BTLA-conjugated (CD272) antibodies for modulating immune responses and treating diseases

Abstract

The present invention provides novel anti-BTLA antibodies, pharmaceutical compositions containing such antibodies, and therapeutic methods using such antibodies and pharmaceutical compositions for the treatment of diseases such as cancer or autoimmune diseases. [Solution] The present invention provides an antibody or antigen-binding fragment thereof that binds to BTLA in both humans and cynomolgus monkeys, wherein the antibody or antigen-binding fragment comprises vhCDR1, vhCDR2, vhCDR3, vlCDR1, vlCDR2, and vlCDR3, each containing a specific amino acid sequence.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority to U.S. Provisional Patent Application No. 62 / 827,744, filed on April 1, 2019, which is incorporated in its entirety by reference herein.

[0002] This disclosure relates to novel anti-BTLA antibodies, as well as pharmaceutical compositions comprising such antibodies for use in modulating immune responses and for the treatment of diseases such as cancer or autoimmune diseases. [Background technology]

[0003] Disease treatments that induce, enhance, or suppress the immune response are called immunotherapy. Immunotherapy has been demonstrated to increase efficacy in the treatment of cancer. Generally, the tumor microenvironment allows cancer cells to evade detection by the immune system through upregulation of immunosuppressive proteins (e.g., PDL1, HVEM) expressed by the tumor cells themselves. Leukocytes express specific receptors for these inhibitory proteins (e.g., PD1 for PDL1 and BTLA for HVEM) (also known as immune checkpoint receptors), and tumor cells can bind these immune checkpoint receptors to suppress the immune response against themselves.

[0004] Many successful immunotherapies in cancer treatment are based on the use of immunomodulatory antibodies targeting immune checkpoints CTLA-4 and PD-1 / PD-L1. However, many FDA-approved antibodies that block immune checkpoint pairs are effective in only about 20% of cancer patients. The treatment is highly effective and durable in responders. However, for the remaining 80% of cancer patients who are non-responders, new immunotherapies targeting other immune checkpoints are needed.

[0005] BTLA (B and T lymphocyte attenuator) is a transmembrane protein whose expression is induced during T cell activation. BTLA is also expressed in B cells and dendritic cells. Similar to PD1 and CTLA4, BTLA interacts with B7H4, a B7 homolog. However, unlike PD-1 and CTLA-4, BTLA also interacts with tumor necrosis family receptors. One such receptor is tumor necrosis factor (receptor) superfamily member 14 (TNFRSF14), also known as herpesvirus entry mediator (HVEM). HVEM is a receptor for HSV glycoprotein D, which is involved in viral entry, and is expressed in hematopoietic cells, including B and T cells, as well as non-hematopoietic cells (parenchymal cells). In addition, HVEM is expressed in various blood and solid tumors. BTLA-HVEM interaction downregulates T cell responses, such as CD8+ T cell responses. Upregulation of BTLA and / or HVEM has been found to be a mechanism exerted by tumor cells to evade immune recognition and destruction. For example, BTLA and HVEM are highly expressed in B-cell chronic lymphocytic leukemia and gastric cancer; BTLA expression is also upregulated in cytotoxic CD8+ T cells in the peripheral blood of patients with Hodgkin lymphoma, B-cell non-Hodgkin lymphoma and several T-cell non-Hodgkin lymphomas, as well as hepatocellular carcinoma. In addition, the absence of HVEM-BTLA signaling leads to an excessive immune response that results in dysregulated inflammation and autoimmune diseases. Therefore, modulation of BTLA signaling can be a target for both cancer and autoimmune diseases. [Overview of the project]

[0006] In one embodiment, the present invention relates to a novel anti-BTLA antibody. In some embodiments, the anti-BTLA antibody includes a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 1 and a light chain variable region containing the amino acid sequence of SEQ ID NO: 2. In some embodiments, the anti-BTLA antibody includes a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 3 and a light chain variable region containing the amino acid sequence of SEQ ID NO: 4. In some embodiments, the anti-BTLA antibody includes a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 5 and a light chain variable region containing the amino acid sequence of SEQ ID NO: 6. In some embodiments, the anti-BTLA antibody includes a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 7 and a light chain variable region containing the amino acid sequence of SEQ ID NO: 8. In some embodiments, the anti-BTLA antibody includes a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 9 and a light chain variable region containing the amino acid sequence of SEQ ID NO: 10. In some embodiments, the anti-BTLA antibody includes a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 11 and a light chain variable region containing the amino acid sequence of SEQ ID NO: 12. In some embodiments, the anti-BTLA antibody includes a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 13 and a light chain variable region containing the amino acid sequence of SEQ ID NO: 14. In some embodiments, the anti-BTLA antibody includes a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 15 and a light chain variable region containing the amino acid sequence of SEQ ID NO: 16. In some embodiments, the anti-BTLA antibody includes a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 17 and a light chain variable region containing the amino acid sequence of SEQ ID NO: 18. In some embodiments, the anti-BTLA antibody includes a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 19 and a light chain variable region containing the amino acid sequence of SEQ ID NO: 20. In some embodiments, the anti-BTLA antibody includes a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 21 and a light chain variable region containing the amino acid sequence of SEQ ID NO: 22. In some embodiments, the anti-BTLA antibody includes a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 23 and a light chain variable region containing the amino acid sequence of SEQ ID NO: 24. In some embodiments, the anti-BTLA antibody includes a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 25 and a light chain variable region containing the amino acid sequence of SEQ ID NO: 26.In some embodiments, the anti-BTLA antibody includes a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 27 and a light chain variable region containing the amino acid sequence of SEQ ID NO: 28. In some embodiments, the anti-BTLA antibody includes a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 29 and a light chain variable region containing the amino acid sequence of SEQ ID NO: 30. In some embodiments, the anti-BTLA antibody includes a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 31 and a light chain variable region containing the amino acid sequence of SEQ ID NO: 32. In some embodiments, the anti-BTLA antibody includes a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 33 and a light chain variable region containing the amino acid sequence of SEQ ID NO: 34. In some embodiments, the anti-BTLA antibody includes a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 35 and a light chain variable region containing the amino acid sequence of SEQ ID NO: 36. In some embodiments, the anti-BTLA antibody includes a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 37 and a light chain variable region containing the amino acid sequence of SEQ ID NO: 38. In some embodiments, the anti-BTLA antibody includes a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 39 and a light chain variable region containing the amino acid sequence of SEQ ID NO: 40. In some embodiments, the anti-BTLA antibody includes a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 41 and a light chain variable region containing the amino acid sequence of SEQ ID NO: 42.

[0007] In some embodiments, the anti-BTLA antibody includes vhCDR1 containing SEQ ID NO: 47, vhCDR2 containing SEQ ID NO: 48, vhCDR3 containing SEQ ID NO: 49, vlCDR1 containing SEQ ID NO: 50, vlCDR2 containing SEQ ID NO: 51, and vlCDR3 containing SEQ ID NO: 52. In some embodiments, the anti-BTLA antibody includes vhCDR1 containing SEQ ID NO: 53, vhCDR2 containing SEQ ID NO: 54, vhCDR3 containing SEQ ID NO: 55, vlCDR1 containing SEQ ID NO: 56, vlCDR2 containing SEQ ID NO: 57, and vlCDR3 containing SEQ ID NO: 58. In some embodiments, the anti-BTLA antibody includes vhCDR1 containing SEQ ID NO: 59, vhCDR2 containing SEQ ID NO: 60, vhCDR3 containing SEQ ID NO: 61, vlCDR1 containing SEQ ID NO: 62, vlCDR2 containing SEQ ID NO: 63, and vlCDR3 containing SEQ ID NO: 64. In some embodiments, the anti-BTLA antibody includes vhCDR1 containing SEQ ID NO: 65, vhCDR2 containing SEQ ID NO: 66, vhCDR3 containing SEQ ID NO: 67, vlCDR1 containing SEQ ID NO: 68, vlCDR2 containing SEQ ID NO: 69, and vlCDR3 containing SEQ ID NO: 70. In some embodiments, the anti-BTLA antibody includes vhCDR1 containing SEQ ID NO: 71, vhCDR2 containing SEQ ID NO: 72, vhCDR3 containing SEQ ID NO: 73, vlCDR1 containing SEQ ID NO: 74, vlCDR2 containing SEQ ID NO: 75, and vlCDR3 containing SEQ ID NO: 76. In some embodiments, the anti-BTLA antibody includes vhCDR1 containing SEQ ID NO: 77, vhCDR2 containing SEQ ID NO: 78, vhCDR3 containing SEQ ID NO: 79, vlCDR1 containing SEQ ID NO: 80, vlCDR2 containing SEQ ID NO: 81, and vlCDR3 containing SEQ ID NO: 82. In some embodiments, the anti-BTLA antibody includes vhCDR1 containing SEQ ID NO: 83, vhCDR2 containing SEQ ID NO: 84, vhCDR3 containing SEQ ID NO: 85, vlCDR1 containing SEQ ID NO: 86, vlCDR2 containing SEQ ID NO: 87, and vlCDR3 containing SEQ ID NO: 88. In some embodiments, the anti-BTLA antibody includes vhCDR1 containing SEQ ID NO: 89, vhCDR2 containing SEQ ID NO: 90, vhCDR3 containing SEQ ID NO: 91, vlCDR1 containing SEQ ID NO: 92, vlCDR2 containing SEQ ID NO: 93, and vlCDR3 containing SEQ ID NO: 94.In some embodiments, the anti-BTLA antibody includes vhCDR1 containing SEQ ID NO: 95, vhCDR2 containing SEQ ID NO: 96, vhCDR3 containing SEQ ID NO: 97, vlCDR1 containing SEQ ID NO: 98, vlCDR2 containing SEQ ID NO: 99, and vlCDR3 containing SEQ ID NO: 100. In some embodiments, the anti-BTLA antibody includes vhCDR1 containing SEQ ID NO: 101, vhCDR2 containing SEQ ID NO: 102, vhCDR3 containing SEQ ID NO: 103, vlCDR1 containing SEQ ID NO: 104, vlCDR2 containing SEQ ID NO: 105, and vlCDR3 containing SEQ ID NO: 106. In some embodiments, the anti-BTLA antibody includes vhCDR1 containing SEQ ID NO: 107, vhCDR2 containing SEQ ID NO: 108, vhCDR3 containing SEQ ID NO: 109, vlCDR1 containing SEQ ID NO: 110, vlCDR2 containing SEQ ID NO: 111, and vlCDR3 containing SEQ ID NO: 112. In some embodiments, the anti-BTLA antibody includes vhCDR1 containing SEQ ID NO: 113, vhCDR2 containing SEQ ID NO: 114, vhCDR3 containing SEQ ID NO: 115, vlCDR1 containing SEQ ID NO: 116, vlCDR2 containing SEQ ID NO: 117, and vlCDR3 containing SEQ ID NO: 118. In some embodiments, the anti-BTLA antibody includes vhCDR1 containing SEQ ID NO: 119, vhCDR2 containing SEQ ID NO: 120, vhCDR3 containing SEQ ID NO: 121, vlCDR1 containing SEQ ID NO: 122, vlCDR2 containing SEQ ID NO: 123, and vlCDR3 containing SEQ ID NO: 124. In some embodiments, the anti-BTLA antibody includes vhCDR1 containing SEQ ID NO: 125, vhCDR2 containing SEQ ID NO: 126, vhCDR3 containing SEQ ID NO: 127, vlCDR1 containing SEQ ID NO: 128, vlCDR2 containing SEQ ID NO: 129, and vlCDR3 containing SEQ ID NO: 130. In some embodiments, the anti-BTLA antibody includes vhCDR1 containing SEQ ID NO: 131, vhCDR2 containing SEQ ID NO: 132, vhCDR3 containing SEQ ID NO: 133, vlCDR1 containing SEQ ID NO: 134, vlCDR2 containing SEQ ID NO: 135, and vlCDR3 containing SEQ ID NO: 136.In some embodiments, the anti-BTLA antibody includes vhCDR1 containing SEQ ID NO: 137, vhCDR2 containing SEQ ID NO: 138, vhCDR3 containing SEQ ID NO: 139, vlCDR1 containing SEQ ID NO: 140, vlCDR2 containing SEQ ID NO: 141, and vlCDR3 containing SEQ ID NO: 142. In some embodiments, the anti-BTLA antibody includes vhCDR1 containing SEQ ID NO: 143, vhCDR2 containing SEQ ID NO: 144, vhCDR3 containing SEQ ID NO: 145, vlCDR1 containing SEQ ID NO: 146, vlCDR2 containing SEQ ID NO: 147, and vlCDR3 containing SEQ ID NO: 148. In some embodiments, the anti-BTLA antibody includes vhCDR1 containing SEQ ID NO: 149, vhCDR2 containing SEQ ID NO: 150, vhCDR3 containing SEQ ID NO: 151, vlCDR1 containing SEQ ID NO: 152, vlCDR2 containing SEQ ID NO: 153, and vlCDR3 containing SEQ ID NO: 154. In some embodiments, the anti-BTLA antibody includes vhCDR1 containing SEQ ID NO: 155, vhCDR2 containing SEQ ID NO: 156, vhCDR3 containing SEQ ID NO: 157, vlCDR1 containing SEQ ID NO: 158, vlCDR2 containing SEQ ID NO: 159, and vlCDR3 containing SEQ ID NO: 160. In some embodiments, the anti-BTLA antibody includes vhCDR1 containing SEQ ID NO: 161, vhCDR2 containing SEQ ID NO: 162, vhCDR3 containing SEQ ID NO: 163, vlCDR1 containing SEQ ID NO: 164, vlCDR2 containing SEQ ID NO: 165, and vlCDR3 containing SEQ ID NO: 166. In some embodiments, the anti-BTLA antibody includes vhCDR1 containing SEQ ID NO: 167, vhCDR2 containing SEQ ID NO: 168, vhCDR3 containing SEQ ID NO: 169, vlCDR1 containing SEQ ID NO: 170, vlCDR2 containing SEQ ID NO: 171, and vlCDR3 containing SEQ ID NO: 172.

