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

Abstract

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

Description

[Technical Field]

[0001] (Cross-reference of related applications) This application is based on U.S. Provisional Patent Application No. 63 / 047, 490, filed on 2 July 2020. This asserts priority. The entirety of the disclosure of the above application is provided herein by reference. It will be used.

[0002] (Statement regarding sequence listings) This application includes a sequence listing submitted electronically in ASCII format, and this sequence listing The entire document is incorporated herein by reference. This A document was created on June 28, 2021. The SCII copy is named "P-597451-PC_SL.txt", and its size The file size is 573,679 bytes.

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

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

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

[0006] TNFR1 is constitutively expressed in virtually all nuclear cell types. TNFR1 is expressed in sT It has been reported that it binds to NFα and mTNFα and is activated by both. One of the main functions of FR1 is to mediate TNF-induced apoptosis via the NF-κB pathway. This is the case. Furthermore, in certain situations, activation of the NF-κB pathway can lead to anti-apoptotic effects. Proteins and inflammatory cytokines such as IL-1 and IL-6 are produced.

[0007] TNFR2 is a recently discovered and well-defined gene primarily associated with activated T cells. It is expressed in bone marrow cells and glial cells. TNFR2 is a 75 kDa transmembrane receptor. Yes, it has an extracellular domain composed of four cysteine-rich domains (CRDs), and a single The transmembrane domain and the cytoplasm that interacts with TNF receptor-related factor 2 (TRAF2) It has a domain. Recruitment of TRAF2 activates the alternative NF-kB pathway. A cascade of events leading to sexualization is promoted. TNF2 promotes sTNFα and mTN It can firmly bind to both Fα and is mainly activated by mTNFα, and sTN It is not activated by Fα.

[0008] TNFR2 is CD4 + CD25 + Foxp3+ Regulatory T cells (Treg) and bone marrow origin It has been shown to be expressed at high levels on the surface of mesenteric cell-modulating cells (MDSCs). Activation of TNFR2 in this context promotes immunosuppressive activity and increases the proliferation of Treg and MDSCs. It is extremely important for survival. In addition, TNFR2 is CD8 + Effector T cells ( It plays two roles in Teff. One role is CD8 during the initial immune response. + Te Another role is to mediate the activation signal to ff, and CD8 + To Teff This involves transmitting apoptosis signals to terminate the immune response.

[0009] The important role of TNFR2 in regulating the immune system is reflected in several pathological conditions. In cancer, TNFR2 is used to protect invasive Tregs, MDSCs, and tumor cells from their own tumor microstructure. Highly expressed in the environment (TME). TNFR2 receptor activity in Tregs and MDSCs. This process suppresses the immune system of TME. In addition, it blocks TNFR2 in in vitro cancer cell lines. It has been shown to have a dose-dependent cytotoxic effect.

[0010] Autoimmune diseases include rheumatoid arthritis (RA), Crohn's disease (CD), and multiple sclerosis (MS). ), abnormalities in the TNFα signaling pathway in various autoimmune diseases such as type 1 diabetes It has been reported that in many of these conditions where TNFR1 is more dominant, the majority of TNFα Blocking it in its soluble form is sufficient to produce an effective effect. (Infliximia) TNFα blockers such as bladderwrack and adalimumab are used to treat rheumatoid arthritis (RA) and Crohn's disease (CD). It has been successfully applied to ulcerative colitis (UC) and other conditions. In diseases such as ) and multiple sclerosis, the above approaches have been shown to be particularly ineffective. .

[0011] In certain cases, specific inhibition or activation of the TNFR2 pathway has been proven to be beneficial. For example, in a mouse cancer model, specific inhibition of TNFR2 directly affects cancer cells. In addition to acting indirectly, it also exhibits robust effects on the activation of the immune system. TNFR 2. In several autoimmune diseases in which Treg and MDCS play a major role, It is strongly involved as a driving force. Experimental autoimmune encephalomyelitis (EA) in multiple sclerosis (MS) E) In mouse models, deletion of the TNFR2 gene has been shown to have adverse effects. In graft-versus-host disease (GvHD) models, agonizing TNFR2 leads to G It has been shown that the severity of vHD decreases. In addition, TNFR2 activation is associated with Calmetre. It is also related to novel treatments for type 1 diabetes (T1D) using Bacillus toxin (BCG).

[0012] Considering that TNFR2 plays an important role in regulating immune responses in various pathological conditions, Considering this, improved treatments for diseases such as cancer and autoimmune diseases As a strategy, improved targeting of TNFR2 with antagonists or agonists. Development of this is required. [Overview of the project] [Means for solving the problem]

[0013] This disclosure provides various anti-TNFR2 (tumor necrosis factor receptor 2) antibodies. One embodiment Each of the anti-TNFR2 antibodies disclosed herein has three complementarity-determining regions (CDRs) on the heavy chain. A set of (HCDR1, HCDR2, and HCDR3) and the three CDRs on the light chain Includes a set (LCDR1, LCDR2, and LCDR3). In one embodiment, HCDR1, The HCDR2 and HCDR3 sets include the amino acid sequence combinations shown in Table 7 below. The corresponding sets of LCDR1, LCDR2, and LCDR3 on the light chain are as follows: This includes the amino acid sequence combinations shown in Table 8. In another embodiment, HCDR1 on the heavy chain, A set of HCDR2 and HCDR3, and their corresponding LCDR1 and LCDR on the light chain. Sets 2 and LCDR3 include 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 this disclosure has 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 are as follows: SEQ ID NOs. 289 and 29 0, SEQ ID NOs: 3 and 4, SEQ ID NOs: 7 and 8, SEQ ID NOs: 11 and 12, SEQ ID NOs: 15 and 1 6, Sequence IDs 19 and 20, 23 and 24, 27 and 28, Sequence ID 3 1 and 32, SEQ ID NOs: 35 and 36, SEQ ID NOs: 39 and 40, SEQ ID NOs: 43 and 44, Column numbers 47 and 48, sequence numbers 51 and 52, sequence numbers 55 and 56, sequence number 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, Sequence IDs 79 and 80, Sequence IDs 83 and 84, Sequence IDs 87 and 88, Sequence IDs 91 and 92, 95 and 96, 99 and 100, and 10 3 and 104, SEQ ID NOs: 107 and 108, SEQ ID NOs: 111 and 112, SEQ ID NOs: 115 and 116, sequence numbers 119 and 120, sequence numbers 123 and 124, sequence number 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 1 52, Sequence IDs 157 and 158, Sequence IDs 163 and 164, Sequence IDs 169 and 17 0, SEQ ID NOs. 175 and 176, SEQ ID NOs. 181 and 182, SEQ ID NOs. 193 and 194 , Sequence IDs 199 and 200, Sequence IDs 205 and 206, Sequence IDs 211 and 212, Sequence IDs 217 and 218, 223 and 224, 229 and 230, Column numbers 233 and 234, sequence numbers 237 and 238, sequence numbers 241 and 242, array Numbers 245 and 246, Sequence numbers 249 and 250, Sequence numbers 253 and 254, Sequence number Sequence numbers 257 and 258, Sequence numbers 261 and 262, Sequence numbers 265 and 266, Sequence number 269 ​​and 270, Sequence IDs 273 and 274, Sequence IDs 277 and 278, Sequence ID 2 81 and 282, Sequence IDs 285 and 286, Sequence IDs 293 and 294, Sequence ID 29 7 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 The group consisting of sequence numbers 322, 325 and 326, and 329 and 330 It has an amino sequence that is selected from among them.

[0024] In another embodiment, each of the anti-TNFR2 antibodies of the present disclosure comprises a heavy chain and a light chain, The chain and light chain are sequence numbers 438 and 292, 291 and 292, and 235. and 236, Sequence IDs 239 and 240, Sequence IDs 243 and 244, Sequence ID 247 and 248, Sequence IDs 251 and 252, Sequence IDs 255 and 256, Sequence ID 259 and 260, Sequence IDs 263 and 264, Sequence IDs 267 and 268, Sequence IDs 271 and 2 72, Sequence IDs 275 and 276, Sequence IDs 279 and 280, Sequence IDs 283 and 28 4. Sequence IDs 287 and 288, 295 and 296, 299 and 300 , Sequence IDs 303 and 304, Sequence IDs 307 and 308, Sequence IDs 311 and 312, Sequence IDs 315 and 316, 319 and 320, 323 and 324, Column numbers 327 and 328, sequence numbers 331 and 332, sequence numbers 432 and 260, array Numbers 433 and 304, Sequence numbers 434 and 308, Sequence numbers 435 and 236, Sequence number Sequence numbers 436 and 244, sequence numbers 437 and 284, and sequence numbers 439 and 296 It has an amino sequence selected from the following group.

[0025] In yet another embodiment, the Disclosure relates to a pharmaceutically acceptable carrier and the anti-TNFR of the Disclosure. The present invention provides a composition containing two antibodies.

[0026] This disclosure also includes the polynucleotide sequence encoding the anti-TNFR2 antibody of this disclosure, and The present invention provides vectors and host cells containing such polynucleotide sequences.

[0027] In further embodiments, the anti-TNFR2 antibody of the present disclosure promotes the proliferation of regulatory T cells and / or This can be used to adjust the function. In another embodiment, the anti-TNFR of the present disclosure 2. Antibodies are used to modulate the proliferation and / or function of myeloid-derived immunosuppressive cells. It is possible.

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

[0029] These embodiments and other embodiments of the anti-TNFR2 antibody disclosed herein are described in the following drawings. This will also be understood from the detailed description of the anti-TNFR2 antibody in 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, HCDR1, HCDR2, and HCDR3, and a set of three CDRs on the light chain, (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, sequence number Numbers 247 and 248, Sequence IDs 251 and 252, Sequence IDs 255 and 256, Sequence IDs 259 and 260, Sequence IDs 263 and 264, Sequence IDs 267 and 268, Sequence IDs 271 and 272, Sequence IDs 275 and 276, Sequence IDs 279 and 280, Sequence IDs 283 and 284, Sequence IDs 287 and 288, Sequence IDs 295 and 296, Sequence IDs 299 and 300, Sequence IDs 303 and 304, Sequence IDs 307 and 308, Sequence ID 3 An anti-TNFR2 antibody having an amino sequence selected from the group consisting of 11 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 antibody in question is an anti-TNFR2 antibody, which may be IgG, Fv, scFv, Fab, F(ab')2, minibody, diabody, tribody, nanobody, bispecific antibody, or single-domain antibody. (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 + and the method. (Item 18) The method according to item 16, stimulating the proliferation of the regulatory T cells, the method. (Item 19) The method according to item 16, activating the regulatory T cells, the method. (Item 20) The method according to item 16, regulating the immune response mediated by the regulatory T cells, the method. (Item 21) A method for regulating the activity or function of myeloid-derived suppressor cells (MDSC) in a subject, comprising administering to the subject a composition comprising an effective amount of the anti-TNFR2 antibody according to any one of items 1 to 3, the method. (Item 22) The method according to item 21, stimulating the proliferation of the myeloid-derived suppressor cells (MDSC), the method. (Item 23) The method according to item 21, activating the myeloid-derived suppressor cells (MDSC), the method. (Item 24) A method for treating a disease in a subject, comprising administering to the subject a composition comprising an effective amount of the anti-TNFR2 antibody according to any one of items 1 to 3, the method. (Item 25) The method according to item 24, where the disease is cancer, autoimmune disease, GvHD, viral infection, or bacterial infection, the method.

Brief Description of the Drawings

[0030] In this specification, several embodiments of anti-TNFR2 antibodies and their uses are described with reference to the accompanying drawings The surface is shown here as an example. The details are illustrative and intended to describe the anti-TNFR2 antibody and embodiments of its use. It is important to emphasize that this is the case. In this regard, the explanation given with reference to the drawings is anti-TNFR The two antibodies and embodiments of their use will be made clear to those skilled in the art.

[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 high affinity receptor that can agonize the TNFR2 receptor in an Fc-independent manner. We provide a panel of antibody agonists that require activation of the TNFR2 pathway (and It can potentially be used to treat patients with the condition (from which one can benefit). When used in detail, the term "agonist antibody" of the TNFR2 receptor refers to the activity of the natural ligand. This refers to an antibody that can essentially mimic a physiological ligand. Agonist activity is the ability of an antibody to mimic a physiological ligand. By binding to the TNF receptor in a manner that mimics the binding of antibodies, antibody-mediated agonism ( It can bring about an agonism. When used herein, "natural ligand" and The term "ordinary ligand" has exactly the same meaning and properties and is used interchangeably.

[0033] In some embodiments, the agonist antibodies of this disclosure mimic the activity of a natural ligand. In some embodiments, the agonist antibodies of this disclosure completely suppress the activity of the conventional ligand. In some embodiments, the agonist antibody of this disclosure replaces the TNFR2 receptor. It is specifically activated. In some embodiments, the agonist antibody of this disclosure is a normal rigor. It completely replaces the activity of the agonist antibody. In some embodiments, the agonist antibody of this disclosure is used It replaces the activity of the normal ligand and specifically activates the TNFR2 receptor.

[0034] In some embodiments, the activity is compared to that of a normal ligand in the presence of a natural ligand. 100% of the activity of the ligand is replaced. In some embodiments, in the presence of a native ligand Compared to the activity of the normal ligand, 50-100% of its activity is substituted. In the application form, the activity was 75% of that of a normal ligand compared to the activity in the presence of a natural ligand. ~100% will be replaced.

[0035] When used in this specification, "includes" means "comprises, comprising, includes, including" Alternatively, the term "having" and its conjugations can mean "including but not limited to." It means "not done."

[0036] When used in this specification, the singular forms "a", "an", and "that "The" includes the plural form of the object it refers to, unless the context clearly indicates a different meaning. For example, the terms "one antibody" or "at least one antibody" refer to combinations of those antibodies. It may contain multiple antibodies, including se.

[0037] Throughout this application, various embodiments of the present invention may be described in scope form. The formulaic representation is merely for convenience and brevity, and does not impose flexibility on the scope of the invention. It should be understood that this should not be interpreted as an limitation. Therefore, the description in range format is: Not only the individual numbers within that range, but also all possible parts within that range It should be considered that the specific range has been disclosed. For example, from 1 to 6. The ranges are described as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6. So, a partial range from 3 to 6, and the individual items included within that range. It should be considered that the numerical values ​​(e.g., 1, 2, 3, 4, 5, and 6) have been specifically disclosed. This applies regardless of the range.

[0038] Whenever a numerical range is specified in this specification, it is always within the specified numerical range. This means including all numerical values ​​(fractions or integers). The first nominal number and the second nominal number The expression "the range between" and "the range from the first specified number to the second specified number" The expression is used interchangeably in this specification, with respect to the first designation number and the second designation number. This means including all fractions and integers between these.

[0039] When a value is expressed as an approximation, the use of "approximately" implies that the specific value is different in other embodiments. It will be understood that this forms the whole. All ranges are comprehensive and can be combined. This is possible. In one embodiment, the term "approximately" means that 0 of the indicated number or numerical range is zero. This refers to a deviation of 0.1-5%. In another embodiment, the term "approximately" means the given number or This refers to a deviation of 1-10% from the numerical range. In another embodiment, the term “approximately” indicates This refers to a deviation of up to 20% from a given numerical value or range. In one embodiment, "approximately" The term refers to a deviation of ±10% from the indicated numerical value or range. In 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 This has the same meaning as that generally understood by those skilled in the art in which the present invention pertains. To do so. To use in the implementation or testing of the anti-TNFR2 antibody and its use embodiments according to the present invention. The following describes methods and materials that can be used, but are the same as those described in this specification. Methods and materials of the same or equivalent nature may also be used. In case of any inconsistencies, the definitions may be included. This specification shall take precedence. In addition, materials, methods, and examples are for illustrative purposes only. This is not intended to be an limitation of the literature or other publications referenced herein. Its entirety is incorporated herein by reference.

[0041] The description presented herein concerns the manufacture and use of anti-TNFR2 antibodies and their variations. This section describes each step of the process. This description is not intended to be restrictive, but rather to describe the components. Changes to the order of steps and other variations of the anti-TNFR2 antibody of this disclosure, Please understand that this is included within the scope of the manufacturing method and its use.

[0042] For clarity, anti-TNFR2 antibodies and their respective embodiments are described in the context of separate embodiments. It is understood that specific features of use may be provided in combination in a single embodiment. Conversely, for the sake of brevity, the anti-TNFR2 described in the context of a single embodiment is Various characteristics of the antibody and its use are described in relation to any other embodiments of the anti-TNFR2 antibody and its use. In this context, they may be appropriately provided separately or in any suitable subcombination. Various Certain features described in the context of the embodiment are such that the embodiment would not function without those elements. Unless otherwise specified, these embodiments should not be considered essential features.

[0043] As used herein, the term “antibody” is interchangeable with the term “immunoglobulin.” They have the same properties and meaning and are used interchangeably. Antibody-binding domain or antigen-binding site This refers to a fragment of an antibody, or one or more fragments of an antibody, that are involved in specific binding to a target antigen. It may be a genetically modified product. "Specific binding" means that it selectively binds to the target antigen, and This means that essential interactions and nonspecific interactions can be distinguished. For example, the equilibrium dissociation constant. 10 -5 M or less, 10 -6 M or less, or 10 -7 If the M value is less than or equal to M, the antibody is It is said to bind specifically to the IL-2 epitope. In some embodiments, The dissociation constant is 10 -8 M or less or 10 -9 It may be less than M. In the application form, 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 condition in which a drug, antibody, or toxin is present after a specified exposure time. This refers to the concentration that represents 50% of the maximum reaction from the baseline. In some embodiments, the reaction This includes binding affinity. In some embodiments, the reaction is a functional reaction, e.g., agonism. This includes a reaction. Those skilled in the art will know that when used in a particular embodiment, the anti-TNFR2 of this disclosure Antibody EC 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 Measurement of binding affinity is performed in the example. As illustrated in Tables 2 and 5, the binding of the anti-TNFR2 antibody of this disclosure to the TNFR2 antigen. This includes measuring the anti-T of the present disclosure when used in a particular embodiment. Those skilled in the art will know that when used in a particular embodiment, the anti-T of the present disclosure EC of NFR2 antibody 50 The measurement is performed using an agonist reaction (EC). 50 Inducing functional agonism It will be understood that this provides the measurement of the 50% effective concentration of an anti-TNFR2 antibody for this purpose. EC 50 Measurement of functional agonism is illustrated in Table 6 of the Examples, using the anti-TNF of the Disclosure. This includes measuring the effect of the R2 antibody on TNFR2 cellular signaling.

[0045] In some embodiments, EC 50 This is the 50% that will be observed when TNFα is bound. Includes the concentration of antibody necessary to obtain the agonist reaction. In certain embodiments, EC 50 of Measurements are commonly used as a measure of drug efficacy, and in some embodiments, the antibody's response This can reflect binding to the container. In some embodiments, the EC of nanomoles 50 Conclusion Anti-TNFR2 antibodies with combined concentration measurements contain anti-TNFR2 antibodies that bind strongly. In some embodiments, the EC of nanomoles 50 Anti-T with functional agonism concentration measurement NFR2 antibodies include functionally effective agonist antibodies. In certain embodiments, this opening The anti-TNFR2 antibody shown exhibits strong binding to the TNFR2 receptor. Specific embodiments Therefore, the anti-TNFR2 antibody of this disclosure comprises a TNFR2 receptor agonist. Specific implementation Morphologically, the anti-TNFR2 antibody of this disclosure is a strongly bound agonist to the TNFR2 receptor. include.

[0046] In some embodiments, the conjugation of anti-TNFR2 antibody EC 50 This is within the range of nM (nanomoles) In some embodiments, the conjugation of the anti-TNFR2 antibody is performed. 50 It is approximately 0.05~ The range is 100 nM. In some embodiments, the conjugated EC of an anti-TNFR2 antibody is used. 50 teeth The concentration is in the range of approximately 0.05 to 50 nM. In some embodiments, the concentration of the anti-TNFR2 antibody is Combined EC 50 This ranges from approximately 0.05 to 20 nM. In some embodiments, it is anti-TNF. R2 antibody binding EC 50 This ranges from approximately 0.05 to 10 nM. In some embodiments, This is the binding of anti-TNFR2 antibodies to EC 50 The range is approximately 0.1 to 100 nM. In this embodiment, the conjugated EC of the anti-TNFR2 antibody 50 The range is approximately 0.1 to 50 nM. In some embodiments, conjugated EC of an anti-TNFR2 antibody 50 It is approximately 0.1 to 20 nM This is the range. In some embodiments, the conjugation of the anti-TNFR2 antibody to EC 50 It is approximately 0.1~ The range is 10 nM. In some embodiments, the conjugated EC of the anti-TNFR2 antibody is 50 teeth, The range is approximately 1 to 100 nM. In some embodiments, the conjugated EC of an anti-TNFR2 antibody is used. 50 The concentration is in the range of approximately 1 to 20 nM. In some embodiments, the concentration of the anti-TNFR2 antibody is Combined EC 50 The range is approximately 20-40 nM. In some embodiments, the anti-TNFR2 Antibody binding EC 50 The range is approximately 40-60 nM. In some embodiments, the anti-T NFR2 antibody-conjugated EC50 This is in the range of approximately 60-80 nM. In some embodiments, This is the binding of anti-TNFR2 antibodies to EC 50 The range is approximately 80-100 nM. In this embodiment, the conjugated EC of the anti-TNFR2 antibody 50 The range is approximately 1 to 40 nM. In one embodiment, the conjugated EC of the anti-TNFR2 antibody 50 The range is approximately 1 to 60 nM. In some embodiments, conjugated EC of an anti-TNFR2 antibody 50 The range is approximately 1 to 80 nM. In some embodiments, the conjugation of the anti-TNFR2 antibody to EC is performed. 50 It is approximately 1-50 nM This is within the range. In some embodiments, the conjugation EC of the anti-TNFR2 antibody 50 It is approximately 0.0 The concentration is in the range of 5 to 5 nM. In some embodiments, the conjugated EC of the anti-TNFR2 antibody is... 50 teeth The concentration is in the range of approximately 0.1 to 5 nM. In some embodiments, the binding of the anti-TNFR2 antibody is C 50 This range is approximately 0.05 to 20 nM.

