respectively, and the sets of LCDR1, LCDR2, and LCDR3 of the same antibody include the amino acid sequences described in SEQ ID NOs. 371, 418, and 427, respectively. In some embodiments, the sets of HCDR1, HCDR2, and HCDR3 include the amino acid sequences described in SEQ ID NOs. 398, 346, and 407, respectively, and the sets of LCDR1, LCDR2, and LCDR3 of the same antibody include the amino acid sequences described in SEQ ID NOs. 371, 377, and 419, respectively. In some embodiments, the sets of HCDR1, HCDR2, and HCDR3 include the amino acid sequences described in SEQ ID NOs. 187, 346, and 408, respectively, and the sets of LCDR1, LCDR2, and LCDR3 of the same antibody include the amino acid sequences described in SEQ ID NOs. 371, 377, and 420, respectively. In some embodiments, the sets of HCDR1, HCDR2, and HCDR3 include the amino acid sequences described in SEQ ID NOs: 189, 406, and 409, respectively, and the sets of LCDR1, LCDR2, and LCDR3 of the same antibody include the amino acid sequences described in SEQ ID NOs: 371, 377, and 421, respectively. In some embodiments, the sets of HCDR1, HCDR2, and HCDR3 include the amino acid sequences described in SEQ ID NOs: 187, 345, and 410, respectively, and the sets of LCDR1, LCDR2, and LCDR3 of the same antibody include the amino acid sequences described in SEQ ID NOs: 371, 418, and 422, respectively. In some embodiments, the sets of HCDR1, HCDR2, and HCDR3 include the amino acid sequences described in SEQ ID NOs: 399, 346, and 411, respectively, and the sets of LCDR1, LCDR2, and LCDR3 of the same antibody include the amino acid sequences described in SEQ ID NOs: 371, 418, and 423, respectively. In some embodiments, the sets of HCDR1, HCDR2, and HCDR3 include the amino acid sequences described in SEQ ID NOs: 400, 346, and 412, respectively, and the sets of LCDR1, LCDR2, and LCDR3 of the same antibody include the amino acid sequences described in SEQ ID NOs: 417, 377, and 424, respectively.In some embodiments, the sets of HCDR1, HCDR2, and HCDR3 include the amino acid sequences described in SEQ ID NOs: 401, 346, and 413, respectively, and the sets of LCDR1, LCDR2, and LCDR3 of the same antibody include the amino acid sequences described in SEQ ID NOs: 417, 418, and 425, respectively. In some embodiments, the sets of HCDR1, HCDR2, and HCDR3 include the amino acid sequences described in SEQ ID NOs: 401, 346, and 414, respectively, and the sets of LCDR1, LCDR2, and LCDR3 of the same antibody include the amino acid sequences described in SEQ ID NOs: 417, 418, and 426, respectively. In some embodiments, the sets of HCDR1, HCDR2, and HCDR3 include the amino acid sequences described in SEQ ID NOs: 398, 346, and 415, respectively, and the sets of LCDR1, LCDR2, and LCDR3 of the same antibody include the amino acid sequences described in SEQ ID NOs: 417, 377, and 428, respectively. In some embodiments, the sets of HCDR1, HCDR2, and HCDR3 include the amino acid sequences described in SEQ ID NOs. 403, 346, and 412, respectively, and the sets of LCDR1, LCDR2, and LCDR3 of the same antibody include the amino acid sequences described in SEQ ID NOs. 371, 418, and 429, respectively. In some embodiments, the sets of HCDR1, HCDR2, and HCDR3 include the amino acid sequences described in SEQ ID NOs. 404, 346, and 412, respectively, and the sets of LCDR1, LCDR2, and LCDR3 of the same antibody include the amino acid sequences described in SEQ ID NOs. 371, 418, and 425, respectively. In some embodiments, the sets of HCDR1, HCDR2, and HCDR3 include the amino acid sequences described in SEQ ID NOs. 401, 346, and 414, respectively, and the sets of LCDR1, LCDR2, and LCDR3 of the same antibody include the amino acid sequences described in SEQ ID NOs. 417, 418, and 430, respectively. In some embodiments, the sets of HCDR1, HCDR2, and HCDR3 include the amino acid sequences described in SEQ ID NOs: 405, 406, and 416, respectively, and the sets of LCDR1, LCDR2, and LCDR3 of the same antibody include the amino acid sequences described in SEQ ID NOs: 371, 377, and 431, respectively.

[0072] In another embodiment, the anti-TNFR2 antibody comprises a heavy chain CDR sequence and a light chain CDR sequence that are at least 80% (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99% or more) identical to the amino acid sequences described above, not limited to the identity determined using, for example, the BlastP software of the National Center for Biotechnology Information (NCBI) with default parameters.

[0073] In one embodiment, each of the anti-TNFR2 antibodies disclosed herein comprises a heavy chain variable region (VH) and a light chain variable region (VL), the amino acid sequences of the heavy chain variable region and the light chain variable region may be one of the following pairs: SEQ ID NOs: 289 and 290, SEQ ID NOs: 3 and 4, SEQ ID NOs: 7 and 8, SEQ ID NOs: 11 and 12, SEQ ID NOs: 15 and 16, SEQ ID NOs: 19 and 20, SEQ ID NOs: 23 and 24, SEQ ID NOs: 27 and 28, SEQ ID NOs: 31 and 32, SEQ ID NOs: 35 and 36, SEQ ID NOs: 39 and 40, SEQ ID NOs: 43 and 44, SEQ ID NOs: 47 and 48, SEQ ID NOs: 51 and 52 , SEQ ID NOs. 55 and 56, SEQ ID NOs. 59 and 60, SEQ ID NOs. 63 and 64, SEQ ID NOs. 67 and 68, SEQ ID NOs. 71 and 72, SEQ ID NOs. 75 and 76, SEQ ID NOs. 79 and 80, SEQ ID NOs. 83 and 84, SEQ ID NOs. 87 and 88, SEQ ID NOs. 91 and 92, SEQ ID NOs. 95 and 96, SEQ ID NOs. 99 and 100, SEQ ID NOs. 103 and 104, SEQ ID NOs. 107 and 108, SEQ ID NOs. 111 and 112, SEQ ID NOs. 115 and 116, SEQ ID NOs. 119 and 120, SEQ ID NOs. 123 and 124, SEQ ID NOs. 127 and 128, SEQ ID NOs. 131 and 132, SEQ ID NOs. 135 and 1 36, SEQ ID NOs: 139 and 140, 143 and 144, 147 and 148, 151 and 152, 157 and 158, 163 and 164, 169 and 170, 175 and 176, 181 and 182, 193 and 194, 199 and 200, 205 and 206, 211 and 212, 217 and 218, 223 and 224, 229 and 230, 233 and 234, 237 and 238, 241 and 24 2. Sequence IDs 245 and 246, 249 and 250, 253 and 254, 257 and 258, 261 and 262, 265 and 266, 269 and 270, 273 and 274, 277 and 278, 281 and 282, 285 and 286, 293 and 294, 297 and 298, 301 and 302, 305 and 306, 309 and 310, 313 and 314, 317 and 318, 321 and 322,Sequence IDs 325 and 326, or sequence IDs 329 and 330.

[0074] In some embodiments, the amino acid sequences of the heavy chain variable region and light chain variable region of the anti-TNFR2 antibody disclosed herein are described in SEQ ID NOs: 289 and 290. In some embodiments, the amino acid sequences of the heavy chain variable region and light chain variable region of the anti-TNFR2 antibody disclosed herein are described in SEQ ID NOs: 3 and 4. In some embodiments, the amino acid sequences of the heavy chain variable region and light chain variable region of the anti-TNFR2 antibody disclosed herein are described in SEQ ID NOs: 7 and 8. In some embodiments, the amino acid sequences of the heavy chain variable region and light chain variable region of the anti-TNFR2 antibody disclosed herein are described in SEQ ID NOs: 11 and 12. In some embodiments, the amino acid sequences of the heavy chain variable region and light chain variable region of the anti-TNFR2 antibody disclosed herein are described in SEQ ID NOs: 15 and 16. In some embodiments, the amino acid sequences of the heavy chain variable region and light chain variable region of the anti-TNFR2 antibody disclosed herein are described in SEQ ID NOs: 19 and 20. In some embodiments, the amino acid sequences of the heavy chain variable region and light chain variable region of the anti-TNFR2 antibody disclosed herein are described in SEQ ID NOs: 23 and 24. In some embodiments, the amino acid sequences of the heavy chain variable region and light chain variable region of the anti-TNFR2 antibody disclosed herein are described in SEQ ID NOs: 27 and 28. In some embodiments, the amino acid sequences of the heavy chain variable region and light chain variable region of the anti-TNFR2 antibody disclosed herein are described in SEQ ID NOs: 31 and 32. In some embodiments, the amino acid sequences of the heavy chain variable region and light chain variable region of the anti-TNFR2 antibody disclosed herein are described in SEQ ID NOs: 35 and 36. In some embodiments, the amino acid sequences of the heavy chain variable region and light chain variable region of the anti-TNFR2 antibody disclosed herein are described in SEQ ID NOs: 39 and 40. In some embodiments, the amino acid sequences of the heavy chain variable region and light chain variable region of the anti-TNFR2 antibody disclosed herein are described in SEQ ID NOs: 43 and 44. In some embodiments, the amino acid sequences of the heavy chain variable region and light chain variable region of the anti-TNFR2 antibody disclosed herein are described in SEQ ID NOs: 47 and 48.In some embodiments, the amino acid sequences of the heavy chain variable region and light chain variable region of the anti-TNFR2 antibody disclosed herein are described in SEQ ID NOs. 51 and 52. In some embodiments, the amino acid sequences of the heavy chain variable region and light chain variable region of the anti-TNFR2 antibody disclosed herein are described in SEQ ID NOs. 55 and 56. In some embodiments, the amino acid sequences of the heavy chain variable region and light chain variable region of the anti-TNFR2 antibody disclosed herein are described in SEQ ID NOs. 59 and 60. In some embodiments, the amino acid sequences of the heavy chain variable region and light chain variable region of the anti-TNFR2 antibody disclosed herein are described in SEQ ID NOs. 63 and 64. In some embodiments, the amino acid sequences of the heavy chain variable region and light chain variable region of the anti-TNFR2 antibody disclosed herein are described in SEQ ID NOs. 67 and 68. In some embodiments, the amino acid sequences of the heavy chain variable region and light chain variable region of the anti-TNFR2 antibody disclosed herein are described in SEQ ID NOs. 71 and 72. In some embodiments, the amino acid sequences of the heavy chain variable region and light chain variable region of the anti-TNFR2 antibody disclosed herein are described in SEQ ID NOs. 75 and 76. In some embodiments, the amino acid sequences of the heavy chain variable region and light chain variable region of the anti-TNFR2 antibody disclosed herein are described in SEQ ID NOs: 79 and 80. In some embodiments, the amino acid sequences of the heavy chain variable region and light chain variable region of the anti-TNFR2 antibody disclosed herein are described in SEQ ID NOs: 83 and 84. In some embodiments, the amino acid sequences of the heavy chain variable region and light chain variable region of the anti-TNFR2 antibody disclosed herein are described in SEQ ID NOs: 87 and 88. In some embodiments, the amino acid sequences of the heavy chain variable region and light chain variable region of the anti-TNFR2 antibody disclosed herein are described in SEQ ID NOs: 91 and 92. In some embodiments, the amino acid sequences of the heavy chain variable region and light chain variable region of the anti-TNFR2 antibody disclosed herein are described in SEQ ID NOs: 95 and 96. In some embodiments, the amino acid sequences of the heavy chain variable region and light chain variable region of the anti-TNFR2 antibody disclosed herein are described in SEQ ID NOs: 99 and 100.In some embodiments, the amino acid sequences of the heavy chain variable region and light chain variable region of the anti-TNFR2 antibody disclosed herein are described in SEQ ID NOs: 103 and 104. In some embodiments, the amino acid sequences of the heavy chain variable region and light chain variable region of the anti-TNFR2 antibody disclosed herein are described in SEQ ID NOs: 107 and 108. In some embodiments, the amino acid sequences of the heavy chain variable region and light chain variable region of the anti-TNFR2 antibody disclosed herein are described in SEQ ID NOs: 111 and 112. In some embodiments, the amino acid sequences of the heavy chain variable region and light chain variable region of the anti-TNFR2 antibody disclosed herein are described in SEQ ID NOs: 115 and 116. In some embodiments, the amino acid sequences of the heavy chain variable region and light chain variable region of the anti-TNFR2 antibody disclosed herein are described in SEQ ID NOs: 119 and 120. In some embodiments, the amino acid sequences of the heavy chain variable region and light chain variable region of the anti-TNFR2 antibody disclosed herein are described in SEQ ID NOs: 123 and 124. In some embodiments, the amino acid sequences of the heavy chain variable region and light chain variable region of the anti-TNFR2 antibody disclosed herein are described in SEQ ID NOs: 127 and 128. In some embodiments, the amino acid sequences of the heavy chain variable region and light chain variable region of the anti-TNFR2 antibody disclosed herein are described in SEQ ID NOs: 131 and 132. In some embodiments, the amino acid sequences of the heavy chain variable region and light chain variable region of the anti-TNFR2 antibody disclosed herein are described in SEQ ID NOs: 135 and 136. In some embodiments, the amino acid sequences of the heavy chain variable region and light chain variable region of the anti-TNFR2 antibody disclosed herein are described in SEQ ID NOs: 139 and 140. In some embodiments, the amino acid sequences of the heavy chain variable region and light chain variable region of the anti-TNFR2 antibody disclosed herein are described in SEQ ID NOs: 143 and 144. In some embodiments, the amino acid sequences of the heavy chain variable region and light chain variable region of the anti-TNFR2 antibody disclosed herein are described in SEQ ID NOs: 147 and 148. In some embodiments, the amino acid sequences of the heavy chain variable region and light chain variable region of the anti-TNFR2 antibody disclosed herein are described in SEQ ID NOs: 151 and 152.In some embodiments, the amino acid sequences of the heavy chain variable region and light chain variable region of the anti-TNFR2 antibody disclosed herein are described in SEQ ID NOs: 157 and 158. In some embodiments, the amino acid sequences of the heavy chain variable region and light chain variable region of the anti-TNFR2 antibody disclosed herein are described in SEQ ID NOs: 163 and 164. In some embodiments, the amino acid sequences of the heavy chain variable region and light chain variable region of the anti-TNFR2 antibody disclosed herein are described in SEQ ID NOs: 169 and 170. In some embodiments, the amino acid sequences of the heavy chain variable region and light chain variable region of the anti-TNFR2 antibody disclosed herein are described in SEQ ID NOs: 175 and 176. In some embodiments, the amino acid sequences of the heavy chain variable region and light chain variable region of the anti-TNFR2 antibody disclosed herein are described in SEQ ID NOs: 181 and 182. In some embodiments, the amino acid sequences of the heavy chain variable region and light chain variable region of the anti-TNFR2 antibody disclosed herein are described in SEQ ID NOs: 193 and 194. In some embodiments, the amino acid sequences of the heavy chain variable region and light chain variable region of the anti-TNFR2 antibody disclosed herein are described in SEQ ID NOs: 199 and 200. In some embodiments, the amino acid sequences of the heavy chain variable region and light chain variable region of the anti-TNFR2 antibody disclosed herein are described in SEQ ID NOs: 205 and 206. In some embodiments, the amino acid sequences of the heavy chain variable region and light chain variable region of the anti-TNFR2 antibody disclosed herein are described in SEQ ID NOs: 211 and 212. In some embodiments, the amino acid sequences of the heavy chain variable region and light chain variable region of the anti-TNFR2 antibody disclosed herein are described in SEQ ID NOs: 217 and 218. In some embodiments, the amino acid sequences of the heavy chain variable region and light chain variable region of the anti-TNFR2 antibody disclosed herein are described in SEQ ID NOs: 223 and 224. In some embodiments, the amino acid sequences of the heavy chain variable region and light chain variable region of the anti-TNFR2 antibody disclosed herein are described in SEQ ID NOs: 229 and 230. In some embodiments, the amino acid sequences of the heavy chain variable region and light chain variable region of the anti-TNFR2 antibody disclosed herein are described in SEQ ID NOs. 233 and 234.In some embodiments, the amino acid sequences of the heavy chain variable region and light chain variable region of the anti-TNFR2 antibody disclosed herein are described in SEQ ID NOs: 237 and 238. In some embodiments, the amino acid sequences of the heavy chain variable region and light chain variable region of the anti-TNFR2 antibody disclosed herein are described in SEQ ID NOs: 241 and 242. In some embodiments, the amino acid sequences of the heavy chain variable region and light chain variable region of the anti-TNFR2 antibody disclosed herein are described in SEQ ID NOs: 245 and 246. In some embodiments, the amino acid sequences of the heavy chain variable region and light chain variable region of the anti-TNFR2 antibody disclosed herein are described in SEQ ID NOs: 249 and 250. In some embodiments, the amino acid sequences of the heavy chain variable region and light chain variable region of the anti-TNFR2 antibody disclosed herein are described in SEQ ID NOs: 253 and 254. In some embodiments, the amino acid sequences of the heavy chain variable region and light chain variable region of the anti-TNFR2 antibody disclosed herein are described in SEQ ID NOs: 257 and 258. In some embodiments, the amino acid sequences of the heavy chain variable region and light chain variable region of the anti-TNFR2 antibody disclosed herein are described in SEQ ID NOs: 261 and 262. In some embodiments, the amino acid sequences of the heavy chain variable region and light chain variable region of the anti-TNFR2 antibody disclosed herein are described in SEQ ID NOs: 265 and 266. In some embodiments, the amino acid sequences of the heavy chain variable region and light chain variable region of the anti-TNFR2 antibody disclosed herein are described in SEQ ID NOs: 269 and 270. In some embodiments, the amino acid sequences of the heavy chain variable region and light chain variable region of the anti-TNFR2 antibody disclosed herein are described in SEQ ID NOs: 273 and 274. In some embodiments, the amino acid sequences of the heavy chain variable region and light chain variable region of the anti-TNFR2 antibody disclosed herein are described in SEQ ID NOs: 277 and 278. In some embodiments, the amino acid sequences of the heavy chain variable region and light chain variable region of the anti-TNFR2 antibody disclosed herein are described in SEQ ID NOs: 281 and 282. In some embodiments, the amino acid sequences of the heavy chain variable region and light chain variable region of the anti-TNFR2 antibody disclosed herein are described in SEQ ID NOs. 285 and 286.In some embodiments, the amino acid sequences of the heavy chain variable region and light chain variable region of the anti-TNFR2 antibody disclosed herein are described in SEQ ID NOs: 293 and 294. In some embodiments, the amino acid sequences of the heavy chain variable region and light chain variable region of the anti-TNFR2 antibody disclosed herein are described in SEQ ID NOs: 297 and 298. In some embodiments, the amino acid sequences of the heavy chain variable region and light chain variable region of the anti-TNFR2 antibody disclosed herein are described in SEQ ID NOs: 301 and 302. In some embodiments, the amino acid sequences of the heavy chain variable region and light chain variable region of the anti-TNFR2 antibody disclosed herein are described in SEQ ID NOs: 305 and 306. In some embodiments, the amino acid sequences of the heavy chain variable region and light chain variable region of the anti-TNFR2 antibody disclosed herein are described in SEQ ID NOs: 309 and 310. In some embodiments, the amino acid sequences of the heavy chain variable region and light chain variable region of the anti-TNFR2 antibody disclosed herein are described in SEQ ID NOs: 313 and 314. In some embodiments, the amino acid sequences of the heavy chain variable region and light chain variable region of the anti-TNFR2 antibody disclosed herein are described in SEQ ID NOs: 317 and 318. In some embodiments, the amino acid sequences of the heavy chain variable region and light chain variable region of the anti-TNFR2 antibody disclosed herein are described in SEQ ID NOs: 321 and 322. In some embodiments, the amino acid sequences of the heavy chain variable region and light chain variable region of the anti-TNFR2 antibody disclosed herein are described in SEQ ID NOs: 325 and 326. In some embodiments, the amino acid sequences of the heavy chain variable region and light chain variable region of the anti-TNFR2 antibody disclosed herein are described in SEQ ID NOs: 329 and 330.