[0008] In some embodiments, the anti-BTLA antibodies described herein bind to human and / or cynomolgus monkey BTLA.

[0009] In some embodiments, anti-BTLA antibodies act as BTLA antagonists, blocking BTLA binding to HVEM and HVEM-mediated repression of T cell function. Examples of such antibodies include the heavy chain variable region containing the amino acid sequence of SEQ ID NO: 21 and the light chain variable region containing the amino acid sequence of SEQ ID NO: 22; the heavy chain variable region containing the amino acid sequence of SEQ ID NO: 31 and the light chain variable region containing the amino acid sequence of SEQ ID NO: 32; the heavy chain variable region containing the amino acid sequence of SEQ ID NO: 35 and the light chain variable region containing the amino acid sequence of SEQ ID NO: 36; vhCDR1 containing SEQ ID NO: 107, vhCDR2 containing SEQ ID NO: 108, vhCDR3 containing SEQ ID NO: 109, vlCDR1 containing SEQ ID NO: 110, vlCDR2 containing SEQ ID NO: 111, and The antibodies include vlCDR3 containing SEQ ID NO: 112; vhCDR1 containing SEQ ID NO: 137, vhCDR2 containing SEQ ID NO: 138, vhCDR3 containing SEQ ID NO: 139, vlCDR1 containing SEQ ID NO: 140, vlCDR2 containing SEQ ID NO: 141, and vlCDR3 containing SEQ ID NO: 142; or vhCDR1 containing SEQ ID NO: 149, vhCDR2 containing SEQ ID NO: 150, vhCDR3 containing SEQ ID NO: 151, vlCDR1 containing SEQ ID NO: 152, vlCDR2 containing SEQ ID NO: 153, and vlCDR3 containing SEQ ID NO: 154.

[0010] In some embodiments, anti-BTLA antibodies act as BTLA agonists, suppressing pro-inflammatory immune cell function. Examples of such antibodies include a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 19 and a light chain variable region containing the amino acid sequence of SEQ ID NO: 20; a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 25 and a light chain variable region containing the amino acid sequence of SEQ ID NO: 26; a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 29 and a light chain variable region containing the amino acid sequence of SEQ ID NO: 30; vhCDR1 containing SEQ ID NO: 101, vhCDR2 containing SEQ ID NO: 102, vhCDR3 containing SEQ ID NO: 103, vlCDR1 containing SEQ ID NO: 104, vlCDR2 containing SEQ ID NO: 105, and The antibodies include vlCDR3 containing SEQ ID NO: 106; vhCDR1 containing SEQ ID NO: 119, vhCDR2 containing SEQ ID NO: 120, vhCDR3 containing SEQ ID NO: 121, vlCDR1 containing SEQ ID NO: 122, vlCDR2 containing SEQ ID NO: 123, and vlCDR3 containing SEQ ID NO: 124; or vhCDR1 containing SEQ ID NO: 131, vhCDR2 containing SEQ ID NO: 132, vhCDR3 containing SEQ ID NO: 133, vlCDR1 containing SEQ ID NO: 134, vlCDR2 containing SEQ ID NO: 135, and vlCDR3 containing SEQ ID NO: 136.

[0011] In some embodiments, the anti-BTLA antibodies described herein include a constant region having an amino acid sequence that is at least 90% identical to human IgG. In some embodiments, IgG is selected from IgG1, IgG2, IgG3, or IgG4. In some embodiments, IgG is IgG2.

[0012] In another embodiment, the present invention relates to a nucleic acid composition encoding any one of the anti-BTLA antibodies described herein. In some embodiments, the nucleic acid composition includes a first nucleic acid containing SEQ ID NO: 185 and a second nucleic acid containing SEQ ID NO: 186. In some embodiments, the nucleic acid composition includes a first nucleic acid containing SEQ ID NO: 187 and a second nucleic acid containing SEQ ID NO: 188. In some embodiments, the nucleic acid composition includes a first nucleic acid containing SEQ ID NO: 189 and a second nucleic acid containing SEQ ID NO: 190. In some embodiments, the nucleic acid composition includes a first nucleic acid containing SEQ ID NO: 191 and a second nucleic acid containing SEQ ID NO: 192. In some embodiments, the nucleic acid composition includes a first nucleic acid containing SEQ ID NO: 193 and a second nucleic acid containing SEQ ID NO: 194. In some embodiments, the nucleic acid composition includes a first nucleic acid containing SEQ ID NO: 195 and a second nucleic acid containing SEQ ID NO: 196. In some embodiments, the nucleic acid composition includes a first nucleic acid containing SEQ ID NO: 197 and a second nucleic acid containing SEQ ID NO: 198. In some embodiments, the nucleic acid composition includes a first nucleic acid containing SEQ ID NO: 199 and a second nucleic acid containing SEQ ID NO: 200. In some embodiments, the nucleic acid composition includes a first nucleic acid containing SEQ ID NO: 201 and a second nucleic acid containing SEQ ID NO: 202. In some embodiments, the nucleic acid composition includes a first nucleic acid containing SEQ ID NO: 203 and a second nucleic acid containing SEQ ID NO: 204. In some embodiments, the nucleic acid composition includes a first nucleic acid containing SEQ ID NO: 205 and a second nucleic acid containing SEQ ID NO: 206. In some embodiments, the nucleic acid composition includes a first nucleic acid containing SEQ ID NO: 207 and a second nucleic acid containing SEQ ID NO: 208. In some embodiments, the nucleic acid composition includes a first nucleic acid containing SEQ ID NO: 209 and a second nucleic acid containing SEQ ID NO: 210. In some embodiments, the nucleic acid composition includes a first nucleic acid containing SEQ ID NO: 211 and a second nucleic acid containing SEQ ID NO: 212. In some embodiments, the nucleic acid composition includes a first nucleic acid containing SEQ ID NO: 213 and a second nucleic acid containing SEQ ID NO: 214. In some embodiments, the nucleic acid composition includes a first nucleic acid containing SEQ ID NO: 215 and a second nucleic acid containing SEQ ID NO: 216.In some embodiments, the nucleic acid composition comprises a first nucleic acid comprising SEQ ID NO: 217 and a second nucleic acid comprising SEQ ID NO: 218. In some embodiments, the nucleic acid composition comprises a first nucleic acid comprising SEQ ID NO: 219 and a second nucleic acid comprising SEQ ID NO: 220. In some embodiments, the nucleic acid composition comprises a first nucleic acid comprising SEQ ID NO: 221 and a second nucleic acid comprising SEQ ID NO: 222. In some embodiments, the nucleic acid composition comprises a first nucleic acid comprising SEQ ID NO: 223 and a second nucleic acid comprising SEQ ID NO: 224. In some embodiments, the nucleic acid composition comprises a first nucleic acid comprising SEQ ID NO: 225 and a second nucleic acid comprising SEQ ID NO: 226.

[0013] Another aspect of the invention relates to an expression vector composition comprising any one of the nucleic acid compositions described herein. In some embodiments, the first nucleic acid is contained in a first expression vector and the second nucleic acid is contained in a second expression vector. In some other embodiments, the first nucleic acid and the second nucleic acid are contained in a single expression vector.