[0047] In some embodiments, the conjugation of anti-TNFR2 antibody EC 50 It is approximately 0.05 to 5 nM This is the range. In some embodiments, the conjugation of the anti-TNFR2 antibody to EC 50 It is approximately 0.1~ The range is 5 nM. In some embodiments, the conjugated EC of the anti-TNFR2 antibody is 50 It is approximately The concentration is in the range of 1 to 5 nM. In some embodiments, the conjugated EC of the anti-TNFR2 antibody is... 50 teeth The concentration is in the range of approximately 0.05 to 10 nM. In some embodiments, the concentration of the anti-TNFR2 antibody is Combined EC 50 The range is approximately 0.1 to 10 nM. In some embodiments, the anti-TNFR 2 antibody-conjugated EC 50 The range is approximately 1 to 10 nM. In some embodiments, the anti-T NFR2 antibody-conjugated EC 50 This range is approximately 5 to 10 nM. In some embodiments, , conjugated EC of anti-TNFR2 antibody 50 The range is approximately 0.05 to 15 nM. In this embodiment, the conjugated EC of the anti-TNFR2 antibody 50 The range is approximately 0.01 to 15 nM. In some embodiments, conjugated EC of an anti-TNFR2 antibody 50 The range is approximately 1 to 15 nM. That is the case.

[0048] In some embodiments, EC measures functional agonism. 50 In this specification, Functional ECs that all have the same properties 50 It is called anti-TNFR. In some embodiments, it is called anti-TNFR. Functional EC of 2 antibodies 50 This is in the range of nM (nanomoles). In some embodiments, Functional EC of anti-TNFR2 antibodies 50 The range is approximately 0.05 to 100 nM. In this embodiment, an anti-TNFR2 antibody is used in functional EC 50 It is in the range of approximately 0.05 to 50 nM. Yes. In some embodiments, the functional EC of anti-TNFR2 antibodies 50 It is approximately 0.05 to 2 The range is 0 nM. In some embodiments, the functional EC of the anti-TNFR2 antibody 50 teeth, The range is approximately 0.05 to 10 nM. In some embodiments, the function of the anti-TNFR2 antibody EC 50 This ranges from approximately 0.1 to 100 nM. In some embodiments, it is anti-TNF. Functional EC of R2 antibodies 50 This ranges from approximately 0.1 to 50 nM. In some embodiments, This is functional EC of anti-TNFR2 antibodies. 50 The range is approximately 0.1 to 20 nM. In this embodiment, an anti-TNFR2 antibody is used in functional EC 50 It is in the range of approximately 0.1 to 10 nM. In some embodiments, the functional EC of anti-TNFR2 antibodies 50 It is approximately 1-100 nM This is within the range. In some embodiments, the functional EC of anti-TNFR2 antibodies 50 It is approximately 1~ The range is 20 nM. In some embodiments, the functional EC of the anti-TNFR2 antibody 50 teeth , in the range of approximately 20-40 nM. In some embodiments, the functional anti-TNFR2 antibody EC 50 The range is approximately 40-60 nM. In some embodiments, anti-TNFR2 anti Functional EC of the body 50 The range is approximately 60-80 nM. In some embodiments, the anti-T Functional EC of NFR2 antibodies 50 The range is approximately 80-100 nM. Several implementations In this state, the functional EC of anti-TNFR2 antibodies 50 The range is approximately 1 to 40 nM. In this embodiment, an anti-TNFR2 antibody is used in functional EC 50 This range is approximately 1 to 60 nM. In some embodiments, functional EC of anti-TNFR2 antibody 50 The range is approximately 1 to 80 nM. In some embodiments, the functional EC of anti-TNFR2 antibodies 50 It is approximately 1-50n This is in the range of M. 50 It is approximately 0 The range is 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 anti-TNFR2 antibody Functional EC 50 is in the range of about 0.05 to 20 nM.

[0049] In some embodiments, the functional EC of the anti-TNFR2 antibody 50 is in the range of about 0.05 to 5 nM In some embodiments, the functional EC of the anti-TNFR2 antibody 50 is 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 E of the anti-TNFR2 antibody C 50 is in the range of about 0.05 to 10 nM. In some embodiments, anti-TNFR2 antibody functional EC 50 is in the range of about 0.1 to 10 nM. In some embodiments, anti-TNFR2 antibody functional EC 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 about 0.01 to 1 5 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" includes, but is not limited to, for example, IgG , heavy chain variable region (VH), light chain variable region (VL), Fab fragment, F(ab')2 fragment, s cFv fragment, Fv fragment, nanobody, minibody, diabody, triabody, tetra It includes antibody fragments that retain binding specificity, including bodies and single-domain antibodies (for example) See also "Hudson and Souriau, Nature Med. 9: 129-134 (2003)". As is commonly understood in the field, these terms are humanized antibodies, primate antibodies, and It also includes chimeric antibodies.

[0051] As used herein, the term "heavy chain variable region (VH)" is used interchangeably with "VH domain". Alternatively, it has the exact same meaning and characteristics as the term "VH" and is used interchangeably. When used in writing, the term "VL (VL)" is sometimes referred to as "VL domain" or " The term "VL" has exactly the same meaning and properties and is used interchangeably. The "heavy chain variable region (VH)" related to antibodies, or "VH," is used as the framework region. Heavy chains containing three complementarity-determining regions (CDRs) interposed between adjacent stretches known as You will recognize that it includes fragments of the framework domain. The framework domain is the complementarity determination domain (CD). It is more highly conserved than R) and forms a scaffold for supporting the complementary determination region (CDR). To accomplish. Similarly, a person skilled in the art would know the “light chain variable region (VL)” of an antibody, i.e., “V "L" is a light chain containing three complementarity determination regions (CDRs) that interpose between framework domains. They will also recognize that it includes fragments.

[0052] As used herein, the term “complementarity-determining region,” or “CDR,” refers to heavy chain This refers to the hypervariable region of the variable region (VH) or light chain variable region (VL). Starting from the N-terminus, the heavy Each of the chain or light chain polypeptides is "CDR1", "CDR2", and "CDR It has three complementarity-determining regions (CDRs) labeled "3". Crystal structure analysis revealed that amino acid residues in the complementarity-determining region (CDR) have a broad spectrum with respect to the bound antigen. It forms a wide range of contacts, and among them, the most extensive antigenic contact is with the heavy chain CDR3. This has been clarified. Thus, the complementarity-determining region (CDR) region is a characteristic of the antigen-binding site. The main factor is heterosexuality. In one embodiment, the antigen-binding site is the heavy chain variable region (VH) and Six complementarities, including the complementarity determination region (CDR) from each of the light chain variable regions (VLs) Includes the decision region (CDR).

[0053] As used herein, the term "framework area," or "FR," is a heavy Four elements that form the complementarity determination region (CDR) of the chain variable region (VH) or light chain variable region (VL). It refers to two adjacent amino acid sequences. Some of the FR residues come into contact with the bound antigen, but the FR residues The base primarily plays a role in folding the variable region into the antigen-binding site. In some embodiments, FR residues, which play a role in folding the mutational region, are residues directly adjacent to the complementarity-determining region (CDR). This includes. Within the framework region (FR), specific amino residues and specific structural features are , is highly conserved. In this regard, all variable region sequences are approximately 90 It contains an internal disulfide loop within an amino acid residue. The variable region folds into the antigen-binding site. When folded, the complementarity-determining region (CDR) forms a protruding loop motif that creates an antigen-binding surface. It is represented as 'Fu'. Generally, it is not related to the exact amino acid sequence of the complementarity-determining region (CDR). Furthermore, the folded shape of the complementarity-determining region (CDR) loop is converted to a specific "standard" structure. It is recognized that there is a preserved structure area for the framework area (FR). Furthermore, certain FR residues stabilize the interaction between the heavy and light chains of antibodies through non-covalent bonds. It is known to be involved in contact between main groups.

[0054] Wu and Kabat are pioneers in antibody peptide sequence alignment and in this field. Their contribution was very significant ("An analysis of the sequences of Wu and Kabat, "An analysis of the sequences of thevariable regions of Bence Jones proteins and myeloma light chains and their implications for antibody complementarity", Journal of Experimental Medicine, 1 32, 2, 8(1970); Kabat et al., "Sequence of proteins of immunological interest", Bethesda: National Institutes of Health; 323 (1983). Firstly, between variable domains By examining the similarity of their sequences, we can identify elements that adopt similar three-dimensional structures and perform similar functional roles. And, insofar as they interact similarly with adjacent residues and exist in similar chemical environments, , corresponding antibodies that have more or less homology in all antibodies of all vertebrate species. The residues were identified. Secondly, the same positional number was assigned to immunoglobulin residues that had homology. He devised a peptide sequence numbering system. Those skilled in the art would know the sequence itself. Without relying on experimental data other than the current Kabat numbering The number can be clearly assigned to any variable domain sequence. Thirdly, Kaba t and Wu indicate whether there are too few or too many amino acids when the variable domain sequence is aligned. To find out, we calculated the variability for each Kabat-numbered sequence position. This means that within four consecutive regions of low variability, there are three consecutive regions of high variability. This was confirmed. Kabat and Wu formally defined the residues that make up these variable regions. These are called "complementarity-determining regions" (CDRs) due to the chemical complementarity between antibodies and antigens. The variable region is not involved in antigen recognition, but it is involved in three-dimensional folding. It is thought that this is the case, and is now called the "framework domain". Fourthly, Kabat and Wu established a public database of antibody peptide and nucleic acid sequences, which is still maintained today. It is possessed and is well known to those skilled in the art.

[0055] Chothia and her collaborators have identified specific areas within Kabat's complementarity determination regions (CDRs). Although the subregion exhibits great diversity at the amino acid sequence level, They found that they have the same peptide backbone structure ("Cyrus Chothia, Arthur M. Les k (1987), Journal of Molecular Biology. 196(4): 901-917). These subregions are They are named L1, L2 and L3, or H1, H2 and H3, with "L" and "H" being These represent the light chain region and the heavy chain region, respectively. These regions represent the complementarity of Kabat. Chothia's complementary decision region (CDR) has a boundary that overlaps with the decision region (CDR). It is also known as

[0056] Recent studies have shown that virtually all antibody-binding residues are included in the structural consensus region. It has become clear that this is the case ("Kunik, V., et al., PloS Computational Biology 8( 2): el002388 (February 2012). In some embodiments, these regions are antibody-bound. These regions are called synthetic regions. It has been shown that these regions can also be identified from antibody sequences. For the purpose of this, in implementing a structural approach to identify the structural consensus of antibodies A certain "Paratome" was used ("Ofran, Y. et al., J. Immunol. 757:6230-6"). 235 (2008). Paratome identified residues that bind to virtually all antibodies. Although it encompasses the region, (according to commonly used complementary determination area (CDR) identification tools) The complementary determination regions (CDRs) identified as such miss a significant portion of the problem. It is identified by ratome, but is not particularly useful in general complementarity-determining region (CDR) identification methods. Antibody-binding residues that are not defined are called paratome-unique residues. Similarly, general While it is identified by conventional complementary determination region (CDR) identification methods, it is not identified by Paratome. Antibody-binding residues that cannot bind are called CDR-unique residues. The energetic contribution is large in Paratome-unique residues, but CDR-unique The size is quite small at the K residue. These results make the identification of antigen-binding sites more reliable. .

[0057] IMGT (registered trademark) is an international ImMunoGeneTics (registered trademark) information system. "NucleicAcids Res. 2015 Jan; 43 (Database issue):D413-22. doi: 10.1093 / nar / gku 1056. Epub 2014Nov 5 Free article. PMID: 25378316 LIGM:441 and Dev Comp Immunol . See "Lefranc et al., Dev Comp Immunol. 27: 55-77 (2003)". IMGT is a unique numbering system for immunoglobulin and T cell receptor variable domains main, and Ig superfamily V-like domains (see "Lefranc et al., Dev Comp Immunol. 27: 55-77 (2003)"). IMGT (registered trademark) presents a unified numbering system for these IG and TcR variable domain sequences, considering the definition of FR and complementarity determining regions (CDRs) by Kabat , structural data, and the characterization of Chothia's hypervariable loops, based on the alignment of five or more IG and TcR variable region sequences. IMGT (registered trademark) is considered well-known in the art as a universal numbering scheme for antibodies . In the description of the mutated amino acid positions present in the heavy chain variable region (VH) and light chain variable region (VL) domains, in some embodiments, to describe the CDR regions, any common CDR definition, including but not limited to the methods used by IMGT (registered trademark), Kabat, Chothia , or Paratome, is used . Antibodies exist in various forms and have various domains, including but not limited to, for example

[0058] complementarity determining regions (CDRs), variable regions (Fv), heavy chain variable region (VH) domains, light chain variable regions (VL) domains, single chain variable regions (scFv), and Fab fragments . .

[0059] . . .

[0060] One skilled in the art would 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. Also, one skilled in the art would understand that the term "Fab" with respect to an antibody generally encompasses the portion of an antibody consisting of a single heavy chain variable region and a single light chain (both variable region and constant region) linked by a disulfide bond to the first constant region, while the term "F(ab')2"

[0061] would be understood to include fragments containing a heavy chain with a heavy chain variable region (VH) and a light chain with a light chain variable region (VL) domain. In some embodiments, the antibody encompasses the entire antibody molecule, including monoclonal and polyclonal antibodies. In some embodiments, the antibody includes, without limitation, a heavy chain variable region (VH) fragment, a light chain variable region (VL) fragment, a Fab fragment, an F(ab')2 fragment, an scFv fragment, an Fv fragment, a minibody, a diabody, a triabody, and a tetrabody, and one or more antibody fragments that retain binding specificity, such as those mentioned above. In one embodiment, the anti-TNFR2 antibody of the present disclosure can be incorporated as part of a bispecific antibody. As is generally known in the art, a bispecific antibody is a recombinant protein that includes antigen-binding fragments of two different monoclonal antibodies, thereby

[0062] being able to bind two different antigens. In some embodiments, the bispecific antibody is, for example, a CTL (such as a CTL receptor component like CD3) or an effector

[0063]

[0064] Cancer immunotherapy targets both lucidator (NK) cells and tumor antigens simultaneously. It is used for this purpose. Similarly, multiple specific antibodies are, for example, antibodies that target two, three, or four different substances. It contains antigen-binding fragments of at least two different monoclonal antibodies, such as clonal antibodies. It is a recombinant protein.

[0064] Anti-TNFR2 antibody

[0065] This disclosure provides various anti-TNFR2 (tumor necrosis factor receptor 2) antibodies. One embodiment So, each anti-TNFR2 antibody has 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 (LC Includes DR1, LCDR2 and LCDR3). In one embodiment, HCDR1 on the heavy chain, H The CDR2 and HCDR3 sets include the amino acid sequence combinations shown in Table 7 below. The corresponding sets of LCDR1, LCDR2, and LCDR3 on the light chain are described below. This includes the amino acid sequence combinations shown in Table 8. Clone CID251 is used as an example for explanation. As for this (see Table 7), the set of HCDR1, HCDR2, and HCDR3 antibodies is, Each contains the amino acid sequences of SEQ ID NO: 185, SEQ ID NO: 190, and SEQ ID NO: 347, respectively. (See Table 7) The sets of LCDR1, LCDR2, and LCDR3 of the same antibody are distributed as follows: Includes the amino acid sequences of sequence number 359, sequence number 372, and sequence number 378 (see Table 8). .