[0075] In another embodiment, the anti-TNFR2 antibody includes VH and VL sequences that are at least 80% (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99% or more) identical to the amino acid sequences described above, and are not limited to those determined using, for example, the BlastP software of the National Center for Biotechnology Information (NCBI) with default parameters.

[0076] In further embodiments, the disclosure provides anti-TNFR2scFv. Given the arrangement of the heavy-chain variable region (VH) and light-chain variable region (VL) disclosed herein, it would be easy for those skilled in the art to construct anti-IL-2scFv by employing standard techniques known in the art.

[0077] In certain embodiments, the disclosure provides polypeptides comprising a VH domain and a VL domain that can be dimerized under appropriate conditions. For example, the VH domain and the VL domain are conjugated in an appropriate buffer and dimerized by an appropriate interaction, such as a hydrophobic interaction. Alternatively, the VH domain and the VL domain may be conjugated in an appropriate buffer containing an enzyme and / or cofactor that can promote the dimerization of the VH domain and the VL domain. In yet another approach, the VH domain and the VL domain may be conjugated in an appropriate vehicle that allows both domains to react with each other in the presence of an appropriate reagent and / or catalyst.

[0078] In certain embodiments, the VH domain and VL domain may be contained in a long polypeptide sequence, but not limited to, a constant region, a hinge region, a linker region, an Fc region, a disulfide bond region, or any combination thereof. The constant region is the immunoglobulin folding unit of the constant portion of the immunoglobulin molecule and is also called the domain of the constant region (e.g., CH1, CH2, CH3, CH4, Ck, Cl). In some embodiments, the long polypeptide may contain multiple copies of one or both of the VH domain and VL domain produced according to the methods disclosed herein. For example, a diabody or triabody can be formed using a polypeptide produced according to the methods disclosed herein.

[0079] In another embodiment, each of the anti-TNFR2 antibodies disclosed herein comprises a heavy chain and a light chain, the amino acid sequences of the heavy chain and light chain may be one of the following pairs: SEQ ID NOs: 438 and 292, 291 and 292, 235 and 236, 239 and 240, 243 and 244, 247 and 248, 251 and 252, 255 and 256, 259 and 260, 263 and 264, 267 and 268, 271 and 272, 275 and 276, 279 and 2 80, SEQ ID NOs. 283 and 284, SEQ ID NOs. 287 and 288, SEQ ID NOs. 295 and 296, SEQ ID NOs. 299 and 300, SEQ ID NOs. 303 and 304, SEQ ID NOs. 307 and 308, SEQ ID NOs. 311 and 312, SEQ ID NOs. 315 and 316, SEQ ID NOs. 319 and 320, SEQ ID NOs. 323 and 324, SEQ ID NOs. 327 and 328, SEQ ID NOs. 331 and 332, SEQ ID NOs. 432 and 260, SEQ ID NOs. 433 and 304, SEQ ID NOs. 434 and 308, SEQ ID NOs. 435 and 236, SEQ ID NOs. 436 and 244, SEQ ID NOs. 437 and 284, or SEQ ID NOs. 439 and 296.

[0080] In some embodiments, the anti-TNFR2 antibody comprises a heavy chain and a light chain, the amino acid sequences of the heavy chain and light chain described in SEQ ID NOs. 438 and 292. In some embodiments, the anti-TNFR2 antibody comprises a heavy chain and a light chain, the amino acid sequences of the heavy chain and light chain described in SEQ ID NOs. 291 and 292. In some embodiments, the anti-TNFR2 antibody comprises a heavy chain and a light chain, the amino acid sequences of the heavy chain and light chain described in SEQ ID NOs. 235 and 236. In some embodiments, the anti-TNFR2 antibody comprises a heavy chain and a light chain, the amino acid sequences of the heavy chain and light chain described in SEQ ID NOs. 239 and 240. In some embodiments, the anti-TNFR2 antibody comprises a heavy chain and a light chain, the amino acid sequences of the heavy chain and light chain described in SEQ ID NOs. 243 and 244. In some embodiments, the anti-TNFR2 antibody comprises a heavy chain and a light chain, the amino acid sequences of the heavy chain and light chain described in SEQ ID NOs. 247 and 248. In some embodiments, the anti-TNFR2 antibody comprises a heavy chain and a light chain, the amino acid sequences of the heavy chain and light chain described in SEQ ID NOs. 251 and 252. In some embodiments, the anti-TNFR2 antibody comprises a heavy chain and a light chain, the amino acid sequences of the heavy chain and light chain described in SEQ ID NOs. 255 and 256. In some embodiments, the anti-TNFR2 antibody comprises a heavy chain and a light chain, the amino acid sequences of the heavy chain and light chain described in SEQ ID NOs. 259 and 260. In some embodiments, the anti-TNFR2 antibody comprises a heavy chain and a light chain, the amino acid sequences of the heavy chain and light chain described in SEQ ID NOs. 263 and 264. In some embodiments, the anti-TNFR2 antibody comprises a heavy chain and a light chain, the amino acid sequences of the heavy chain and light chain described in SEQ ID NOs. 267 and 268. In some embodiments, the anti-TNFR2 antibody comprises a heavy chain and a light chain, the amino acid sequences of the heavy chain and light chain described in SEQ ID NOs. 271 and 272. In some embodiments, the anti-TNFR2 antibody comprises a heavy chain and a light chain, the amino acid sequences of the heavy chain and light chain described in SEQ ID NOs. 275 and 276. In some embodiments, the anti-TNFR2 antibody comprises a heavy chain and a light chain, the amino acid sequences of the heavy chain and light chain described in SEQ ID NOs. 279 and 280.In some embodiments, the anti-TNFR2 antibody comprises a heavy chain and a light chain, the amino acid sequences of the heavy chain and light chain described in SEQ ID NOs. 283 and 284. In some embodiments, the anti-TNFR2 antibody comprises a heavy chain and a light chain, the amino acid sequences of the heavy chain and light chain described in SEQ ID NOs. 287 and 288. In some embodiments, the anti-TNFR2 antibody comprises a heavy chain and a light chain, the amino acid sequences of the heavy chain and light chain described in SEQ ID NOs. 295 and 296. In some embodiments, the anti-TNFR2 antibody comprises a heavy chain and a light chain, the amino acid sequences of the heavy chain and light chain described in SEQ ID NOs. 299 and 300. In some embodiments, the anti-TNFR2 antibody comprises a heavy chain and a light chain, the amino acid sequences of the heavy chain and light chain described in SEQ ID NOs. 303 and 304. In some embodiments, the anti-TNFR2 antibody comprises a heavy chain and a light chain, the amino acid sequences of the heavy chain and light chain described in SEQ ID NOs. 307 and 308. In some embodiments, the anti-TNFR2 antibody comprises a heavy chain and a light chain, the amino acid sequences of the heavy chain and light chain described in SEQ ID NOs: 311 and 312. In some embodiments, the anti-TNFR2 antibody comprises a heavy chain and a light chain, the amino acid sequences of the heavy chain and light chain described in SEQ ID NOs: 315 and 316. In some embodiments, the anti-TNFR2 antibody comprises a heavy chain and a light chain, the amino acid sequences of the heavy chain and light chain described in SEQ ID NOs: 319 and 320. In some embodiments, the anti-TNFR2 antibody comprises a heavy chain and a light chain, the amino acid sequences of the heavy chain and light chain described in SEQ ID NOs: 323 and 324. In some embodiments, the anti-TNFR2 antibody comprises a heavy chain and a light chain, the amino acid sequences of the heavy chain and light chain described in SEQ ID NOs: 327 and 328. In some embodiments, the anti-TNFR2 antibody comprises a heavy chain and a light chain, the amino acid sequences of the heavy chain and light chain described in SEQ ID NOs: 331 and 332. In some embodiments, the anti-TNFR2 antibody comprises a heavy chain and a light chain, the amino acid sequences of the heavy chain and light chain described in SEQ ID NOs. 432 and 260. In some embodiments, the anti-TNFR2 antibody comprises a heavy chain and a light chain, the amino acid sequences of the heavy chain and light chain described in SEQ ID NOs. 433 and 304.In some embodiments, the anti-TNFR2 antibody comprises a heavy chain and a light chain, the amino acid sequences of the heavy chain and light chain described in SEQ ID NOs. 434 and 308. In some embodiments, the anti-TNFR2 antibody comprises a heavy chain and a light chain, the amino acid sequences of the heavy chain and light chain described in SEQ ID NOs. 435 and 236. In some embodiments, the anti-TNFR2 antibody comprises a heavy chain and a light chain, the amino acid sequences of the heavy chain and light chain described in SEQ ID NOs. 436 and 244. In some embodiments, the anti-TNFR2 antibody comprises a heavy chain and a light chain, the amino acid sequences of the heavy chain and light chain described in SEQ ID NOs. 437 and 284. In some embodiments, the anti-TNFR2 antibody comprises a heavy chain and a light chain, the amino acid sequences of the heavy chain and light chain described in SEQ ID NOs. 439 and 296.

[0081] In another embodiment, the anti-TNFR2 antibody includes heavy and light chain amino acid sequences that are at least 80% (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99% or more) identical to the amino acid sequences described above, and is not limited to those determined using, for example, the BlastP software of the National Center for Biotechnology Information (NCBI) with default parameters.

[0082] In one embodiment, the anti-TNFR2 antibody of the Disclosure may be IgG, Fv, scFv, Fab, F(ab')2, minibody, diabody, triabody, nanobody, bispecific antibody, or single-domain antibody. In some embodiments, the anti-TNFR2 antibody of the Disclosure is an IgG1 antibody. In some other embodiments, the anti-TNFR2 antibody of the Disclosure is an IgG2 antibody. In some yet another embodiment, the anti-TNFR2 antibody of the Disclosure is an IgG3 antibody. In some further embodiments, the anti-TNFR2 antibody of the Disclosure is an IgG4 antibody.

[0083] In some embodiments, the anti-TNFR2 antibodies of the Disclosure can agonize TNFR2. In some embodiments, the anti-TNFR2 antibodies of the Disclosure can agonize TNFR2 in an Fc-independent manner.