[0014] Another aspect of the invention relates to a host cell comprising any one of the expression vectors described herein. A method of making an anti-BTLA antibody is also provided, which method comprises culturing the host cell under conditions in which the antibody is expressed and recovering the antibody.

[0015] In another aspect, the invention relates to a composition comprising any one of the anti-BTLA antibodies described herein and a pharmaceutically acceptable carrier or diluent.

[0016] Methods of modulating an immune response in a subject, and methods comprising administering to a subject an effective amount of any one of the anti-BTLA antibodies described herein, or any one of the compositions described herein, are also described. In some embodiments, the method stimulates an immune response in the subject, and the method comprises administering to the subject an effective amount of an anti-BTLA antibody that acts as a BTLA antagonist, or a pharmaceutical composition thereof. In some embodiments, the method suppresses an immune response in the subject, and the method comprises administering to the subject an effective amount of an anti-BTLA antibody that acts as a BTLA agonist, or a pharmaceutical composition thereof.

[0017] In some embodiments, the method stimulates an immune response in the subject, and the method comprises administering to the subject an effective amount of an anti-BTLA antibody comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 21 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 22; and / or comprising vhCDR1 comprising SEQ ID NO: 107, vhCDR2 comprising SEQ ID NO: 108, vhCDR3 comprising SEQ ID NO: 109, vlCDR1 comprising SEQ ID NO: 110, vlCDR2 comprising SEQ ID NO: 111, and vlCDR3 comprising SEQ ID NO: 112.

[0018] In some embodiments, the method stimulates an immune response in the subject, and the method comprises administering to the subject an effective amount of an anti-BTLA antibody comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 31 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 32; and / or comprising vhCDR1 comprising SEQ ID NO: 137, vhCDR2 comprising SEQ ID NO: 138, vhCDR3 comprising SEQ ID NO: 139, vlCDR1 comprising SEQ ID NO: 140, vlCDR2 comprising SEQ ID NO: 141, and vlCDR3 comprising SEQ ID NO: 142.

[0019] In some embodiments, the method stimulates an immune response in a subject, and the method comprises administering an effective amount of anti-BTLA antibody to the subject, the anti-BTLA antibody comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 35 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 36; and / or vhCDR1 comprising SEQ ID NO: 149, vhCDR2 comprising SEQ ID NO: 150, vhCDR3 comprising SEQ ID NO: 151, vlCDR1 comprising SEQ ID NO: 152, vlCDR2 comprising SEQ ID NO: 153, and vlCDR3 comprising SEQ ID NO: 154.

[0020] In some embodiments, the method suppresses the immune response in a subject, and the method comprises administering an effective amount of anti-BTLA antibody to the subject, the anti-BTLA antibody comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 19 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 20; and / or vhCDR1 comprising SEQ ID NO: 101, vhCDR2 comprising SEQ ID NO: 102, vhCDR3 comprising SEQ ID NO: 103, vlCDR1 comprising SEQ ID NO: 104, vlCDR2 comprising SEQ ID NO: 105, and vlCDR3 comprising SEQ ID NO: 106.

[0021] In some embodiments, the method suppresses the immune response in a subject, and the method comprises administering an effective amount of anti-BTLA antibody to the subject, the anti-BTLA antibody comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 25 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 26; and / or vhCDR1 comprising SEQ ID NO: 119, vhCDR2 comprising SEQ ID NO: 120, vhCDR3 comprising SEQ ID NO: 121, vlCDR1 comprising SEQ ID NO: 122, vlCDR2 comprising SEQ ID NO: 123, and vlCDR3 comprising SEQ ID NO: 124.

[0022] In some embodiments, the method suppresses the immune response in a subject, and the method comprises administering an effective amount of anti-BTLA antibody to the subject, the anti-BTLA antibody comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 29 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 30; and / or vhCDR1 comprising SEQ ID NO: 131, vhCDR2 comprising SEQ ID NO: 132, vhCDR3 comprising SEQ ID NO: 133, vlCDR1 comprising SEQ ID NO: 134, vlCDR2 comprising SEQ ID NO: 135, and vlCDR3 comprising SEQ ID NO: 136.

[0023] In another embodiment, the present invention relates to a method for treating cancer in a subject, comprising administering to the subject an effective amount of an anti-BTLA antibody or composition thereof described herein, wherein the anti-BTLA antibody acts as a BTLA antagonist. In some embodiments, the treated cancer upregulates HVEM compared to the corresponding non-cancerous tissue. In some embodiments, the treated subject expresses high levels of BTLA on T cells. The treated cancer may be gastric cancer. In some embodiments, the anti-BTLA antibody is used in combination with one or more additional therapeutic agents that treat the cancer. In some embodiments, such anti-cancer therapeutic agents are other immune checkpoint inhibitors, such as ipilimumab, nivolumab, pembrolizumab, avelumab, durvalumab, and atezolizumab.

[0024] In another embodiment, the present invention relates to a method for treating an autoimmune disease in a subject, comprising administering to the subject an effective amount of an anti-BTLA antibody or a composition thereof as described herein, wherein the anti-BTLA antibody acts as a BTLA agonist. In some embodiments, the subject to be treated expresses low levels of HVEM in autoreactive T cells present at the site where the autoimmune disease occurs. In some embodiments, the autoimmune disease is multiple sclerosis.

[0025] The present invention can be fully understood from the following detailed description, which is to be read in conjunction with the accompanying drawings. The following figures are included in the drawings: [Brief explanation of the drawing]

[0026] [Figure 1] Figure 1 shows the binding of anti-BTLA antibody 13-F7A to human BTLA by ELISA. Anti-BTLA antibodies 4C7 and 8D5 were used as controls. Human IgG was used as a negative control. Relative light units (RLU) were calculated. [Figure 2A] Figures 2A-2C are histograms showing the binding of the anti-BTLA antibody 13-F7A to BTLA by FACS analysis. Figure 2A shows the binding of 13-F7A to human BTLA expressed in HEK-293 cells. Antibodies at concentrations of 10 μg / ml and 1 μg / ml were tested together with a control consisting of only the secondary antibody. [Figure 2B] Figures 2A-2C are histograms showing the binding of the anti-BTLA antibody 13-F7A to BTLA by FACS analysis. Figure 2B shows the binding of 13-F7A to cynomolgus monkey BTLA expressed in HEK-293 cells. Antibodies at concentrations of 10 μg / ml and 1 μg / ml were tested together with a control consisting of only the secondary antibody. [Figure 2C] Figures 2A-2C are histograms showing the binding of the anti-BTLA antibody 13-F7A to BTLA by FACS analysis. Figure 2C shows the binding of 13-F7A to HEK-293 cells as a negative control. Antibodies at concentrations of 10 μg / ml and 1 μg / ml were tested together with a control consisting of only the secondary antibody. [Figure 3] Figure 3 shows that the anti-BTLA antibody 13-F7A inhibits the human BTLA / HVEM interaction as determined by ELISA. Anti-BTLA antibodies 4C7 and 8D5, as well as human IgG, were used as controls. [Figure 4] Figure 4 shows the binding affinity of the anti-BTLA antibody 13-F7A to human BTLA as measured by bio-layer interferometry. 13-F7A was immobilized on a biosensor chip and recombinant human BTLA of 166.7, 55.6, 18.5, and 6.17 nM was tested. The measured dissociation constant KD was 5.4 x 10⁻⁹ M. [Figure 5A]Figures 5A–5F show dose-response curves of SEB-stimulated IL-2 secretion by primary T cells in the presence of anti-BTLA antibodies. Human IgG2 and IgG4 were used as controls. Figures 5A–5C show the anti-BTLA antibodies 16-I20A, 15-C19A, and 16-H16A, as well as their antagonist effects on T cell function. [Figure 5B] Figures 5A–5F show dose-response curves of SEB-stimulated IL-2 secretion by primary T cells in the presence of anti-BTLA antibodies. Human IgG2 and IgG4 were used as controls. Figures 5A–5C show the anti-BTLA antibodies 16-I20A, 15-C19A, and 16-H16A, as well as their antagonist effects on T cell function. [Figure 5C] Figures 5A–5F show dose-response curves of SEB-stimulated IL-2 secretion by primary T cells in the presence of anti-BTLA antibodies. Human IgG2 and IgG4 were used as controls. Figures 5A–5C show the anti-BTLA antibodies 16-I20A, 15-C19A, and 16-H16A, as well as their antagonist effects on T cell function. [Figure 5D] Figures 5A–5F show dose-response curves of SEB-stimulated IL-2 secretion by primary T cells in the presence of anti-BTLA antibodies. Human IgG2 and IgG4 were used as controls. Figures 5D–5F show the anti-BTLA antibodies 12-I8A, 8-M23A, and 13-F7A, as well as their agonist effects on T cell function. [Figure 5E] Figures 5A–5F show dose-response curves of SEB-stimulated IL-2 secretion by primary T cells in the presence of anti-BTLA antibodies. Human IgG2 and IgG4 were used as controls. Figures 5D–5F show the anti-BTLA antibodies 12-I8A, 8-M23A, and 13-F7A, as well as their agonist effects on T cell function. [Figure 5F] Figures 5A–5F show dose-response curves of SEB-stimulated IL-2 secretion by primary T cells in the presence of anti-BTLA antibodies. Human IgG2 and IgG4 were used as controls. Figures 5D–5F show the anti-BTLA antibodies 12-I8A, 8-M23A, and 13-F7A, as well as their agonist effects on T cell function. [Modes for carrying out the invention]

[0027] This disclosure provides novel anti-BTLA antibodies. The anti-BTLA antibodies described herein bind to human and / or cynomolgus monkey BTLA. In some embodiments, the anti-BTLA antibodies bind to human and / or cynomolgus monkey BTLA with high affinity. In some embodiments, the anti-BTLA antibodies act as functional BTLA antagonists, and upon binding to BTLA, they block the interaction between BTLA and HVEM, thereby blocking HVEM-mediated repression of T cell function. In some embodiments, the anti-BTLA antibodies act as functional BTLA agonists, and upon binding to BTLA, they suppress T cell function. Methods of using antibodies to modulate the immune response in a subject, for example, to treat cancer or autoimmune diseases, are also provided in this disclosure. In addition, nucleic acids encoding these antibodies and host cells containing such nucleic acids are described in this disclosure.

[0028] To facilitate understanding of the present invention, several terms and phrases are defined below.

[0029] Each of the following terms used herein has the meaning associated with it in this section.