[0066] In some embodiments, the set of HCDR1, HCDR2, and HCDR3 is each This contains the amino acid sequences described in SEQ ID NOs. 185, 190, and 347, and is the same antibody as LC. The set of DR1, LCDR2, and LCDR3 each contains the amino acid sequences set forth in SEQ ID NOs: 359, 372, and 3 78. In some embodiments, the set of HCDR1, HCDR 2, and HCDR3 each contains the amino acid sequences set forth in SEQ ID NOs: 185, 334, 347, and the set of LCDR1, LCDR2, and LCDR3 of the same antibody each contains the amino acid sequences set forth in SEQ ID NOs: 360, 372, and 379. In some embodiments, the set of HCDR1, HCDR2, and HCDR3 each contains the amino acid sequences set forth in SEQ ID NOs: 1 85, 190, and 347, and the set of LCDR1, LCDR2, and LCDR3 of the same antibody each contains the amino acid sequences set forth in SEQ ID NOs: 359, 372, and 380. In some embodiments, the set of HCDR1, HCDR2, and HCDR3 each contains the amino acid sequences set forth in SEQ ID NOs: 1 85, 190, and 347, and the set of LCDR1, LCDR2, and LCDR3 of the same antibody each contains the amino acid sequences set forth in SEQ ID NOs: 359, 372, and 381. In some embodiments, the set of HCDR1, HCDR2, and HCDR3 each contains the amino acid sequences set forth in SEQ ID NOs: 185, 190, and 347, and the set of LCDR1, LCDR2, and LCDR3 of the same antibody each contains the amino acid sequences set forth in SEQ ID NOs: 359, 372, and 382. In some embodiments, the set of HCDR1, HCDR2, and HCDR3 each contains the amino acid sequences set forth in SEQ ID NOs: 185, 190, and 347, and the set of LCDR1, LCDR2, and LCDR3 of the same antibody each contains the amino acid sequences set forth in SEQ ID NOs: 361, 372, and 381. In some embodiments, the set of HCDR1, HCDR2, and HCDR3 each contains the amino acid sequences set forth in SEQ ID NOs: 185, 190, and 347, and the set of LCDR1, LCDR2, and LCDR3 of the same antibody each contains the amino acid sequences set forth in SEQ ID NOs: 362, 3[[ID=I9]] 72, and 382. In some embodiments, the set of HCDR1, HCDR2, and HCDR3 each contains the amino acid sequences set forth in SEQ ID NOs: 185, 190, and 347, and the set of LCDR1, LCDR2, and LCDR3 of the same antibody each contains the amino acid sequences set forth in SEQ ID NOs: 359, 372, and 381. In some embodiments, the set of HCDR1, HCDR2, and HCDR3 each contains the amino acid sequences set forth in SEQ ID NOs: 185, 190, and 347, and the set of LCDR1, LCDR2, and LCDR3 of the same antibody each contains the amino acid sequences set forth in SEQ ID NOs: 361, 372, and 381. In some embodiments, the set of HCDR1, HCDR2, and HCDR3 each contains the amino acid sequences set forth in SEQ ID NOs: 185, 190, and 347, and the set of LCDR1, LCDR2, and LCDR3 of the same antibody each contains the amino acid sequences set forth in SEQ ID NOs: 362, 3 72, and 382. In some embodiments, the set of HCDR1, HCDR2, and HCDR3 each contains the amino acid sequences set forth in SEQ ID NOs: 185, 190, and 347, and the set of LCDR1, LCDR2, and LCDR3 of the same antibody each contains the amino acid sequences set forth in SEQ ID NOs: 359, 372, and 381. In some embodiments, the set of HCDR1, HCDR2, and HCDR3 each contains the amino acid sequences set forth in SEQ ID NOs: 185, 190, and 347, and the set of LCDR1, LCDR2, and LCDR3 of the same antibody each contains the amino acid sequences set forth in SEQ ID NOs: 362, 3 72, and 382. In some embodiments, HCDR1 The HCDR2 and HCDR3 sets are numbered 185, 335, and 347 respectively. The described amino acid sequence is included in the cells of LCDR1, LCDR2, and LCDR3 of the same antibody. Each packet contains the amino acid sequences described in SEQ ID NOs. 363, 372, and 383, respectively. In some embodiments, the sets HCDR1, HCDR2, and HCDR3 are, respectively The LCDR of the same antibody contains the amino acid sequences described in SEQ ID NOs: 185, 190, and 347. 1. Sets LCDR2 and LCDR3 are sequence numbers 364, 372, and 384, respectively. It includes the amine acid sequence described in [reference]. In some embodiments, HCDR1, HCDR2 and The HCDR3 set is described in sequence numbers 185, 336, and 348, respectively. The set of LCDR1, LCDR2, and LCDR3 antibodies, each containing an acid sequence, This includes the amino acid sequences described in SEQ ID NOs. 365, 372, and 378. Several implementations In terms of form, the set of HCDR1, HCDR2, and HCDR3 is each sequence number 185 , containing the amino acid sequences described in 337 and 347, the same antibody LCDR1, LCDR Sets 2 and LCDR3 are described in sequence numbers 359, 373, and 385, respectively. Includes an amino acid sequence. In some embodiments, HCDR1, HCDR2 and HCDR3 Each set contains the amino acid sequences described in SEQ ID NOs. 185, 338, and 348, respectively. Furthermore, sets of the same antibody, LCDR1, LCDR2, and LCDR3, are each assigned the same sequence number 3. The amino acid sequences include those described in 59, 372 and 386. In some embodiments, H The sets CDR1, HCDR2, and HCDR3 are sequence numbers 185, 339, and The amino acid sequence described in 349 is included in the same antibody LCDR1, LCDR2 and LCD The R3 set consists of the amino acid sequences described in SEQ ID NOs. 359, 372, and 378, respectively. Includes. In some embodiments, the set of HCDR1, HCDR2 and HCDR3 is Each contains the amino acid sequence described in SEQ ID NOs. 185, 190, and 350, and is the same antibody. The LCDR1, LCDR2, and LCDR3 set are sequence numbers 366 and 372, respectively. and includes the amino acid sequence described in 387. In some embodiments, HCDR1, H The CDR2 and HCDR3 sets are described in sequence numbers 185, 190, and 351, respectively. A set of LCDR1, LCDR2, and LCDR3 antibodies containing the specified amino acid sequence. These contain the amino acid sequences described in SEQ ID NOs. 359, 372, and 378, respectively. In one embodiment, the sets of HCDR1, HCDR2, and HCDR3 are arranged in an array. The LCDR1 of the same antibody contains the amino acid sequences described in numbers 185, 334, and 347. The LCDR2 and LCDR3 sets are numbered 360, 372, and 388 respectively. Includes the amino acid sequence described. In some embodiments, HCDR1, HCDR2 and The HCDR3 set consists of amino acids described in SEQ ID NOs. 185, 334, and 352, respectively. The sets of LCDR1, LCDR2, and LCDR3, which contain acid sequences and are the same antibody, are respectively Includes the amino acid sequences described in SEQ ID NOs. 359, 372, and 378. Several implementation forms In this state, the HCDR1, HCDR2, and HCDR3 sets each have sequence number 185, LCDR1 and LCDR2 contain the amino acid sequences described in 190 and 352, and are the same antibodies. The sets of LCDR3 and LCDR3 are as described in sequence numbers 359, 372, and 389, respectively. It contains a mino acid sequence. In some embodiments, HCDR1, HCDR2 and HCDR3 Each set contains the amino acid sequences described in SEQ ID NOs: 185, 334, and 347, respectively. The set of LCDR1, LCDR2, and LCDR3 antibodies is each numbered SEQ ID NO: 35 The amino acid sequences described in 9, 372 and 378 are included. In some embodiments, HC The DR1, HCDR2, and HCDR3 sets are sequence numbers 185, 190, and 3, respectively. The amino acid sequence described in 47 is included in the same antibody LCDR1, LCDR2 and LCDR The three sets contain the amino acid sequences described in SEQ ID NOs. 365, 372, and 378, respectively. Includes. In some embodiments, the set of HCDR1, HCDR2 and HCDR3 is Each contains the amino acid sequences described in SEQ ID NOs. 185, 336, and 352, and is the same antibody. The LCDR1, LCDR2, and LCDR3 sets correspond to sequence numbers 365, 372, and It includes the amino acid sequence described in 390. In some embodiments, HCDR1, HC The DR2 and HCDR3 sets are described in sequence numbers 185, 336, and 352, respectively. The set of LCDR1, LCDR2, and LCDR3 antibodies, which contain the same amino acid sequence, , each containing the amino acid sequences described in SEQ ID NOs. 365, 374, and 391, respectively. In that embodiment, the sets HCDR1, HCDR2, and HCDR3 are each numbered by sequence number The amino acid sequence described in issues 186, 340, and 353, and the same antibody LCDR1, The LCDR2 and LCDR3 sets are described in sequence numbers 367, 375, and 392, respectively. Includes the amino acid sequence shown. In some embodiments, HCDR1, HCDR2 and H The CDR3 set consists of the amino acids described in SEQ ID NOs: 186, 341, and 354, respectively. The sets of LCDR1, LCDR2, and LCDR3, which contain the sequence and are the same antibody, are each distributed Includes the amino acid sequences described in columns 367, 375, and 392. In some embodiments, The sets HCDR1, HCDR2, and HCDR3 are sequence numbers 186 and 34, respectively. The amino acid sequences described in 1 and 354 are included in the same antibody LCDR1, LCDR2 and The LCDR3 set contains amino acids described in Sequence IDs 368, 375, and 392, respectively. It contains an acid sequence. In some embodiments, a set of HCDR1, HCDR2 and HCDR3 Each of these contains the amino acid sequences described in SEQ ID NOs. 186, 342, and 354, respectively. The set of antibodies LCDR1, LCDR2, and LCDR3 each have sequence numbers 367, The amino acid sequences include those described in 375 and 392. In some embodiments, HCDR 1. The HCDR2 and HCDR3 sets are sequence numbers 186, 343, and 354, respectively. It contains the amino acid sequence described, and the same antibody LCDR1, LCDR2, and LCDR3 The set contains the amino acid sequences described in SEQ ID NOs. 369, 375, and 393, respectively. In some embodiments, the sets HCDR1, HCDR2 and HCDR3 are each This contains the amino acid sequences described in SEQ ID NOs. 186, 343, and 355, and the LC of the same antibody The sets DR1, LCDR2, and LCDR3 are sequence numbers 367, 375, and 3, respectively. It includes the amino acid sequence described in 92. In some embodiments, HCDR1, HCDR Sets 2 and HCDR3 are described in Sequence IDs 186, 341, and 354, respectively. The set of LCDR1, LCDR2, and LCDR3 antibodies, which contain the amino acid sequence, Each contains the amino acid sequences described in SEQ ID NOs. 367, 375, and 392, respectively. In this embodiment, the set of HCDR1, HCDR2, and HCDR3 is represented by Sequence ID No. 1 The same antibody LCDR1, LC, contains the amino acid sequences described in 86, 341, and 355. The DR2 and LCDR3 sets are described in sequence numbers 367, 376, and 392, respectively. It contains the amino acid sequence. In some embodiments, HCDR1, HCDR2 and HCD The R3 set consists of the amino acid sequences described in SEQ ID NOs. 186, 344, and 354, respectively. The set of LCDR1, LCDR2, and LCDR3 antibodies, each containing the same antibody, has a sequence number. The amino acid sequences include those described in issues 370, 375, and 392. In some embodiments, The sets HCDR1, HCDR2, and HCDR3 are sequence numbers 186 and 341, respectively. and containing the amino acid sequence described in 354, the same antibody LCDR1, LCDR2 and L The CDR3 set consists of the amino acids described in SEQ ID NOs: 367, 375, and 394, respectively. Includes an array. In some embodiments, a set of HCDR1, HCDR2 and HCDR3. These contain the amino acid sequences described in SEQ ID NOs. 186, 344, and 354, respectively, and are the same The set of antibodies LCDR1, LCDR2, and LCDR3 are sequence numbers 367 and 3, respectively. The amino acid sequence includes those described in 75 and 392. In some embodiments, HCDR1 The HCDR2 and HCDR3 sets are numbered 186, 341, and 359 respectively. The described amino acid sequence is included in the cells of LCDR1, LCDR2, and LCDR3 of the same antibody. Each packet contains the amino acid sequences described in SEQ ID NOs. 367, 375, and 393, respectively. In some embodiments, the sets HCDR1, HCDR2, and HCDR3 are, respectively LCD of the same antibody, containing the amino acid sequences described in SEQ ID NOs: 187, 345, and 356. The sets R1, LCDR2, and LCDR3 are sequence numbers 371, 377, and 39, respectively. It includes the amino acid sequence described in 5. In some embodiments, HCDR1, HCDR2 The sets of HCDR3 and HCDR3 are described in sequence numbers 188, 345, and 357, respectively. The set of LCDR1, LCDR2, and LCDR3 antibodies, which contain the mino acid sequence, Each contains the amino acid sequences described in SEQ ID NOs. 371, 377, and 396. In the application configuration, the sets HCDR1, HCDR2, and HCDR3 are each sequence number 18. LCDR1, LCD, which contain the amino acid sequences described in 9, 346 and 358, and the same antibodies The sets of R2 and LCDR3 are described in sequence numbers 371, 377, and 397, respectively. It contains the amino acid sequence.

[0067] In another embodiment, the anti-TNFR2 antibody is used, for example, with default parameters. Determined using BlastP software from the National Center for Biotechnology Information (NCBI). Not limited to the same identity, the amino acid sequence described above is at least 80% (for example, (85%, 90%, 95%, 96%, 97%, 98%, 99% or more) identical heavy chain CDR Includes sequence and light chain CDR sequence.

[0068] Those skilled in the art will know the degree to which the identity percentage (identity%) of the query sequence differs from the target sequence. A numerical value is provided to indicate the degree of similarity (i.e., how identical the amino acids in each sequence are). You will understand that this is provided. The higher the identity percentage, the greater the degree of agreement. Yes.

[0069] When used in relation to polypeptide (or protein) sequences, the term "identity" is used. This refers to the degree of identity between two or more polypeptide (or protein) sequences or fragments. It refers to the similarity between two or more polypeptide (or protein) sequences. Generally, the similarity between two or more polypeptide (or protein) sequences is defined as the similarity between two or more polypeptide sequences. The composition, order, or sequence of two or more amino acids of the polypeptide (or protein) above This refers to the degree of similarity.

[0070] In some further embodiments, HCDR1, HCDR2, and HCDR3 on the heavy chain The set and the corresponding sets of LCDR1, LCDR2, and LCDR3 on the light chain are It contains the amino acid sequence shown in Table 9 below. For illustrative purposes, clone CID_264 is used as an example. When taken (see Table 9), the set of HCDR1, HCDR2, and HCDR3 antibodies is, Each contains the amino acid sequences of SEQ ID NO: 402, SEQ ID NO: 345, and SEQ ID NO: 413, respectively (Table 9) (Reference) The LCDR1, LCDR2, and LCDR3 sets of the same antibody are, respectively, sequence numbers Contains the amino acid sequences of number 371, sequence number 418, and sequence number 427 (see Table 9).

[0071] In some embodiments, the set of HCDR1, HCDR2, and HCDR3 is each This contains the amino acid sequences described in SEQ ID NOs. 402, 345, and 413, and the LC of the same antibody The sets DR1, LCDR2, and LCDR3 are sequence numbers 371, 418, and 4, respectively. It includes the amino acid sequence described in 27. In some embodiments, HCDR1, HCDR Sets 2 and HCDR3 are described in Sequence IDs 398, 346, and 407, respectively. The set of LCDR1, LCDR2, and LCDR3 antibodies, which contain the amino acid sequence, Each contains the amino acid sequences described in SEQ ID NOs: 371, 377, and 419. In this embodiment, the set of HCDR1, HCDR2, and HCDR3 is represented by Sequence ID No. 1 The same antibody LCDR1, LC, contains the amino acid sequences described in 87, 346, and 408. The DR2 and LCDR3 sets are described in sequence numbers 371, 377, and 420, respectively. It contains the amino acid sequence. In some embodiments, HCDR1, HCDR2 and HCD The R3 set consists of the amino acid sequences described in SEQ ID NOs: 189, 406, and 409, respectively. The set of LCDR1, LCDR2, and LCDR3 antibodies, each containing the same antibody, has a sequence number. Includes amino acid sequences described in issues 371, 377, and 421. In some embodiments, The sets HCDR1, HCDR2, and HCDR3 are sequence numbers 187 and 345, respectively. and containing the amino acid sequence described in 410, the same antibody LCDR1, LCDR2 and L The CDR3 set consists of the amino acids described in SEQ ID NOs: 371, 418, and 422, respectively. Includes an array. In some embodiments, a set of HCDR1, HCDR2 and HCDR3. These contain the amino acid sequences described in SEQ ID NOs. 399, 346, and 411, respectively, and are the same The set of antibodies LCDR1, LCDR2, and LCDR3 are sequence numbers 371 and 4, respectively. The amino acid sequence includes those described in 18 and 423. In some embodiments, HCDR1 The HCDR2 and HCDR3 sets correspond to sequence numbers 400, 346, and 412, respectively. The described amino acid sequence is included in the cells of LCDR1, LCDR2, and LCDR3 of the same antibody. Each packet contains the amino acid sequences described in Sequence ID Nos. 417, 377, and 424, respectively. In some embodiments, the sets HCDR1, HCDR2, and HCDR3 are, respectively LCD of the same antibody, containing the amino acid sequences described in SEQ ID NOs: 401, 346, and 413. The sets R1, LCDR2, and LCDR3 correspond to sequence numbers 417, 418, and 42, respectively. It includes the amino acid sequence described in 5. In some embodiments, HCDR1, HCDR2 The sets of HCDR3 and HCDR3 are described in sequence numbers 401, 346, and 414, respectively. The set of LCDR1, LCDR2, and LCDR3 antibodies, which contain the mino acid sequence, Each contains the amino acid sequences described in SEQ ID NOs. 417, 418, and 426. In the application configuration, the sets HCDR1, HCDR2, and HCDR3 are each numbered 39. LCDR1, LCD, which contain the amino acid sequences described in 8, 346 and 415, and the same antibodies The sets of R2 and LCDR3 are described in sequence numbers 417, 377, and 428, respectively. It includes the amino acid sequence. In some embodiments, HCDR1, HCDR2 and HCDR The three sets contain the amino acid sequences described in SEQ ID NOs. 403, 346, and 412, respectively. The set of LCDR1, LCDR2, and LCDR3 antibodies containing the same antibody is called SEQ ID NO: The amino acid sequences include those described in 371, 418, and 429. In some embodiments, The HCDR1, HCDR2, and HCDR3 sets correspond to sequence numbers 404, 346, and The same antibody LCDR1, LCDR2, and LC contain the amino acid sequence described in bi412. The DR3 set consists of amino acid combinations described in SEQ ID NOs. 371, 418, and 425, respectively. Includes columns. In some embodiments, the set of HCDR1, HCDR2 and HCDR3 is Each contains the amino acid sequence described in Sequence ID Nos. 401, 346, and 414, respectively, and has the same anti- The sets of LCDR1, LCDR2, and LCDR3 are sequence numbers 417 and 41, respectively. It includes the amino acid sequences described in 8 and 430. In some embodiments, HCDR1, The HCDR2 and HCDR3 sets are indicated by sequence numbers 405, 406, and 416, respectively. The set of LCDR1, LCDR2, and LCDR3 antibodies contains the listed amino acid sequence. The amino acid sequences described in SEQ ID NOs. 371, 377, and 431, respectively, are included in each of these sequences.

[0072] In another embodiment, the anti-TNFR2 antibody is used, for example, with default parameters. Determined using BlastP software from the National Center for Biotechnology Information (NCBI). Not limited to the same identity, the amino acid sequence described above is at least 80% (for example, (85%, 90%, 95%, 96%, 97%, 98%, 99% or more) identical heavy chain CDR Includes sequence and light chain CDR sequence.

[0073] In one embodiment, each of the anti-TNFR2 antibodies disclosed herein has a heavy chain variable region. (VH) and light chain variable region (VL) are included, and the amino acid distribution of the heavy chain variable region and the light chain variable region The columns may be one of the following pairs: Sequence IDs 289 and 290, Sequence IDs 3 and 4, Sequence IDs 7 and 8, 11 and 12, 15 and 16, 19 and 2 0, SEQ ID NOs. 23 and 24, SEQ ID NOs. 27 and 28, SEQ ID NOs. 31 and 32, SEQ ID NOs. 3 5 and 36, Sequence IDs 39 and 40, Sequence IDs 43 and 44, Sequence IDs 47 and 48, Distribution Column numbers 51 and 52, sequence numbers 55 and 56, sequence numbers 59 and 60, sequence number 63 and 64, Sequence IDs 67 and 68, Sequence IDs 71 and 72, Sequence IDs 75 and 76, Sequence ID 79 and 80, Sequence IDs 83 and 84, Sequence IDs 87 and 88, Sequence IDs 91 and 92, Sequence IDs 95 and 96, 99 and 100, 103 and 104, Sequence ID 107 and 108, SEQ ID NOs: 111 and 112, SEQ ID NOs: 115 and 116, SEQ ID NO: 1 19 and 120, SEQ ID NOs: 123 and 124, SEQ ID NOs: 127 and 128, SEQ ID NO: 13 Sequence IDs 1 and 132, 135 and 136, 139 and 140, and 143 and 144, Sequence IDs 147 and 148, Sequence IDs 151 and 152, Sequence ID 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 2 00, Sequence IDs 205 and 206, Sequence IDs 211 and 212, Sequence IDs 217 and 21 8. Sequence IDs 223 and 224, 229 and 230, 233 and 234 , Sequence IDs 237 and 238, Sequence IDs 241 and 242, Sequence IDs 245 and 246, Sequence IDs 249 and 250, 253 and 254, 257 and 258, Column numbers 261 and 262, sequence numbers 265 and 266, sequence numbers 269 and 270, array Numbers 273 and 274, Sequence numbers 277 and 278, Sequence numbers 281 and 282, Sequence number Sequence numbers 285 and 286, Sequence numbers 293 and 294, Sequence numbers 297 and 298, Sequence number 301 and 302, SEQ ID NOs: 305 and 306, SEQ ID NOs: 309 and 310, SEQ ID NO: 3 13 and 314, SEQ ID NOs: 317 and 318, SEQ ID NOs: 321 and 322, SEQ ID NOs: 32 5 and 326, or sequence numbers 329 and 330.