[0084] In one embodiment, the disclosure provides an antibody that binds to TNFR2 with high affinity. In one embodiment, the binding affinity is calculated by a modified Scatchard method, as described by Frankel et al. ("Mol. Immunol., 16:101-106, 1979"). In another embodiment, the binding affinity is measured by the antigen / antibody dissociation rate. In another embodiment, the binding affinity is measured by competitive radioimmunoassay. In another embodiment, the binding affinity is measured by ELISA. In another embodiment, the antibody affinity is measured by flow cytometry.

[0085] In one embodiment, the anti-TNFR2 antibody of the Disclosure activates TNFR2-mediated signaling at a level of at least 80% compared to signaling activated by TNF. In another embodiment, the anti-TNFR2 antibody of the Disclosure activates TNFR2-mediated signaling at a level of at least 85%, at least 90%, at least 95%, at least 99%, or at least 100% compared to signaling activated by TNF.

[0086] In one embodiment, the Disclosure also provides isolated polynucleotide sequences encoding heavy-chain CDRs and light-chain CDRs disclosed herein. In another embodiment, the Disclosure also provides vectors comprising such polynucleotide sequences. Given the amino acid sequences disclosed herein, those skilled in the art will be able to easily construct vectors or plasmids encoding such amino acid sequences. In another embodiment, the Disclosure also provides host cells comprising the vectors of the Disclosure. Those skilled in the art will be able to easily employ suitable host cells, depending on the application and experimental conditions, to support and / or express the above polynucleotide sequences. In some embodiments, the host cells include, but are not limited to, mammalian host cells such as ExpiCHO(TM) or Expi293F(TM) (ThermoFisher, USA).

[0087] In one embodiment, the Disclosure also provides isolated polynucleotide sequences encoding heavy-chain variable regions and light-chain variable regions disclosed herein. In another embodiment, the Disclosure also provides vectors comprising such polynucleotide sequences. Given the amino acid sequences disclosed herein, those skilled in the art will be able to easily construct vectors or plasmids encoding such amino acid sequences. In another embodiment, the Disclosure also provides host cells comprising the vectors of the Disclosure. Those skilled in the art will be able to easily employ suitable host cells, depending on the application and experimental conditions, to support and / or express the above polynucleotide sequences.

[0088] In one embodiment, the Disclosure also provides isolated polynucleotide sequences encoding heavy and light chains disclosed herein. In another embodiment, the Disclosure also provides vectors comprising such polynucleotide sequences. With regard to the amino acid sequences disclosed herein, those skilled in the art will be able to easily construct vectors or plasmids encoding such amino acid sequences. In another embodiment, the Disclosure also provides host cells comprising the vectors of the Disclosure. Those skilled in the art will be able to easily employ suitable host cells, depending on the application and experimental conditions, to support and / or express the above polynucleotide sequences.

[0089] Use of the composition

[0090] In one embodiment, the Disclosure also provides a composition comprising the anti-TNFR2 antibody disclosed herein and a pharmaceutically acceptable carrier. The use of pharmaceutically acceptable carriers is well known in the Art. For example, "Remington's Pharmaceutical Sciences, by EW Martin, Mack Publishing Co., Easton, PA, 23rd Edition, 2020" describes compositions and formulations suitable for the pharmaceutically acceptable delivery of the antibody disclosed herein.

[0091] Compositions comprising the anti-TNFR2 antibody or its antigen-binding fragment disclosed herein may be administered to a subject (e.g., a human or animal) alone or in combination with a carrier, i.e., a pharmaceutically acceptable carrier. "pharmaceutically acceptable" means a material that can be administered to a subject without causing any biologically or otherwise undesirable material, i.e., without causing any undesirable biological effects or interacting in an adverse manner with any other component of the pharmaceutical composition containing the material. As is well known to those skilled in the art, the carrier is selected to minimize any degradation of the polypeptide disclosed herein and to minimize any adverse side effects in the subject. Pharmaceutical compositions may be prepared by methodologies well known in the pharmaceutical art.

[0092] In one embodiment, the composition of the present disclosure comprises an anti-TNFR2 antibody having a set of three complementarity-determining regions (CDRs) on the heavy chain (HCDR1, HCDR2, and HCDR3) and a set of three CDRs on the light chain (LCDR1, LCDR2, and LCDR3). In one embodiment, as described above, the set of HCDR1, HCDR2, and HCDR3 comprises the amino acid sequence shown in Table 7 below, and the corresponding set of LCDR1, LCDR2, and LCDR3 on the light chain comprises the amino acid sequence shown in Table 8 below.

[0093] In some embodiments, the composition of the present disclosure comprises an anti-TNFR2 antibody having the HCDR1, HCDR2, and HCDR3 amino acid sequences described in SEQ ID NOs. 185, 190, and 347, respectively, and the LCDR1, LCDR2, and LCDR3 amino acid sequences described in SEQ ID NOs. 359, 372, and 378, respectively. In some embodiments, the composition of the present disclosure comprises an anti-TNFR2 antibody having the HCDR1, HCDR2, and HCDR3 amino acid sequences described in SEQ ID NOs. 185, 190, and 347, respectively, and the LCDR1, LCDR2, and LCDR3 amino acid sequences described in SEQ ID NOs. 359, 372, and 378, respectively. In some embodiments, the composition of the present disclosure comprises an anti-TNFR2 antibody having the HCDR1, HCDR2, and HCDR3 amino acid sequences described in SEQ ID NOs. 185, 334, and 347, respectively, and the LCDR1, LCDR2, and LCDR3 amino acid sequences described in SEQ ID NOs. 360, 372, and 379, respectively. In some embodiments, the composition of the present disclosure comprises an anti-TNFR2 antibody having the HCDR1, HCDR2, and HCDR3 amino acid sequences described in SEQ ID NOs. 185, 190, and 347, respectively, and the LCDR1, LCDR2, and LCDR3 amino acid sequences described in SEQ ID NOs. 359, 372, and 380, respectively. In some embodiments, the composition of the present disclosure comprises an anti-TNFR2 antibody having the HCDR1, HCDR2, and HCDR3 amino acid sequences described in SEQ ID NOs. 185, 190, and 347, respectively, and the LCDR1, LCDR2, and LCDR3 amino acid sequences described in SEQ ID NOs. 359, 372, and 381, respectively. In some embodiments, the composition of the present disclosure comprises an anti-TNFR2 antibody having the HCDR1, HCDR2, and HCDR3 amino acid sequences described in SEQ ID NOs. 185, 190, and 347, respectively, and the LCDR1, LCDR2, and LCDR3 amino acid sequences described in SEQ ID NOs. 361, 372, and 381, respectively. In some embodiments, the compositions of the present disclosure include an anti-TNFR2 antibody having the HCDR1, HCDR2, and HCDR3 amino acid sequences described in SEQ ID NOs: 185, 190, and 347, respectively, and the LCDR1, LCDR2, and LCDR3 amino acid sequences described in SEQ ID NOs: 362, 372, and 382, ​​respectively.In some embodiments, the composition of the present disclosure comprises an anti-TNFR2 antibody having the HCDR1, HCDR2, and HCDR3 amino acid sequences described in SEQ ID NOs. 185, 335, and 347, respectively, and the LCDR1, LCDR2, and LCDR3 amino acid sequences described in SEQ ID NOs. 363, 372, and 383, respectively. In some embodiments, the composition of the present disclosure comprises an anti-TNFR2 antibody having the HCDR1, HCDR2, and HCDR3 amino acid sequences described in SEQ ID NOs. 185, 190, and 347, respectively, and the LCDR1, LCDR2, and LCDR3 amino acid sequences described in SEQ ID NOs. 364, 372, and 384, respectively. In some embodiments, the composition of the present disclosure comprises an anti-TNFR2 antibody having the HCDR1, HCDR2, and HCDR3 amino acid sequences described in SEQ ID NOs. 185, 336, and 348, respectively, and the LCDR1, LCDR2, and LCDR3 amino acid sequences described in SEQ ID NOs. 365, 372, and 378, respectively. In some embodiments, the composition of the present disclosure comprises an anti-TNFR2 antibody having the HCDR1, HCDR2, and HCDR3 amino acid sequences described in SEQ ID NOs. 185, 337, and 347, respectively, and the LCDR1, LCDR2, and LCDR3 amino acid sequences described in SEQ ID NOs. 359, 373, and 385, respectively. In some embodiments, the composition of the present disclosure comprises an anti-TNFR2 antibody having the HCDR1, HCDR2, and HCDR3 amino acid sequences described in SEQ ID NOs. 185, 338, and 348, respectively, and the LCDR1, LCDR2, and LCDR3 amino acid sequences described in SEQ ID NOs. 359, 372, and 386, respectively. In some embodiments, the composition of the present disclosure comprises an anti-TNFR2 antibody having the HCDR1, HCDR2, and HCDR3 amino acid sequences described in SEQ ID NOs. 185, 339, and 349, respectively, and the LCDR1, LCDR2, and LCDR3 amino acid sequences described in SEQ ID NOs. 359, 372, and 378, respectively. In some embodiments, the compositions of the present disclosure include an anti-TNFR2 antibody having the HCDR1, HCDR2, and HCDR3 amino acid sequences described in SEQ ID NOs: 185, 190, and 350, respectively, and the LCDR1, LCDR2, and LCDR3 amino acid sequences described in SEQ ID NOs: 366, 372, and 387, respectively.In some embodiments, the composition of the present disclosure comprises an anti-TNFR2 antibody having the HCDR1, HCDR2, and HCDR3 amino acid sequences described in SEQ ID NOs. 185, 190, and 351, respectively, and the LCDR1, LCDR2, and LCDR3 amino acid sequences described in SEQ ID NOs. 359, 372, and 378, respectively. In some embodiments, the composition of the present disclosure comprises an anti-TNFR2 antibody having the HCDR1, HCDR2, and HCDR3 amino acid sequences described in SEQ ID NOs. 185, 334, and 347, respectively, and the LCDR1, LCDR2, and LCDR3 amino acid sequences described in SEQ ID NOs. 360, 372, and 388, respectively. In some embodiments, the composition of the present disclosure comprises an anti-TNFR2 antibody having the HCDR1, HCDR2, and HCDR3 amino acid sequences described in SEQ ID NOs. 185, 334, and 352, respectively, and the LCDR1, LCDR2, and LCDR3 amino acid sequences described in SEQ ID NOs. 359, 372, and 378, respectively. In some embodiments, the composition of the present disclosure comprises an anti-TNFR2 antibody having the HCDR1, HCDR2, and HCDR3 amino acid sequences described in SEQ ID NOs. 185, 190, and 352, respectively, and the LCDR1, LCDR2, and LCDR3 amino acid sequences described in SEQ ID NOs. 359, 372, and 389, respectively. In some embodiments, the composition of the present disclosure comprises an anti-TNFR2 antibody having the HCDR1, HCDR2, and HCDR3 amino acid sequences described in SEQ ID NOs. 185, 334, and 347, respectively, and the LCDR1, LCDR2, and LCDR3 amino acid sequences described in SEQ ID NOs. 359, 372, and 378, respectively. In some embodiments, the composition of the present disclosure comprises an anti-TNFR2 antibody having the HCDR1, HCDR2, and HCDR3 amino acid sequences described in SEQ ID NOs. 185, 190, and 347, respectively, and the LCDR1, LCDR2, and LCDR3 amino acid sequences described in SEQ ID NOs. 365, 372, and 378, respectively. In some embodiments, the compositions of the present disclosure include an anti-TNFR2 antibody having the HCDR1, HCDR2, and HCDR3 amino acid sequences described in SEQ ID NOs: 185, 336, and 352, respectively, and the LCDR1, LCDR2, and LCDR3 amino acid sequences described in SEQ ID NOs: 365, 372, and 390, respectively.In some embodiments, the composition of the present disclosure comprises an anti-TNFR2 antibody having the HCDR1, HCDR2, and HCDR3 amino acid sequences described in SEQ ID NOs: 185, 336, and 352, respectively, and the LCDR1, LCDR2, and LCDR3 amino acid sequences described in SEQ ID NOs: 365, 374, and 391, respectively. In some embodiments, the composition of the present disclosure comprises an anti-TNFR2 antibody having the HCDR1, HCDR2, and HCDR3 amino acid sequences described in SEQ ID NOs: 186, 340, and 353, respectively, and the LCDR1, LCDR2, and LCDR3 amino acid sequences described in SEQ ID NOs: 367, 375, and 392, respectively. In some embodiments, the composition of the present disclosure comprises an anti-TNFR2 antibody having the HCDR1, HCDR2, and HCDR3 amino acid sequences described in SEQ ID NOs: 186, 341, and 354, respectively, and the LCDR1, LCDR2, and LCDR3 amino acid sequences described in SEQ ID NOs: 367, 375, and 392, respectively. In some embodiments, the composition of the present disclosure comprises an anti-TNFR2 antibody having the HCDR1, HCDR2, and HCDR3 amino acid sequences described in SEQ ID NOs: 186, 341, and 354, respectively, and the LCDR1, LCDR2, and LCDR3 amino acid sequences described in SEQ ID NOs: 368, 375, and 392, respectively. In some embodiments, the composition of the present disclosure comprises an anti-TNFR2 antibody having the HCDR1, HCDR2, and HCDR3 amino acid sequences described in SEQ ID NOs: 186, 342, and 354, respectively, and the LCDR1, LCDR2, and LCDR3 amino acid sequences described in SEQ ID NOs: 367, 375, and 392, respectively. In some embodiments, the composition of the present disclosure comprises an anti-TNFR2 antibody having the HCDR1, HCDR2, and HCDR3 amino acid sequences described in SEQ ID NOs: 186, 343, and 354, respectively, and the LCDR1, LCDR2, and LCDR3 amino acid sequences described in SEQ ID NOs: 369, 375, and 393, respectively. In some embodiments, the compositions of the present disclosure include an anti-TNFR2 antibody having the HCDR1, HCDR2, and HCDR3 amino acid sequences described in SEQ ID NOs: 186, 343, and 355, respectively, and the LCDR1, LCDR2, and LCDR3 amino acid sequences described in SEQ ID NOs: 367, 375, and 392, respectively.In some embodiments, the composition of the present disclosure comprises an anti-TNFR2 antibody having the HCDR1, HCDR2, and HCDR3 amino acid sequences described in SEQ ID NOs: 186, 341, and 354, respectively, and the LCDR1, LCDR2, and LCDR3 amino acid sequences described in SEQ ID NOs: 367, 375, and 392, respectively. In some embodiments, the composition of the present disclosure comprises an anti-TNFR2 antibody having the HCDR1, HCDR2, and HCDR3 amino acid sequences described in SEQ ID NOs: 186, 341, and 355, respectively, and the LCDR1, LCDR2, and LCDR3 amino acid sequences described in SEQ ID NOs: 367, 376, and 392, respectively. In some embodiments, the composition of the present disclosure comprises an anti-TNFR2 antibody having the HCDR1, HCDR2, and HCDR3 amino acid sequences described in SEQ ID NOs: 186, 344, and 354, respectively, and the LCDR1, LCDR2, and LCDR3 amino acid sequences described in SEQ ID NOs: 370, 375, and 392, respectively. In some embodiments, the composition of the present disclosure comprises an anti-TNFR2 antibody having the HCDR1, HCDR2, and HCDR3 amino acid sequences described in SEQ ID NOs: 186, 341, and 354, respectively, and the LCDR1, LCDR2, and LCDR3 amino acid sequences described in SEQ ID NOs: 367, 375, and 394, respectively. In some embodiments, the composition of the present disclosure comprises an anti-TNFR2 antibody having the HCDR1, HCDR2, and HCDR3 amino acid sequences described in SEQ ID NOs: 186, 344, and 354, respectively, and the LCDR1, LCDR2, and LCDR3 amino acid sequences described in SEQ ID NOs: 367, 375, and 392, respectively. In some embodiments, the composition of the present disclosure comprises an anti-TNFR2 antibody having the HCDR1, HCDR2, and HCDR3 amino acid sequences described in SEQ ID NOs: 186, 341, and 359, respectively, and the LCDR1, LCDR2, and LCDR3 amino acid sequences described in SEQ ID NOs: 367, 375, and 393, respectively. In some embodiments, the compositions of the present disclosure include an anti-TNFR2 antibody having the HCDR1, HCDR2, and HCDR3 amino acid sequences described in SEQ ID NOs: 187, 345, and 356, respectively, and the LCDR1, LCDR2, and LCDR3 amino acid sequences described in SEQ ID NOs: 371, 377, and 395, respectively.In some embodiments, the composition of the present disclosure comprises an anti-TNFR2 antibody having the HCDR1, HCDR2, and HCDR3 amino acid sequences described in SEQ ID NOs. 188, 345, and 357, respectively, and the LCDR1, LCDR2, and LCDR3 amino acid sequences described in SEQ ID NOs. 371, 377, and 396, respectively. In some embodiments, the composition of the present disclosure comprises the HCDR1, HCDR2, and HCDR3 amino acid sequences described in SEQ ID NOs. 189, 346, and 358, respectively, and SEQ ID NOs. 371, This product contains an anti-TNFR2 antibody having the LCDR1, LCDR2, and LCDR3 amino acid sequences described in 377 and 397, respectively.