[0030] The articles "a" and "an" as used herein refer to one or more (i.e., at least one) grammatical objective cases of the article. For example, "an element" means one or more elements.

[0031] For example, when referring to measurable values ​​such as quantity or duration, "about" as used herein means encompassing variations of ±20% or ±10%, more preferably ±5%, even more preferably ±1%, and even more preferably ±0.1% from the specified value, as appropriate for carrying out the method in which such variations are disclosed.

[0032] In this specification, “ablation” means a reduction or removal of activity. Therefore, for example, “ablation of FcγR binding” means that the Fc region amino acid variant has less than 50% initiation binding compared to an Fc region that does not contain the particular variant, preferably less than 70-80-90-95-98% loss of activity, and generally means that the activity is below the level of binding detectable in the Biacore assay.

[0033] As used herein, “ADCC” or “antibody-dependent cell-mediated cytotoxicity” refers to a cell-mediated reaction in which nonspecific cytotoxic cells expressing FcγR recognize a bound antibody in target cells, subsequently causing lysis of the target cells. ADCC correlates with binding to FcγRIIIa; increased binding to FcγRIIIa leads to increased ADCC activity. As discussed herein, many embodiments of the invention ablate ADCC activity overall.

[0034] As used herein, "ADCP" or "antibody-dependent cell-mediated phagocytosis" refers to a cell-mediated response in which nonspecific cytotoxic cells expressing FcγR recognize a bound antibody in a target cell, and subsequently trigger phagocytosis in the target cell.

[0035] In this specification, “antigen-binding domain” or “ABD” means a set of six complementarity-determining regions (CDRs) that, when present as part of a polypeptide sequence, specifically bind to a target antigen as discussed herein. Thus, the “antigen-binding domain” binds to the target antigen outlined herein. As is known in the art, these CDRs generally exist as a first set of variable heavy-chain CDRs (vhCDR or VHCDR or CDR-HC) and a second set of variable light-chain CDRs (vlCDR or VLCDR or CDR-LC), each containing three CDRs: vhCDR1, vhCDR2, vhCDR3 with respect to the heavy chain and vlCDR1, vlCDR2, and vlCDR3 with respect to the light chain. The CDRs are present in the variable heavy-chain and variable light-chain domains, respectively, and together form the Fv region. Thus, in some cases, the six CDRs of the antigen-binding domain are contributed by the variable heavy-chain and variable light-chain. In the "Fab" format, the set of six CDRs is contributed by two different polypeptide sequences, a variable heavy chain domain (vh or VH; containing vhCDR1, vhCDR2, and vhCDR3) and a variable light chain domain (vl or VL; containing vlCDR1, vlCDR2, and vlCDR3), where the C-terminus of the vh domain binds to the N-terminus of the CH1 domain of the heavy chain, and the C-terminus of the vl domain binds to the N-terminus of the constant light chain domain (thus forming a light chain). In the scFv format, the VH and VL domains are covalently bonded to a single polypeptide sequence by the use of a linker, generally outlined herein, which may be either vh-linker-vl or vl-linker-vh (starting from the N-terminus), with the former generally preferred (a domain linker may be included on each side at will, depending on the format used). As understood in the art, CDRs are separated by framework regions in the variable heavy chain and variable light chain domains, respectively: with respect to the light chain variable region, these are FR1-vlCDR1-FR2-vlCDR2-FR3-vlCDR3-FR4, and with respect to the heavy chain variable region, these are FR1-vhCDR1-FR2-vhCDR2-FR3-vhCDR3-FR4, and the framework regions exhibit high identity with respect to human germline sequences.The antigen-binding domains of the present invention include Fab, Fv, and scFv.

[0036] In this specification, “linker” means a linker used in scFv and / or other antibody structures. In general, several suitable scFv linkers exist that can be used, including traditional peptide bonds produced by recombinant technology. Linker peptides may primarily contain the following amino acid residues: Gly, Ser, Ala, or Thr. The linker peptide should have a length appropriate for linking two molecules so that they assume precise conformations to each other and they retain the desired activity. In one embodiment, the linker is about 1 to 50 amino acid long, preferably about 1 to 30 amino acid long. In one embodiment, linkers of 1 to 20 amino acid long may be used, and about 5 to about 10 amino acids find use in some embodiments. Useful linkers include glycine-serine polymers [wherein n is at least one integer (generally 3 to 4)], glycine-alanine polymers, alanine-serine polymers, and other mobile linkers, including, for example, (GS)n, (GSGGS)n, (GGGGS)n, and (GGGS)n. Alternatively, various non-proteinaceous polymers, including but not limited to polyethylene glycol (PEG), polypropylene glycol, polyoxyalkylene, or copolymers of polyethylene glycol and polypropylene glycol, may be found to be used as linkers. Other linker sequences may include CL / CH1 domains of any length from any sequence (but not all residues of the CL / CH1 domain); for example, the first 5-12 amino acid residues of a CL / CH1 domain may be included. Linkers may be derived from immunoglobulin light chains, e.g., Cκ or Cλ. Linkers may be derived from any isotype of immunoglobulin heavy chains, including, for example, Cγ1, Cγ2, Cγ3, Cγ4, Cα1, Cα2, Cδ, Cε, and Cμ. Linker sequences may also be derived from other proteins, e.g., Ig-like proteins (e.g., TCR, FcR, KIR), hinge region-derived sequences, and other native sequences of other proteins. In some embodiments, the linker is a “domain linker” used to link any two domains outlined together herein.Any suitable linker can be used, but many embodiments utilize glycine-serine polymers including, for example, (GS)n, (GSGGS)n, (GGGGS)n, and (GGGS)n [wherein n is at least one integer (generally 3 to 4 to 5)] as well as any peptide sequence that is of sufficient length and mobility, allows each domain to retain its biological function, and enables recombination of two domains.

[0037] The term “antibody” is used in its broadest sense and includes, for example, intact immunoglobulins or antigen-binding moieties. Antigen-binding moieties can be produced by recombinant DNA technology or by enzymatic or chemical cleavage of intact antibodies. Thus, the term antibody includes traditional tetrameric antibodies with two heavy chains and two light chains, as well as antigen-binding fragments, such as Fv, Fab, and scFv. In some cases, the present invention provides bispecific antibodies comprising at least one antigen-binding domain outlined herein.

[0038] In this specification, “modification” means an amino acid substitution, insertion, and / or deletion in a polypeptide sequence or an alteration of a portion chemically linked to a protein. For example, a modification may be a modified carbohydrate or PEG structure linked to a protein. In this specification, “amino acid modification” means an amino acid substitution, insertion, and / or deletion in a polypeptide sequence. For clarity, unless otherwise noted, amino acid modifications always refer to amino acids encoded in or by DNA (e.g., 20 amino acids with codons in DNA and RNA).

[0039] In this specification, “amino acid substitution” or “substitution” means the replacement of an amino acid with a different amino acid at a specific position in the parent polypeptide sequence. In particular, in some embodiments, the substitution is for an amino acid that is not native at a specific position, either within an organism or in any organism. For example, substitution M252Y refers to a mutant polypeptide, in this case an Fc mutant in which methionine at position 252 is replaced with tyrosine. To clarify, a protein that alters the nucleic acid coding sequence but does not alter the starting amino acid (e.g., replacing CGG (coding arginine) with CGA (still coding arginine) to increase the expression level in the host organism) is not an “amino acid substitution”; in other words, creating a new gene that codes for the same protein if it has the same amino acid at the specific position where the protein starts is not an amino acid substitution.

[0040] As used herein, “mutant protein” or “protein variant” or “variant” means a protein that differs from that of the parent protein by the efficacy of at least one amino acid modification. A protein variant may refer to the protein itself, a composition containing the protein, or the amino sequence encoding it. Preferably, the protein variant has at least one amino acid modification compared to the parent protein, for example, about 1 to about 70 amino acid modifications compared to the parent, preferably about 1 to about 5 amino acid modifications. In some embodiments, as described below, the parent polypeptide, for example, the Fc parent polypeptide, is the Fc region from a human wild-type sequence, for example, IgG1, IgG2, IgG3, or IgG4. The protein variant sequences herein preferably have at least about 80% identity with the parent protein sequence, most preferably at least about 90% identity, and more preferably at least about 95%-98%-99% identity. A mutant protein may refer to the mutant protein itself, a composition containing the protein variant, or the DNA sequence encoding it.

[0041] Therefore, as used herein, “antibody variant” or “mutant antibody” means an antibody that differs from the parent antibody by the efficacy of at least one amino acid modification; “IgG variant” or “mutant IgG” means an antibody that differs from the parent IgG (again, often a human IgG sequence) by the efficacy of at least one amino acid modification; and “immunoglobulin variant” or “mutant immunoglobulin” means an immunoglobulin sequence that differs from that of the parent immunoglobulin sequence by the efficacy of at least one amino acid modification. As used herein, “Fc variant” or “mutant Fc” means a protein that contains amino acid modifications in its Fc domain. The Fc variants of the present invention are defined according to the amino acid modifications that constitute them. For example, M252Y or 252Y is an Fc variant having a substituted tyrosine at position 252 relative to the parent Fc polypeptide (where numbering follows the EU index). Similarly, M252Y / S254T / T256E defines an Fc variant having substitutions M252Y, S254T, and T256E on the parent Fc polypeptide. The wild-type amino acid does not need to be identified, in which case the aforementioned variant is referred to as 252Y / 254T / 256E. It should be noted that the order in which the substitutions are provided is arbitrary, i.e., 252Y / 254T / 256E is the same Fc variant as 254T / 252Y / 256E, etc. With respect to antibodies, for all positions discussed in this invention, unless otherwise noted, amino acid position numbering follows Kabat with respect to variable region numbering and the EU index with respect to constant regions including the Fc region. The EU index, or the EU index as in Kabat or the EU numbering scheme, refers to the numbering of EU antibodies (Edelman et al., 1969, Proc Natl Acad Sci USA 63:78-85, incorporated herein by reference in its entirety). Modifications may be additions, deletions, or substitutions. Substitutions may include natural amino acids, and in some cases, synthetic amino acids.

[0042] As used herein, “protein” means at least two covalently bonded amino acids and includes proteins, polypeptides, oligopeptides, and peptides. Peptidyl groups may include native amino acid bonds and peptide bonds.

[0043] As used herein, “Fab” or “Fab region” means a polypeptide comprising the VH, CH1, VL, and CL immunoglobulin domains. Fab may refer to this region in an isolate, or to this region in relation to a full-length antibody, antibody fragment, or Fab fusion protein. As used herein, “Fv,” “Fv fragment,” or “Fv region” means polypeptides containing the VL and VH domains of a single antigen-binding domain (ABD). As will be understood by those skilled in the art, these can generally consist of two chains or be combined (with a linker as generally discussed herein) to form an scFv.