[0074] In some embodiments, the heavy chain variable region of the anti-TNFR2 antibody disclosed herein and The amino acid sequences of the light chain variable region are described in SEQ ID NOs. 289 and 290. In this embodiment, the heavy chain variable region and light chain variable region of the anti-TNFR2 antibody disclosed herein The amino acid sequences of the region 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 in the specification are: , as described in Sequence IDs 7 and 8. In some embodiments, as disclosed herein The amino acid sequences of the heavy chain variable region and light chain variable region of the anti-TNFR2 antibody are as shown in SEQ ID NO: 11 and As described in 12. In some embodiments, the anti-TNFR2 anti- The amino acid sequences of the heavy chain variable region and light chain variable region of the body are described in SEQ ID NOs. 15 and 16. In some embodiments, the heavy chain variable region of the anti-TNFR2 antibody disclosed herein The amino acid sequences of the region and light chain variable region are described in SEQ ID NOs: 19 and 20. In that embodiment, the heavy chain variable region and light chain variable region of the anti-TNFR2 antibody disclosed herein The amino acid sequences of the region are described in SEQ ID NOs: 23 and 24. In some embodiments The amino acids in the heavy chain variable region and light chain variable region of the anti-TNFR2 antibody disclosed herein are... The sequences are described in Sequence IDs 27 and 28. In some embodiments, as specified herein The amino acid sequences of the heavy chain variable region and light chain variable region of the disclosed anti-TNFR2 antibody are, As described in issues 31 and 32. In some embodiments, the anti-T disclosed herein The amino acid sequences of the heavy chain variable region and light chain variable region of the NFR2 antibody are as follows: SEQ ID NOs. 35 and 36 It is described in [the relevant section]. In some embodiments, the anti-TNFR2 antibody disclosed herein is The amino acid sequences of the heavy chain variable region and the light chain variable region are described in SEQ ID NOs. 39 and 40. In some embodiments, the heavy chain variable region of the anti-TNFR2 antibody disclosed herein and The amino acid sequences of the light chain variable region are described in SEQ ID NOs. 43 and 44. In embodiments, the heavy chain variable region and light chain variable region of the anti-TNFR2 antibody disclosed herein The amino acid sequences are described in SEQ ID NOs. 47 and 48. In some embodiments, Amino acid sequences of the heavy chain variable region and light chain variable region of the anti-TNFR2 antibody disclosed herein These are described in Sequence IDs 51 and 52. In some embodiments, they are disclosed herein. The amino acid sequences of the heavy chain variable region and light chain variable region of the anti-TNFR2 antibody are as follows: SEQ ID NO: 5 As described in sections 5 and 56. In some embodiments, the anti-TNF disclosed herein The amino acid sequences of the heavy chain variable region and light chain variable region of the R2 antibody are described in SEQ ID NOs. 59 and 60. It is described. In some embodiments, the heavy chain of the anti-TNFR2 antibody disclosed herein The amino acid sequences of the variable region and the light chain variable region are described in SEQ ID NOs. 63 and 64. In some embodiments, the heavy chain variable region and light chain of the anti-TNFR2 antibody disclosed herein The amino acid sequences of the chain variable region are described in SEQ ID NOs. 67 and 68. Several implementations In terms of form, the heavy chain variable region and light chain variable region of the anti-TNFR2 antibody disclosed herein The mino acid sequences are described in SEQ ID NOs. 71 and 72. In some embodiments, the present invention The amino acid sequences of the heavy chain variable region and light chain variable region of the anti-TNFR2 antibody disclosed in this document are: As described in Sequence IDs 75 and 76. In some embodiments, as disclosed herein. The amino acid sequences of the heavy chain variable region and light chain variable region of the anti-TNFR2 antibody are as follows: As described in 80. In some embodiments, the anti-TNFR2 disclosed herein The amino acid sequences of the heavy chain variable region and light chain variable region of the antibody are described in SEQ ID NOs. 83 and 84. In some embodiments, the heavy chain variable of the anti-TNFR2 antibody disclosed herein. The amino acid sequences of the region and light chain variable region are described in SEQ ID NOs. 87 and 88. In several embodiments, the heavy chain variable region and light chain variable region of the anti-TNFR2 antibody disclosed herein The amino acid sequences of the variant region are described in SEQ ID NOs: 91 and 92. Several embodiments The amino acids of the heavy chain variable region and light chain variable region of the anti-TNFR2 antibody disclosed herein. The acid sequences are described in SEQ ID NOs: 95 and 96. In some embodiments, as specified herein. The amino acid sequences of the heavy chain variable region and light chain variable region of the anti-TNFR2 antibody disclosed herein are: As described in numbers 99 and 100. In some embodiments, as disclosed herein. The amino acid sequences of the heavy chain variable region and light chain variable region of the anti-TNFR2 antibody are as follows: As described in pp. 104. In some embodiments, the anti-TNFR disclosed herein The amino acid sequences of the heavy chain variable region and light chain variable region of the two antibodies are shown in SEQ ID NOs: 107 and 108. It is described. In some embodiments, the weight of the anti-TNFR2 antibody disclosed herein The amino acid sequences of the chain variable region and the light chain variable region are described in SEQ ID NOs: 111 and 112. In some embodiments, the heavy chain variable region of the anti-TNFR2 antibody disclosed herein The amino acid sequences of the light chain variable region are described in SEQ ID NOs: 115 and 116. In several embodiments, the heavy chain variable region and light chain variable region of the anti-TNFR2 antibody disclosed herein The amino acid sequences of the variant regions are described in SEQ ID NOs: 119 and 120. Several implementations In terms of form, the heavy chain variable region and light chain variable region of the anti-TNFR2 antibody disclosed herein Mino acid sequences are described in SEQ ID NOs. 123 and 124. In some embodiments, Amino acid sequences of the heavy chain variable region and light chain variable region of the anti-TNFR2 antibody disclosed herein These are described in Sequence IDs 127 and 128. In some embodiments, the following is described herein. The amino acid sequences of the heavy chain variable region and light chain variable region of the disclosed anti-TNFR2 antibody are, As described in issues 131 and 132. In some embodiments, disclosed herein The amino acid sequences of the heavy chain variable region and light chain variable region of the anti-TNFR2 antibody are as follows: As described in pp. 136. In some embodiments, the anti-TNFR disclosed herein The amino acid sequences of the heavy chain variable region and light chain variable region of the two antibodies are shown in SEQ ID NOs: 139 and 140. It is described. In some embodiments, the weight of the anti-TNFR2 antibody disclosed herein The amino acid sequences of the chain variable region and the light chain variable region are described in SEQ ID NOs: 143 and 144. In some embodiments, the heavy chain variable region of the anti-TNFR2 antibody disclosed herein The amino acid sequences of the light chain variable region are described in SEQ ID NOs. 147 and 148. In several embodiments, the heavy chain variable region and light chain variable region of the anti-TNFR2 antibody disclosed herein The amino acid sequences of the variant regions are described in SEQ ID NOs: 151 and 152. Several implementations In terms of form, the heavy chain variable region and light chain variable region of the anti-TNFR2 antibody disclosed herein Mino acid sequences are described in SEQ ID NOs. 157 and 158. In some embodiments, Amino acid sequences of the heavy chain variable region and light chain variable region of the anti-TNFR2 antibody disclosed herein These are described in Sequence IDs 163 and 164. In some embodiments, the following is described herein. The amino acid sequences of the heavy chain variable region and light chain variable region of the disclosed anti-TNFR2 antibody are, As described in issues 169 and 170. In some embodiments, as disclosed herein. The amino acid sequences of the heavy chain variable region and light chain variable region of the anti-TNFR2 antibody are as follows: As described in pp. 176. In some embodiments, the anti-TNFR disclosed herein The amino acid sequences of the heavy chain variable region and light chain variable region of the two antibodies are shown in SEQ ID NOs: 181 and 182. It is described. In some embodiments, the weight of the anti-TNFR2 antibody disclosed herein The amino acid sequences of the chain variable region and the light chain variable region are described in SEQ ID NOs: 193 and 194. In some embodiments, the heavy chain variable region of the anti-TNFR2 antibody disclosed herein The amino acid sequences of the light chain variable region are described in SEQ ID NOs: 199 and 200. In several embodiments, the heavy chain variable region and light chain variable region of the anti-TNFR2 antibody disclosed herein The amino acid sequences of the variant regions are described in SEQ ID NOs. 205 and 206. Several implementations In terms of form, the heavy chain variable region and light chain variable region of the anti-TNFR2 antibody disclosed herein Mino acid sequences are described in SEQ ID NOs. 211 and 212. In some embodiments, Amino acid sequences of the heavy chain variable region and light chain variable region of the anti-TNFR2 antibody disclosed herein These are described in Sequence IDs 217 and 218. In some embodiments, the following is described herein. The amino acid sequences of the heavy chain variable region and light chain variable region of the disclosed anti-TNFR2 antibody are, As described in issues 223 and 224. In some embodiments, disclosed herein The amino acid sequences of the heavy chain variable region and light chain variable region of the anti-TNFR2 antibody are as follows: As described in 230. In some embodiments, the anti-TNFR disclosed herein The amino acid sequences of the heavy chain variable region and light chain variable region of the two antibodies are shown in SEQ ID NOs. 233 and 234. It is described. In some embodiments, the weight of the anti-TNFR2 antibody disclosed herein The amino acid sequences of the chain variable region and the light chain variable region are described in SEQ ID NOs. 237 and 238. In some embodiments, the heavy chain variable region of the anti-TNFR2 antibody disclosed herein The amino acid sequences of the light chain variable region are described in SEQ ID NOs. 241 and 242. In several embodiments, the heavy chain variable region and light chain variable region of the anti-TNFR2 antibody disclosed herein The amino acid sequences of the variant regions are described in SEQ ID NOs. 245 and 246. Several implementations In terms of form, the heavy chain variable region and light chain variable region of the anti-TNFR2 antibody disclosed herein Mino acid sequences are described in SEQ ID NOs. 249 and 250. In some embodiments, Amino acid sequences of the heavy chain variable region and light chain variable region of the anti-TNFR2 antibody disclosed herein These are described in Sequence IDs 253 and 254. In some embodiments, as described herein The amino acid sequences of the heavy chain variable region and light chain variable region of the disclosed anti-TNFR2 antibody are, As described in issues 257 and 258. In some embodiments, as disclosed herein. The amino acid sequences of the heavy chain variable region and light chain variable region of the anti-TNFR2 antibody are as follows: As described in pp. 262. In some embodiments, the anti-TNFR disclosed herein The amino acid sequences of the heavy chain variable region and light chain variable region of the two antibodies are shown in SEQ ID NOs. 265 and 266. It is described. In some embodiments, the weight of the anti-TNFR2 antibody disclosed herein The amino acid sequences of the chain variable region and the light chain variable region are described in SEQ ID NOs. 269 and 270. In some embodiments, the heavy chain variable region of the anti-TNFR2 antibody disclosed herein The amino acid sequences of the light chain variable region are described in SEQ ID NOs. 273 and 274. In several embodiments, the heavy chain variable region and light chain variable region of the anti-TNFR2 antibody disclosed herein The amino acid sequences of the variant regions are described in SEQ ID NOs. 277 and 278. Several implementations In terms of form, the heavy chain variable region and light chain variable region of the anti-TNFR2 antibody disclosed herein Mino acid sequences are described in SEQ ID NOs. 281 and 282. In some embodiments, Amino acid sequences of the heavy chain variable region and light chain variable region of the anti-TNFR2 antibody disclosed herein These are described in Sequence IDs 285 and 286. In some embodiments, the following is described herein. The amino acid sequences of the heavy chain variable region and light chain variable region of the disclosed anti-TNFR2 antibody are, As described in issues 293 and 294. In some embodiments, as disclosed herein. The amino acid sequences of the heavy chain variable region and light chain variable region of the anti-TNFR2 antibody are as follows: As described in pp. 298. In some embodiments, the anti-TNFR disclosed herein The amino acid sequences of the heavy chain variable region and light chain variable region of the two antibodies are as follows: SEQ ID NOs: 301 and 302 It is described. In some embodiments, the weight of the anti-TNFR2 antibody disclosed herein The amino acid sequences of the chain variable region and the light chain variable region are described in SEQ ID NOs: 305 and 306. stomach In some embodiments, the heavy chain variable region of the anti-TNFR2 antibody disclosed herein and The amino acid sequences of the light chain variable region are described in SEQ ID NOs. 309 and 310. In that embodiment, the heavy chain variable region and light chain variable region of the anti-TNFR2 antibody disclosed herein The amino acid sequences of the region are described in SEQ ID NOs: 313 and 314. Several implementations In this state, the heavy chain variable region and light chain variable region of the anti-TNFR2 antibody disclosed herein The no-acid sequences are described in SEQ ID NOs: 317 and 318. In some embodiments, this The amino acid sequences of the heavy chain variable region and light chain variable region of the anti-TNFR2 antibody disclosed in the specification are: As described in Sequence IDs 321 and 322. In some embodiments, as described herein. The amino acid sequences of the heavy chain variable region and light chain variable region of the anti-TNFR2 antibody shown are as follows: As described in sections 325 and 326. In some embodiments, the anti- The amino acid sequences of the heavy chain variable region and light chain variable region of the TNFR2 antibody are as follows: SEQ ID NO: 329 and It is described in 330.

[0075] In another embodiment, the anti-TNFR2 antibody is used, for example, with default parameters. Determined using BlastP software from the National Center for Biotechnology Information (NCBI). Not limited to those listed above, the amino acid sequences and at least 80% (for example, 8 (5%, 90%, 95%, 96%, 97%, 98%, 99% or more) identical VH sequences and Includes VL sequence

[0076] In further embodiments, the present disclosure provides anti-TNFR2scFv. Considering the arrangement of the heavy chain variable region (VH) and light chain variable region (VL), a person skilled in the art would understand Therefore, we will construct an anti-IL-2scFv by employing standard techniques known in this field. It should be easy.

[0077] In certain embodiments, the Disclosure relates to VH domains that can be dimerized under appropriate conditions. The invention provides polypeptides including a VL domain. For example, a VH domain and a VL domain. The ions are bound in a suitable buffer and dimerize through appropriate interactions such as hydrophobic interactions. Alternatively, the VH domain and VL domain can be used as a combination of the VH domain and VL domain. Bound in a suitable buffer containing an enzyme and / or cofactor that can promote dimerization. It may be done. Another approach is to use the VH domain and VL domain as appropriate reagents. and / or a suitable vial that allows both domains to react with each other in the presence of a catalyst. They can be combined within the group.

[0078] In certain embodiments, the VH domain and the VL domain are long polypeptide sequences, While not limited to these, for example, the steady-state region, hinge region, linker region, Fc region, disulfide region It may be contained in the bond-binding region, or any combination thereof. The constant domain is immunoglobulin. It is the immunoglobulin folding unit of the constant region of the brin molecule, and the domain of the constant region (for example) It is also called CH1, CH2, CH3, CH4, Ck, Cl). Several embodiments Then, the long polypeptide is produced according to the method disclosed herein. It may include multiple copies of one or both of the in and VL domains. For example, as described herein. Using polypeptides generated according to the method shown, diabodies and triabodies It is possible to form an i.

[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 and light chains may be one of the following pairs: SEQ ID NO: 4 38 and 292, Sequence IDs 291 and 292, Sequence IDs 235 and 236, Sequence ID 23 9 and 240, Sequence IDs 243 and 244, Sequence IDs 247 and 248, Sequence ID 251 and 252, Sequence IDs 255 and 256, Sequence IDs 259 and 260, Sequence ID 263 and 264, Sequence IDs 267 and 268, Sequence IDs 271 and 272, Sequence ID 275 and 276, Sequence IDs 279 and 280, Sequence IDs 283 and 284, Sequence IDs 287 and 2 88, Sequence IDs 295 and 296, Sequence IDs 299 and 300, Sequence IDs 303 and 30 4. Sequence IDs 307 and 308, 311 and 312, 315 and 316 , Sequence IDs 319 and 320, Sequence IDs 323 and 324, Sequence IDs 327 and 328, Sequence IDs 331 and 332, 432 and 260, 433 and 304, Column numbers 434 and 308, sequence numbers 435 and 236, sequence numbers 436 and 244, array Numbers 437 and 284, or sequence numbers 439 and 296.

[0080] In some embodiments, the anti-TNFR2 antibody comprises a heavy chain and a light chain, and the heavy chain and light chain The amino acid sequences are described in SEQ ID NOs. 438 and 292. In some embodiments, The anti-TNFR2 antibody contains a heavy chain and a light chain, and the amino acid sequences of the heavy chain and light chain are as follows: As described in 291 and 292. In some embodiments, the anti-TNFR2 antibody is heavy The compound includes a heavy chain and a light chain, and the amino acid sequences of the heavy chain and light chain are described in SEQ ID NOs. 235 and 236. In some embodiments, the anti-TNFR2 antibody comprises a heavy chain and a light chain, and the heavy chain and The amino acid sequences of the light chain are described in SEQ ID NOs. 239 and 240. Several implementations Morphologically, the anti-TNFR2 antibody contains a heavy chain and a light chain, and the amino acid sequences of the heavy chain and light chain are as follows: As described in Sequence IDs 243 and 244, in some embodiments, anti-TNFR2 anti The body contains a heavy chain and a light chain, and the amino acid sequences of the heavy chain and light chain are as follows: SEQ ID NOs. 247 and 248 As described in [reference], in some embodiments, the anti-TNFR2 antibody comprises a heavy chain and a light chain. The amino acid sequences of the heavy and light chains are described in SEQ ID NOs. 251 and 252. In one embodiment, the anti-TNFR2 antibody comprises a heavy chain and a light chain, and the amino acids of the heavy chain and light chain The sequences are described in sequence numbers 255 and 256. In some embodiments, anti-TN The FR2 antibody contains a heavy chain and a light chain, and the amino acid sequences of the heavy chain and light chain are as follows: As described in page 260. In some embodiments, the anti-TNFR2 antibody is heavy chain and light chain The amino acid sequences of the heavy and light chains are described in SEQ ID NOs. 263 and 264. In some embodiments, the anti-TNFR2 antibody comprises a heavy chain and a light chain, and the heavy chain and light chain The amino acid sequences are described in SEQ ID NOs. 267 and 268. In some embodiments, The anti-TNFR2 antibody contains a heavy chain and a light chain, and the amino acid sequences of the heavy chain and light chain are as follows: As described in 271 and 272. In some embodiments, the anti-TNFR2 antibody is heavy The compound includes a chain and a light chain, and the amino acid sequences of the heavy chain and light chain are described in SEQ ID NOs. 275 and 276. In some embodiments, the anti-TNFR2 antibody comprises a heavy chain and a light chain, and the heavy chain and The amino acid sequences of the light chain are described in SEQ ID NOs. 279 and 280. Several implementations Morphologically, the anti-TNFR2 antibody contains a heavy chain and a light chain, and the amino acid sequences of the heavy chain and light chain are as follows: As described in Sequence IDs 283 and 284, in some embodiments, anti-TNFR2 anti The body contains a heavy chain and a light chain, and the amino acid sequences of the heavy chain and light chain are as follows: SEQ ID NOs. 287 and 288 As described in [reference], in some embodiments, the anti-TNFR2 antibody comprises a heavy chain and a light chain. The amino acid sequences of the heavy and light chains are described in SEQ ID NOs. 295 and 296. In one embodiment, the anti-TNFR2 antibody comprises a heavy chain and a light chain, and the amino acids of the heavy chain and light chain The sequences are described in sequence numbers 299 and 300. In some embodiments, anti-TN The FR2 antibody contains a heavy chain and a light chain, and the amino acid sequences of the heavy chain and light chain are as follows: As described in 304. In some embodiments, the anti-TNFR2 antibody is heavy chain and light chain The amino acid sequences of the heavy and light chains are described in SEQ ID NOs: 307 and 308. In some embodiments, the anti-TNFR2 antibody comprises a heavy chain and a light chain, and the heavy chain and light chain The amino acid sequences are described in SEQ ID NOs: 311 and 312. In some embodiments, The anti-TNFR2 antibody contains a heavy chain and a light chain, and the amino acid sequences of the heavy chain and light chain are as follows: As described in 315 and 316. In some embodiments, the anti-TNFR2 antibody is heavy The compound includes a heavy chain and a light chain, and the amino acid sequences of the heavy chain and light chain are described in SEQ ID NOs. 319 and 320. In some embodiments, the anti-TNFR2 antibody comprises a heavy chain and a light chain, and the heavy chain and The amino acid sequences of the light chain are described in SEQ ID NOs. 323 and 324. Several implementations Morphologically, the anti-TNFR2 antibody contains a heavy chain and a light chain, and the amino acid sequences of the heavy chain and light chain are as follows: As described in Sequence IDs 327 and 328. In some embodiments, anti-TNFR2 anti The body contains a heavy chain and a light chain, and the amino acid sequences of the heavy chain and light chain are as follows: SEQ ID NOs. 331 and 332 As described in [reference], in some embodiments, the anti-TNFR2 antibody comprises a heavy chain and a light chain. The amino acid sequences of the heavy and light chains are described in SEQ ID NOs. 432 and 260. In one embodiment, the anti-TNFR2 antibody comprises a heavy chain and a light chain, and the amino acids of the heavy chain and light chain The sequences are described in sequence numbers 433 and 304. In some embodiments, anti-TN The FR2 antibody contains a heavy chain and a light chain, and the amino acid sequences of the heavy chain and light chain are as follows: As described in 308. In some embodiments, the anti-TNFR2 antibody is heavy chain and light chain The amino acid sequences of the heavy and light chains are described in SEQ ID NOs: 435 and 236. In some embodiments, the anti-TNFR2 antibody comprises a heavy chain and a light chain, and the heavy chain and light chain The amino acid sequences are described in SEQ ID NOs: 436 and 244. In some embodiments, The anti-TNFR2 antibody contains a heavy chain and a light chain, and the amino acid sequences of the heavy chain and light chain are as follows: As described in 437 and 284. In some embodiments, the anti-TNFR2 antibody is heavy The compound includes a chain and a light chain, and the amino acid sequences of the heavy chain and light chain are described in SEQ ID NOs. 439 and 296. It is being done.

[0081] In another embodiment, the anti-TNFR2 antibody is used, for example, with default parameters. Determined using BlastP software from the National Center for Biotechnology Information (NCBI). Not limited to those listed above, the amino acid sequences and at least 80% (for example, 8 (5%, 90%, 95%, 96%, 97%, 98%, 99% or more) The heavy and light chains are identical. It contains the amino acid sequence.

[0082] In one embodiment, the anti-TNFR2 antibody of the present disclosure is IgG, Fv, scFv, Fab, F (ab')2, minibody, diabody, triabody, nanobody, bispecific antibody , or it may be a single-domain antibody. In some embodiments, the anti-TNFR2 antibody of the present disclosure The antibody is an IgG1 antibody. In some other embodiments, the anti-TNFR2 antibody of the present disclosure is used. This is an IgG2 antibody. In some yet other embodiments, the anti-TNFR2 antibody of the present disclosure The body is an IgG3 antibody. In some further embodiments, the anti-TNFR2 antibody of the present disclosure The body is producing IgG4 antibodies.

[0083] In some embodiments, the anti-TNFR2 antibody of this disclosure agonizes TNFR2. It is possible. In some embodiments, the anti-TNFR2 antibody of this disclosure is Fc-independent. TNFR2 can be agonized.

[0084] In one embodiment, the present disclosure provides an antibody that binds to TNFR2 with high affinity. In one embodiment, the binding affinity is determined by Frankel et al. ("Mol. Immunol., 16:101-106, 197") Calculated by a modification of the Scatchard method, as described in 9). In another embodiment, binding affinity is measured by the antigen / antibody dissociation rate. Morphologically, binding affinity is measured by competitive radioimmunoassay. In another embodiment, Binding affinity is measured by ELISA. In another embodiment, antibody affinity is measured by flow - Measured by cytometry.

[0085] In one embodiment, the anti-TNFR2 antibody of this disclosure inhibits TNFR2-mediated signaling. Compared to signaling activated by TNF, it is activated at a level of at least 80%. In another embodiment, the anti-TNFR2 antibody of this disclosure transforms the signal via TNFR2. Compared to signaling activated by TNF, the transmission was at least 85% less. At least 90%, at least 95%, at least 99%, or at least 100% level It is activated by [something].

[0086] In one embodiment, the disclosure also includes heavy chain CDRs and light chain CDRs disclosed herein. The present disclosure provides an isolated polynucleotide sequence. In another embodiment, the present disclosure also provides the The present invention provides a vector containing a polynucleotide sequence such as the amino acids disclosed herein. Considering the sequence, a person skilled in the art would know that a vector encoding such an amino acid sequence is also Plasmids can be easily constructed. In another embodiment, the disclosure also The present disclosure provides host cells containing the vector. Those skilled in the art will be able to adapt the application and experimental conditions accordingly. Then, a suitable host cell is selected, and the above polynucleotide sequence is loaded and / or expressed. It would be easy to do so. In some embodiments, the host cell is, but is not limited to, an example. For example, ExpiCHO(TM) and Expi293F(TM) (ThermoFisher, USA) It includes mammalian host cells.