[0094] In another embodiment, as described above, the set of HCDR1, HCDR2, and HCDR3 on the heavy chain and the corresponding set of LCDR1, LCDR2, and LCDR3 on the light chain include the amino acid sequence shown in Table 9 below.

[0095] In some embodiments, the composition of the present disclosure includes an anti-TNFR2 antibody having the HCDR1, HCDR2, and HCDR3 amino acid sequences described in SEQ ID NOs. 402, 345, and 413, respectively, and the LCDR1, LCDR2, and LCDR3 amino acid sequences described in SEQ ID NOs. 371, 418, and 427, respectively. In some embodiments, the composition of the present disclosure includes an anti-TNFR2 antibody having the HCDR1, HCDR2, and HCDR3 amino acid sequences described in SEQ ID NOs. 398, 346, and 407, respectively, and the LCDR1, LCDR2, and LCDR3 amino acid sequences described in SEQ ID NOs. 371, 377, and 419, respectively. In some embodiments, the composition of the present disclosure includes an anti-TNFR2 antibody having the HCDR1, HCDR2, and HCDR3 amino acid sequences described in SEQ ID NOs. 187, 346, and 408, respectively, and the LCDR1, LCDR2, and LCDR3 amino acid sequences described in SEQ ID NOs. 371, 377, and 420, respectively. In some embodiments, the composition of the present disclosure includes an anti-TNFR2 antibody having the HCDR1, HCDR2, and HCDR3 amino acid sequences described in SEQ ID NOs: 189, 406, and 409, respectively, and the LCDR1, LCDR2, and LCDR3 amino acid sequences described in SEQ ID NOs: 371, 377, and 421, respectively. In some embodiments, the composition of the present disclosure includes an anti-TNFR2 antibody having the HCDR1, HCDR2, and HCDR3 amino acid sequences described in SEQ ID NOs: 187, 345, and 410, respectively, and the LCDR1, LCDR2, and LCDR3 amino acid sequences described in SEQ ID NOs: 371, 418, and 422, respectively. In some embodiments, the composition of the present disclosure includes an anti-TNFR2 antibody having the HCDR1, HCDR2, and HCDR3 amino acid sequences described in SEQ ID NOs: 399, 346, and 411, respectively, and the LCDR1, LCDR2, and LCDR3 amino acid sequences described in SEQ ID NOs: 371, 418, and 423, respectively. In some embodiments, the compositions of the present disclosure include an anti-TNFR2 antibody having the HCDR1, HCDR2, and HCDR3 amino acid sequences described in SEQ ID NOs: 400, 346, and 412, respectively, and the LCDR1, LCDR2, and LCDR3 amino acid sequences described in SEQ ID NOs: 417, 377, and 424, respectively.In some embodiments, the composition of the present disclosure comprises an anti-TNFR2 antibody having the HCDR1, HCDR2, and HCDR3 amino acid sequences described in SEQ ID NOs. 401, 346, and 413, respectively, and the LCDR1, LCDR2, and LCDR3 amino acid sequences described in SEQ ID NOs. 417, 418, and 425, respectively. In some embodiments, the composition of the present disclosure comprises an anti-TNFR2 antibody having the HCDR1, HCDR2, and HCDR3 amino acid sequences described in SEQ ID NOs. 401, 346, and 414, respectively, and the LCDR1, LCDR2, and LCDR3 amino acid sequences described in SEQ ID NOs. 417, 418, and 426, respectively. In some embodiments, the composition of the present disclosure comprises an anti-TNFR2 antibody having the HCDR1, HCDR2, and HCDR3 amino acid sequences described in SEQ ID NOs. 398, 346, and 415, respectively, and the LCDR1, LCDR2, and LCDR3 amino acid sequences described in SEQ ID NOs. 417, 377, and 428, respectively. In some embodiments, the composition of the present disclosure comprises an anti-TNFR2 antibody having the HCDR1, HCDR2, and HCDR3 amino acid sequences described in SEQ ID NOs: 403, 346, and 412, respectively, and the LCDR1, LCDR2, and LCDR3 amino acid sequences described in SEQ ID NOs: 371, 418, and 429, respectively. In some embodiments, the composition of the present disclosure comprises an anti-TNFR2 antibody having the HCDR1, HCDR2, and HCDR3 amino acid sequences described in SEQ ID NOs: 404, 346, and 412, respectively, and the LCDR1, LCDR2, and LCDR3 amino acid sequences described in SEQ ID NOs: 371, 418, and 425, respectively. In some embodiments, the composition of the present disclosure comprises an anti-TNFR2 antibody having the HCDR1, HCDR2, and HCDR3 amino acid sequences described in SEQ ID NOs: 401, 346, and 414, respectively, and the LCDR1, LCDR2, and LCDR3 amino acid sequences described in SEQ ID NOs: 417, 418, and 430, respectively. In some embodiments, the compositions of the present disclosure include an anti-TNFR2 antibody having the HCDR1, HCDR2, and HCDR3 amino acid sequences described in SEQ ID NOs. 405, 406, and 416, respectively, and the LCDR1, LCDR2, and LCDR3 amino acid sequences described in SEQ ID NOs. 371, 377, and 431, respectively.

[0096] In another embodiment, the composition of the present disclosure comprises an anti-TNFR2 antibody having a heavy chain CDR sequence and a light chain CDR sequence that are at least 80% (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99% or more) identical to the amino acid sequences described above, for example, and not limited to those determined using the BlastP software of the National Center for Biotechnology Information (NCBI) with default parameters.

[0097] In another embodiment, the composition of the present disclosure comprises an anti-TNFR2 antibody having one of the following pairs of heavy chain variable regions and light chain variable regions: SEQ ID NOs: 289 and 290, SEQ ID NOs: 3 and 4, SEQ ID NOs: 7 and 8, SEQ ID NOs: 11 and 12, SEQ ID NOs: 15 and 16, SEQ ID NOs: 19 and 20, SEQ ID NOs: 23 and 24, SEQ ID NOs: 27 and 28, SEQ ID NOs: 31 and 32, SEQ ID NOs: 35 and 36, SEQ ID NOs: 39 and 40, SEQ ID NOs: 43 and 44, SEQ ID NOs: 47 and 48, SEQ ID NOs: 51 and 52, SEQ ID NOs: 55 and 56, SEQ ID NOs: 59 and 60, SEQ ID NOs: 63 and 64 Numbers 67 and 68, SEQ ID NOs 71 and 72, SEQ ID NOs 75 and 76, SEQ ID NOs 79 and 80, SEQ ID NOs 83 and 84, SEQ ID NOs 87 and 88, SEQ ID NOs 91 and 92, SEQ ID NOs 95 and 96, SEQ ID NOs 99 and 100, SEQ ID NOs 103 and 104, SEQ ID NOs 107 and 108, SEQ ID NOs 111 and 112, SEQ ID NOs 115 and 116, SEQ ID NOs 119 and 120, SEQ ID NOs 123 and 124, SEQ ID NOs 127 and 128, SEQ ID NOs 131 and 132, SEQ ID NOs 135 and 136, SEQ ID NOs 139 and 140, SEQ ID NOs 143 and 144, SEQ ID NOs 1 47 and 148, SEQ ID NOs: 151 and 152, SEQ ID NOs: 157 and 158, SEQ ID NOs: 163 and 164, SEQ ID NOs: 169 and 170, SEQ ID NOs: 175 and 176, SEQ ID NOs: 181 and 182, SEQ ID NOs: 193 and 194, SEQ ID NOs: 199 and 200, SEQ ID NOs: 205 and 206, SEQ ID NOs: 211 and 212, SEQ ID NOs: 217 and 218, SEQ ID NOs: 223 and 224, SEQ ID NOs: 229 and 230, SEQ ID NOs: 233 and 234, SEQ ID NOs: 237 and 238, SEQ ID NOs: 241 and 242, SEQ ID NOs: 245 and 246, SEQ ID NOs: 249 and 250, SEQ ID NOs: Sequence numbers 253 and 254, sequence numbers 257 and 258, sequence numbers 261 and 262, sequence numbers 265 and 266, sequence numbers 269 and 270, sequence numbers 273 and 274, sequence numbers 277 and 278, sequence numbers 281 and 282, sequence numbers 285 and 286, sequence numbers 293 and 294, sequence numbers 297 and 298, sequence numbers 301 and 302, sequence numbers 305 and 306, sequence numbers 309 and 310, sequence numbers 313 and 314, sequence numbers 317 and 318, sequence numbers 321 and 322, sequence numbers 325 and 326, or sequence numbers 329 and 330.