[0044] As used herein, “amino acid” and “amino acid identity” mean one of the 20 natural amino acids encoded by or encoded by DNA and RNA.

[0045] As used herein, “effector function” refers to the biochemical event resulting from the interaction between an antibody Fc region and an Fc receptor or ligand. Effector functions include, but are not limited to, ADCC, ADCP, and CDC.

[0046] As used herein, “Fc gamma receptor,” “FcγR,” or “Fc gamma R” refers to any member of the family of proteins that bind to the Fc region of an IgG antibody, and is encoded by the FcγR gene. In humans, this family includes, but is not limited to, FcγRI(CD64) including isoforms FcγRIa, FcγRIb, and FcγRIc; FcγRII(CD32) including isoforms FcγRIIa (allotypes H131 and R131), FcγRIIb (allotypes FcγRIIb-1 and FcγRIIb-2), and FcγRIIc; and FcγRIII(CD16) including isoforms FcγRIIIa (allotypes V158 and F158) and FcγRIIIb (allotypes FcγRIIb-NA1 and FcγRIIb-NA2) (Jefferis et al., 2002, Immunol Lett 82:57-65, incorporated as a whole by reference), as well as any undiscovered human FcγR or FcγR isoform or allotype. In some cases, as outlined herein, binding to one or more FcγR receptors is reduced or ablated. For example, reduced binding to FcγRIIIa reduces ADCC, and in some cases, reduced binding to both FcγRIIIa and FcγRIIb is desired.

[0047] As used herein, “FcRn” or “neonatal Fc receptor” means a protein that binds to the Fc region of an IgG antibody and is at least partially encoded by the FcRn gene. FcRn may originate from any organism, including, but not limited to, humans, mice, rats, rabbits, and monkeys. As is well known in the art, a functional FcRn protein often comprises two polypeptides, referred to as a heavy chain and a light chain. The light chain is beta-2-microglobulin, and the heavy chain is encoded by the FcRn gene. Unless otherwise noted herein, FcRn or FcRn protein refers to the complex of the FcRn heavy chain and beta-2-microglobulin.

[0048] As used herein, “parent polypeptide” means the initiating polypeptide from which a variant is subsequently produced. The parent polypeptide may be a natural polypeptide, or a modified version of a variant or natural polypeptide. The parent polypeptide may refer to the polypeptide itself, a composition containing the parent polypeptide, or the amino acid sequence encoding it. Therefore, as used herein, “parent immunoglobulin” means an unmodified immunoglobulin polypeptide from which a variant is produced, and as used herein, “parent antibody” means an unmodified antibody from which a variant antibody is produced. It should be noted that “parent antibody” includes known, commercially available recombinantly produced antibodies, as outlined below.

[0049] In this specification, “heavy chain constant region” generally refers to the CH1-hinge-CH2-CH3 portion of antibodies derived from human IgG1, IgG2, or IgG4.

[0050] As used herein, "target antigen" refers to a molecule that is specifically bound by the variable region of a given antibody. In the present case, the target antigen is the BTLA protein.

[0051] As used herein, "target cells" refers to cells that express a target antigen.

[0052] As used herein, “variable region” means a region of immunoglobulin containing one or more Ig domains substantially encoded by any V. kappa, V. lambda, and / or VH gene, which constitute the kappa, lambda, and heavy chain immunoglobulin loci, respectively.

[0053] In this specification, “wild-type” or “WT” means a naturally occurring amino acid or nucleotide sequence, including allelic variations. WT proteins have an amino acid or nucleotide sequence that is not intentionally modified.

[0054] As used herein, “position” refers to a location within a protein sequence. Positions may be numbered sequentially or according to established formats such as the EU Index for antibody numbering.

[0055] As used herein, "residue" refers to its position in a protein and its associated amino acid identity. For example, asparagine 297 (referred to as Asn297 or N297) is the residue at position 297 in the human antibody IgG1.

[0056] The antibodies of the present invention are generally recombinant. "Recombinant" means that the antibody is produced in exogenous host cells using recombinant nucleic acid technology.

[0057] With respect to protein sequences, "percent (%) amino acid sequence identity" is defined as the percentage of amino acid residues in a candidate sequence that are identical to amino acid residues in a specific (parent) sequence, after aligning the sequences and introducing gaps to achieve maximum percentage sequence identity as necessary, and without considering any conservative substitutions, as part of the sequence identity. Alignment aimed at determining percentage amino acid sequence identity can be achieved in various ways within the scope of the art using publicly available computer software such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. A person skilled in the art can determine appropriate parameters for measuring the alignment, including any algorithms necessary to achieve maximum alignment over the entire length of the sequences being compared. One particular program is the ALIGN-2 program outlined in paragraphs

[0279] to

[0280] of U.S. Patent Application Publication No. 20160244525, which is incorporated herein by reference. Another approximate alignment for nucleic acid sequences is provided by the local homology algorithm described by Smith and Waterman in Advances in Applied Mathematics, 2:482-489 (1981). This algorithm can be applied to amino acid sequences by using a scoring matrix developed by Dayhoff in Atlas of Protein Sequences and Structure, MO Dayhoff ed., 5 suppl. 3:353-358, National Biomedical Research Foundation, Washington, DC, USA, and standardized by Gribskov in Nucl. Acids Res. 14(6):6745-6763 (1986).

[0058] An exemplary implementation of this algorithm for determining the percent identity of sequences is demonstrated by the Genetics Computer Group (Madison, WI) in the “BestFit” utility application. Default parameters for this method are described in the Wisconsin Sequence Analysis Package Program Manual, Version 8 (1995) (available from Genetics Computer Group, Madison, WI). Another method for establishing percent identity in connection with the present invention is to use the MPSRCH package of programs developed by John F. Collins and Shane S. Sturrok, distributed by IntelliGenetics, Inc. (Mountain View, CA), and copyrighted by the University of Edinburgh. From this package, the Smith-Waterman algorithm may be employed, with default parameters used for the scoring table (e.g., gap-open penalty 12, gap-extension penalty 1, and gap 6). From the generated data, the “match” values ​​reflect the “sequence identity.” Other programs suitable for calculating the percentage identity or similarity between sequences are generally known in the art, for example, another alignment program is BLAST, which is used with default parameters. For example, BLASTN and BLASTP may be used with the following default parameters: gene code=standard; filter=none; strand=both; cutoff=60; expected value=10; matrix=BLOSUM62; description=50 sequences; screening=high score; database=non-redundant, GenBank+EMBL+DDBJ+PDB+GenBank CDS translation+Swiss protein+Spupdate+PIR. Details of these programs can be found at the internet address located by typing http: / / in front of blast.ncbi.nlm.nih.gov / Blast.cgi.

[0059] The degree of identity between the amino acid sequence of the present invention ("Inventive Sequence") and the parent amino acid sequence is calculated as the number of perfect matches in the alignment of the two sequences, where a perfect match is obtained by dividing the length of the "Inventive Sequence" or the length of the parent sequence, whichever is shorter. The result is expressed as a percentage of identity.

[0060] In some embodiments, two or more amino acid sequences are identical by at least 50%, 60%, 70%, 80%, or 90%. In some embodiments, two or more amino acid sequences are identical by at least 95%, 97%, 98%, 99%, or even 100%.

[0061] "Specific binding," or "specifically binding" to a particular antigen or epitope, or simply "specific," means a binding that is measurably different from nonspecific interactions. Specific binding can be measured by determining the binding of a molecule by comparing it to the binding of a control molecule, which is a similarly structured molecule that generally does not possess binding activity. For example, specific binding can be determined by competition with a control molecule similar to the target.

[0062] As used herein, the terms "Kassoc" or "Ka" are intended to refer to the association rate of a particular antibody-antigen interaction, while the terms "Kdis" or "Kd" are intended to refer to the dissociation rate of a particular antibody-antigen interaction. D The term "Kd" is intended to refer to the dissociation constant, which is derived from the ratio of Kd to Ka (i.e., Kd / Ka) and expressed as molar concentration (M). Kd for antibodies D The value can be determined using methods well established in the art. In some embodiments, the K of the antibody D Methods for determining the K of the antibody include using surface plasmon resonance, for example, by using a biosensor system, such as the BIACORE® system. In some embodiments, the K of the antibody DThis is determined by Bio-Layer Interferometry. In some embodiments, K D The value is fixed and measured. In other embodiments, K D The value is measured using an immobilized antibody (e.g., parental mouse antibody, chimeric antibody, or humanized antibody variant). In certain embodiments, K D The value is measured in bivalent binding mode. In other embodiments, K D The value is measured in monovalent binding mode.

[0063] "Disease" includes a state of animal health, including that of humans, in which the animal is unable to maintain homeostasis and the disease does not go into remission, resulting in a continued deterioration of the animal's health.

[0064] In contrast, “disorder” in animals, including humans, includes health conditions in which the animal can maintain homeostasis, but the animal’s health is less favorable than in the absence of the disorder. Leaving it untreated does not necessarily lead to a further deterioration of the animal’s health.

[0065] Terms such as “treatment,” “treating,” and “treat” refer to obtaining a desired pharmacological and / or physiological effect. The effect may be preventive in relation to completely or partially preventing a disease or its symptoms or reducing the likelihood of a disease or its symptoms, and / or therapeutic in relation to the partial or complete cure of a disease and / or any side effects that may result from the disease. As used herein, “treatment” encompasses any treatment of a disease in mammals, in particular humans, and includes: (a) preventing the occurrence of a disease in a subject who may be predisposed to the disease but has not yet been diagnosed as having it; (b) inhibiting a disease, i.e., stopping its onset or progression; and (c) mitigating a disease, i.e., causing a regression of the disease and / or reducing one or more symptoms of the disease. “Treatment” also means the delivery of a drug to provide a pharmacological effect even in the absence of a disease or condition. For example, “treatment” includes the delivery of a composition (e.g., a vaccine) that can induce an immune response or provide immunity in the absence of a disease condition.

[0066] As used herein, the term “mammal” refers to any mammal, including, but not limited to, rodents such as mice and hamsters, and mammals of the order Logomorpha such as rabbits. In some embodiments, the mammal is from the order Carnivora, including cats and dogs. In some embodiments, the mammal is from the order Artiodactyla, including cattle and pigs, or from the order Persodactyla, including horses. It is most preferable that the mammal is from the order Primates, Ceboids, or Simoids (monkeys) or Anthropoids (humans and apes). In some embodiments, the mammal is a human. In some embodiments, the mammal is a cynomolgus macaque.