[0087] In one embodiment, the disclosure also includes heavy chain variable regions and light chain variable regions disclosed herein. Provides an isolated polynucleotide sequence encoding. In another embodiment, the disclosure provides Furthermore, vectors containing such polynucleotide sequences are provided herein. Considering the amino acid sequence, a person skilled in the art would know that encoding such an amino acid sequence is In another embodiment, this The disclosure also provides host cells containing the vector of this disclosure. Those skilled in the art will be able to use and experiment Depending on the conditions, appropriate host cells are selected to support the above polynucleotide sequence and / or It would be easy to make it manifest.

[0088] In one embodiment, the disclosure also encodes the heavy and light chains disclosed herein. Provides a polynucleotide sequence. In another embodiment, the disclosure also provides such polynucleotide sequences. A vector containing a creotide sequence is provided. Considering the amino acid sequence disclosed herein... Therefore, a person skilled in the art would know of a vector or plasmid encoding such an amino acid sequence. It will be possible to construct it easily. In another embodiment, the Disclosure also relates to the We provide host cells containing a cell. Those skilled in the art will know to choose an appropriate host cell depending on the application and experimental conditions. It is easy to use cells to support and / or express the above polynucleotide sequence. It is likely.

[0089] Use of the composition

[0090] In one embodiment, the present disclosure also includes the anti-TNFR2 antibody disclosed herein and pharmaceutically The present invention provides a composition comprising an acceptable carrier. The use of a pharmaceutically acceptable carrier is permitted in this invention. It is well known in the field. For example, "Remington's Pharmaceutical Sciences, by EW M Artin, MackPublishing Co., Easton, PA, 23rd Edition, 2020, discloses herein Compositions and formulations suitable for the pharmaceutical delivery of antibodies are described.

[0091] A composition comprising an anti-TNFR2 antibody or its antigen-binding fragment disclosed herein may be used alone. Or, in combination with a carrier, i.e., a pharmaceutically acceptable carrier, the target (e.g., human) It may be administered to (or animals). Pharmacologically acceptable means that it is not biologically or otherwise desirable. Undesirable materials, that is, materials that cause undesirable biological effects or contain It does not interact in any harmful form with any of the other components of the pharmaceutical composition in which it is used. It means a material that can be administered to. As is well known to those skilled in the art, the carrier is To minimize any degradation of the polypeptides disclosed herein, and to target The pharmaceutical composition is selected to minimize any harmful side effects in the pharmaceutical. It may be prepared by methodologies well known in the field of technology.

[0092] In one embodiment, the composition disclosed herein comprises a set of three complementarity-determining regions (CDRs) on a heavy chain. (HCDR1, HCDR2, and HCDR3) and a set of three CDRs on the light chain (LC It includes an anti-TNFR2 antibody having DR1, LCDR2, and LCDR3. In one embodiment As mentioned above, the HCDR1, HCDR2, and HCDR3 set is shown in Table 7 below. The amino acid sequence shown is included, and the corresponding light chain contains LCDR1, LCDR2 and LCDR Set 3 contains the amino acid sequence shown in Table 8 below.

[0093] In some embodiments, the compositions of the present disclosure are as described in SEQ ID NOs: 185, 190, and 347. The amino acid sequences of HCDR1, HCDR2, and HCDR3 described respectively, and SEQ ID NO: 35 LCDR1, LCDR2, and LCDR3 are described in 9, 372, and 378 respectively. The present invention comprises an anti-TNFR2 antibody having a noacid sequence. In some embodiments, the composition of the present invention These are HCDR1, HCDR2 and as described in Sequence IDs 185, 190, and 347, respectively. The HCDR3 amino acid sequence and the sequences described in SEQ ID NOs. 359, 372, and 378, respectively. This includes an anti-TNFR2 antibody having the LCDR1, LCDR2, and LCDR3 amino acid sequences. In some embodiments, the compositions of the present disclosure are as described in SEQ ID NOs: 185, 334, and 347. The amino acid sequences of HCDR1, HCDR2, and HCDR3 described respectively, and SEQ ID NO: 36 LCDR1, LCDR2, and LCDR3 are described in 0, 372, and 379 respectively. The present invention comprises an anti-TNFR2 antibody having a noacid sequence. In some embodiments, the composition of the present invention These are HCDR1, HCDR2 and as described in Sequence IDs 185, 190, and 347, respectively. The HCDR3 amino acid sequence and the sequences described in SEQ ID NOs. 359, 372, and 380, respectively. This includes an anti-TNFR2 antibody having the LCDR1, LCDR2, and LCDR3 amino acid sequences. In some embodiments, the compositions of the present disclosure are as described in SEQ ID NOs: 185, 190, and 347. The amino acid sequences of HCDR1, HCDR2, and HCDR3 described respectively, and SEQ ID NO: 35 LCDR1, LCDR2, and LCDR3 are described in 9, 372, and 381 respectively. The present invention comprises an anti-TNFR2 antibody having a noacid sequence. In some embodiments, the composition of the present invention These are HCDR1, HCDR2 and as described in Sequence IDs 185, 190, and 347, respectively. The HCDR3 amino acid sequence and the sequences described in SEQ ID NOs. 361, 372, and 381, respectively. This includes an anti-TNFR2 antibody having the LCDR1, LCDR2, and LCDR3 amino acid sequences. In some embodiments, the compositions of the present disclosure are as described in SEQ ID NOs: 185, 190, and 347. The amino acid sequences of HCDR1, HCDR2, and HCDR3 described respectively, and SEQ ID NO: 36 LCDR1, LCDR2, and LCDR3 are described in 2, 372, and 382 respectively. The present invention comprises an anti-TNFR2 antibody having a noacid sequence. In some embodiments, the composition of the present invention These are HCDR1, HCDR2 and as described in Sequence IDs 185, 335, and 347, respectively. The HCDR3 amino acid sequence and the sequences described in SEQ ID NOs. 363, 372, and 383, respectively. This includes an anti-TNFR2 antibody having the LCDR1, LCDR2, and LCDR3 amino acid sequences. In some embodiments, the compositions of the present disclosure are as described in SEQ ID NOs: 185, 190, and 347. The amino acid sequences of HCDR1, HCDR2, and HCDR3 described respectively, and SEQ ID NO: 36 LCDR1, LCDR2, and LCDR3 are described in 4, 372, and 384 respectively. The present invention comprises an anti-TNFR2 antibody having a noacid sequence. In some embodiments, the composition of the present invention These are HCDR1, HCDR2 and as described in Sequence IDs 185, 336, and 348, respectively. The HCDR3 amino acid sequence and the sequences described in SEQ ID NOs. 365, 372, and 378, respectively. This includes an anti-TNFR2 antibody having the LCDR1, LCDR2, and LCDR3 amino acid sequences. In some embodiments, the compositions of the present disclosure are as described in SEQ ID NOs: 185, 337, and 347. The amino acid sequences of HCDR1, HCDR2, and HCDR3 described respectively, and SEQ ID NO: 35 LCDR1, LCDR2, and LCDR3 are described in 9, 373, and 385 respectively. The present invention comprises an anti-TNFR2 antibody having a noacid sequence. In some embodiments, the composition of the present invention These are HCDR1, HCDR2 and as described in Sequence IDs 185, 338, and 348, respectively. The HCDR3 amino acid sequence and the sequences described in SEQ ID NOs. 359, 372, and 386, respectively. This includes an anti-TNFR2 antibody having the LCDR1, LCDR2, and LCDR3 amino acid sequences. In some embodiments, the compositions of the present disclosure are as described in SEQ ID NOs: 185, 339, and 349. The amino acid sequences of HCDR1, HCDR2, and HCDR3 described respectively, and SEQ ID NO: 35 LCDR1, LCDR2, and LCDR3 are described in 9, 372, and 378 respectively. The present invention comprises an anti-TNFR2 antibody having a noacid sequence. In some embodiments, the composition of the present invention These are HCDR1, HCDR2 and as described in sequence numbers 185, 190, and 350, respectively. The HCDR3 amino acid sequence and the sequences described in SEQ ID NOs. 366, 372, and 387, respectively. This includes an anti-TNFR2 antibody having the LCDR1, LCDR2, and LCDR3 amino acid sequences. In some embodiments, the compositions of the present disclosure are as described in SEQ ID NOs: 185, 190, and 351. The amino acid sequences of HCDR1, HCDR2, and HCDR3 described respectively, and SEQ ID NO: 35 LCDR1, LCDR2, and LCDR3 are described in 9, 372, and 378 respectively. The present invention comprises an anti-TNFR2 antibody having a noacid sequence. In some embodiments, the composition of the present invention These are HCDR1, HCDR2 and as described in Sequence IDs 185, 334, and 347, respectively. The HCDR3 amino acid sequence and the sequences described in SEQ ID NOs. 360, 372, and 388, respectively. This includes an anti-TNFR2 antibody having the LCDR1, LCDR2, and LCDR3 amino acid sequences. In some embodiments, the compositions of the present disclosure are as described in SEQ ID NOs: 185, 334, and 352. The amino acid sequences of HCDR1, HCDR2, and HCDR3 described respectively, and SEQ ID NO: 35 LCDR1, LCDR2, and LCDR3 are described in 9, 372, and 378 respectively. The present invention comprises an anti-TNFR2 antibody having a noacid sequence. In some embodiments, the composition of the present invention These are HCDR1, HCDR2 and as described in sequence numbers 185, 190, and 352, respectively. The HCDR3 amino acid sequence and the sequences described in SEQ ID NOs. 359, 372, and 389, respectively. This includes an anti-TNFR2 antibody having the LCDR1, LCDR2, and LCDR3 amino acid sequences. In some embodiments, the compositions of the present disclosure are as described in SEQ ID NOs: 185, 334, and 347. The amino acid sequences of HCDR1, HCDR2, and HCDR3 described respectively, and SEQ ID NO: 35 LCDR1, LCDR2, and LCDR3 are described in 9, 372, and 378 respectively. The present invention comprises an anti-TNFR2 antibody having a noacid sequence. In some embodiments, the composition of the present invention These are HCDR1, HCDR2 and as described in Sequence IDs 185, 190, and 347, respectively. The HCDR3 amino acid sequence and the sequences described in SEQ ID NOs. 365, 372, and 378, respectively. This includes an anti-TNFR2 antibody having the LCDR1, LCDR2, and LCDR3 amino acid sequences. In some embodiments, the compositions of the present disclosure are as described in SEQ ID NOs: 185, 336, and 352. The amino acid sequences of HCDR1, HCDR2, and HCDR3 described respectively, and SEQ ID NO: 36 LCDR1, LCDR2, and LCDR3 are described in 5, 372, and 390 respectively. The present invention comprises an anti-TNFR2 antibody having a noacid sequence. In some embodiments, the composition of the present invention These are HCDR1, HCDR2 and as described in Sequence IDs 185, 336, and 352, respectively. The HCDR3 amino acid sequence and the sequences described in SEQ ID NOs. 365, 374, and 391, respectively. This includes an anti-TNFR2 antibody having the LCDR1, LCDR2, and LCDR3 amino acid sequences. In some embodiments, the compositions of the present disclosure are as described in SEQ ID NOs: 186, 340, and 353. The amino acid sequences of HCDR1, HCDR2, and HCDR3 described respectively, and SEQ ID NO: 36 LCDR1, LCDR2, and LCDR3 are described in 7, 375, and 392 respectively. The present invention comprises an anti-TNFR2 antibody having a noacid sequence. In some embodiments, the composition of the present invention These are HCDR1, HCDR2 and as described in Sequence IDs 186, 341, and 354, respectively. The HCDR3 amino acid sequence and the L sequences described in SEQ ID NOs. 367, 375, and 392, respectively. This product contains an anti-TNFR2 antibody having the CDR1, LCDR2, and LCDR3 amino acid sequences. In some embodiments, the compositions of the present disclosure are as shown in SEQ ID NOs: 186, 341, and 354. The HCDR1, HCDR2, and HCDR3 amino acid sequences described therein, and SEQ ID NO: 368 LCDR1, LCDR2, and LCDR3 amino acids described in 375 and 392, respectively. The present invention comprises an anti-TNFR2 antibody having an acid sequence. In some embodiments, the composition of the present invention , HCDR1, HCDR2 and as described in Sequence IDs 186, 342 and 354 respectively. The HCDR3 amino acid sequence and the L sequences described in SEQ ID NOs. 367, 375, and 392, respectively. This product contains an anti-TNFR2 antibody having the CDR1, LCDR2, and LCDR3 amino acid sequences. In some embodiments, the compositions of the present disclosure are as shown in SEQ ID NOs: 186, 343, and 354. The HCDR1, HCDR2, and HCDR3 amino acid sequences described therein, and SEQ ID NO: 369 LCDR1, LCDR2, and LCDR3 amino acids described in 375 and 393, respectively. The present invention comprises an anti-TNFR2 antibody having an acid sequence. In some embodiments, the composition of the present invention , HCDR1, HCDR2 and as described in Sequence IDs 186, 343 and 355 respectively The HCDR3 amino acid sequence and the L sequences described in SEQ ID NOs. 367, 375, and 392, respectively. This product contains an anti-TNFR2 antibody having the CDR1, LCDR2, and LCDR3 amino acid sequences. In some embodiments, the compositions of the present disclosure are as shown in SEQ ID NOs: 186, 341, and 354. The HCDR1, HCDR2, and HCDR3 amino acid sequences described therein, and SEQ ID NO: 367 LCDR1, LCDR2, and LCDR3 amino acids described in 375 and 392, respectively. The present invention comprises an anti-TNFR2 antibody having an acid sequence. In some embodiments, the composition of the present invention , HCDR1, HCDR2 and as described in Sequence IDs 186, 341 and 355 respectively The HCDR3 amino acid sequence and the L sequences described in SEQ ID NOs. 367, 376, and 392, respectively. This product contains an anti-TNFR2 antibody having the CDR1, LCDR2, and LCDR3 amino acid sequences. In some embodiments, the compositions of the present disclosure are as shown in SEQ ID NOs: 186, 344, and 354. The amino acid sequences of HCDR1, HCDR2, and HCDR3 described therein, and SEQ ID NO: 370 LCDR1, LCDR2, and LCDR3 amino acids described in 375 and 392, respectively. The present invention comprises an anti-TNFR2 antibody having an acid sequence. In some embodiments, the composition of the present invention , HCDR1, HCDR2 and as described in Sequence IDs 186, 341 and 354 respectively The HCDR3 amino acid sequence and the L sequences described in SEQ ID NOs. 367, 375, and 394, respectively. This product contains an anti-TNFR2 antibody having the CDR1, LCDR2, and LCDR3 amino acid sequences. In some embodiments, the compositions of the present disclosure are as shown in SEQ ID NOs: 186, 344, and 354. The HCDR1, HCDR2, and HCDR3 amino acid sequences described therein, and SEQ ID NO: 367 LCDR1, LCDR2, and LCDR3 amino acids described in 375 and 392, respectively. The present invention comprises an anti-TNFR2 antibody having an acid sequence. In some embodiments, the composition of the present invention , HCDR1, HCDR2 and as described in Sequence IDs 186, 341 and 359 respectively The HCDR3 amino acid sequence and the L sequences described in SEQ ID NOs. 367, 375, and 393, respectively. This product contains an anti-TNFR2 antibody having the CDR1, LCDR2, and LCDR3 amino acid sequences. In some embodiments, the compositions of the present disclosure are as described in SEQ ID NOs: 187, 345, and 356. The HCDR1, HCDR2, and HCDR3 amino acid sequences described therein, and SEQ ID NO: 371 LCDR1, LCDR2, and LCDR3 amino acids described in 377 and 395, respectively. The present invention comprises an anti-TNFR2 antibody having an acid sequence. In some embodiments, the composition of the present invention , HCDR1, HCDR2 and as described in Sequence IDs 188, 345 and 357 respectively The HCDR3 amino acid sequence and the L sequences described in SEQ ID NOs. 371, 377, and 396, respectively. This product contains an anti-TNFR2 antibody having the CDR1, LCDR2, and LCDR3 amino acid sequences. In some embodiments, the compositions of the present disclosure are as shown in SEQ ID NOs: 189, 346, and 358. The HCDR1, HCDR2, and HCDR3 amino acid sequences described therein, and SEQ ID NO: 371 , The LCDR1, LCDR2, and LCDR3 amino acids described in 377 and 397, respectively. It contains an anti-TNFR2 antibody having the sequence.

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

[0095] In some embodiments, the compositions of the present disclosure are as follows: SEQ ID NOs: 402, 345, and 413 The amino acid sequences of HCDR1, HCDR2, and HCDR3 described respectively, and SEQ ID NO: 37 LCDR1, LCDR2, and LCDR3 described in 1, 418, and 427 respectively The present invention comprises an anti-TNFR2 antibody having a noacid sequence. In some embodiments, the composition of the present invention These are HCDR1, HCDR2 and as described in Sequence IDs 398, 346, and 407, respectively. The HCDR3 amino acid sequence and the sequences described in SEQ ID NOs. 371, 377, and 419, respectively. This includes an anti-TNFR2 antibody having the LCDR1, LCDR2, and LCDR3 amino acid sequences. In some embodiments, the compositions of the present disclosure are as described in SEQ ID NOs: 187, 346, and 408. The amino acid sequences of HCDR1, HCDR2, and HCDR3 described respectively, and SEQ ID NO: 37 LCDR1, LCDR2 and LCDR3 are described in 1, 377 and 420 respectively. The present invention comprises an anti-TNFR2 antibody having a noacid sequence. In some embodiments, the composition of the present invention These are HCDR1, HCDR2 and as described in Sequence IDs 189, 406, and 409, respectively. The HCDR3 amino acid sequence and the sequences described in SEQ ID NOs. 371, 377, and 421, respectively. This includes an anti-TNFR2 antibody having the LCDR1, LCDR2, and LCDR3 amino acid sequences. In some embodiments, the compositions of the present disclosure are as described in SEQ ID NOs: 187, 345, and 410. The amino acid sequences of HCDR1, HCDR2, and HCDR3 described respectively, and SEQ ID NO: 37 LCDR1, LCDR2, and LCDR3 are described in 1, 418, and 422 respectively. The present invention comprises an anti-TNFR2 antibody having a noacid sequence. In some embodiments, the composition of the present invention These are HCDR1, HCDR2 and as described in sequence numbers 399, 346, and 411, respectively. The HCDR3 amino acid sequence and the sequences described in SEQ ID NOs. 371, 418, and 423, respectively. This includes an anti-TNFR2 antibody having the LCDR1, LCDR2, and LCDR3 amino acid sequences. In some embodiments, the compositions of the present disclosure are as follows: The amino acid sequences of HCDR1, HCDR2, and HCDR3 described respectively, and SEQ ID NO: 41 LCDR1, LCDR2, and LCDR3 are described in 7, 377, and 424 respectively. The present invention comprises an anti-TNFR2 antibody having a noacid sequence. In some embodiments, the composition of the present invention These are HCDR1, HCDR2 and as described in Sequence IDs 401, 346, and 413, respectively. The HCDR3 amino acid sequence and the sequences described in SEQ ID NOs. 417, 418, and 425, respectively. This includes an anti-TNFR2 antibody having the LCDR1, LCDR2, and LCDR3 amino acid sequences. In some embodiments, the compositions of the present disclosure are as follows: The amino acid sequences of HCDR1, HCDR2, and HCDR3 described respectively, and SEQ ID NO: 41 LCDR1, LCDR2, and LCDR3 mesh described in 7, 418, and 426 respectively The present invention comprises an anti-TNFR2 antibody having a noacid sequence. In some embodiments, the composition of the present invention These are HCDR1, HCDR2 and as described in Sequence IDs 398, 346, and 415, respectively. The HCDR3 amino acid sequence and the sequences described in SEQ ID NOs. 417, 377, and 428, respectively. This includes an anti-TNFR2 antibody having the LCDR1, LCDR2, and LCDR3 amino acid sequences. In some embodiments, the compositions of the present disclosure are as follows: The amino acid sequences of HCDR1, HCDR2, and HCDR3 described respectively, and SEQ ID NO: 37 LCDR1, LCDR2, and LCDR3 are described in 1, 418, and 429 respectively. The present invention comprises an anti-TNFR2 antibody having a noacid sequence. In some embodiments, the composition of the present invention These are HCDR1, HCDR2 and as described in Sequence IDs 404, 346, and 412, respectively. The HCDR3 amino acid sequence and the sequences described in SEQ ID NOs. 371, 418, and 425, respectively. This includes an anti-TNFR2 antibody having the LCDR1, LCDR2, and LCDR3 amino acid sequences. In some embodiments, the compositions of the present disclosure are as follows: The amino acid sequences of HCDR1, HCDR2, and HCDR3 described respectively, and SEQ ID NO: 41 LCDR1, LCDR2, and LCDR3 are described in 7, 418, and 430 respectively. The present invention comprises an anti-TNFR2 antibody having a noacid sequence. In some embodiments, the composition of the present invention These are HCDR1, HCDR2 and as described in Sequence IDs 405, 406, and 416, respectively. The HCDR3 amino acid sequence and the sequences described in SEQ ID NOs. 371, 377, and 431, respectively. This includes an anti-TNFR2 antibody having the LCDR1, LCDR2, and LCDR3 amino acid sequences. .

[0096] In another embodiment, the compositions of the present disclosure are used, for example, with default parameters in the United States Determined using BlastP software from the National Center for Biotechnology Information (NCBI) Not limited to those, but including the amino acid sequences described above and at least 80% (for example, 85%) (Identical heavy chain CDR sequences) %, 90%, 95%, 96%, 97%, 98%, 99% or more It also includes an anti-TNFR2 antibody having a light chain CDR sequence.