[0098] In certain embodiments, the composition of the Disclosure includes a TNFR2 antibody having a heavy chain variable region and a light chain variable region as described in SEQ ID NOs: 289 and 290. In certain embodiments, the composition of the Disclosure includes a TNFR2 antibody having a heavy chain variable region and a light chain variable region as described in SEQ ID NOs: 3 and 4. In certain embodiments, the composition of the Disclosure includes a TNFR2 antibody having a heavy chain variable region and a light chain variable region as described in SEQ ID NOs: 7 and 8. In certain embodiments, the composition of the Disclosure includes a TNFR2 antibody having a heavy chain variable region and a light chain variable region as described in SEQ ID NOs: 11 and 12. In certain embodiments, the composition of the Disclosure includes a TNFR2 antibody having a heavy chain variable region and a light chain variable region as described in SEQ ID NOs: 15 and 16. In certain embodiments, the composition of the Disclosure includes a TNFR2 antibody having a heavy chain variable region and a light chain variable region as described in SEQ ID NOs: 19 and 20. In certain embodiments, the composition of the Disclosure includes a TNFR2 antibody having a heavy chain variable region and a light chain variable region as described in SEQ ID NOs: 23 and 24. In certain embodiments, the composition of the Disclosure includes a TNFR2 antibody having a heavy chain variable region and a light chain variable region as described in SEQ ID NOs. 27 and 28. In certain embodiments, the composition of the Disclosure includes a TNFR2 antibody having a heavy chain variable region and a light chain variable region as described in SEQ ID NOs. 31 and 32. In certain embodiments, the composition of the Disclosure includes a TNFR2 antibody having a heavy chain variable region and a light chain variable region as described in SEQ ID NOs. 35 and 36. In certain embodiments, the composition of the Disclosure includes a TNFR2 antibody having a heavy chain variable region and a light chain variable region as described in SEQ ID NOs. 39 and 40. In certain embodiments, the composition of the Disclosure includes a TNFR2 antibody having a heavy chain variable region and a light chain variable region as described in SEQ ID NOs. 43 and 44. In certain embodiments, the composition of the Disclosure includes a TNFR2 antibody having a heavy chain variable region and a light chain variable region as described in SEQ ID NOs. 47 and 48. In certain embodiments, the composition of the Disclosure includes a TNFR2 antibody having a heavy chain variable region and a light chain variable region as described in SEQ ID NOs. 51 and 52. In certain embodiments, the compositions of the present disclosure include a TNFR2 antibody having heavy chain variable regions and light chain variable regions as described in SEQ ID NOs. 55 and 56.In certain embodiments, the composition of the Disclosure includes a TNFR2 antibody having a heavy chain variable region and a light chain variable region as described in SEQ ID NOs. 59 and 60. In certain embodiments, the composition of the Disclosure includes a TNFR2 antibody having a heavy chain variable region and a light chain variable region as described in SEQ ID NOs. 63 and 64. In certain embodiments, the composition of the Disclosure includes a TNFR2 antibody having a heavy chain variable region and a light chain variable region as described in SEQ ID NOs. 67 and 68. In certain embodiments, the composition of the Disclosure includes a TNFR2 antibody having a heavy chain variable region and a light chain variable region as described in SEQ ID NOs. 71 and 72. In certain embodiments, the composition of the Disclosure includes a TNFR2 antibody having a heavy chain variable region and a light chain variable region as described in SEQ ID NOs. 75 and 76. In certain embodiments, the composition of the Disclosure includes a TNFR2 antibody having a heavy chain variable region and a light chain variable region as described in SEQ ID NOs. 79 and 80. In certain embodiments, the composition of the Disclosure includes a TNFR2 antibody having a heavy chain variable region and a light chain variable region as described in SEQ ID NOs. 83 and 84. In certain embodiments, the composition of the Disclosure includes a TNFR2 antibody having a heavy chain variable region and a light chain variable region as described in SEQ ID NOs: 87 and 88. In certain embodiments, the composition of the Disclosure includes a TNFR2 antibody having a heavy chain variable region and a light chain variable region as described in SEQ ID NOs: 91 and 92. In certain embodiments, the composition of the Disclosure includes a TNFR2 antibody having a heavy chain variable region and a light chain variable region as described in SEQ ID NOs: 95 and 96. In certain embodiments, the composition of the Disclosure includes a TNFR2 antibody having a heavy chain variable region and a light chain variable region as described in SEQ ID NOs: 99 and 100. In certain embodiments, the composition of the Disclosure includes a TNFR2 antibody having a heavy chain variable region and a light chain variable region as described in SEQ ID NOs: 103 and 104. In certain embodiments, the composition of the Disclosure includes a TNFR2 antibody having a heavy chain variable region and a light chain variable region as described in SEQ ID NOs: 107 and 108. In certain embodiments, the composition of the Disclosure includes a TNFR2 antibody having a heavy chain variable region and a light chain variable region as described in SEQ ID NOs: 111 and 112. In certain embodiments, the compositions of the present disclosure include a TNFR2 antibody having a heavy chain variable region and a light chain variable region as described in SEQ ID NOs: 115 and 116.In certain embodiments, the composition of the Disclosure includes a TNFR2 antibody having a heavy chain variable region and a light chain variable region as described in SEQ ID NOs: 119 and 120. In certain embodiments, the composition of the Disclosure includes a TNFR2 antibody having a heavy chain variable region and a light chain variable region as described in SEQ ID NOs: 123 and 124. In certain embodiments, the composition of the Disclosure includes a TNFR2 antibody having a heavy chain variable region and a light chain variable region as described in SEQ ID NOs: 127 and 128. In certain embodiments, the composition of the Disclosure includes a TNFR2 antibody having a heavy chain variable region and a light chain variable region as described in SEQ ID NOs: 131 and 132. In certain embodiments, the composition of the Disclosure includes a TNFR2 antibody having a heavy chain variable region and a light chain variable region as described in SEQ ID NOs: 135 and 136. In certain embodiments, the composition of the Disclosure includes a TNFR2 antibody having a heavy chain variable region and a light chain variable region as described in SEQ ID NOs: 139 and 140. In certain embodiments, the composition of the Disclosure includes a TNFR2 antibody having a heavy chain variable region and a light chain variable region as described in SEQ ID NOs: 143 and 144. In certain embodiments, the composition of the Disclosure includes a TNFR2 antibody having a heavy chain variable region and a light chain variable region as described in SEQ ID NOs: 147 and 148. In certain embodiments, the composition of the Disclosure includes a TNFR2 antibody having a heavy chain variable region and a light chain variable region as described in SEQ ID NOs: 151 and 152. In certain embodiments, the composition of the Disclosure includes a TNFR2 antibody having a heavy chain variable region and a light chain variable region as described in SEQ ID NOs: 157 and 158. In certain embodiments, the composition of the Disclosure includes a TNFR2 antibody having a heavy chain variable region and a light chain variable region as described in SEQ ID NOs: 163 and 164. In certain embodiments, the composition of the Disclosure includes a TNFR2 antibody having a heavy chain variable region and a light chain variable region as described in SEQ ID NOs: 169 and 170. In certain embodiments, the composition of the Disclosure includes a TNFR2 antibody having a heavy chain variable region and a light chain variable region as described in SEQ ID NOs: 175 and 176. In certain embodiments, the composition of the Disclosure includes a TNFR2 antibody having a heavy chain variable region and a light chain variable region as described in SEQ ID NOs: 181 and 182. In certain embodiments, the compositions of the present disclosure include a TNFR2 antibody having heavy chain variable regions and light chain variable regions as described in SEQ ID NOs: 193 and 194.In certain embodiments, the composition of the Disclosure includes a TNFR2 antibody having a heavy chain variable region and a light chain variable region as described in SEQ ID NOs: 199 and 200. In certain embodiments, the composition of the Disclosure includes a TNFR2 antibody having a heavy chain variable region and a light chain variable region as described in SEQ ID NOs: 205 and 206. In certain embodiments, the composition of the Disclosure includes a TNFR2 antibody having a heavy chain variable region and a light chain variable region as described in SEQ ID NOs: 211 and 212. In certain embodiments, the composition of the Disclosure includes a TNFR2 antibody having a heavy chain variable region and a light chain variable region as described in SEQ ID NOs: 217 and 218. In certain embodiments, the composition of the Disclosure includes a TNFR2 antibody having a heavy chain variable region and a light chain variable region as described in SEQ ID NOs: 223 and 224. In certain embodiments, the composition of the Disclosure includes a TNFR2 antibody having a heavy chain variable region and a light chain variable region as described in SEQ ID NOs: 229 and 230. In certain embodiments, the composition of the Disclosure includes a TNFR2 antibody having a heavy chain variable region and a light chain variable region as described in SEQ ID NOs: 233 and 234. In certain embodiments, the composition of the Disclosure includes a TNFR2 antibody having a heavy chain variable region and a light chain variable region as described in SEQ ID NOs. 237 and 238. In certain embodiments, the composition of the Disclosure includes a TNFR2 antibody having a heavy chain variable region and a light chain variable region as described in SEQ ID NOs. 241 and 242. In certain embodiments, the composition of the Disclosure includes a TNFR2 antibody having a heavy chain variable region and a light chain variable region as described in SEQ ID NOs. 245 and 246. In certain embodiments, the composition of the Disclosure includes a TNFR2 antibody having a heavy chain variable region and a light chain variable region as described in SEQ ID NOs. 249 and 250. In certain embodiments, the composition of the Disclosure includes a TNFR2 antibody having a heavy chain variable region and a light chain variable region as described in SEQ ID NOs. 253 and 254. In certain embodiments, the composition of the Disclosure includes a TNFR2 antibody having a heavy chain variable region and a light chain variable region as described in SEQ ID NOs. 257 and 258. In certain embodiments, the composition of the Disclosure includes a TNFR2 antibody having a heavy chain variable region and a light chain variable region as described in SEQ ID NOs. 261 and 262. In certain embodiments, the compositions of the present disclosure include a TNFR2 antibody having heavy chain variable regions and light chain variable regions as described in SEQ ID NOs. 265 and 266.In certain embodiments, the composition of the Disclosure includes a TNFR2 antibody having a heavy chain variable region and a light chain variable region as described in SEQ ID NOs. 269 and 270. In certain embodiments, the composition of the Disclosure includes a TNFR2 antibody having a heavy chain variable region and a light chain variable region as described in SEQ ID NOs. 273 and 274. In certain embodiments, the composition of the Disclosure includes a TNFR2 antibody having a heavy chain variable region and a light chain variable region as described in SEQ ID NOs. 277 and 278. In certain embodiments, the composition of the Disclosure includes a TNFR2 antibody having a heavy chain variable region and a light chain variable region as described in SEQ ID NOs. 281 and 282. In certain embodiments, the composition of the Disclosure includes a TNFR2 antibody having a heavy chain variable region and a light chain variable region as described in SEQ ID NOs. 285 and 286. In certain embodiments, the composition of the Disclosure includes a TNFR2 antibody having a heavy chain variable region and a light chain variable region as described in SEQ ID NOs. 293 and 294. In certain embodiments, the composition of the Disclosure includes a TNFR2 antibody having a heavy chain variable region and a light chain variable region as described in SEQ ID NOs. 297 and 298. In certain embodiments, the composition of the Disclosure includes a TNFR2 antibody having a heavy chain variable region and a light chain variable region as described in SEQ ID NOs. 301 and 302. In certain embodiments, the composition of the Disclosure includes a TNFR2 antibody having a heavy chain variable region and a light chain variable region as described in SEQ ID NOs. 305 and 306. In certain embodiments, the composition of the Disclosure includes a TNFR2 antibody having a heavy chain variable region and a light chain variable region as described in SEQ ID NOs. 309 and 310. In certain embodiments, the composition of the Disclosure includes a TNFR2 antibody having a heavy chain variable region and a light chain variable region as described in SEQ ID NOs. 313 and 314. In certain embodiments, the composition of the Disclosure includes a TNFR2 antibody having a heavy chain variable region and a light chain variable region as described in SEQ ID NOs. 317 and 318. In certain embodiments, the composition of the Disclosure includes a TNFR2 antibody having a heavy chain variable region and a light chain variable region as described in SEQ ID NOs. 321 and 322. In certain embodiments, the composition of the Disclosure includes a TNFR2 antibody having a heavy chain variable region and a light chain variable region as described in SEQ ID NOs. 325 and 326. In certain embodiments, the compositions of the present disclosure include a TNFR2 antibody having heavy chain variable regions and light chain variable regions as described in SEQ ID NOs. 329 and 330.

[0099] In another embodiment, the composition of the present disclosure comprises an anti-TNFR2 antibody having VH and VL sequences that are at least 80% (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99% or more) identical to the amino acid sequences described above, for example, and not limited to those determined using the BlastP software of the National Center for Biotechnology Information (NCBI) with default parameters.

[0100] In yet another embodiment, the composition of the present disclosure comprises an anti-TNFR2 antibody having one of the following pairs of heavy chain and light chain: SEQ ID NOs: 438 and 292, 291 and 292, 235 and 236, 239 and 240, 243 and 244, 247 and 248, 251 and 252, 255 and 256, 259 and 260, 263 and 264, 267 and 268, 271 and 272, 275 and 276, 279 and 280, 28 3 and 284, SEQ ID NOs: 287 and 288, SEQ ID NOs: 295 and 296, SEQ ID NOs: 299 and 300, SEQ ID NOs: 303 and 304, SEQ ID NOs: 307 and 308, SEQ ID NOs: 311 and 312, SEQ ID NOs: 315 and 316, SEQ ID NOs: 319 and 320, SEQ ID NOs: 323 and 324, SEQ ID NOs: 327 and 328, SEQ ID NOs: 331 and 332, SEQ ID NOs: 432 and 260, SEQ ID NOs: 433 and 304, SEQ ID NOs: 434 and 308, SEQ ID NOs: 435 and 236, SEQ ID NOs: 436 and 244, SEQ ID NOs: 437 and 284, SEQ ID NOs: 439 and 296.

[0101] In some embodiments, the compositions of the present disclosure include an anti-TNFR2 antibody having a heavy chain and a light chain as described in SEQ ID NOs: 438 and 292. In some embodiments, the compositions of the present disclosure include an anti-TNFR2 antibody having a heavy chain and a light chain as described in SEQ ID NOs: 291 and 292. In some embodiments, the compositions of the present disclosure include an anti-TNFR2 antibody having a heavy chain and a light chain as described in SEQ ID NOs: 235 and 236. In some embodiments, the compositions of the present disclosure include an anti-TNFR2 antibody having a heavy chain and a light chain as described in SEQ ID NOs: 239 and 240. In some embodiments, the compositions of the present disclosure include an anti-TNFR2 antibody having a heavy chain and a light chain as described in SEQ ID NOs: 243 and 244. In some embodiments, the compositions of the present disclosure include an anti-TNFR2 antibody having a heavy chain and a light chain as described in SEQ ID NOs: 247 and 248. In some embodiments, the compositions of the present disclosure include an anti-TNFR2 antibody having a heavy chain and a light chain as described in SEQ ID NOs: 251 and 252. In some embodiments, the compositions of the present disclosure include an anti-TNFR2 antibody having a heavy chain and a light chain as described in SEQ ID NOs: 255 and 256. In some embodiments, the compositions of the present disclosure include an anti-TNFR2 antibody having a heavy chain and a light chain as described in SEQ ID NOs. 259 and 260. In some embodiments, the compositions of the present disclosure include an anti-TNFR2 antibody having a heavy chain and a light chain as described in SEQ ID NOs. 263 and 264. In some embodiments, the compositions of the present disclosure include an anti-TNFR2 antibody having a heavy chain and a light chain as described in SEQ ID NOs. 267 and 268. In some embodiments, the compositions of the present disclosure include an anti-TNFR2 antibody having a heavy chain and a light chain as described in SEQ ID NOs. 271 and 272. In some embodiments, the compositions of the present disclosure include an anti-TNFR2 antibody having a heavy chain and a light chain as described in SEQ ID NOs. 275 and 276. In some embodiments, the compositions of the present disclosure include an anti-TNFR2 antibody having a heavy chain and a light chain as described in SEQ ID NOs. 279 and 280. In some embodiments, the compositions of the present disclosure include an anti-TNFR2 antibody having a heavy chain and a light chain as described in SEQ ID NOs. 283 and 284.In some embodiments, the compositions of the present disclosure include an anti-TNFR2 antibody having a heavy chain and a light chain as described in SEQ ID NOs. 287 and 288. In some embodiments, the compositions of the present disclosure include an anti-TNFR2 antibody having a heavy chain and a light chain as described in SEQ ID NOs. 295 and 296. In some embodiments, the compositions of the present disclosure include an anti-TNFR2 antibody having a heavy chain and a light chain as described in SEQ ID NOs. 299 and 300. In some embodiments, the compositions of the present disclosure include an anti-TNFR2 antibody having a heavy chain and a light chain as described in SEQ ID NOs. 303 and 304. In some embodiments, the compositions of the present disclosure include an anti-TNFR2 antibody having a heavy chain and a light chain as described in SEQ ID NOs. 307 and 308. In some embodiments, the compositions of the present disclosure include an anti-TNFR2 antibody having a heavy chain and a light chain as described in SEQ ID NOs. 311 and 312. In some embodiments, the compositions of the present disclosure include an anti-TNFR2 antibody having a heavy chain and a light chain as described in SEQ ID NOs. 315 and 316. In some embodiments, the compositions of the present disclosure include an anti-TNFR2 antibody having a heavy chain and a light chain as described in SEQ ID NOs. 319 and 320. In some embodiments, the compositions of the present disclosure include an anti-TNFR2 antibody having a heavy chain and a light chain as described in SEQ ID NOs. 323 and 324. In some embodiments, the compositions of the present disclosure include an anti-TNFR2 antibody having a heavy chain and a light chain as described in SEQ ID NOs. 327 and 328. In some embodiments, the compositions of the present disclosure include an anti-TNFR2 antibody having a heavy chain and a light chain as described in SEQ ID NOs. 331 and 332. In some embodiments, the compositions of the present disclosure include an anti-TNFR2 antibody having a heavy chain and a light chain as described in SEQ ID NOs. 432 and 260. In some embodiments, the compositions of the present disclosure include an anti-TNFR2 antibody having a heavy chain and a light chain as described in SEQ ID NOs. 433 and 304. In some embodiments, the compositions of the present disclosure include an anti-TNFR2 antibody having a heavy chain and a light chain as described in SEQ ID NOs. 434 and 308. In some embodiments, the compositions of the present disclosure include an anti-TNFR2 antibody having a heavy chain and a light chain as described in SEQ ID NOs. 435 and 236. In some embodiments, the compositions of the present disclosure include an anti-TNFR2 antibody having a heavy chain and a light chain as described in SEQ ID NOs. 436 and 244.In some embodiments, the compositions of the present disclosure include an anti-TNFR2 antibody having a heavy chain and a light chain as described in SEQ ID NOs: 437 and 284. In some embodiments, the compositions include an anti-TNFR2 antibody having a heavy chain and a light chain as described in SEQ ID NOs: 439 and 296.

[0102] In another embodiment, the composition of the present disclosure comprises an anti-TNFR2 antibody having heavy and light chains that are at least 80% (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99% or more) identical to the amino acid sequences described above, not limited to the identity determined using, for example, the BlastP software of the National Center for Biotechnology Information (NCBI) with default parameters.