[0067] As used herein, the term “regression” and stemmings thereof do not necessarily mean 100% or complete regression. Rather, there are various degrees of regression that a person skilled in the art would recognize as having potential benefits or therapeutic effects. In this regard, the methods disclosed can provide any level and amount of cancer regression in mammals. Furthermore, the regression provided by the methods of the present invention may include regression of one or more states or symptoms of a disease, e.g., cancer. Also, for the purposes of this specification, “regression” may include delaying the onset of a disease, delaying the onset of symptoms, and / or delaying the onset of a state. With respect to progressive diseases and disorders, “regression” may include slowing the progression of a disease or disorder, slowing the progression of symptoms of a disease or disorder, and / or slowing the progression of a state.

[0068] The “effective amount” or “therapeutic effective amount” of a composition includes an amount of the composition sufficient to provide a beneficial effect to the subject to which the composition is administered. The “effective amount” of a delivery medium includes an amount sufficient to effectively bind to or deliver the composition.

[0069] "Individual," "host," "subject," or "patient" means any mammalian subject, especially humans, to whom diagnosis, treatment, or therapy is desired. Other subjects may include cynomolgus macaques, cattle, dogs, cats, guinea pigs, rabbits, rats, mice, horses, and so on.

[0070] As used herein, the term “in combination” refers to a use in which, for example, the first treatment is administered during the entire course of administration of the second treatment; the first treatment is administered over a period that overlaps with the administration of the second treatment, for example, the administration of the first treatment begins before the administration of the second treatment and ends before the administration of the second treatment ends; the administration of the second treatment begins before the administration of the first treatment and ends before the administration of the first treatment ends; the administration of the first treatment begins before the administration of the second treatment begins and ends before the administration of the first treatment ends; or the administration of the second treatment begins before the administration of the first treatment begins and ends before the administration of the second treatment ends. Thus, “in combination” may also refer to a regimen involving the administration of two or more treatments. As used herein, “in combination” also refers to the administration of two or more treatments that may be administered in the same or different formulations, by the same or different routes, and in the same or different dosage form types.

[0071] "Code" includes the inherent properties of a specific sequence of nucleotides in a polynucleotide, such as a gene, cDNA, or mRNA, and the biological properties derived therefrom, which serve as a template for the synthesis of other polymers and macromolecules in biological processes, having either a defined sequence of nucleotides (i.e., rRNA, tRNA, and mRNA) or a defined sequence of amino acids. Therefore, for example, if the transcription and translation of mRNA corresponding to a gene produces a protein in a cell or other biological system, that gene codes for that protein. Both the coding strand, whose nucleotide sequence is identical to the mRNA sequence and is usually provided in a sequence listing, and the non-coding strand, used as a template for the transcription of a gene or cDNA, can be said to code for a protein or other product of that gene or cDNA.

[0072] The term "nucleic acid" includes RNA or DNA molecules having multiple nucleotides in any form, including single-stranded, double-stranded, oligonucleotides, or polynucleotides. The term "nucleotide sequence" includes the ordering of nucleotides in oligonucleotides or polynucleotides in single-stranded nucleic acids.

[0073] A "nucleic acid construct" refers to a nucleic acid sequence that consists of one or more functional units that are constructed and not found together in nature. Examples include circular, linear, and double-stranded extrachromosomal DNA molecules (plasmids) containing non-natural nucleic acid sequences, cosmids (plasmids containing COS sequences derived from lambda phages), and viral genomes.

[0074] As used herein, the term “operatably linked” includes a second polynucleotide, e.g., a single-stranded or double-stranded nucleic acid moiety, in which at least one of two polynucleotides is functionally related to a nucleic acid moiety, the other of which includes two polynucleotides positioned within the nucleic acid moiety in a manner that enables it to exert the physiological effect being characterized. For example, a promoter operatably linked to the coding region of a gene can promote transcription of the coding region. The order in which the terms are specified is not important when referring to operatable linkage. For example, the phrases “promoter operatably linked to a nucleotide sequence” and “nucleotide sequence operatably linked to a promoter” are used interchangeably and are considered equivalent herein. In some cases, the nucleic acid encoding the desired protein further includes a promoter / control sequence, which is positioned at the 5' end of the desired protein-coding sequence, and as a result, it drives the expression of the desired protein in the cell.

[0075] As used interchangeably herein, the terms “oligonucleotide,” “polynucleotide,” and “nucleic acid molecule” refer to polymeric forms of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. Therefore, these terms include, but are not limited to, single-stranded, double-stranded, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or polymers containing purine and pyrimidine bases, or other natural, chemically or biochemically modified, unnatural, or derivatized nucleotide bases. The polynucleotide backbone may include sugars and phosphate groups (typically as can be found in RNA or DNA), or modified or substituted sugars or phosphate groups.

[0076] The term "recombinant" applied to polynucleotides means that the polynucleotide is the product of various combinations of cloning, restriction, or ligation steps, as well as other procedures that result in a construct distinct from and / or different from naturally occurring polynucleotides. The term includes the original polynucleotide construct and the derivative copies of the original viral construct, respectively.

[0077] As used herein, the term “promoter” includes a DNA sequence operably ligated to a nucleic acid sequence to be transcribed, such as a nucleic acid sequence encoding a desired molecule. Promoters are generally located upstream of the nucleic acid sequence to be transcribed and provide a site for specific binding by RNA polymerase and other transcription factors.

[0078] A "vector" is capable of transferring a gene sequence to a target cell. Typically, "vector construct," "expression vector," and "gene transfer vector" refer to any nucleic acid construct capable of directing the expression of a desired gene, which can transfer the gene sequence to a target cell, and this can be achieved by genomic integration of all or part of the vector or by transient or genetic maintenance of the vector as an extrachromosomal element. Therefore, this term includes cloning and expression media, as well as integration vectors.

[0079] As used herein, the term “regulatory element” includes nucleotide sequences that control several aspects of nucleic acid sequence expression. Exemplary examples of regulatory elements include enhancers, internal ribosome entry sites (IRESs), introns, origins of replication, polyadenylation signals (pA), promoters, enhancers, transcription termination sequences, and upstream regulatory domains, which contribute to the replication, transcription, and / or post-transcriptional processing of nucleic acid sequences. In this case, regulatory elements may also include cis-regulatory DNA elements and transposition elements (TEs). Those skilled in the art can select these and other regulatory elements using routine experimental methods and use them in expression constructs. Expression constructs can be generated synthetically using genetic recombination approaches or known methodologies.

[0080] A “control element” or “control sequence” is a nucleotide sequence involved in molecular interactions that contribute to the control of polynucleotide function, including replication, duplication, transcription, splicing, translation, or degradation. The control may affect the frequency, rate, or specificity of a process, and may be enhancing or inhibiting in its properties. Control elements known in the art include, for example, transcriptional control sequences, such as promoters and enhancers. A promoter is a DNA region that, under certain conditions, can bind to RNA polymerase and initiate transcription of a coding region typically located downstream (3' direction) of the promoter.

[0081] As used herein, the term "phosphorylated" refers to the esterification of an amino acid residue by a phosphate group to the side chain of the amino acid residue. Typical amino acid residues that can be phosphorylated include serine (Ser), threonine (Thr), and tyrosine (Tyr).

[0082] As used herein, the term “pharmaceutical composition” refers to a combination of an activator and a carrier (inactive or active) that makes a composition particularly suitable for in vivo or ex vivo diagnostic or therapeutic use.

[0083] As used herein, the term “pharmaceutically acceptable carrier” refers to any standard pharmaceutically acceptable carrier, such as phosphate-buffered saline, water, emulsions (e.g., oil / water or water / oil emulsions), and various types of wetting agents. The composition may also contain stabilizers and preservatives. For examples of carriers, stabilizers, and adjuvants, see, for example, Martin, Remington's Pharmaceutical Sciences, 15th Ed., Mack Publ. Co., Easton, PA

[1975] .

[0084] Throughout the description, when a composition is described as having, including, or comprising a particular component, or when a process and method is described as having, including, or comprising a particular step, it is intended that there be a composition of the present invention that is essentially composed of or consists of a reference component, and a process and method according to the present invention that is essentially composed of or consists of a reference processing step.

[0085] As a general rule, unless otherwise specified, percentages of compositions are given by weight. Furthermore, if a variable is not defined, the definition preceding the variable takes precedence.

[0086] Various aspects of the present invention are described in the following sections; however, the aspects of the present invention described in one particular section are not limited to any particular section.

[0087] I. Antibodies This disclosure provides novel anti-BTLA antibodies. Such antibodies can bind to human and / or cynomolgus monkey BTLA. Table 1 lists the heavy chain variable region and light chain variable region peptide sequences that can bind to human and / or cynomolgus monkey BTLA in the combinations shown in Table 1. In some embodiments, the heavy chain variable region and light chain variable region are arranged in a Fab format. In some embodiments, the heavy chain variable region and light chain variable region are fused together to form an scFv (from).

[0088] [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 1-6] [Table 1-7] [Table 1-8]

[0089] In some embodiments, the anti-BTLA antibody in this disclosure includes a heavy chain variable region having an amino acid sequence identical to at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) of SEQ ID NO: 1 and a light chain variable region having an amino acid sequence identical to at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) of SEQ ID NO: 2.

[0090] In some embodiments, the anti-BTLA antibody includes vhCDR1 containing SEQ ID NO: 47, vhCDR2 containing SEQ ID NO: 48, vhCDR3 containing SEQ ID NO: 49, vlCDR1 containing SEQ ID NO: 50, vlCDR2 containing SEQ ID NO: 51, and vlCDR3 containing SEQ ID NO: 52. In some embodiments, one or more such six CDRs have 1, 2, 3, 4, or 5 amino acid modifications. In further embodiments, a single CDR contains 1 or 2 amino acid substitutions, and the modified anti-BTLA antibody retains binding to human and / or cynomolgus monkey BTLA.

[0091] In some embodiments, the anti-BTLA antibody in this disclosure includes a heavy chain variable region having an amino acid sequence identical to at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) of SEQ ID NO: 3 and a light chain variable region having an amino acid sequence identical to at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) of SEQ ID NO: 4.

[0092] In some embodiments, the anti-BTLA antibody includes vhCDR1 containing SEQ ID NO: 53, vhCDR2 containing SEQ ID NO: 54, vhCDR3 containing SEQ ID NO: 55, vlCDR1 containing SEQ ID NO: 56, vlCDR2 containing SEQ ID NO: 57, and vlCDR3 containing SEQ ID NO: 58. In further embodiments, a single CDR contains one or two amino acid substitutions, and the modified anti-BTLA antibody retains binding to human and / or cynomolgus monkey BTLA.