[0097] In another embodiment, the composition of the present disclosure comprises the following pairs of heavy chain variable regions and light chain variable regions. Contains anti-TNFR2 antibodies with either: 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, Sequence IDs 23 and 24, Sequence IDs 27 and 28, Sequence IDs 31 and 32, Sequence ID 35 and 36, SEQ ID NOs: 39 and 40, SEQ ID NOs: 43 and 44, SEQ ID NOs: 47 and 48, Sequence IDs 51 and 52, 55 and 56, 59 and 60, 63 and 64, Sequence IDs 67 and 68, Sequence IDs 71 and 72, Sequence IDs 75 and 76, Sequence No. Sequence numbers 79 and 80, Sequence numbers 83 and 84, Sequence numbers 87 and 88, Sequence numbers 91 and 92 , Sequence IDs 95 and 96, Sequence IDs 99 and 100, Sequence IDs 103 and 104, Sequence No. Sequence numbers 107 and 108, Sequence numbers 111 and 112, Sequence numbers 115 and 116, Sequence number 119 and 120, SEQ ID NOs: 123 and 124, SEQ ID NOs: 127 and 128, SEQ ID NO: 1 31 and 132, SEQ ID NOs: 135 and 136, SEQ ID NOs: 139 and 140, SEQ ID NO: 14 3 and 144, SEQ ID NOs: 147 and 148, SEQ ID NOs: 151 and 152, SEQ ID NO: 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, Sequence IDs 205 and 206, Sequence IDs 211 and 212, Sequence IDs 217 and 2 18, Sequence IDs 223 and 224, Sequence IDs 229 and 230, Sequence IDs 233 and 23 4. Sequence IDs 237 and 238, 241 and 242, 245 and 246 , Sequence IDs 249 and 250, Sequence IDs 253 and 254, Sequence IDs 257 and 258, Sequence IDs 261 and 262, 265 and 266, 269 and 270, Column numbers 273 and 274, sequence numbers 277 and 278, sequence numbers 281 and 282, array Numbers 285 and 286, Sequence numbers 293 and 294, Sequence numbers 297 and 298, Sequence number Sequence numbers 301 and 302, Sequence numbers 305 and 306, Sequence numbers 309 and 310, Sequence number 313 and 314, SEQ ID NOs: 317 and 318, SEQ ID NOs: 321 and 322, SEQ ID NO: 3 25 and 326, or sequence numbers 329 and 330.

[0098] In certain embodiments, the compositions of the present disclosure are heavy chains as described in Sequence IDs 289 and 290. This disclosure includes a TNFR2 antibody having a variable region and a light chain variable region. In certain embodiments, this disclosure The composition has a heavy chain variable region and a light chain variable region as described in Sequence ID Nos. 3 and 4. Contains FR2 antibody. In certain embodiments, the compositions of this disclosure are as described in SEQ ID NOs: 7 and 8. The present invention includes a TNFR2 antibody having a heavy chain variable region and a light chain variable region. In certain embodiments, The compositions of this disclosure include the heavy chain variable region and the light chain variable region described in Sequence IDs 11 and 12. The composition comprises a TNFR2 antibody having the following characteristics: In certain embodiments, the composition of the Disclosure is: SEQ ID NO: 15 and includes a TNFR2 antibody having a heavy chain variable region and a light chain variable region as described in 16. In a specific embodiment, the composition of the present disclosure is a heavy chain variable region as described in SEQ ID NOs: 19 and 20. and a TNFR2 antibody having a light chain variable region. In a particular embodiment, the composition of the present disclosure This refers to TNFR having heavy chain variable regions and light chain variable regions as described in Sequence IDs 23 and 24. Contains 2 antibodies. In certain embodiments, the compositions of this disclosure are as described in SEQ ID NOs: 27 and 28. The present invention includes a TNFR2 antibody having a heavy chain variable region and a light chain variable region. In certain embodiments, The compositions of this disclosure include the heavy chain variable region and the light chain variable region described in Sequence ID Nos. 31 and 32. It comprises a TNFR2 antibody having the following characteristics. In certain embodiments, the composition of this disclosure is SEQ ID NO: 35 and includes a TNFR2 antibody having a heavy chain variable region and a light chain variable region as described in 36. In certain embodiments, the compositions of the present disclosure are heavy chain variable regions as described in SEQ ID NOs: 39 and 40. and a TNFR2 antibody having a light chain variable region. In a particular embodiment, the composition of the present disclosure This refers to TNFR having heavy chain variable regions and light chain variable regions as described in Sequence IDs 43 and 44. Contains 2 antibodies. In certain embodiments, the compositions of this disclosure are as described in SEQ ID NOs: 47 and 48. The present invention includes a TNFR2 antibody having a heavy chain variable region and a light chain variable region. In certain embodiments, The compositions of this disclosure include the heavy chain variable region and the light chain variable region described in Sequence IDs 51 and 52. The composition comprises a TNFR2 antibody having the following characteristics: In certain embodiments, the composition of the Disclosure is: SEQ ID NO: 55 and includes a TNFR2 antibody having a heavy chain variable region and a light chain variable region as described in 56. In a specific embodiment, the composition of the present disclosure is a heavy chain variable region as described in SEQ ID NOs. 59 and 60. and a TNFR2 antibody having a light chain variable region. In a particular embodiment, the composition of the present disclosure This refers to a TNFR having heavy chain variable regions and light chain variable regions as described in Sequence IDs 63 and 64. Contains 2 antibodies. In certain embodiments, the compositions of this disclosure are as described in SEQ ID NOs. 67 and 68. The present invention includes a TNFR2 antibody having a heavy chain variable region and a light chain variable region. In certain embodiments, The compositions of this disclosure include the heavy chain variable region and the light chain variable region described in Sequence IDs 71 and 72. The composition comprises a TNFR2 antibody having the following characteristics: In certain embodiments, the composition of the Disclosure is: SEQ ID NO: 75 and includes a TNFR2 antibody having a heavy chain variable region and a light chain variable region as described in 76. In a specific embodiment, the composition of the present disclosure is a heavy chain variable region as described in SEQ ID NOs: 79 and 80. and a TNFR2 antibody having a light chain variable region. In a particular embodiment, the composition of the present disclosure This refers to TNFR having heavy chain variable regions and light chain variable regions as described in Sequence IDs 83 and 84. Contains 2 antibodies. In certain embodiments, the compositions of this disclosure are as described in SEQ ID NOs: 87 and 88. The present invention includes a TNFR2 antibody having a heavy chain variable region and a light chain variable region. In certain embodiments, The compositions of this disclosure include the heavy chain variable region and the light chain variable region described in Sequence IDs 91 and 92. The composition comprises a TNFR2 antibody having the following characteristics: In certain embodiments, the composition of the present disclosure is: SEQ ID NO: 95 and includes a TNFR2 antibody having a heavy chain variable region and a light chain variable region as described in 96. In a specific embodiment, the composition of the present disclosure is a heavy chain variable region as described in SEQ ID NOs: 99 and 100. The present invention includes a TNFR2 antibody having a region and a light chain variable region. In certain embodiments, the composition of the present invention The object has a heavy chain variable region and a light chain variable region as described in Sequence IDs 103 and 104. Contains an NFR2 antibody. In certain embodiments, the compositions of this disclosure include SEQ ID NOs: 107 and 10 Includes a TNFR2 antibody having the heavy chain variable region and light chain variable region described in 8. In the application, the composition of this disclosure includes the heavy chain variable region described in Sequence IDs 111 and 112 and The present invention includes a TNFR2 antibody having a light chain variable region. In certain embodiments, the composition of the present invention TNF having heavy chain variable regions and light chain variable regions as described in Sequence IDs 115 and 116 Contains R2 antibody. In certain embodiments, the composition of this disclosure contains SEQ ID NOs: 119 and 120 Includes a TNFR2 antibody having the described heavy chain variable region and light chain variable region. Specific implementation In this state, the compositions of the present disclosure are heavy-chain variable regions and light-chain variable regions as described in Sequence IDs 123 and 124. It comprises a TNFR2 antibody having a chain variable region. In certain embodiments, the composition of this disclosure comprises TNFR2 having heavy chain variable region and light chain variable region as described in rows 127 and 128 Contains antibodies. In certain embodiments, the compositions of this disclosure are described in SEQ ID NOs: 131 and 132. The TNFR2 antibody comprises a heavy chain variable region and a light chain variable region. In certain embodiments, The compositions of this disclosure include the heavy chain variable region and light chain variable region described in Sequence IDs 135 and 136. The present invention comprises a TNFR2 antibody having a variant region. In certain embodiments, the composition of the present invention is sequence number TNFR2 antibodies having heavy chain variable regions and light chain variable regions as described in Nos. 139 and 140 Includes. In certain embodiments, the compositions of this disclosure are as described in SEQ ID NOs: 143 and 144. The TNFR2 antibody comprises a heavy chain variable region and a light chain variable region. In a particular embodiment, The compositions disclosed herein include the heavy chain variable region and light chain variable region described in Sequence IDs 147 and 148. It contains a TNFR2 antibody having a region. In certain embodiments, the composition of this disclosure is SEQ ID NO: 1 This includes a TNFR2 antibody having heavy chain variable regions and light chain variable regions as described in 51 and 152. In certain embodiments, the compositions of this disclosure are heavy compositions as described in Sequence IDs 157 and 158. The present invention comprises a TNFR2 antibody having a chain-variable region and a light chain-variable region. In certain embodiments, The composition shown comprises the heavy chain variable region and light chain variable region described in Sequence ID Nos. 163 and 164. It contains a TNFR2 antibody. In certain embodiments, the composition of this disclosure contains SEQ ID NO: 169 The present invention also includes a TNFR2 antibody having a heavy chain variable region and a light chain variable region as described in paragraph 170. In certain embodiments, the compositions of this disclosure are heavy chains as described in SEQ ID NOs: 175 and 176. The present invention includes a TNFR2 antibody having a variable region and a light chain variable region. In certain embodiments, the present invention The composition has heavy chain variable regions and light chain variable regions as described in Sequence IDs 181 and 182. It contains a TNFR2 antibody. In certain embodiments, the composition of this disclosure is SEQ ID NO: 193 and Contains a TNFR2 antibody having heavy chain variable regions and light chain variable regions as described in 194. In one embodiment, the composition of the present disclosure is a heavy chain variable region as described in Sequence IDs 199 and 200. The present invention includes a TNFR2 antibody having a region and a light chain variable region. In certain embodiments, the composition of the present invention The object has a heavy chain variable region and a light chain variable region as described in Sequence IDs 205 and 206. Contains an NFR2 antibody. In certain embodiments, the compositions of this disclosure include SEQ ID NOs. 211 and 21 Includes a TNFR2 antibody having the heavy chain variable region and light chain variable region described in 2. In the application, the composition of this disclosure includes the heavy chain variable region described in Sequence IDs 217 and 218 and The present invention includes a TNFR2 antibody having a light chain variable region. In certain embodiments, the composition of the present invention , TNF having heavy chain variable region and light chain variable region as described in Sequence IDs 223 and 224 Contains R2 antibody. In certain embodiments, the composition of this disclosure is as described in SEQ ID NOs: 229 and 230. Includes a TNFR2 antibody having the described heavy chain variable region and light chain variable region. Specific implementation In this state, the compositions of the present disclosure are heavy chain variable regions and light chains as described in Sequence IDs 233 and 234. It comprises a TNFR2 antibody having a chain variable region. In certain embodiments, the composition of this disclosure comprises TNFR2 having heavy chain variable region and light chain variable region as described in columns 237 and 238 Contains antibodies. In certain embodiments, the compositions of this disclosure are described in SEQ ID NOs: 241 and 242. The TNFR2 antibody comprises a heavy chain variable region and a light chain variable region. In certain embodiments, The compositions of this disclosure are heavy-chain variable regions and light-chain variable regions as described in Sequence IDs 245 and 246. The present invention comprises a TNFR2 antibody having a variant region. In certain embodiments, the composition of the present invention is sequence number TNFR2 antibodies having heavy chain variable regions and light chain variable regions as described in Nos. 249 and 250 This includes. In certain embodiments, the compositions of this disclosure are as described in Sequence IDs 253 and 254. The TNFR2 antibody comprises a heavy chain variable region and a light chain variable region. In a particular embodiment, The compositions disclosed herein include the heavy chain variable region and light chain variable region described in Sequence IDs 257 and 258. It contains a TNFR2 antibody having a region. In certain embodiments, the composition of the present disclosure is SEQ ID NO: 2 This includes a TNFR2 antibody having heavy chain variable regions and light chain variable regions as described in 61 and 262. In certain embodiments, the compositions of this disclosure are heavy compositions as described in Sequence IDs 265 and 266. The present invention comprises a TNFR2 antibody having a chain-variable region and a light chain-variable region. In certain embodiments, The composition shown contains the heavy chain variable region and light chain variable region described in Sequence ID Nos. 269 and 270. It contains a TNFR2 antibody. In certain embodiments, the composition of this disclosure is SEQ ID NO: 273 The present invention also includes a TNFR2 antibody having a heavy chain variable region and a light chain variable region as described in 274. In certain embodiments, the compositions of the present disclosure are heavy-chain compounds as described in Sequence IDs 277 and 278. The present invention includes a TNFR2 antibody having a variable region and a light chain variable region. In certain embodiments, the present invention The composition has heavy chain variable regions and light chain variable regions as described in Sequence IDs 281 and 282. It contains a TNFR2 antibody. In certain embodiments, the composition of this disclosure contains SEQ ID NO: 285 and Contains a TNFR2 antibody having heavy chain variable regions and light chain variable regions as described in 286. In one embodiment, the composition of the present disclosure is a heavy chain variable region as described in Sequence IDs 293 and 294. The present invention includes a TNFR2 antibody having a region and a light chain variable region. In certain embodiments, the composition of the present invention The object has a heavy chain variable region and a light chain variable region as described in Sequence IDs 297 and 298. Contains an NFR2 antibody. In certain embodiments, the compositions of this disclosure include SEQ ID NOs. 301 and 30 Includes a TNFR2 antibody having the heavy chain variable region and light chain variable region described in 2. In the application, the composition of this disclosure includes the heavy chain variable region described in Sequence IDs 305 and 306 and The present invention includes a TNFR2 antibody having a light chain variable region. In certain embodiments, the composition of the present invention TNF having heavy chain variable regions and light chain variable regions as described in Sequence IDs 309 and 310 Contains R2 antibody. In certain embodiments, the composition of this disclosure contains SEQ ID NOs: 313 and 314 Includes a TNFR2 antibody having the described heavy chain variable region and light chain variable region. Specific implementation In this state, the compositions of the present disclosure are heavy-chain variable regions and light-chain variable regions as described in Sequence IDs 317 and 318. It comprises a TNFR2 antibody having a chain variable region. In certain embodiments, the composition of this disclosure comprises TNFR2 having heavy chain variable region and light chain variable region as described in columns 321 and 322 Contains antibodies. In certain embodiments, the compositions of this disclosure are described in SEQ ID NOs. 325 and 326. The TNFR2 antibody comprises a heavy chain variable region and a light chain variable region. In certain embodiments, The compositions of this disclosure are heavy-chain variable regions and light-chain variable regions as described in Sequence IDs 329 and 330. Contains a TNFR2 antibody with a variant region.

[0099] In another embodiment, the compositions of the present disclosure are used, for example, with default parameters in the United States Determined using BlastP software from the National Center for Biotechnology Information (NCBI) Not limited to those, but including the amino acid sequences described above and at least 80% (for example, 85%) (90%, 95%, 96%, 97%, 98%, 99% or more) The VH sequence and V are identical. Contains an anti-TNFR2 antibody having an L sequence.

[0100] In yet another embodiment, the composition disclosed herein comprises one of the following pairs of heavy chains and light chains. Contains anti-TNFR2 antibodies: SEQ ID NOs. 438 and 292, SEQ ID NOs. 291 and 292, Sequence IDs 235 and 236, 239 and 240, 243 and 244, Column numbers 247 and 248, sequence numbers 251 and 252, sequence numbers 255 and 256, array Numbers 259 and 260, Sequence numbers 263 and 264, Sequence numbers 267 and 268, Sequence number Sequence numbers 271 and 272, Sequence numbers 275 and 276, Sequence numbers 279 and 280, Sequence number Sequence IDs 283 and 284, Sequence IDs 287 and 288, Sequence IDs 295 and 296, Sequence ID 2 99 and 300, SEQ ID NOs: 303 and 304, SEQ ID NOs: 307 and 308, SEQ ID NOs: 31 1 and 312, SEQ ID NOs: 315 and 316, SEQ ID NOs: 319 and 320, SEQ ID NOs: 323 and 324, Sequence IDs 327 and 328, Sequence IDs 331 and 332, Sequence ID 432 and bi260, SEQ ID NOs. 433 and 304, SEQ ID NOs. 434 and 308, SEQ ID NOs. 435 and 236, Sequence IDs 436 and 244, Sequence IDs 437 and 284, Sequence IDs 439 and 2 96.

[0101] In some embodiments, the compositions described herein are as described in Sequence IDs 438 and 292. The present invention comprises an anti-TNFR2 antibody having a heavy chain and a light chain. In some embodiments, the composition of the present invention The substance is an anti-TNFR2 agent having heavy and light chains as described in Sequence IDs 291 and 292. Including bodies. In some embodiments, the compositions of the present disclosure are described in SEQ ID NOs: 235 and 236. It contains an anti-TNFR2 antibody having the heavy and light chains listed. In some embodiments, The compositions disclosed herein include the heavy and light chain antibodies described in Sequence IDs 239 and 240. Contains a TNFR2 antibody. In some embodiments, the composition of this disclosure is SEQ ID NO: 243 and It contains an anti-TNFR2 antibody having heavy and light chains as described in 244. In application, the compositions of this disclosure include the heavy and light chains described in Sequence IDs 247 and 248. The present invention comprises an anti-TNFR2 antibody having a chain. In some embodiments, the composition of the present disclosure is sequence This product contains anti-TNFR2 antibodies having heavy and light chains as described in numbers 251 and 252. In some embodiments, the compositions of this disclosure are described in SEQ ID NOs: 255 and 256. It includes an anti-TNFR2 antibody having heavy and light chains. In some embodiments, the combination of the present disclosure The product is an anti-TNFR2 having heavy and light chains as described in Sequence IDs 259 and 260. Contains an antibody. In some embodiments, the compositions of this disclosure are as follows: The present invention includes an anti-TNFR2 antibody having the heavy and light chains described. In some embodiments, The compositions of this disclosure have heavy and light chains as described in Sequence IDs 267 and 268. Contains an anti-TNFR2 antibody. In some embodiments, the composition of this disclosure is SEQ ID NO: 271 and include anti-TNFR2 antibodies having heavy and light chains as described in 272. In embodiments, the compositions of the present disclosure include the heavy chains described in SEQ ID NOs. 275 and 276. It contains an anti-TNFR2 antibody having a light chain. In some embodiments, the composition of the present disclosure is Includes the heavy and light chain anti-TNFR2 antibodies described in columns 279 and 280. In some embodiments, the compositions of the present disclosure are as described in SEQ ID NOs: 283 and 284. It includes an anti-TNFR2 antibody having heavy and light chains. In some embodiments of this disclosure The composition has the heavy and light chains described in Sequence ID Nos. 287 and 288, and is an anti-TNFR. Contains two antibodies. In some embodiments, the compositions of this disclosure include SEQ ID NOs: 295 and 296 The present invention includes an anti-TNFR2 antibody having heavy and light chains as described in some embodiments. The compositions of this disclosure have the heavy and light chains described in Sequence IDs 299 and 300. It contains an anti-TNFR2 antibody. In some embodiments, the composition of the present disclosure is SEQ ID NO: 30 It contains anti-TNFR2 antibodies having heavy and light chains as described in 3 and 304. In the embodiments described herein, the compositions are heavy chains and as described in SEQ ID NOs: 307 and 308. It contains an anti-TNFR2 antibody having a light chain. In some embodiments, the composition of the present disclosure is Contains the heavy and light chain anti-TNFR2 antibodies described in SEQ ID NOs. 311 and 312. In some embodiments, the compositions of this disclosure are as described in SEQ ID NOs: 315 and 316. The present invention includes an anti-TNFR2 antibody having heavy and light chains. In some embodiments, the present disclosure The composition is an anti-TNF having heavy and light chains as described in Sequence ID Nos. 319 and 320. Contains R2 antibody. In some embodiments, the composition of this disclosure contains SEQ ID NOs. 323 and 32 The present invention includes an anti-TNFR2 antibody having the heavy and light chains described in 4. The compositions of this disclosure have the heavy and light chains described in Sequence IDs 327 and 328. It contains an anti-TNFR2 antibody. In some embodiments, the composition of the present disclosure is as follows: Contains anti-TNFR2 antibodies having heavy and light chains as described in 31 and 332. In that embodiment, the composition of the present disclosure is heavy chain and as described in SEQ ID NOs: 432 and 260. It contains an anti-TNFR2 antibody having a light chain. In some embodiments, the composition of the present disclosure is It contains anti-TNFR2 antibodies having heavy and light chains as described in columns 433 and 304. In some embodiments, the compositions of the present disclosure are heavy chains as described in SEQ ID NOs: 434 and 308. and an anti-TNFR2 antibody having a light chain. In some embodiments, the composition of the present disclosure This includes an anti-TNFR2 antibody having heavy and light chains as described in SEQ ID NOs. 435 and 236. In some embodiments, the compositions of the present disclosure are as described in Sequence IDs 436 and 244. The present invention comprises an anti-TNFR2 antibody having a heavy chain and a light chain. In some embodiments, the composition of the present invention The substance is an anti-TNFR2 antibody having heavy and light chains as described in SEQ ID NOs. 437 and 284. Includes. In some embodiments, the composition is as described in Sequence IDs 439 and 296. It contains an anti-TNFR2 antibody having heavy and light chains.