[0103] In one embodiment, the antibodies disclosed herein may take the form of a conjugate. As used herein, “conjugate” refers to an antibody or antibody fragment (e.g., an antigen-binding fragment) covalently bound to an effector molecule or a second protein (e.g., a second antibody). The effector molecule may be, for example, a drug, a toxin, a therapeutic agent, a detectable label, a protein, a nucleic acid, a lipid, a nanoparticle, a carbohydrate, or a recombinant virus. Antibody conjugates are also called “immunoconjugates.” If the conjugate contains an antibody bound to a drug (e.g., a cytotoxic drug), the conjugate may be called an “antibody-drug conjugate.” Other antibody conjugates include, for example, multispecific (e.g., bispecific or tripspecific) antibodies and chimeric antigen receptors (CARs).

[0104] The pharmaceutical compositions of this disclosure, comprising antibodies or antigen-binding fragments thereof, may be administered by any suitable method (e.g., to mammals, cells, or tissues) depending on whether topical or systemic treatment is desired. For example, the compositions of this disclosure may be administered topically (e.g., by intraocular, intravaginal, intrarectal, intranasal, or transdermal administration), orally, by inhalation, or parenterally (e.g., by intravenous infusion, or by subcutaneous, intrathoracic, intraperitoneal, intradermal, or intramuscular injection). Topical intranasal administration means delivering the compositions of this disclosure into the nasal cavity through one or both nostrils. The compositions of this disclosure may be delivered by a spray mechanism, a droplet mechanism, or by aerosolization. Alternatively, administration may be intratumoral, e.g., by local or intravenous injection.

[0105] When the compositions of this disclosure are administered parenterally, administration is generally carried out by injection. Injectable preparations may be prepared in conventional forms, such as liquid solutions or suspensions, solid forms suitable for suspension in liquid before injection, or emulsions. In addition, oral administration may involve the preparation of a sustained-release or sustained-release system to maintain a constant dose.

[0106] How to use

[0107] In one embodiment, the anti-TNFR2 antibody of the present disclosure can be used to modulate the proliferation and / or function of regulatory T cells (T-reg cells) or myeloid-derived suppressor cells.

[0108] T-reg cells represent a heterogeneous class of T cells that can be distinguished based on the presentation of their unique surface proteins. The most studied T-reg cells are CD4 + CD25 + FoxP3 + T-reg cells, and CD17 +T-reg cells are one example. Certain classes of T-reg cells have been shown to inhibit the production of interleukin-2 (IL-2), a proliferation-inducing cytokine, in target T cells, and further to sequester IL-2 from autoreactive cells through the affinity of CD25 (a subdomain of the IL-2 receptor) for IL-2. + CD25 + FoxP3 + T-reg cells are also present in B-cell-rich regions and have been shown to directly suppress immunoglobulin production without relying on their ability to weaken TH2 cell activity.

[0109] As used herein, the terms “myelo-derived suppressor cells” or “MDSCs” refer to immune system cells capable of regulating the activity of various effector cells and antigen-presenting cells, particularly T cells, NK cells, dendritic cells, and macrophages. Myelo-derived suppressor cells are distinguished by their gene expression profiles.

[0110] In another embodiment, the anti-TNFR2 antibody of this disclosure can be used to treat diseases such as cancer, autoimmune diseases, GvHd, viral infections, or bacterial infections.

[0111] As used herein, the term “method” means a technique, means, art, and procedure for achieving a given objective, and such include, but are not limited to, techniques, means, art, and procedures known to those skilled in the art in the fields of chemistry, pharmacology, biology, biochemistry, and medicine, or techniques, means, art, and procedures readily developed therefrom by those skilled in the art.

[0112] As used herein, the term “treat, treatment, therapy” (and its variations) refers to therapeutic actions, including prophylactic measures, aimed at preventing or delaying (mitigating) undesirable physiological changes associated with a disease or condition. Beneficial or desirable clinical outcomes include, but are not limited to, symptom relief, reduction of the scope of the disease or condition, stabilization of the disease or condition (i.e., no worsening of the disease or condition), delay or reduction of the progression of the disease or condition, improvement or mitigation of the disease or condition, and remission of the disease or condition (whether partial or complete), whether detectable or not. People who need treatment include not only those who already have a disease or condition, but also those who are susceptible to the disease or condition, and those who should be prevented from developing the disease or condition.

[0113] The terms “subject,” “individual,” and “patient” are used interchangeably herein and refer to human or non-human animals to which treatment with a composition or formulation comprising the anti-TNFR2 antibody according to the present invention is provided. The terms “non-human animal” and “non-human mammal” are also used interchangeably herein and include all vertebrates, e.g., non-human primates (e.g., higher primates), mammals such as sheep, dogs, rodents (e.g., mice, rats), guinea pigs, goats, pigs, cats, rabbits, cattle, and horses, and non-mammals such as reptiles, amphibians, chickens, and turkeys. The compositions described herein can be used to treat any suitable mammal, including primates (e.g., monkeys, humans), horses, cattle, dogs, rabbits, and rodents (e.g., mice, rats). In one embodiment, the mammal being treated is a human. The human may be a human of any age. In one embodiment, the human is an adult. In another embodiment, the human is a child. A human being can be male, female, pregnant, middle-aged, young, or elderly.

[0114] Pharmaceutical compositions suitable for use in the methods of this disclosure include compositions containing an active ingredient in an amount effective to achieve the intended purpose. In one embodiment, a therapeutically effective amount means an amount of one or more active ingredients (e.g., an anti-TNFR2 antibody) that is effective in preventing, alleviating, or improving the symptoms of a disease, or in extending the survival of the subject of treatment. Determining a therapeutically effective amount is well within the capabilities of those skilled in the art.

[0115] In one embodiment, the Disclosure provides a method for modulating the activity or function of regulatory T cells in a subject. As used herein, “modulate” means to “stimulate” or “inhibit” the activity of a molecular target or pathway. For example, the compositions of the Disclosure modulate the activity of a molecular target or pathway to stimulate or inhibit it by at least 10%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 90%, at least about 95%, at least about 98%, or about 99% or more compared to the activity of a molecular target or pathway under conditions identical except that the compositions of the Disclosure are not used. In another example, the compositions of the Disclosure modulate the activity of a molecular target or pathway to stimulate or inhibit it by at least 2 times, at least 5 times, at least 10 times, at least 20 times, at least 50 times, or at least 100 times compared to the activity of a molecular target or pathway under identical conditions except that the compositions of the Disclosure are not used. The activity of a molecular target or molecular pathway can be measured in a reproducible manner. The activity of a molecular target or molecular pathway can be measured in vitro or in vitro. For example, the activity of a molecular target or molecular pathway can be measured in vitro or in vitro by a suitable assay known in the art of activity measurement. A control sample (untreated with the compositions of the Disclosure) shall be assigned a relative activity value of 100%.

[0116] In one embodiment, the method of the Disclosure comprises the step of administering a composition containing an effective amount of the anti-TNFR2 antibody of the Disclosure to a subject. In one embodiment, the composition of the Disclosure comprises an anti-TNFR2 antibody having the heavy chain CDR sequence and the light chain CDR sequence disclosed herein. In another embodiment, the composition of the Disclosure comprises an anti-TNFR2 antibody having the VH sequence and the VL sequence disclosed herein. In yet another embodiment, the composition of the Disclosure comprises an anti-TNFR2 antibody having the heavy chain sequence and the light chain sequence disclosed herein. In one embodiment, regulatory T cells are CD4 + CD25 + Foxp3 + , or alternatively, CD4 + CD25 + C127 low In one embodiment, the method of the present disclosure stimulates the proliferation of regulatory T cells. In another embodiment, the method of the present disclosure can activate regulatory T cells. In yet another embodiment, the method of the present disclosure can modulate the immune response mediated by the above-mentioned regulatory T cells.

[0117] Those skilled in the art will understand that in some embodiments, the activation of regulatory T cells includes suppressing the proliferation of immune system effector cells, including Teff and NK cells; reducing the cytotoxic activity of effector cells, including Teff and NK cells; reducing the production of inflammatory cytokines by immune system effector cells, including Teff and NK cells; or any combination thereof. In some embodiments, target administration of an anti-TNFR2 antibody by the method of the present disclosure suppresses the proliferation of immune system effector cells, including Teff and NK cells. In some embodiments, target administration of an anti-TNFR2 antibody by the method of the present disclosure reduces the cytotoxic activity of immune system effector cells, including Teff and NK cells. In some embodiments, target administration of an anti-TNFR2 antibody by the method of the present disclosure reduces the production of inflammatory cytokines by immune system effector cells, including Teff and NK cells.

[0118] Those skilled in the art will understand that in some embodiments, the modulation of the immune response includes reducing or eliminating inflammation in situations where, without the use of the anti-TNFR2 antibody of this disclosure, the outcome would have been expected to be inflammation.

[0119] In one embodiment, the Disclosure also provides the use of a composition comprising an anti-TNFR2 antibody of a subject for modulating the activity or function of regulatory T cells in the subject. In one embodiment, the composition of the Disclosure comprises an anti-TNFR2 antibody having a heavy-chain CDR sequence and a light-chain CDR sequence as disclosed herein. In another embodiment, the composition of the Disclosure comprises an anti-TNFR2 antibody having a VH sequence and a VL sequence as disclosed herein. In yet another embodiment, the composition of the Disclosure comprises an anti-TNFR2 antibody having a heavy-chain sequence and a light-chain sequence as disclosed herein.

[0120] In one embodiment, the Disclosure provides a method for modulating the activity or function of myeloid-derived suppressor cells (MDSCs) in a subject. In one embodiment, the method of the Disclosure comprises administering a composition containing an effective amount of the anti-TNFR2 antibody of the Disclosure to a subject. In one embodiment, the composition of the Disclosure comprises an anti-TNFR2 antibody having the heavy-chain CDR sequence and the light-chain CDR sequence disclosed herein. In another embodiment, the composition of the Disclosure comprises an anti-TNFR2 antibody having the VH sequence and the VL sequence disclosed herein. In yet another embodiment, the composition of the Disclosure comprises an anti-TNFR2 antibody having the heavy-chain sequence and the light-chain sequence disclosed herein. In one embodiment, the method of the Disclosure stimulates the proliferation of MDSCs. In another embodiment, the method of the Disclosure can activate MDSCs.

[0121] Those skilled in the art will understand that in some embodiments, MDSC activation includes suppression of the proliferation of immune system effector cells, including Teff and NK cells; reduction of the cytotoxic activity of immune system effector cells, including Teff and NK cells; downregulation of the production of pro-inflammatory cytokines by immune system effector cells, including Teff and NK cells; or any combination thereof. In some embodiments, target administration of an anti-TNFR2 antibody by the method of the present disclosure suppresses the proliferation of immune system effector cells, including Teff and NK cells. In some embodiments, target administration of an anti-TNFR2 antibody by the method of the present disclosure reduces the cytotoxic activity of immune system effector cells, including Teff and NK cells. In some embodiments, target administration of an anti-TNFR2 antibody by the method of the present disclosure reduces the production of inflammatory cytokines by immune system effector cells, including Teff and NK cells.

[0122] In one embodiment, the Disclosure also provides the use of a composition comprising an anti-TNFR2 antibody for modulating the activity or function of MDSCs in a subject. In one embodiment, the composition of the Disclosure comprises an anti-TNFR2 antibody having a heavy-chain CDR sequence and a light-chain CDR sequence as disclosed herein. In another embodiment, the composition of the Disclosure comprises an anti-TNFR2 antibody having a VH sequence and a VL sequence as disclosed herein. In yet another embodiment, the composition of the Disclosure comprises an anti-TNFR2 antibody having a heavy-chain sequence and a light-chain sequence as disclosed herein.

[0123] In another embodiment, the Disclosure provides a method for treating a disease of interest, comprising the step of administering to an interest a composition containing an effective amount of the anti-TNFR2 antibody of the Disclosure. In one embodiment, the composition of the Disclosure comprises an anti-TNFR2 antibody having a heavy-chain CDR sequence and a light-chain CDR sequence as disclosed herein. In another embodiment, the composition of the Disclosure comprises an anti-TNFR2 antibody having a VH sequence and a VL sequence as disclosed herein. In yet another embodiment, the composition of the Disclosure comprises an anti-TNFR2 antibody having a heavy-chain sequence and a light-chain sequence as disclosed herein.

[0124] In one embodiment, the Disclosure also provides the use of a composition comprising an anti-TNFR2 antibody for treating a disease of interest. In one embodiment, the composition of the Disclosure comprises an anti-TNFR2 antibody having a heavy-chain CDR sequence and a light-chain CDR sequence as disclosed herein. In another embodiment, the composition of the Disclosure comprises an anti-TNFR2 antibody having a VH sequence and a VL sequence as disclosed herein. In yet another embodiment, the composition of the Disclosure comprises an anti-TNFR2 antibody having a heavy-chain sequence and a light-chain sequence as disclosed herein.

[0125] In one embodiment, the exact amount of polypeptide or composition of the present invention required to produce the desired effect varies from subject to subject, depending on the species, age, sex, weight and general condition of the subject, the specific polypeptide, the route of administration, and whether other drugs are included in the regimen. Therefore, it is not possible to specify an exact amount for all compositions. However, those skilled in the art can determine the appropriate amount through routine experimentation. Dosages vary, and polypeptides can be administered once or more times a day (e.g., two or more, three or more, four or more, or five or more times) over a period of one day or more. Guidance for selecting an appropriate dose of antibody is readily available in the literature.

[0126] In one embodiment, the disease may be a viral infection, a bacterial infection, cancer, an autoimmune disease, or an immune disorder. In one embodiment, the disease may be an upper respiratory tract viral infection, an early lung infection, or an end-stage lung infection. Various diseases and cancers are known to be caused by viruses. Examples of disease-causing viruses include, but are not limited to, norovirus; rotavirus; hepatitis viruses of type 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; polyomavirus (JC virus, BK virus, etc.); Ebola virus; dengue virus; or any combination thereof.

[0127] In another embodiment, the disease may be cancer. Cancers include, but are not limited to, carcinoma, sarcoma, lymphoma, leukemia, germ cell tumor, blastoma, chondrosarcoma, Ewing's sarcoma, malignant fibrous histiocytoma of bone, osteosarcoma, rhabdomyosarcoma, cardiac cancer, brain tumor, astrocytoma, glioma, medulloblastoma, neuroblastoma, breast cancer, medullary carcinoma, adrenocortical carcinoma, thyroid cancer, Merkel cell carcinoma, ocular cancer, gastrointestinal cancer, colon cancer, gallbladder cancer, gastric cancer, gastrointestinal carcinoid tumor, hepatocellular carcinoma, 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, cervical cancer, nasopharyngeal cancer, hematopoietic tumors, non-Hodgkin lymphoma, skin cancer, basal cell carcinoma, melanoma, small cell lung cancer, non-small cell lung cancer, and any combination thereof.

[0128] In another embodiment, the disease may be an autoimmune disease. Examples of autoimmune diseases include, but are 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, Behçet's disease, celiac disease, Chagas disease, chronic inflammatory demyelinating polyneuropathy, Cogan syndrome, congenital heart block, Crohn's disease, dermatitis, dermatomyositis, lupus discoid, Dressler syndrome, endometriosis, fibromyalgia, fibrotic alveolitis, granulomatosis with polyangiitis, Graves' disease, Guillain-Barré syndrome, herpes zoster of pregnancy, and immune thrombocytopenic purpura. These include 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, post-myocardial infarction syndrome, post-pericardiotomy 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, Sjögren's syndrome, thrombocytopenic purpura, type 1 diabetes, ulcerative colitis, uveitis, vasculitis, and vitiligo.