[0093] In some embodiments, the anti-BTLA antibody in this disclosure includes a heavy chain variable region having an amino acid sequence identical to at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) of SEQ ID NO: 5 and a light chain variable region having an amino acid sequence identical to at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) of SEQ ID NO: 6.

[0094] In some embodiments, the anti-BTLA antibody includes vhCDR1 containing SEQ ID NO: 59, vhCDR2 containing SEQ ID NO: 60, vhCDR3 containing SEQ ID NO: 61, vlCDR1 containing SEQ ID NO: 62, vlCDR2 containing SEQ ID NO: 63, and vlCDR3 containing SEQ ID NO: 64. In some embodiments, one or more such six CDRs have 1, 2, 3, 4, or 5 amino acid modifications. In further embodiments, a single CDR contains 1 or 2 amino acid substitutions, and the modified anti-BTLA antibody retains binding to human and / or cynomolgus monkey BTLA.

[0095] In some embodiments, the anti-BTLA antibody in this disclosure includes a heavy chain variable region having an amino acid sequence identical to at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) of SEQ ID NO: 7 and a light chain variable region having an amino acid sequence identical to at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) of SEQ ID NO: 8.

[0096] In some embodiments, the anti-BTLA antibody includes vhCDR1 containing SEQ ID NO: 65, vhCDR2 containing SEQ ID NO: 66, vhCDR3 containing SEQ ID NO: 67, vlCDR1 containing SEQ ID NO: 68, vlCDR2 containing SEQ ID NO: 69, and vlCDR3 containing SEQ ID NO: 70. In some embodiments, one or more such six CDRs have 1, 2, 3, 4, or 5 amino acid modifications. In further embodiments, a single CDR contains 1 or 2 amino acid substitutions, and the modified anti-BTLA antibody retains binding to human and / or cynomolgus monkey BTLA.

[0097] In some embodiments, the anti-BTLA antibody in this disclosure includes a heavy chain variable region having an amino acid sequence identical to at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) of SEQ ID NO: 9 and a light chain variable region having an amino acid sequence identical to at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) of SEQ ID NO: 10.

[0098] In some embodiments, the anti-BTLA antibody includes vhCDR1 containing SEQ ID NO: 71, vhCDR2 containing SEQ ID NO: 72, vhCDR3 containing SEQ ID NO: 73, vlCDR1 containing SEQ ID NO: 74, vlCDR2 containing SEQ ID NO: 75, and vlCDR3 containing SEQ ID NO: 76. In some embodiments, one or more such six CDRs have 1, 2, 3, 4, or 5 amino acid modifications. In further embodiments, a single CDR contains 1 or 2 amino acid substitutions, and the modified anti-BTLA antibody retains binding to human and / or cynomolgus monkey BTLA.

[0099] In some embodiments, the anti-BTLA antibody in this disclosure includes a heavy chain variable region having an amino acid sequence identical to at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) of SEQ ID NO: 11 and a light chain variable region having an amino acid sequence identical to at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) of SEQ ID NO: 12.

[0100] In some embodiments, the anti-BTLA antibody includes vhCDR1 containing SEQ ID NO: 77, vhCDR2 containing SEQ ID NO: 78, vhCDR3 containing SEQ ID NO: 79, vlCDR1 containing SEQ ID NO: 80, vlCDR2 containing SEQ ID NO: 81, and vlCDR3 containing SEQ ID NO: 82. In some embodiments, one or more such six CDRs have 1, 2, 3, 4, or 5 amino acid modifications. In further embodiments, a single CDR contains 1 or 2 amino acid substitutions, and the modified anti-BTLA antibody retains binding to human and / or cynomolgus monkey BTLA.

[0101] In some embodiments, the anti-BTLA antibody in this disclosure includes a heavy chain variable region having an amino acid sequence identical to at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) of SEQ ID NO: 13 and a light chain variable region having an amino acid sequence identical to at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) of SEQ ID NO: 14.

[0102] In some embodiments, the anti-BTLA antibody includes vhCDR1 containing SEQ ID NO: 83, vhCDR2 containing SEQ ID NO: 84, vhCDR3 containing SEQ ID NO: 85, vlCDR1 containing SEQ ID NO: 86, vlCDR2 containing SEQ ID NO: 87, and vlCDR3 containing SEQ ID NO: 88. In some embodiments, one or more such six CDRs have 1, 2, 3, 4, or 5 amino acid modifications. In further embodiments, a single CDR contains 1 or 2 amino acid substitutions, and the modified anti-BTLA antibody retains binding to human and / or cynomolgus monkey BTLA.

[0103] In some embodiments, the anti-BTLA antibody in this disclosure includes a heavy chain variable region having an amino acid sequence identical to at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) of SEQ ID NO: 15 and a light chain variable region having an amino acid sequence identical to at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) of SEQ ID NO: 16.

[0104] In some embodiments, the anti-BTLA antibody includes vhCDR1 containing SEQ ID NO: 89, vhCDR2 containing SEQ ID NO: 90, vhCDR3 containing SEQ ID NO: 91, vlCDR1 containing SEQ ID NO: 92, vlCDR2 containing SEQ ID NO: 93, and vlCDR3 containing SEQ ID NO: 94. In some embodiments, one or more such six CDRs have 1, 2, 3, 4, or 5 amino acid modifications. In further embodiments, a single CDR contains 1 or 2 amino acid substitutions, and the modified anti-BTLA antibody retains binding to human and / or cynomolgus monkey BTLA.

[0105] In some embodiments, the anti-BTLA antibody in this disclosure includes a heavy chain variable region having an amino acid sequence identical to at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) of SEQ ID NO: 17 and a light chain variable region having an amino acid sequence identical to at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) of SEQ ID NO: 18.

[0106] In some embodiments, the anti-BTLA antibody includes vhCDR1 containing SEQ ID NO: 95, vhCDR2 containing SEQ ID NO: 96, vhCDR3 containing SEQ ID NO: 97, vlCDR1 containing SEQ ID NO: 98, vlCDR2 containing SEQ ID NO: 99, and vlCDR3 containing SEQ ID NO: 100. In some embodiments, one or more such six CDRs have 1, 2, 3, 4, or 5 amino acid modifications. In further embodiments, a single CDR contains 1 or 2 amino acid substitutions, and the modified anti-BTLA antibody retains binding to human and / or cynomolgus monkey BTLA.

[0107] In some embodiments, the anti-BTLA antibody in this disclosure includes a heavy chain variable region having an amino acid sequence identical to at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) of SEQ ID NO: 19 and a light chain variable region having an amino acid sequence identical to at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) of SEQ ID NO: 20.

[0108] In some embodiments, the anti-BTLA antibody includes vhCDR1 containing SEQ ID NO: 101, vhCDR2 containing SEQ ID NO: 102, vhCDR3 containing SEQ ID NO: 103, vlCDR1 containing SEQ ID NO: 104, vlCDR2 containing SEQ ID NO: 105, and vlCDR3 containing SEQ ID NO: 106. In some embodiments, one or more such six CDRs have 1, 2, 3, 4, or 5 amino acid modifications. In further embodiments, a single CDR contains 1 or 2 amino acid substitutions, and the modified anti-BTLA antibody retains binding to human and / or cynomolgus monkey BTLA.

[0109] In some embodiments, the anti-BTLA antibody in this disclosure includes a heavy chain variable region having an amino acid sequence identical to at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) of SEQ ID NO: 21 and a light chain variable region having an amino acid sequence identical to at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) of SEQ ID NO: 22.

[0110] In some embodiments, the anti-BTLA antibody includes vhCDR1 containing SEQ ID NO: 107, vhCDR2 containing SEQ ID NO: 108, vhCDR3 containing SEQ ID NO: 109, vlCDR1 containing SEQ ID NO: 110, vlCDR2 containing SEQ ID NO: 111, and vlCDR3 containing SEQ ID NO: 112. In some embodiments, one or more such six CDRs have 1, 2, 3, 4, or 5 amino acid modifications. In further embodiments, a single CDR contains 1 or 2 amino acid substitutions, and the modified anti-BTLA antibody retains binding to human and / or cynomolgus monkey BTLA.

[0111] In some embodiments, the anti-BTLA antibody in this disclosure includes a heavy chain variable region having an amino acid sequence identical to at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) of SEQ ID NO: 23 and a light chain variable region having an amino acid sequence identical to at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) of SEQ ID NO: 24.

[0112] In some embodiments, the anti-BTLA antibody includes vhCDR1 containing SEQ ID NO: 113, vhCDR2 containing SEQ ID NO: 114, vhCDR3 containing SEQ ID NO: 115, vlCDR1 containing SEQ ID NO: 116, vlCDR2 containing SEQ ID NO: 117, and vlCDR3 containing SEQ ID NO: 118. In some embodiments, one or more such six CDRs have 1, 2, 3, 4, or 5 amino acid modifications. In further embodiments, a single CDR contains 1 or 2 amino acid substitutions, and the modified anti-BTLA antibody retains binding to human and / or cynomolgus monkey BTLA.

[0113] In some embodiments, the anti-BTLA antibody in this disclosure includes a heavy chain variable region having an amino acid sequence identical to at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) of SEQ ID NO: 25 and a light chain variable region having an amino acid sequence identical to at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) of SEQ ID NO: 26.

[0114] In some embodiments, the anti-BTLA antibody includes vhCDR1 containing SEQ ID NO: 119, vhCDR2 containing SEQ ID NO: 120, vhCDR3 containing SEQ ID NO: 121, vlCDR1 containing SEQ ID NO: 122, vlCDR2 containing SEQ ID NO: 123, and vlCDR3 containing SEQ ID NO: 124. In some embodiments, one or more such six CDRs have 1, 2, 3, 4, or 5 amino acid modifications. In further embodiments, a single CDR contains 1 or 2 amino acid substitutions, and the modified anti-BTLA antibody retains binding to human and / or cynomolgus monkey BTLA.

[0115] In some embodiments, the anti-BTLA antibody in this disclosure includes a heavy chain variable region having an amino acid sequence identical to at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) of SEQ ID NO: 27 and a light chain variable region having an amino acid sequence identical to at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) of SEQ ID NO: 28.