[0102] In another embodiment, the compositions of the present disclosure are used, for example, with default parameters in the United States Determined using BlastP software from the National Center for Biotechnology Information (NCBI) Not limited to the sameness, at least 80% (for example, 8) of the amino acid sequence described above. (5%, 90%, 95%, 96%, 97%, 98%, 99% or more) The heavy and light chains are identical. It contains an anti-TNFR2 antibody.

[0103] In one embodiment, the antibody disclosed herein may take the form of a conjugate. When used herein, "conjugate" means an effector molecule or a second An antibody or antibody fragment (e.g., antigen) covalently bound to a protein (e.g., a second antibody) This refers to a composite fragment. Effector molecules are, for example, drugs, toxins, therapeutic agents, detectable labels, etc. It may be protein, nucleic acid, lipid, nanoparticle, carbohydrate, or recombinant virus. A conjugate is also called an "immune conjugate." A conjugate is a drug (for example) If the conjugate contains an antibody bound to a cytotoxic drug, the conjugate is called an "antibody-drug conjugate." It can be called an antibody conjugate. Other antibody conjugates include, for example, multispecificity ( Examples include bispecific and trispecific antibodies, and chimeric antigen receptors (CARs). It can be done.

[0104] The pharmaceutical compositions of this disclosure, comprising antibodies or antigen-binding fragments thereof, are topical. Depending on whether treatment is desired or systemic treatment is desired, use any appropriate method (e.g., breastfeeding) It may be administered to animals, cells, or tissues. For example, the compositions of this disclosure may be administered topically (e.g. (By intraocular, vaginal, rectal, nasal, or transdermal administration), orally, or by inhalation. Alternatively, it can be administered parenterally (for example, by intravenous infusion, or subcutaneously, intrathoracically, intraperitoneally, or cutaneously). It may be administered (by intravenous or intramuscular injection). Local intranasal administration is as described in this disclosure. This refers to delivering the composition into the nasal cavity through one or both nostrils. It can be delivered by a spray mechanism, a droplet mechanism, or by aerosolization. The administration may be intratumoral, for example, by local injection or intravenous injection.

[0105] When the compositions of this disclosure are administered parenterally, the administration is generally carried out by injection. Injectable drugs may be liquid solutions or suspensions, or solid forms suitable for suspension in liquid before injection. Alternatively, it can be prepared in conventional forms as an emulsion. In addition, with oral administration, To maintain the dosage, this may include preparing a sustained-release or long-lasting release system.

[0106] How to use

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

[0108] T-reg cells can be distinguished based on the presentation of their unique surface proteins. This represents a heterogeneous class of T cells. 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 are proliferative cytokines in target T cells. It inhibits the production of a certain interleukin-2 (IL-2), and furthermore, it inhibits IL-2. IL-2 is released from autoreactive cells by affinity for IL-25 (a subdomain of the IL-2 receptor). It has been indicated that isolation is necessary. Furthermore, CD4 + CD25 + FoxP3 + T-reg The cells are also present in areas rich in B cells, and do not depend on their ability to weaken the activity of TH2 cells. It has been shown that it can directly suppress the production of immunoglobulins.

[0109] As used herein, the terms "myelo-derived suppressor cells" or "MDSCs" are used in a variety of ways. Various effector cells and antigen-presenting cells, particularly T cells, NK cells, dendritic cells, and ma These refer to immune system cells that can regulate the activity of cells such as clophages. These are distinguished by their gene expression profiles.

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

[0111] As used herein, the term "method" means a technique for achieving a given objective. This refers to means, techniques, and procedures, and includes, but is not limited to, chemistry, medicine, etc. Methods, means, techniques, and procedures known to those skilled in the art in the fields of physics, biology, biochemistry, and medicine. This includes methods, means, techniques, and procedures that are readily developed from those by those skilled in the art.

[0112] When used herein, the term "treatment (treat, treatment, therapy)" (and (These deformities) prevent or delay undesirable physiological changes associated with disease or condition. This refers to therapeutic measures, including preventive measures, aimed at reducing (or alleviating) the clinically beneficial or desirable clinical effects. As a result, although not limited to this, regardless of whether it is detectable or not, symptom relief, disease Reduction in the extent of the affected area or condition, stabilization of the disease or condition (i.e., worsening of the disease or condition) (If not done), delay or reduce the progression of the disease or condition, improve or slow the disease or condition. Remission of the disease or condition (whether partial or complete) is also included. Those who need treatment include not only those who already have a disease or condition, This includes people who are susceptible to disease or condition, or who should take preventative measures against disease or condition.

[0113] The terms “subject,” “individual,” and “patient” are used interchangeably in this specification. Treatment with a composition or formulation containing the anti-TNFR2 antibody according to the invention is provided to human or animal. "T" refers to non-human animals. Furthermore, the terms "non-human animals" and "non-human mammals" are used in the context of the original text. In the details, it is used interchangeably for all vertebrates, e.g., non-human primates (e.g., higher primates). Longnidulates, sheep, dogs, rodents (e.g., mice, rats), guinea pigs, goats, pigs Mammals such as cats, rabbits, cows, and horses, as well as, for example, reptiles, amphibians, and chickens. This includes non-mammals such as turkeys. The compositions described herein are for primates (e.g., monkeys, Humans, horses, cattle, cats, dogs, rabbits, and rodents (e.g., mice, rats) It can be used to treat any suitable mammal, including, The mammal being treated is a human. The human can be a human of any age. In one embodiment... In one embodiment, the human is an adult. In another embodiment, the human is a child. The human is male, female. This could include pregnant women, middle-aged people, young people, and the elderly.

[0114] Pharmaceutical compositions suitable for use in the manner disclosed herein include active ingredients that achieve the intended purpose. The composition contains an effective amount for the disease. In one embodiment, an effective therapeutic amount is defined as the amount of the disease Effective in preventing, alleviating, or improving the symptoms of, or in extending the survival of the patient being treated. This refers to the amount of one or more effective active ingredients (e.g., anti-TNFR2 antibody). The determination of the quantity is entirely possible within the capabilities of those skilled in the art.

[0115] In one embodiment, the present disclosure modulates the activity or function of regulatory T cells in a subject. Provides a method. As used herein, “modulate” means the activity of a molecular target or pathway. This refers to "stimulating" or "inhibiting" something. For example, the compositions of this disclosure are... Except for not using the indicated composition, the activity of the molecular target or pathway under the same conditions is compared to that of other compositions. , the activity of the molecular target or pathway, 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 of stimulation or inhibition To modify the activity of a molecular target or pathway. In another example, the compositions of the present disclosure modify the activity of a molecular target or pathway. Aside from not using the disclosed composition, the activity of the molecular target or pathway is compared to that under the same conditions. The activity of the molecular target or pathway is increased by at least 2 times, at least 5 times, and at least 10 times. , stimulate or inhibit at least 20 times, at least 50 times, or at least 100 times Thus, it modulates the activity of a molecular target or pathway. The activity of the molecular target or molecular pathway is reproduced. It can be measured by a specific method. The activity of a molecular target or molecular pathway can be measured in vitro. Alternatively, it can be measured using in vitro. For example, the activity of a molecular target or molecular pathway is measured using in vitro. Measurements can be taken in vitro or in vitro by appropriate assays known in the technical field of measurement. It can be determined. The control sample (untreated with the composition of this disclosure) has 100% relative activity. A gender value shall be assigned.

[0116] In one embodiment, the method of the present disclosure comprises a composition containing an effective amount of the anti-TNFR2 antibody of the present disclosure. The method includes the step of administering a substance to a target. In one embodiment, the composition disclosed herein is described herein. The present invention includes an anti-TNFR2 antibody having the indicated heavy chain CDR sequence and light chain CDR sequence. In form, the compositions disclosed herein are anti-T2 compositions having the VH sequence and VL sequence disclosed herein. Contains an NFR2 antibody. In yet another embodiment, the composition disclosed herein is It contains an anti-TNFR2 antibody having a heavy chain sequence and a light chain sequence. In one embodiment, adjustable T-cell The cell is CD4 + CD25 + Foxp3 + , or alternatively, CD4 + CD25 + C127 low In one embodiment, the method disclosed herein stimulates the proliferation of regulatory T cells. In one embodiment, the method disclosed herein can activate regulatory T cells. Another embodiment Therefore, the method of this disclosure modulates the immune response mediated by regulatory T cells as described above. It is possible.

[0117] Those skilled in the art will know that in some embodiments, the activation of regulatory T cells involves Teff and Suppression of the proliferation of immune system effector cells, including NK cells, Teff and effector cells including NK cells Reduced cytotoxic activity of kuta cells, by immune system effector cells including Teff and NK cells This includes reducing the production of inflammatory cytokines, or any combination thereof. This will be understood. In some embodiments, an anti-TNFR2 antibody by the method of the present disclosure is When administered to the target, it promotes the proliferation of immune system effector cells, including Teff and NK cells. It is suppressed. In some embodiments, the anti-TNFR2 antibody administered by the method of the present disclosure is targeted By administering it, the cytotoxic activity of immune system effector cells, including Teff cells and NK cells, is activated. The properties are reduced. In some embodiments, the anti-TNFR2 antibody by the method of the present disclosure is targeted. By administration, inflammatory stimuli are induced by immune system effector cells, including Teff and NK cells. Itokine production is reduced.

[0118] Those skilled in the art will know that in some embodiments, the modulation of the immune response is the anti-TNFR2 of the Disclosure. In situations where inflammation would be expected if antibodies were not used, mild inflammation You will understand that this includes reducing or eliminating inflammation.

[0119] In one embodiment, the disclosure also modulates the activity or function of regulatory T cells in a subject. The present invention provides the use of a composition containing the target anti-TNFR2 antibody for this purpose. The disclosed composition is an anti-T2 having the heavy chain CDR sequence and light chain CDR sequence disclosed herein. Contains an NFR2 antibody. In another embodiment, the composition disclosed herein is VH The present invention includes an anti-TNFR2 antibody having a sequence and a VL sequence. In yet another embodiment, the present disclosure The composition comprises an anti-TNFR2 antibody having the heavy chain sequence and light chain sequence disclosed herein. .

[0120] In one embodiment, the present disclosure relates to the activity of myeloid-derived suppressor cells (MDSCs) in a subject or A method for adjusting the function is provided. In one embodiment, the method of the present disclosure provides the anti-TNFR of the present disclosure. The method includes the step of administering a composition containing an effective amount of two antibodies to a target. In one embodiment, The disclosed composition is an anti-T2 having the heavy chain CDR sequence and light chain CDR sequence disclosed herein. Contains an NFR2 antibody. In another embodiment, the composition disclosed herein is VH The present invention includes an anti-TNFR2 antibody having a sequence and a VL sequence. In yet another embodiment, the present disclosure The composition comprises an anti-TNFR2 antibody having the heavy chain sequence and light chain sequence disclosed herein. In one embodiment, the method disclosed herein stimulates the proliferation of MDSCs. In another embodiment, The method of disclosure can activate MDSC.

[0121] Those skilled in the art will know that in some embodiments, MDSC activation involves Teff and NK Suppression of the proliferation of immune system effector cells, including Teff and NK cells Reduced cytotoxic activity of effector cells, including Teff and NK cells in the immune system. Downregulation of pro-inflammatory cytokine production, or any combination thereof. You will understand that this is possible. In some embodiments, anti-TNF by the methods of the present disclosure By targeting R2 antibodies with administration, immune system effector cells, including Teff and NK cells, are targeted. Cell proliferation is suppressed. In some embodiments, an anti-TNFR2 antibody is produced by the method of the present disclosure. By administering it to the target, immune system effector cells, including Teff cells and NK cells, are targeted. Cytotoxic activity is reduced. In some embodiments, the anti-TNFR2 activity is reduced by the method disclosed herein. By administering it to the body, immune system effector cells, including Teff and NK cells, The production of inflammatory cytokines is reduced.

[0122] In one embodiment, the disclosure also modulates the activity or function of MDSCs in a subject. The present disclosure provides the use of a composition containing an anti-TNFR2 antibody. In one embodiment, the composition of the present disclosure The substance is an anti-TNFR2 agent having a heavy chain CDR sequence and a light chain CDR sequence disclosed herein. Includes the body. In another embodiment, the composition disclosed herein includes the VH sequence and V The present invention comprises an anti-TNFR2 antibody having an L sequence. In yet another embodiment, the present invention comprises This specification includes an anti-TNFR2 antibody having the heavy chain sequence and light chain sequence disclosed herein.

[0123] In another embodiment, the present disclosure is a method for treating a disease in question, wherein the anti-TNF of the present disclosure The present invention provides a method comprising the step of administering a composition containing an effective amount of R2 antibody to a target. In embodiments, the compositions disclosed herein include heavy chain CDR sequences and light chain CDR sequences disclosed herein. It comprises an anti-TNFR2 antibody having a sequence. In another embodiment, the composition of this disclosure is as specified herein. This includes an anti-TNFR2 antibody having the VH sequence and VL sequence disclosed herein. In this state, the compositions of this disclosure have the heavy chain sequence and light chain sequence disclosed herein as anti-TN Contains FR2 antibody.

[0124] In one embodiment, the disclosure also includes an anti-TNFR2 antibody for treating the disease in question. The use of the composition is provided. In one embodiment, the composition of the Disclosure is disclosed herein. The present embodiment includes an anti-TNFR2 antibody having a heavy chain CDR sequence and a light chain CDR sequence. The compositions disclosed herein are anti-TNFR2 having the VH sequence and VL sequence disclosed herein. Contains antibodies. In yet another embodiment, the composition of the Disclosure contains heavy chain compounds disclosed herein. It contains an anti-TNFR2 antibody having a column and light chain sequence.

[0125] In one embodiment, the polypeptide of the present invention or the polypeptide necessary to achieve the desired effect The exact amount of the composition depends on the target species, age, sex, weight and overall condition, and the specific polypeptide. Depending on the route of administration and whether other drugs are included in the regimen, the treatment will vary from patient to patient. Therefore, it is not possible to specify the exact amount for all compositions. Furthermore, a person skilled in the art can determine the appropriate amount through routine experimentation. Dosage The requirements vary, and polypeptides are taken more than once a day (for example, more than twice, more than three times, more than four times). The appropriate antibody can be administered over a period of one day or more (or five or more times). Guidance for selecting dosages can be easily found in the literature.

[0126] In one embodiment, the disease is a viral infection, a bacterial infection, cancer, an autoimmune disease, or an immune disease. It can be an infectious disease. In one embodiment, the disease is an upper respiratory tract viral infection, an early lung infection, and It could be a terminal lung infection. Various diseases and cancers are caused by viruses. This is known. Examples of disease-causing viruses are not limited to this, but include, for example, Norovirus; rotavirus; hepatitis viruses of type A, B, C, D, or E; Rabies virus, West Nile virus, Enterovirus, Echovirus, Coxsackievirus Virus, herpes simplex virus (HSV), HSV-2, varicella-zoster virus, mosquito-borne 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) (e.g., BK virus), Ebola virus, dengue virus, or any combination thereof) A combination was mentioned.

[0127] In another embodiment, the disease may be cancer. Cancer is not limited to cancer, but for example, 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, eye cancer, digestive cancer ductal cancer, colon cancer, gallbladder cancer, stomach cancer, gastrointestinal carcinoid tumors, 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 malignancies, non-Hodgkin lymphoma, skin cancer, basal cell carcinoma Examples include cancer, 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. While not limited to these, examples include achalasia, amyloidosis, ankylosing spondylitis, and anti-GBM / anti-T. BM nephritis, antiphospholipid syndrome, arthritis, autoimmune angioedema, autoimmune encephalomyelitis, auto Immune 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 disease, dermatitis, dermatomyositis, discoid lupus, Dressler syndrome, endometriosis, fibromyalgia, fibrosis alveolitis pallidum, granulomatosis with polyangiitis, Graves' disease, Guillain-Barré syndrome, herpes zoster of pregnancy Immunotoxic thrombocytopenic purpura, interstitial cystitis, juvenile arthritis, juvenile diabetes (type 1 diabetes) ), juvenile myositis, Kawasaki disease, Lambert-Eaton syndrome, lichen planus, lupus, Lyme disease Multiple sclerosis, myasthenia gravis, myositis, neonatal lupus, neutropenia, palindromic rheumatism Peripheral neuropathy, polyarteritis nodosa, polymyalgia rheumatica, polymyositis, post-myocardial infarction Syndrome, post-pericardiotomy syndrome, primary biliary cirrhosis, primary sclerosing cholangitis, progesterone-induced ulcers Dermatitis, psoriasis, psoriatic arthritis, reactive arthritis, retroperitoneal fibrosis, rheumatic fever, rheumatoid arthritis, Sarcoidosis, Schmidt syndrome, scleritis, scleroderma, Sjögren's syndrome, thrombocytopenia Examples include purpura minor, 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). Yes. In one embodiment, GVHD is acute GVHD. In another embodiment, GVHD This is chronic GVHD.

[0130] The agonist anti-TNFR2 antibody (or its antigen-binding fragment) of this disclosure further affects organs It can be used to treat patients who require repair or regeneration. For example, the jaw of the present disclosure The TNFR2 antibody or its antigen-binding fragment is, for example, TRAF2 / 3- and / Alternatively, TNFR2 can be added to the surface of cells in damaged tissue to induce NFκB-mediated cell proliferation. By combining them, they can be used to promote organ repair or regeneration. Inducing regeneration by administering an agonist TNFR2 antibody or its antigen-binding fragment. Examples of tissues and organs that can produce this include, but are not limited to, the pancreas, salivary glands, pituitary gland, and kidneys. heart, lungs, hematopoietic system, cranial nerves, blood vessels including the aorta, olfactory glands, ears, nerves, head structure, Eyes, thymus, tongue, bones, liver, small intestine, large intestine, intestines, lungs, brain, skin, peripheral nervous system, central nervous system, spine These include the marrow, mammary gland, embryonic structures, embryo, and testes.

[0131] The agonist anti-TNFR2 antibody (or its antigen-binding fragment) of this disclosure also affects neurological diseases. It can be administered to an subject (e.g., a human) to treat a disease or condition. (Neurology) Examples of diseases or conditions include, but are not limited to, brain tumors, brain metastases, spinal cord injuries, Schizophrenia, epilepsy, amyotrophic lateral sclerosis (ALS), Parkinson's disease, Alzheimer's disease Examples include cerebrospinal fluid, Huntington's disease, and stroke.

[0132] The agonist TNFR2 antibody (or its antigen-binding fragment) of this disclosure also affects regulatory T cells. It may be mixed, conjugated, or administered together with another drug that promotes growth, or administered separately. It may be administered. Additional drugs that can be used to promote the expansion of regulatory T cells and However, although not limited to this, IL-2 and TNFα, which are homologous ligands of TNFR2, It can be listed.

[0133] In another embodiment, the present disclosure relates to a polynucleotide encoding an anti-TNFR2 antibody. This provides a method for treating the diseases or conditions described above using [a specific method / technology].

[0134] The anti-TNFR2 Various embodiments and forms of the body are experimentally supported in the following examples. The example given in the detailed document is that it exists as a non-aggregated species and is generally folded as SE IgG1. This involves the generation of an anti-TNFR2 agonist antibody that moves on a C column. IgG1 agonist antibodies 2 have an EC of approximately 1.8 nM to 66 nM. 50 Binding solubility within the range These antibodies bound to TNFR2-His, demonstrating their robust binder properties. Furthermore, the antibody specifically bound to TNFR2 but not to TNFR1. The developed anti-TNFR2 antibody contains a high-affinity functional agonist, and EC 50 range This agonizes the TNFR2 receptor without the addition of other molecules ranging from 0.5 nM to 277 nM. Furthermore, the activation of TNFR2 by the generated anti-TNFR2 antibody is linked to the IgG-Fc class It is thought to be independent of teration. Regarding the activation properties of the generated anti-TNFR2 antibody When the effects of TNFα were investigated, these antibodies almost completely activated the TNFR2 receptor. The most likely possibility of activation is with epitopes that do not block the TNFα binding site. This was shown.

[0135] Examples

[0136] Example 1

[0137] Fabrication of a TNFR2 agonist

[0138] Experimental Procedure

[0139] Library creation

[0140] (Building a library)

[0141] The library was obtained by overlap extension PCR using denatured oligonucleotides. Constructed based on three template antibodies (PDB: 2I5Y, 4IOI, and 3E8U) The PCR used to introduce diversity was Phusion high fidelity DNA polymerase. Using -se (NewEngland Biolabs USA, Cat: M0530), follow the manufacturer's instructions. The reaction was carried out in three stages (30 seconds at 98°C, 20 seconds at 65°C, 30 seconds at 72°C, 30 seconds). (Cycle). PCR products are gel-purified using a gel purification kit, and primers are present. Except for the absence of certain components, the assembly was carried out in equimolar ratios using a three-step PCR reaction, in the same manner as described above. The resulting PCR product is used as a PCR template to amplify the complete scFv library, as described above. Reuse as a rate, and display the yeast surface in the scFv library at 5' and 3' (Y Using forward and reverse primers with added SD expression vector homologous sequences, yeast Homologous recombination within cells was efficiently performed.