[0129] In some embodiments, the disease is a transplant-related disease such as graft-versus-host disease (GvHD). In one embodiment, GvHD is acute GvHD. In another embodiment, GvHD is chronic GvHD.

[0130] The agonist anti-TNFR2 antibodies (or their antigen-binding fragments) of this disclosure can also be used to treat patients requiring organ repair or regeneration. For example, the agonist TNFR2 antibodies or their antigen-binding fragments of this disclosure can be used to promote organ repair or regeneration, for example, by binding TNFR2 to the surface of cells in damaged tissue to induce TRAF2 / 3- and / or NFκB-mediated cell proliferation. Examples of tissues and organs that can be regenerated by administration of agonist TNFR2 antibodies or their antigen-binding fragments include, but are not limited to, the pancreas, salivary glands, pituitary gland, kidney, heart, lung, hematopoietic system, cranial nerves, blood vessels including the aorta, olfactory glands, ear, nerves, head structures, eyes, thymus, tongue, bone, liver, small intestine, large intestine, intestine, lung, brain, skin, peripheral nervous system, central nervous system, spinal cord, breast, embryonic structures, embryo, and testes.

[0131] The agonist anti-TNFR2 antibody (or its antigen-binding fragment) of this disclosure may also be administered to subjects (e.g., humans) to treat neurological disorders or conditions. Neurological disorders or conditions include, but are not limited to, brain tumors, brain metastases, spinal cord injury, schizophrenia, epilepsy, amyotrophic lateral sclerosis (ALS), Parkinson's disease, Alzheimer's disease, Huntington's disease, and stroke.

[0132] The agonist TNFR2 antibody (or its antigen-binding fragment) of this disclosure may also be administered mixed, conjugated, or together with another agent that promotes regulatory T cell proliferation, or separately. Additional agents that may be used to promote regulatory T cell expansion include, but are not limited to, IL-2 and TNFα, which are homologous ligands of TNFR2.

[0133] In another embodiment, the Disclosure provides a method for treating such diseases or conditions using a polynucleotide encoding the anti-TNFR2 antibody of the Disclosure.

[0134] The various embodiments and representations of the anti-TNFR2 antibody of this disclosure, as described in detail above and claimed in the claims, are experimentally supported in the following examples. Illustrated herein is the production of an anti-TNFR2 agonist antibody that exists as a non-aggregated species and generally migrates on an SEC column as folded IgG1. The generated anti-TNFR2 IgG1 agonist antibody is EC1 at approximately 1.8 nM to 66 nM. 50 These antibodies bound to soluble TNFR2-His within a certain range, demonstrating their robust binder properties. Furthermore, the antibodies specifically bound to TNFR2 but not to TNFR1. The generated anti-TNFR2 antibodies contained high-affinity functional agonists and were EC 50 The antibodies agonize the TNFR2 receptor without the addition of other molecules with a range of 0.5 nM to 277 nM. Furthermore, the activation of TNFR2 by the generated anti-TNFR2 antibodies appears to be independent of IgG-Fc clustering. When the effect of TNFα on the activation properties of the generated anti-TNFR2 antibodies was investigated, these antibodies were shown to almost completely activate the TNFR2 receptor and are most likely to activate in epitopes that do not block the TNFα binding site.

[0135] Examples

[0136] Example 1

[0137] Fabrication of a TNFR2 agonist

[0138] Experimental Procedure

[0139] Library creation

[0140] (Building a library)

[0141] The libraries were constructed based on three template antibodies (PDB: 2I5Y, 4IOI, and 3E8U) by overlap extension PCR using denatured oligonucleotides. PCR used to introduce diversity was performed in three steps using Phusion high fidelity DNA polymerase (New England Biolabs USA, Cat: M0530) according to the manufacturer's instructions (30 seconds at 98°C, 20 seconds at 65°C, 30 seconds at 72°C, 30 cycles). The PCR products were gel-purified using a gel purification kit and assembled in equimolar ratios in three steps, as described above, except for the absence of primers. The assembled PCR products were reused as templates for PCR to amplify the complete scFv library, and homologous recombination in yeast cells was efficiently performed using forward and reverse primers with yeast surface display (YSD) expression vector homologous sequences added at 5' and 3' to the scFv library.

[0142] The scFv library was constructed by repeating the flexible linker Gly-Gly-Gly-Gly-Ser (sequence number 333) three times between the VH and VL.

[0143] The Fab display library was constructed in a similar manner to the scFv library, except that VL and VH were constructed separately and cloned under two promoters. VH was cloned under the Gal-10 promoter within a frame between the aga2 gene and the constant heavy chain domain 1 (CH1). VL was cloned under the Gal-1 promoter within a frame between the signal peptide and the constant light chain domain (CL). The Fab fragments were combined into a single fragment using PCR and cloned into the pFAB1 expression vector in a similar manner to the scFv library.

[0144] (Transfection of the library)

[0145] Library transfection (transformation) was performed using a publicly available method ("Benatuil et al., An improved yeast transformation method for the generation of very large human antibody libraries. Protein Eng. Des. Sel. 23, 155-159 (2010)"). 400 μl of yeast suspension (EBY100, ATCC, USA) per 0.2 cm cuvette (cell projects) was electroporated (BioRad, USA, GenePulser) using 4 μg of linearization vector (pCTcon3 or pFAB1) and 12 μg of DNA insert (scFv fragment or assembled Fab) in a vector:insert ratio of 1:3 ("Chao, G. et al., Isolating and engineering human antibody using yeast surface display. Nat. Protoc. 1, 755-768 (2006)"). The average number of transformants in the library was approximately 1 × 10⁶ by serial dilution of transformed cells. 8 I did.

[0146] Library screening

[0147] (Screening and selection using yeast surface displays)

[0148] Yeast display libraries were grown in SDCAA selective medium, and expression was induced overnight at 30°C with 2% w / v galactose according to an established protocol ("Chao, G. et al., (2006)"). In short, the library was incubated for 1 hour in PBS (0.1% BSA) with 1000nM to 0.1nM recombinant human TNFR2 containing a 6xhis tag or TNFR2-Fc fusion (Reprokine, Israel), then washed three times with PBS (0.1% BSA), and subsequently labeled with mouse anti-c-MycFITC (Miltenyi Biotec, cat #130-116-485) or mouse anti-c-Myc (Santa Cruze, USA cat # sc-40), as well as goat anti-mouse IgG-FITC (Sigma-Aldrich, cat # F4143-1ml), monoclonal anti-HisAPC (Miltenyi Biotec, Germany, cat 0020130-119-782), or anti-FcAPC (Jackson ImmunoResearch, USA, cat # It was fluorescently labeled with (109-135-098).

[0149] When nonspecific binding needed to be avoided, fluorescently labeled antibodies were substituted with rabbit anti-c-Myc (Abcam, cat# ab9106), goat anti-rabbit APC (Abcam, Cat# ab130805), and anti-HisAlexa488 (Qiagen, cat# 20-35310).

[0150] After labeling, the library has a library size of 1 x 10 6 The cells were selected on MACS beads until they were reduced in size, and then sorted for high affinity binders for recombinant human TNFR2 on a BioRad S3e fluorescence-activated cell sorter or a BD ARIA III fluorescence-activated cell sorter (FACS). The clones isolated from the final sort were sequenced by extracting plasmid DNA from the yeast clones using the Zymoprep kit (Zymo Research, USA), thereby determining the DNA sequence.

[0151] When applicable, yeast-displayed Fabs were labeled and selected under the same conditions as scFv selection, in which anti-FLAG-PE (Miltenyi Biotec, cat #130-101-576) was added for detection of Fab light chain display.

[0152] (Selection of Koff)

[0153] To select binders with improved off-rate, yeast was incubated with 10 nM to 1 nM of TNFR2-His for 30 minutes. Subsequently, the yeast was washed three times with 1 mL of PBS (0.1% BSA), and incubated with 100 nM of TNFR2-Fc for 4 hours, 6 hours, and 24 hours. Alternatively, after washing, the cells were diluted 10-fold in PBS (0.1% BSA) and incubated until the specified time point. Next, the yeast was washed three times, labeled with anti-Myc-FITC (Santa Cruz, USA, Cat# 9E10) and monoclonal anti-His APC (Miltenyi Biotec, Germany, cat 0020130-119-782), and sorted on a SE3 FACS as described above.

[0154] Furthermore, two rounds of Koff selection were performed at the optimal time point when approximately 50% to 70% of the initial binding was lost.

[0155] (Analysis of clones by yeast surface display EC 50 )

[0156] To determine the 50% effective concentration (EC 50 ) for TNFR2 binding to yeast scFv or Fab clones (EC 50 -TNFR2 binding), specific clones were labeled with TNFR concentrations ranging from 0.1 nM to 1000 nM and analyzed by FACS. The median fluorescence intensity (MFI) was measured for each TNFR2 concentration, and EC 50 was calculated using Prism 8 GraphPad (GraphPad, San Diego, USA) software.

[0157] IgG production

[0158] (Reformat)

[0159] The selected scFv clones were reformatted to human IgG1 format. The sequences of the light chain (LC) and heavy chain (HC) variable regions were optimized for the use of mammalian codons and ordered as GenBlock (GB) from IDT (Integrated DNA Technologies, Coralville, Iowa, USA). GenBlock (GB) was cloned into pSF-CMV-HuIgG1_HC (HC plasmid) and pSF-CMV-HuLambda_LC (LC plasmid) (Oxford Genetics, Oxford UK) using standard cloning techniques. Where indicated, pSF-CMV-HuIgG1_HC_LALA (HC plasmid) with the L234A / L235A (LALA) mutation was used.

[0160] (IgG expression)

[0161] Expi-CHO cells (Thermo Fisher Scientific, USA) were transfected with LC plasmid and HC plasmid in a 2:1 ratio and expressed according to the manufacturer's instructions. In short, 50 ml of Expi-CHO cells were expressed at 37°C, 120 rpm, and 8% CO2 at a rate of 6 × 10⁶ 6The cells were incubated to a density of cells / ml. Next, 50 μg of heavy chain expression plasmid and light chain expression plasmid were transfected into the CHO cells in a 1:2 ratio. After transfection, booster and feed were added to the culture, and the growth conditions were changed to 32°C, 120 rpm, and 5% CO2. Cells were harvested 10 days after transfection. IgG was purified from the supernatant using proteinA beads (Tosoh Bioscience GmbH, Germany), and then purified by size exclusion chromatography (SEC) on a SUPERDEX200™ 10 / 300 increasing column using PBS as the mobile phase (GE healthcare, USA).

[0162] (Size exclusion chromatography)

[0163] To analyze and purify IgG, samples were loaded onto a SUPERDEX200™ 10 / 300 increment column (GE Healthcare, USA) using a GE AKTAE xplorer chromatography system (GE Healthcare, USA) at a flow rate of 0.8 ml / min. Alternatively, if instructed, up to 12 mg of IgG was loaded onto a Waters ACQUITY arc HPLC with a BioResolve™ SECmAb column (Waters, USA) at a flow rate of 0.5 ml / min for a run time of 20 minutes. PBS served as the mobile phase, and retention was monitored at 280 nM on both columns.

[0164] (Ligand-binding ELISA)

[0165] The IgG binding affinity to TNFR2 was investigated by ELISA. The analyzed antibody was coated (50 ng / well) onto a 96-well plate (Greiner Bio-One high binding) and incubated overnight at 4°C. The plate was then washed three times with 300 μl of PBS buffer containing 0.05% Tween20 (PBS-T), blocked with 300 μl of PBS-T supplemented with 1%-2% BSA, and incubated at room temperature for 1 hour. The antibody-coated plate was washed three times with 300 μl of PBS-T and incubated with a final volume of 50 ml of serial dilution of the test ligand hTNFR2-His (Reprokine, Israel) for 1-2 hours. Next, the plate was washed three times with 300 μl of PBS-T and incubated with 50 μl of anti-HIS-HRP (Santa Cruz Biotechnology, USA, SC-8036HRP) conjugate diluted 1:250 in PBS. After six further washing steps, the reaction was expanded with 50 ml of tetramethylbenzidine (TMB) reagent (Southern Biotech, USA), and the reaction was stopped with 50 ml of 0.5 N H2SO4. Detection was performed using a Synergy LX BioTek (BioTek, USA) plate reader with the absorbance filter set to 450 nM. Binding affinity was determined by fitting the data to a specific binding nonlinear regression model on Prisma 8 GraphPad software.

[0166] TNFR2 / TNFR1 specificity ELISA is used in EC 50 ELISA analysis (EC 50 The procedure was carried out in the same manner as with (TNFR2 binding), but with both TNFR1 and TNFR2 at concentrations of 100-1000 nM.

[0167] (FACS analysis)

[0168] LALA-mutated Fc format anti-TNFR2 antibody (30.116) was incubated with HEK-TNFR1 (InvivoGen, Cat: hkb-tnfdmyd) and HEK-TNFR2 (200 nM Ab, 1 million cells / well) cell lines on ice for 15 minutes. Cells were then stained with goat anti-human Fc-APC conjugate on ice for 30 minutes according to the manufacturer's instructions (Jackson Immunological Research, cat. # 109-135-098). Cells were analyzed using a CytoFLEX flow cytometer (Beckman, USA). Gating was determined individually for each cell population based on secondary control only.

[0169] result

[0170] Library design

[0171] To produce antibodies that bind to TNFR2, the “re-epitope” method was applied to existing antibodies. The re-epitope method allows for the introduction of new specificity into existing antibodies and enables the selection of known antibodies with desirable biophysical and biochemical properties as templates. Thus, re-epitope antibodies possess both new specificity and a desirable developmentability profile. The computational process of the re-epitope method requires two steps: (i) using any computer analysis to identify the estimated complementarity between the existing antibody and the new epitope, and (ii) applying any computer analysis or tool that can propose the introduction of specific mutations predicted to enhance antibody binding to the new desired epitope. Examples of such computer analyses are described in U.S. Patent Application Publication No. 2018 / 0068055 and “Nimrod et al., Cell Rep. 25(8):2121-2131 (2018)”. In one embodiment, three libraries were designed using the variable domain sequences of re-epitope template antibodies 2I5Y, 4IOI, and 3E8U, which are predicted to be promising candidates for introducing novel specificity to TNFR2. Some of these template sequences have been further modified in later generations of libraries to improve developability and humanization. The re-epitope libraries were constructed in this manner and introduced into yeast.

[0172] YSD screening for TNFR2 binders

[0173] After introducing mutations, the library was screened in yeast surface display (YSD) format to identify clones that specifically bind to TNFR2. First, the library was subjected to MACS (magnetic bead) selection, and then to FCAS selection. All clones from the three re-epitope templates showed relative binding.

[0174] To further improve affinity, an affinity maturation library was constructed by the same method as the construction of the above library. The affinity maturation library was subjected to conventional and specific Koff improvement selection as described above. The best binders were gated, yeast clones were isolated and sequenced. These clones are shown in Table 1.

[0175]

Table 1-1

[0176]

Table 1-2

[0177]

Table 1-3

[0178]

Table 1-4

[0179]

Table 1-5

[0180]

Table 1-6

[0181]

Table 1-7

[0182]

Table 1-8

[0183] EC against TNFR2 in yeast surface display 50 Binding value

[0184] To confirm that TNFR2-binding clones exhibit at least one to two orders of magnitude nanomolar affinity for TNFR2, a limited set of clones were subjected to yeast surface display (YSD) EC as described above. 50 The clones were subjected to binding assays. As shown in Figures 1A to 1D, these clones showed affinity for TNFR2-His in the range of 1 nM to 20 nM. This indicates that scFv and Fab in yeast displays are robust binders for human TNFR2. YSD-EC of these clones. 50 Table 2 shows an overview of the values.

[0185] [Table 2]

[0186] Analysis of clones binding to TNFR2 in IgG format

[0187] (SEC analysis)

[0188] To further clarify the characteristics of clones that showed binding to TNFR2, the clones shown in Tables 1 and 2, which showed the most promising binding to scFv and Fab, were reformatted to human IgG1, transiently expressed in expi-CHO cells according to the manufacturer's instructions, and purified as described herein. In addition, DNA of clones 30.032, 30.046, and 30.033 was synthesized, and IgG was produced in HEK cells by Genscript Antibody Services (Genscript, USA).

[0189] Clones 30.113, 30.114, 30.116, 30.117, 30.118, and 30.119 were reformatted to IgG1 containing the heavy chain L234A / L235A mutation (LALA mutation), designed to reduce IgG binding to Fc and Fc-γ receptors. Additionally, IgG1 clones containing the heavy chain L234A / L235A mutation (LALA mutation) were generated. A list of IgG1 anti-TNFR2 antibody clones containing the heavy chain L234A / L235A mutation includes BDG30.113, BDG30.114, BDG30.115, BDG30.116, BDG30.117, BDG30.118, BDG30.119, BDG30.122, BDG30.123, BDG30.200, BDG30.201, BDG30.202, BDG30.203, and BDG30.204.

[0190] Table 3 shows summaries of scFv and Fab files reformatted to IgG format. Unless otherwise noted, the heavy chain contains LALA mutations.

[0191] [Table 3-1]

[0192] [Table 3-2]

[0193] [Table 3-3]

[0194] [Table 3-4]

[0195] [Table 3-5]

[0196] Examples of amino acid sequences of complementarity-determining regions (CDRs) are shown in Tables 7 to 9 below. Table 7 shows an example of a set of three CDRs on the heavy chain (HCDR1, HCDR2, and HCDR3), and Table 8 shows the corresponding set of three CDRs on the light chain (LCDR1, LCDR2, and LCDR3). Table 9 shows yet another example of the set of HCDR1, HCDR2, and HCDR3 and the set of LCDR1, LCDR2, and LCDR3 of the anti-TNFR2 antibody disclosed herein.

[0197] To test the aggregation tendency of these antibodies, IgG was subjected to size exclusion chromatography (SEC) using PBS as the mobile phase, as described above, with Akta, SUPERDEX10 / 300 increasing columns, or Waters ACQUITY arc HPLC, BioResolve SEC mAb columns. Examples of SEC analysis of selected antibodies are shown in Figures 2A–2R, and a summary of the complete SEC analysis of clones is shown in Table 4. These results indicate that IgG 30.086–30.113, 30.115, 30.117–119, 30.122–30.123, 30.202, and 30.204 eluted from protein A as mostly non-aggregating species and generally migrated to the SEC column as folded IgG1.

[0198] [Table 4-1]

[0199] [Table 4-2]

[0200] (specificity ELISA)

[0201] The extracellular domains of TNFR2 and TNFR1 share 27% homology. To test the specificity for TNFR2, antibodies were analyzed by ELISA analysis for TNFR1 and TNFR2 as described herein. Briefly, antibodies were directly coated into ELISA plate wells, the wells were blocked, 100 nM TNFR1-His-Fc or TNFR2-His-Fc was added to the wells, washed, and detected using anti-His-HRP. As shown in Figures 3A and 3B, IgG 30.092, 30.093, 30.094, 30.095, 30.085, 30.089, 30.086, 30.116, 30.118, 30.119, 30.111, 30.113, and 30.114 specifically bound to TNFR2 but not to TNFR1.

[0202] IgG binding affinity to TNFR2

[0203] (IgG ELISA EC 50 )

[0204] To test the binding affinity of clones reformatted as IgG1 and IgG1LALA to TNFR2, the EC of the ELISA was performed for each antibody. 50 Binding experiments were performed. As shown in Figures 4A to 4F, these antibodies were bound to EC2 at concentrations of 1.8 nM to 66 nM. 50 They bind to soluble TNFR2-His within a certain range, indicating that these antibodies have a robust binder. Table 5 shows the EC2 of TNFR2 for the antibodies shown. 50 The values ​​are shown. These values ​​are the mean values ​​from at least two biological replicates.

[0205] [Table 5-1]

[0206] [Table 5-2]

[0207] Establishment of a TNFR2-dependent NFκB cell-based assay

[0208] Reporter cell lines were created to investigate whether the antibody functionally affects TNFR2 cell signaling, and to determine whether the antibody exhibits agonist, antagonist, or inaction effects on TNFR2.

[0209] HEK-Blue(TM) Null cells were purchased from InvivoGen (Toulouse France). These cells contained a TNFR1 null mutation and a plasmid encoding soluble embryonic alkaline phosphatase (SEAP) under the control of an IFN-β minimal promoter fused to five NF-κB and AP-1 binding sites. A pcDNA3.1 plasmid encoding human TNFR2 (residues 1-461) under a CMV promoter was introduced into the HEK-Blue(TM) Null cells. The cells were stored for 14 days under selection with 50 μg / ml hygromycin B. After the selection period, TNFR2 expression was confirmed by Western blotting (Figure 5).

[0210] The cells were then diluted to a critical dilution of 0.5 cells / well in a 96-well plate and grown in growth medium containing DMEM supplemented with 10% FBS, L-glutamine, PEN / Strep, and 50 μg / ml hygromycin B. To identify TNFα-responsive clones, replica plates were prepared with single clones, and TNFα-dependent NFκB activation was tested using Quanti-Blue (QB, InvivoGen) substrate according to the manufacturer's instructions (Figures 6A-6C).

[0211] Figures 7A and 7B show the results of the TNFα-dependent NFκB response of a specific clone in a dynamic range of 20 pM to 1000 pM. Activation was dose-dependently inhibited by soluble TNFR2 (Figure 7B) but unaffected by isotype control antibodies (Figure 7A). Clone G6 was selected to evaluate target antibodies for agonizing or antagonistizing the TNFR2 receptor.

[0212] Testing the function of IgG in HEK-TNFR2 reporter cell lines

[0213] To test the effect of anti-TNFR2 antibodies on TNFR2 cellular signaling, IgG was incubated overnight with HEK-TNFR2 reporter cell lines at a maximum concentration of 600 nM, and antibody-dependent NFκB activation was tested as detailed herein (Figures 8A–8D).

[0214] The IgGs shown in Table 6, with the exception of 30.088 and 30.117, exhibited dose-dependent NFκB activation and functional agonism in the EC. 50 The values ​​range from 0.5 nM to 25.7 nM, indicating that these antibodies are high-affinity functional agonists capable of agonizing receptors without the addition of other molecules.

[0215] To verify that NFκB activation depends on direct activation of the TNFR2 receptor by antibodies and is not activated via the TNFR1 alternative pathway, we tested antibodies 30.113, 30.114, 30.115, 30.116, 30.117, 30.118, 30.119, 30.123, 30.200, 30.201, 30.202, 30.203, and 30.204 using HEK-Blue-TNFa cells. HEK-Blue-TNFa cells do not express TNFR2 but spontaneously express TNFR1 and contain plasmids encoding soluble embryonic alkaline phosphatase (SEAP) under the control of IFN-β minimal promoters fused to five NF-κB and AP-1 binding sites.

[0216] As shown in Figure 9A, HEK-Blue-TNFa cells do not express TNFR2, as revealed by FACS analysis of these cells labeled with 200 nM 30.116 anti-TNFR2Ab. On the other hand, NFκB is readily activated by the addition of 11 nM TNFa (Figure 9B), suggesting that NFκB activation is via the TNFR1 pathway. In HEK-TNFR2 cell lines, the antibodies shown activated NFκB, but in HEK-TNFR1 cell lines, NFκB was not activated upon addition of 1 mM antibodies 30.113, 30.114, 30.115, 30.116, 30.117, 10.118, 30.119, 30.123, 30.200, 30.201, 30.202, 30.203, or 30.204. This suggests that the activation is specific to TNFR2 (Figures 9B-9C).

[0217] TNFR2 activation is independent of IgG-Fc clustering. This is supported by the fact that the HEK293 reporter cell line does not express the Fcγ receptor, and that the IgG in the above experiment was added to the cells in a soluble form and did not bind to the plate. Furthermore, IgG with LALA mutations designed to reduce IgG binding to the Fcγ receptor (30.113-30.204) was expressed. Taken together, these data suggest that TNFR2 activation is independent of IgG-Fc clustering. Table 6 shows the EC of functional agonism of TNFR2 in the HEK293-NFκB reporter cell line. 50 The values ​​are summarized below. These values ​​are the mean values ​​from at least two biological replicates.

[0218] [Table 6-1]

[0219] [Table 6-2]

[0220] [Table 6-3]

[0221] To investigate the effect of TNFα on antibody-dependent NFκB activation, HEK-TNFR2 cells were incubated with 200 nM anti-TNF2 antibody, and TNFα was added to a concentration of 100 nM. As shown in Figures 10A–10D, TNFα had little effect on the maximal activation induced by antibodies 30.116, 30.111, 30.086, and 30.119. This indicates that these antibodies activate the TNFR2 receptor to near its maximum capacity in epitopes that do not block the TNFα binding site.

[0222] In summary, the results of this disclosure demonstrate that the antibody binds strongly to TNFR2 in the sub-nanomole to low two-order-of-magnitude nanomolar range, is specific to TNFR2, and agonizes TNFR2 in an Fc-independent manner.

[0223] While this specification has illustrated and described specific features of the anti-TNFR2 antibody according to the present invention, various modifications, substitutions, alterations, and equivalents may be conceivable to those skilled in the art. Therefore, it should be understood that the appended claims are intended to encompass all such modifications and alterations that fall within the scope of the present invention.

[0224] [Table 7-1]

[0225] [Table 7-2]

[0226] [Table 7-3]

[0227] Table 7-4

[0228] Table 8-1

[0229] Table 8-2

[0230] Table 8-3

[0231] Table 9-1

[0232] Table 9-2

Claims

1. An isolated anti-TNFR2 antibody (anti-tumor necrosis factor receptor 2 antibody) comprising a set of three CDRs (complementarity-determining regions) on the heavy chain, namely HCDR1, HCDR2, and HCDR3, and a set of three CDRs on the light chain, namely LCDR1, LCDR2, and LCDR3, wherein the CDRs have the amino acid sequences of SEQ ID NO: 187, SEQ ID NO: 345, and SEQ ID NO: 356 (HCDR1, HCDR2, and HCDR3, respectively), and SEQ ID NO: 371, SEQ ID NO: 377, and SEQ ID NO: 395 (LCDR1, LCDR2, and LCDR3, respectively). Anti-TNFR2 antibody.

2. The anti-TNFR2 antibody according to claim 1, It includes a heavy chain variable region (VH) having at least 90% sequence identity with SEQ ID NO: 87 and a light chain variable region (VL) having at least 90% sequence identity with SEQ ID NO:

88. Anti-TNFR2 antibody.

3. The anti-TNFR2 antibody according to claim 1, The antibody in question contains a heavy chain and a light chain. The heavy chain and the light chain are, (a) A heavy chain having at least 90% sequence identity with SEQ ID NO: 432 and a light chain sequence having at least 90% sequence identity with SEQ ID NO: 260, and (b) A heavy chain having at least 90% sequence identity with SEQ ID NO: 259 and a light chain sequence having at least 90% sequence identity with SEQ ID NO: 260 An anti-TNFR2 antibody having an amino acid sequence selected from the following.

4. An anti-TNFR2 antibody according to any one of claims 1 to 3, The antibodies in question are IgG, Fv, scFv, Fab, and F(ab'). ２ Anti-TNFR2 antibodies, which are mini-bodies, dia-bodies, tri-bodies, or bispecific antibodies.

5. The anti-TNFR2 antibody according to claim 4, The IgG is an anti-TNFR2 antibody, which is IgG1, IgG2, IgG3, or IgG4.

6. The anti-TNFR2 antibody according to claim 2, This antibody is an anti-TNFR2 antibody that agonizes TNFR2.

7. The anti-TNFR2 antibody according to claim 6, This antibody is an anti-TNFR2 antibody that agonizes TNFR2 in an Fc-independent manner.

8. The anti-TNFR2 antibody according to claim 2, The antibody has an EC of approximately 0.05 to 100 nM. ５０ Anti-TNFR2 antibody with binding measurement value.

9. The anti-TNFR2 antibody according to claim 2, The antibody has an EC of approximately 0.05 to 100 nM. ５０ Anti-TNFR2 antibody with functional agonism measurement capabilities.

10. The anti-TNFR2 antibody according to claim 2, An anti-TNFR2 antibody that activates TNFR2-mediated signaling at at least 80% of the level compared to signaling activated by TNF.

11. The anti-TNFR2 antibody according to claim 2, An anti-TNFR2 antibody that activates TNFR2-mediated signaling at a level of at least 95% compared to signaling activated by TNF.

12. An anti-TNFR2 antibody according to any one of claims 1 to 11, A composition comprising a pharmaceutically acceptable carrier.

13. An isolated polynucleotide encoding an anti-TNFR2 antibody according to any one of claims 1 to 11.

14. A vector comprising a polynucleotide as described in claim 13.

15. A host cell comprising the vector according to claim 14.

16. A composition for regulating the activity or function of regulatory T cells in a subject, A composition comprising the anti-TNFR2 antibody according to any one of claims 1 to 11.

17. The composition according to claim 16, The regulatory T cells are CD4 ＋ CD25 ＋ Foxp3 ＋ A composition.

18. The composition according to claim 16, A composition that stimulates the proliferation of the aforementioned regulatory T cells.

19. The composition according to claim 16, A composition that activates the aforementioned regulatory T cells.

20. The composition according to claim 16, A composition that modulates the immune response mediated by the regulatory T cells.

21. A composition for regulating the activity or function of myeloid-derived immunosuppressive cells (MDSCs) in a subject, A composition comprising the anti-TNFR2 antibody according to any one of claims 1 to 11.

22. The composition according to claim 21, A composition that stimulates the proliferation of the myeloid-derived immunosuppressive cells (MDSCs).

23. The composition according to claim 21, A composition that activates the myeloid-derived immunosuppressive cells (MDSCs).

24. A composition for treating a disease in a subject, A composition comprising the anti-TNFR2 antibody according to any one of claims 1 to 11.

25. The composition according to claim 24, The disease is cancer, autoimmune disease, graft-versus-host disease (GvHD), viral infection, bacterial infection, organ repair or regeneration, and / or neurological disease or condition, and is a composition.

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