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

[0143] The Fab display library was built in a similar way to the scFv library, but V L and VH were built separately and cloned under two promoters. VH was Gal-1 Under promoter 0, within the frame between the aga2 gene and the constant heavy chain domain 1 (CH1) It was cloned using the Gal-1 promoter. VL was converted to a constant light chain under the Gal-1 promoter. Cloned within a frame between the main (CL) and the Fab fragment using PCR. It binds to a single fragment and clones into the pFAB1 expression vector in a similar manner to the scFv library. It became a rut.

[0144] (Transfection of the library)

[0145] The library transfection (transformation) was performed using publicly available methods. (“Benatuilet al., An improved yeast transformation method for the generation n of very largehuman antibody libraries. Protein Eng. Des. Sel. 23, 155-159 (20 10) ). 400 μl of yeast suspension per 0.2 cm cuvette (cell projects) (EBY1 (00, ATCC, USA) is mixed with 4 μg of linearization vector (pCTcon3 or pFAB1) and 1 2 μg of DNA insert (scFv fragment or assembled Fab) in a 1:3 vector -: Used in insert ratio and electroporated (BioRad, USA, GenePulser) ("Chao, G." et al., Isolating and engineering human antibodies using yeast surface display. Nat. Protoc. 1,755-768 (2006). The average number of transformants in the library is the number of transformants. By serial dilution of cells, approximately 1 × 10⁻⁶ 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 established prototypes Following the instructions, expression was induced overnight at 30°C with 2% w / v galactose (" Chao, G. et al., (2006). To put it simply, the library uses 6xhis tags or These are pairs of TNFR2-Fc fusions (Reprokine, Israel) with a molecular weight of 1000 nM to 0.1 nM. Incubate the replacement human TNFR2 in PBS (0.1% BSA) for 1 hour, then proceed to... Then, wash three times with PBS (0.1% BSA), and then use mouse anti-c-MycFITC ( Miltenyi Biotec, cat #130-116-485), or mouse anti-c-Myc (Santa Cruz, USA) Labeled with cat# sc-40), and also labeled with goat anti-mouse IgG-FITC (Sigma-Aldrich, cat# F 4143-1ml), monoclonal anti-HisAPC (Miltenyi Biotec, Germany. cat 0020130- 119-782), or anti-FcAPC (Jackson Immuno Research, USA. cat. 109-135-098) It was fluorescently labeled.

[0149] If nonspecific binding needs to be avoided, use a fluorescently labeled antibody against rabbit anti-c-Myc(Abca m, cat# ab9106), goat anti-rabbit APC (Abcam, Cat# ab130805), anti-HisAlexa Replaced with 488 (Qiagen, cat# 20-35310).

[0150] After labeling, the library has a library size of 1 x 10 6 MACS Select on the range, then use the BioRad S3e fluorescence-activated cell sorter, or BD A On RIA III fluorescence-activated cell sorter (FACS), recombinant human TNFR2 The high-affinity binders were sorted. The sequences of the clones separated from the final sort were then determined. Using the Zymoprep kit (Zymo Research, USA), plus This was done by extracting mid-DNA, which was then used to determine the DNA sequence.

[0151] When applied, yeast display Fab is used for detection of Fab light chain displays. - Selective selection of scFv with the addition of FLAG-PE (Miltenyi Biotec, cat #130-101-576) The labels and selections were made under the same conditions.

[0152] (Koff's choice)

[0153] To select a binder with improved off-rate, use yeast in a TN blender of 10nM to 1nM. The yeast was incubated with FR2-His for 30 minutes. Then, the yeast was added to 1 ml of PBS (0 Wash three times with 0.1% BSA, then sterilize with 100nM TNFR2-Fc for 4 hours, 6 hours, and 2 hours. Incubate for 4 hours. Alternatively, after washing, soak the cells in PBS (0.1% BSA) for 10 minutes. Diluted twice and incubated until the specified time. Next, the yeast was washed three times and treated with anti-Myc. c-FITC (SantaCruze, USA, Cat# 9E10), and monoclonal anti-HisAPC Labeled as (MiltenyiBiotec, Germany. cat 0020130-119-782), and as shown above, Se3 Sorted using FACS.

[0154] Furthermore, at the optimal point when approximately 50% to 70% of the initial bonds have been lost, two Koff selections are performed. went.

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

[0156] 50% effective concentration (EC5) of TNFR2 binding to yeast scFv or Fab clones (EC) in order to determine 0) 50 -TNFR2 binding), specific clones 0.1nM~1 Samples were labeled with TNFR concentrations in the range of 000 nM and analyzed by FACS. Median fluorescence intensity (MF) I) Measure EC for each TNFR2 concentration, 50 Prisma8GraphPad( The calculations were performed using GraphPad (San Diego, USA) software.

[0157] IgG production

[0158] (Reformat)

[0159] The selected scFv clone was reformatted to human IgG1 format. Light chain The sequences of the (LC) variable region and the heavy chain (HC) variable region are optimized for the use of mammalian codons. IDT (IntegratedDNA Technologies, Coralville, Iowa USA) has released Genblock. We ordered it as GB. Genbrok (GB) was created using standard cloning techniques. And, pSF-CMV-HuIgG1_HC (HC plasmid), and pSF-CMV- HuLambda_LC (LC plasmid) (Oxford genetics, Oxford UK) If instructed, pS with L234A / L235A(LALA) mutations F-CMV-HuIgG1_HC_LALA (HC plasmid) was used.

[0160] (IgG expression)

[0161] Expi-CHO cells (Thermo Fisher Scientific, USA) were subjected to LC plasmid and H Transfect the C plasmid in a 2:1 ratio and express it according to the manufacturer's instructions. To put it simply, 50 ml of Expi-CHO cells were subjected to a procedure at 37°C and 120 rpm. , with 8% CO2, 6 × 10 6 The cells were incubated to a density of cells / ml. In CHO cells, 50 μg of heavy chain expression plasmid and light chain expression plasmid were administered in a 1:2 ratio. Then, transfection was performed. Booster and feed were added to the transfected culture. The following was added, and the growth conditions were changed to 32°C, 120 rpm, and 5% CO2. Cells were collected 10 days after the procedure. (protein A beads (Tosoh Bioscience GmbH,) IgG was purified from the supernatant using (Germany), and then PBS was used as the mobile phase (GEhealthcar e, USA) used on SUPERDEX200(TM) 10 / 300 augmented columns Size exclusion chromatography (SEC) purification was performed.

[0162] (Size exclusion chromatography)

[0163] To analyze and purify IgG, the sample is placed in a SUPERDEX200(TM)10 / On a 300-increase column (GEhealthcare, USA), GE AKTAE explorer cross The sample was loaded at a flow rate of 0.8 ml / min using a matrixing system (GE Healthcare, USA). Alternatively, if instructed, take up to 12 mg of IgG in BioResolve™ Waters ACQUITY arc with SECmAb column (Waters, USA) The sample was loaded on an HPLC at a flow rate of 0.5 ml / min for a run time of 20 minutes. PBS was used as the mobile phase. It functioned as such, and retention was monitored at 280 nM in both columns.

[0164] (Ligand-binding ELISA)

[0165] The IgG binding affinity to TNFR2 was investigated by ELISA. (96-well plate (Gr)) The analyzed antibody was coated (50 ng / well) on Bio-One highbinding. The plates were incubated overnight at 4°C. Then, the plates were treated with 0.05% Tween20. Wash three times with 300 μl of PBS buffer containing PBS-T, and then add 1% to 2% BSA. The sample was blocked with 300 μl of PBS-T and incubated at room temperature for 1 hour. Wash the antibody-coated plate three times with 300 μl of PBS-T, then add the test ligand. Combine with a sequential dilution of hTNFR2-His (Reprokine, Israel) in a final volume of 50 ml. Incubate for 1-2 hours. Then, wash the plate three times with 300 μl of PBS-T. 50 μl of anti-HIS-HRP (Santa Cruz Biotechnolo) diluted 1:250 with PBS. Incubated with gy, USA, SC-8036HRP) conjugate. Six washing steps. After further processing, add 50 ml of tetramethylbenzidine (TMB) reagent (Southern biote The reaction was carried out using (ch, USA) and stopped with 50 ml of 0.5 N H2SO4. Detection This is the Synergy LX BioTek (BioT) with the absorbance filter set to 450nM. (ek, USA) The procedure was performed using a plate reader. Binding affinity was determined using Prisma 8 Grap By fitting the data to a specific coupled nonlinear regression model on the hPad software... And so it was decided.

[0166] TNFR2 / TNFR1 specificity ELISA is used in EC 50 ELISA analysis (EC 50 -T The same method was used as for NFR2 binding, but both TNFR1 and TNFR2 were increased to 100-1 The test was performed at a concentration of 000 nM.

[0167] (FACS analysis)

[0168] LALA mutation Fc format anti-TNFR2 antibody (30.116) HEK-TN FR1 (InvivoGen, Cat: hkb-tnfdmyd) and HEK-TNFR2 (200nM Ab, Cell lines (1 million cells / well) were incubated on ice for 15 minutes. Then, production Following the manufacturer's instructions, cells were incubated with goat anti-human Fc-APC conjugate on ice for 3 minutes. Cells were stained for 0 minutes (Jackson Immune Research, cat. # 109-135-098). Analysis was performed using a FLEX flow cytometer (Beckman, USA) based on secondary controls only. The gate was determined individually for each cell population.

[0169] result

[0170] Library design

[0171] To produce antibodies that bind to TNFR2, the "re-epitope" method is applied to existing antibodies. The re-epitope method makes it possible to introduce new specificity into existing antibodies, and is preferable. Select a known antibody with biophysical and biochemical properties as a template. This makes it possible. Therefore, re-epitope antibodies offer new specificity and desirable development possibilities. It has both a profile and a re-epitope calculation process, (i) existing antibodies Using any computer analysis to identify the estimated complementarity between the new epitope and the new epitope (ii) a specific mutation that is predicted to enhance antibody binding to a new desired epitope. Two steps: one is to apply any computer analysis or tool that can be used to introduce a different approach. The following steps are required. An example of such computer analysis is found in U.S. Patent Application Publication No. 2. Issue 018 / 0068055, and "Nimrod et al., Cell Rep. 25(8):2121-2131 (2018 ) is described in one embodiment, to introduce a new specificity for TNFR2 The following are predicted to be promising candidates for the re-epitope template antibodies: 2I5Y, 4IOI, and 3 We designed three libraries using the variable domain array of E8U. Some of the rate sequences are designed with future generations of libraries in mind, considering improved developability and humanization. It has been further modified. In this way, a library of re-epitopes is constructed and applied to yeast. It was implemented.

[0172] YSD screening for TNFR2 binders

[0173] After introducing the mutation, in order to identify clones that specifically bind to TNFR2, live Larri were screened using the Yeast Surface Display (YSD) format. First, the library was... First, MACS (magnetic beads) were used for selection, and then FCAS was used for selection. Three re-epistols All clones from the clone template showed relative bonding.

[0174] To further enhance affinity, affinity maturation libraries are created using a similar method to the library construction described above. A library was built. The affinity-mature library was configured as described above, using the standard and specific Koff libraries. The best binder was used for selection. The best binder was gated, and yeast clones were isolated and sequenced. These clones were determined. They 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] EC2 for TNFR2 in yeast surface displays 50 Join value

[0184] TNFR2-binding clones have at least one to two orders of magnitude nanomolar affinity to TNFR2. To confirm the sex, a limited set of clones were diatomized on the yeast surface as described above. Spray (YSD) EC 50 The assay was performed. As shown in Figures 1A to 1D, Our clones showed affinity for TNFR2-His in the range of 1 nM to 20 nM. This indicates that the scFv and Fab of yeast displays robust binding of human TNFR2. This indicates that these clones are YSD-EC 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 the clone that showed binding with TNFR2, scFv and The clones shown in Tables 1 and 2 that showed the most promising binding in Fab were re-treated with human IgG1. Format the cells and temporarily express them in expi-CHO cells according to the manufacturer's instructions. Purified as described herein. In addition, clones 30.032 and 30.04 6. Synthesize 30.033 DNA and use Genscript Antibody Service (Genscript, USA) IgG was produced in HEK cells using the following method.

[0189] Clones 30.113, 30.114, 30.116, 30.117, 30.118, 30.119 is a heavy chain L designed to reduce IgG binding to Fc and Fc-γ receptors. IgG1 was reformatted to have the 234A / L235A mutation (LALA mutation). Furthermore, additional IgG1 clones containing heavy chain L234A / 235A mutations (LALA mutations) were added. We created an IgG1 anti-TNFR2 antibody clone containing the heavy chain L234A / L235A mutation. The list includes BDG30.113, BDG30.114, BDG30.115, BD G30.116, BDG30.117, BDG30.118, BDG30.119, BD G30.122, BDG30.123, BDG30.200, BDG30.201, BD Examples include G30.202, BDG30.203, and BDG30.204.

[0190] Table 3 shows an overview of the scFv and Fab files reformatted to IgG format. If present, the heavy chain contains the LALA mutation.

[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 is: An example of a set of three CDRs on a heavy chain (HCDR1, HCDR2, and HCDR3) is shown. Table 8 shows the set of three CDRs on the corresponding light chain (LCDR1, LCDR2, and LCDR 3) is shown. Table 9 shows the HCDR1 and HCDR2 anti-TNFR2 antibodies disclosed herein. And the set of HCDR3 and the set of LCDR1, LCDR2 and LCDR3 Here's another example.

[0197] To test the aggregation tendency of these antibodies, IgG was used as the mobile phase with PBS, as described above. For example, Akta, SUPERDEX10 / 300 augmented column, or Waters A Using CQUITY arc HPLC and BioResolve SEC mAb columns Size exclusion chromatography (SEC) was then performed. Example of SEC analysis of selected antibodies. Figures 2A to 2R show the results, and Table 4 outlines the complete SEC analysis of the clones. The results were IgG 30.086-30.113, 30.115, 30.117-119, 3 0.122-30.123, 30.202, and 30.204 are mostly non-aggregated species. It then elutes from protein A and migrates to the SEC column as generally folded IgG1. This indicates that.

[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, the antibody described herein is... The sea urchin was analyzed by ELISA for TNFR1 and TNFR2. Brief explanation: Then, the antibody is directly coated onto the ELISA plate wells, blocking the wells. Add 100 nM of TNFR1-His-Fc or TNFR2-His-Fc to the wells. The samples were washed and detected using anti-His-HRP. As shown in Figures 3A and 3B, Ig G 30.092, 30.093, 30.094, 30.095, 30.085, 30. 089, 30.086, 30.116, 30.118, 30.119, 30.111, 3 0.113 and 30.114 specifically bound to TNFR2, but not to TNFR1. I didn't.

[0202] IgG binding affinity to TNFR2

[0203] (IgG ELISA EC 50 )

[0204] TNFR2 of clones reformatted as IgG1 and IgG1LALA To test the binding affinity, the ELISA EC was performed for each antibody. 50 Conduct a bonding experiment. As shown in Figures 4A to 4F, these antibodies were EC13-66 nM. 50 They bind to soluble TNFR2-His within a certain range, which means these antibodies have a strong binder. This indicates that... Table 5 shows the EC2 of TNFR2 for the antibodies shown. 50 This shows the value. The 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] To investigate whether antibodies functionally affect TNFR2 cell signaling Furthermore, the antibody exhibits agonist, antagonist, or no effect on TNFR2. To investigate whether or not this was observed, a reporter cell line was created.

[0209] HEK-Blue(TM) Null cells, InvivoGen (Toulouse France) These cells were purchased from [source]. These cells have a TNFR1 null mutation and five NF-κB and AP-1 mutations. Soluble embryonic alkaline phosphine under the control of the IFN-β minimal promoter fused to the binding site. Contains a plasmid encoding an atease (SEAP). Human TN under CMV promoter. The pcDNA3.1 plasmid encoding FR2 (residues 1-461) is processed by HEK-Blu The cells were introduced into e(TM)Null cells. The cells were selected for 50 μg / ml hygromycin B. The samples were stored under selection conditions for 14 days. After the selection period, TNFR was determined by Western blot analysis. Expression of gene 2 was confirmed (Figure 5).

[0210] Then, the cells were diluted to a critical dilution of 0.5 cells / well in a 96-well plate. In addition, 10% FBS, L-glutamine, Pen / Strep, and 50 μg / ml Hygro The TNFα-reactive clones were grown in a growth medium containing DMEM supplemented with mycin B. To identify the plate, a replica plate was made as a single clone, following the manufacturer's instructions. Using the Quanti-Blue (QB, InvivoGen) substrate, TNFα-dependent NFκB Activation was tested (Figures 6A-6C).

[0211] Figures 7A and 7B show specific crows in a dynamic range of 20 pM to 1000 pM. The results of the TNFα-dependent NFκB reaction of are shown. Activation is performed by soluble TNFR2. It was inhibited in a dose-dependent manner (Figure 7B), but was not affected by the isotype control antibody. (Figure 7A). Clone G6 agonizes or antagonistizes the TNFR2 receptor. It was selected to evaluate the target antibody.

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

[0213] To test the effect of anti-TNFR2 antibodies on TNFR2 cell signaling, I gG was incubated overnight with the HEK-TNFR2 reporter cell line at a maximum concentration of 600 nM. We then tested antibody-dependent NFκB activation as detailed herein (Figure 8A). (Figure 8D).

[0214] The IgG values ​​shown in Table 6, with the exception of 30.088 and 30.117, exhibit dose-dependent NFκ B activation is observed, and EC exhibits functional agonism. 50 The value is in the range of 0.5 nM to 25.7 nM. Therefore, this means that these antibodies are high-affinity functional agonists and add other molecules This demonstrates that receptors can be agonized without any intervention.

[0215] NFκB activation depends on direct activation of the TNFR2 receptor by antibodies, To verify that the NFR1 alternative pathway is not activated, HEK-Blue-TNF Using α cells (InvivoGen, Cat: hkb-tnfdmyd), antibodies 30.113 and 30.114 were used. , 30.115, 30.116, 30.117, 30.118, 30.119, 30.1 23, 30.200, 30.201, 30.202, 30.203, and 30.204 The test showed that HEK-Blue-TNFa cells do not express TNFR2, but do express TNFR1. Spontaneously expressed, IFN-β minimal particles fused to five NF-κB and AP-1 binding sites. Under the control of romorta, a plastic that encodes soluble embryonic alkaline phosphatase (SEAP) Contains sumidol.

[0216] As shown in Figure 9A, HEK-Blue-TNFa cells have a concentration of 30.116 at 200 nM. As revealed by FACS analysis of these cells labeled with anti-TNFR2Ab TNFR2 is not expressed. On the other hand, NFκB is readily expressed by the addition of 11 nM TNFα. Activation occurs (Figure 9B), indicating that NFκB activation is mediated by the TNFR1 pathway. Inducing... In the HEK-TNFR2 cell line, the shown antibody was shown to activate NFκB. However, 1 mM antibodies 30.113, 30.114, 30.115, 30.116, 3 0.117, 10.118, 30.119, 30.123, 30.200, 30.201 Adding 30.202, 30.203, or 30.204 will not affect HEK-TNFR1 NFκB was not activated in the cell line. This suggests that the activation is specific to TNFR2. This suggests a certain possibility (Figures 9B-9C).

[0217] TNFR2 activation is independent of IgG-Fc clustering. This indicates that H The EK293 reporter cell line does not express the Fcγ receptor, and in the above experiment... This is further supported by the fact that the IgG is added to the cells in a soluble form and does not bind to the plate. Furthermore, LALA is designed to reduce IgG binding to the Fc-γ receptor. IgG with mutations at 30.113–30.204 was expressed. Combining these data... This suggests that TNFR2 activation is independent of IgG-Fc clustering. Table 6 shows the functional agonist properties of TNFR2 in the HEK293-NFκB reporter cell line. Zoom's EC 50 The values ​​are summarized below. These values ​​are the mean values ​​from at least two biological replicates. That is the case.

[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-TNFR Two cells were incubated with a 200 nM anti-TNF2 antibody, and TNFα was added at a 100 nM level. The substance was added up to the specified concentration. As shown in Figures 10A to 10D, TNFα was 30.116, 30. Most of the maximum activation induced by antibodies 111, 30.086, and 30.119 It had no effect. This suggests that these antibodies blocked the TNFα binding site. This study demonstrates that the absence of an epitope activates the TNFR2 receptor to nearly its maximum capacity.

[0222] In summary, the antibodies of this disclosure are in the sub-nanomole to low two-order-of-eight nanomolar range. It binds strongly to TNFR2, is specific to TNFR2, and is Fc-independent. It was shown to substantially agonize TNFR2.

[0223] In this specification, specific characteristics of the anti-TNFR2 antibody according to the present invention have been illustrated and described. Various modifications, substitutions, alterations, and equivalents may be conceivable to those skilled in the art. However, Therefore, the attached claims include all such modifications within the scope of the gist of the present invention. Please understand that this is intended to include changes and modifications.

[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

[Claim 1] The invention described in the specification.

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