[0116] In some embodiments, the anti-BTLA antibody includes vhCDR1 containing SEQ ID NO: 125, vhCDR2 containing SEQ ID NO: 126, vhCDR3 containing SEQ ID NO: 127, vlCDR1 containing SEQ ID NO: 128, vlCDR2 containing SEQ ID NO: 129, and vlCDR3 containing SEQ ID NO: 130. In some embodiments, one or more such six CDRs have 1, 2, 3, 4, or 5 amino acid modifications. In further embodiments, a single CDR contains 1 or 2 amino acid substitutions, and the modified anti-BTLA antibody retains binding to human and / or cynomolgus monkey BTLA.

[0117] In some embodiments, the anti-BTLA antibody in this disclosure includes a heavy chain variable region having an amino acid sequence identical to at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) of SEQ ID NO: 29 and a light chain variable region having an amino acid sequence identical to at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) of SEQ ID NO: 30.

[0118] In some embodiments, the anti-BTLA antibody includes vhCDR1 containing SEQ ID NO: 131, vhCDR2 containing SEQ ID NO: 132, vhCDR3 containing SEQ ID NO: 133, vlCDR1 containing SEQ ID NO: 134, vlCDR2 containing SEQ ID NO: 135, and vlCDR3 containing SEQ ID NO: 136. In some embodiments, one or more such six CDRs have 1, 2, 3, 4, or 5 amino acid modifications. In further embodiments, a single CDR contains 1 or 2 amino acid substitutions, and the modified anti-BTLA antibody retains binding to human and / or cynomolgus monkey BTLA.

[0119] In some embodiments, the anti-BTLA antibody in this disclosure includes a heavy chain variable region having an amino acid sequence identical to at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) of SEQ ID NO: 31 and a light chain variable region having an amino acid sequence identical to at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) of SEQ ID NO: 32.

[0120] In some embodiments, the anti-BTLA antibody includes vhCDR1 containing SEQ ID NO: 137, vhCDR2 containing SEQ ID NO: 138, vhCDR3 containing SEQ ID NO: 139, vlCDR1 containing SEQ ID NO: 140, vlCDR2 containing SEQ ID NO: 141, and vlCDR3 containing SEQ ID NO: 142. In some embodiments, one or more such six CDRs have 1, 2, 3, 4, or 5 amino acid modifications. In further embodiments, a single CDR contains 1 or 2 amino acid substitutions, and the modified anti-BTLA antibody retains binding to human and / or cynomolgus monkey BTLA.

[0121] In some embodiments, the anti-BTLA antibody in this disclosure includes a heavy chain variable region having an amino acid sequence identical to at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) of SEQ ID NO: 33 and a light chain variable region having an amino acid sequence identical to at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) of SEQ ID NO: 34.

[0122] In some embodiments, the anti-BTLA antibody includes vhCDR1 containing SEQ ID NO: 143, vhCDR2 containing SEQ ID NO: 144, vhCDR3 containing SEQ ID NO: 145, vlCDR1 containing SEQ ID NO: 146, vlCDR2 containing SEQ ID NO: 147, and vlCDR3 containing SEQ ID NO: 148. In some embodiments, one or more such six CDRs have 1, 2, 3, 4, or 5 amino acid modifications. In further embodiments, a single CDR contains 1 or 2 amino acid substitutions, and the modified anti-BTLA antibody retains binding to human and / or cynomolgus monkey BTLA.

[0123] In some embodiments, the anti-BTLA antibody in this disclosure includes a heavy chain variable region having an amino acid sequence identical to at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) of SEQ ID NO: 35 and a light chain variable region having an amino acid sequence identical to at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) of SEQ ID NO: 36.

[0124] In some embodiments, the anti-BTLA antibody includes vhCDR1 containing SEQ ID NO: 149, vhCDR2 containing SEQ ID NO: 150, vhCDR3 containing SEQ ID NO: 151, vlCDR1 containing SEQ ID NO: 152, vlCDR2 containing SEQ ID NO: 153, and vlCDR3 containing SEQ ID NO: 154. In some embodiments, one or more such six CDRs have 1, 2, 3, 4, or 5 amino acid modifications. In further embodiments, a single CDR contains 1 or 2 amino acid substitutions, and the modified anti-BTLA antibody retains binding to human and / or cynomolgus monkey BTLA.

[0125] In some embodiments, the anti-BTLA antibody in this disclosure includes a heavy chain variable region having an amino acid sequence identical to at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) of SEQ ID NO: 37 and a light chain variable region having an amino acid sequence identical to at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) of SEQ ID NO: 38.

[0126] In some embodiments, the anti-BTLA antibody includes vhCDR1 containing SEQ ID NO: 155, vhCDR2 containing SEQ ID NO: 156, vhCDR3 containing SEQ ID NO: 157, vlCDR1 containing SEQ ID NO: 158, vlCDR2 containing SEQ ID NO: 159, and vlCDR3 containing SEQ ID NO: 160. In some embodiments, one or more such six CDRs have 1, 2, 3, 4, or 5 amino acid modifications. In further embodiments, a single CDR contains 1 or 2 amino acid substitutions, and the modified anti-BTLA antibody retains binding to human and / or cynomolgus monkey BTLA.

[0127] In some embodiments, the anti-BTLA antibody in this disclosure includes a heavy chain variable region having an amino acid sequence identical to at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) of SEQ ID NO: 39 and a light chain variable region having an amino acid sequence identical to at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) of SEQ ID NO: 40.

[0128] In some embodiments, the anti-BTLA antibody includes vhCDR1 containing SEQ ID NO: 161, vhCDR2 containing SEQ ID NO: 162, vhCDR3 containing SEQ ID NO: 163, vlCDR1 containing SEQ ID NO: 164, vlCDR2 containing SEQ ID NO: 165, and vlCDR3 containing SEQ ID NO: 166. In some embodiments, one or more such six CDRs have 1, 2, 3, 4, or 5 amino acid modifications. In further embodiments, a single CDR contains 1 or 2 amino acid substitutions, and the modified anti-BTLA antibody retains binding to human and / or cynomolgus monkey BTLA.

[0129] In some embodiments, the anti-BTLA antibody in this disclosure includes a heavy chain variable region having an amino acid sequence identical to at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) of SEQ ID NO: 41 and a light chain variable region having an amino acid sequence identical to at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) of SEQ ID NO: 42.

[0130] In some embodiments, the anti-BTLA antibody includes vhCDR1 containing SEQ ID NO: 167, vhCDR2 containing SEQ ID NO: 168, vhCDR3 containing SEQ ID NO: 169, vlCDR1 containing SEQ ID NO: 170, vlCDR2 containing SEQ ID NO: 171, and vlCDR3 containing SEQ ID NO: 172. In some embodiments, one or more such six CDRs have 1, 2, 3, 4, or 5 amino acid modifications. In further embodiments, a single CDR contains 1 or 2 amino acid substitutions, and the modified anti-BTLA antibody retains binding to human and / or cynomolgus monkey BTLA.

[0131] In addition to the sequence variants described herein in the heavy chain and light chain variable regions and / or CDR, changes can be made in the framework regions of the heavy chain and / or light chain variable regions. In some embodiments, the framework region variant (e.g., elimination of the CDR) retains at least about 80, 85, 90, or 95% identity with the germline sequence. Table 2 lists the germline gene alleles encoding the anti-BTLA antibodies described herein. The variants can be made to retain at least about 80, 85, 90, or 95% identity with any one of the alleles of the light chain V gene, light chain J gene, heavy chain V gene, heavy chain J gene, and heavy chain D gene.

[0132] [Table 2-1] [Table 2-2]

[0133] In some embodiments, variations are created in a framework region that maintains at least 80, 85, 90, or 95% identity with the germline gene sequences listed in Table 2, while the six CDRs remain immutable.

[0134] In some embodiments, variations are made in both the framework region (which maintains at least 80, 85, 90, or 95% identity with the germline gene sequences listed in Table 2) and the six CDRs. The CDRs may have amino acid modifications (e.g., 1, 2, 3, 4, or 5 amino acid modifications in a set of CDRs) (i.e., the CDRs may be modified as long as the total number of changes in the set of six CDRs is less than 6 amino acid modifications, and any combination of CDRs may change; for example, one change may be present in vlCDR1, two changes in vhCDR2, no change in vhCDR3, etc.).

[0135] A person skilled in the art can design an anti-BTLA antibody according to the present invention by selecting the amino acid sequences of the heavy chain and light chain CDR and / or variable regions from those described herein and, if necessary, combining them with the amino acid sequences of the heavy chain and light chain framework regions and / or constant regions of the antibody. The antibody framework regions and / or constant regions (Fc domains) described herein may be derived from any species, e.g., human, rabbit, dog, cat, mouse, horse, or monkey.

[0136] In some embodiments, the constant region includes a heavy chain constant region derived from human IgG, IgA, IgM, IgE, and IgD subtypes or their variants, and a light chain constant region derived from kappa or lambda subtypes or their variants. In some embodiments, the heavy chain constant region is derived from human IgG, including IgG1, IgG2, IgG3, and IgG4. In some embodiments, the amino acid sequence of the heavy chain constant region is at least 80%, 85%, 90%, or 95% identical to that of the human IgG1, IgG2, IgG3, or IgG4 constant region. In some other embodiments, the amino acid sequence of the constant region is at least 80%, 85%, 90%, or 95% identical to that of the antibody constant region from another mammal, e.g., rabbit, dog, cat, mouse, horse, or monkey. In some embodiments, the antibody constant region includes a hinge, a CH2 domain, a CH3 domain, and optionally a CH1 domain.

[0137] In some embodiments, the antibodies described herein may originate from mixtures of different species (e.g., forming chimeric antibodies and / or humanized antibodies). Generally, both “chimeric antibodies” and “humanized antibodies” refer to antibodies that combine regions from multiple species. For example, a “chimeric antibody” traditionally contains a variable region from mouse (or, in some cases, rat) and a constant region from human. A “humanized antibody” generally refers to a non-human antibody that had a variable domain framework region swapped with sequences found in human antibodies. Generally, in humanized antibodies, the entire antibody (excluding the CDR) is identical to such an antibody except that it is encoded by a polynucleotide of human origin or within its CDR. The CDR (some or all of which are encoded by nucleic acids of non-human origin) is transplanted into the beta-sheet framework of the human antibody variable region to produce an antibody, and its specificity is determined by the transplanted CDR. The creation of such antibodies is described, for example, in WO92 / 11018, Jones, 1986, Nature 321:522-525, and Verhoeyen et al., 1988, Science 239:1534-1536, where all are incorporated holistically by reference. A "reverse mutation" of selected acceptor framework residues relative to the corresponding donor residue is often required to restore the affinity lost in the initial transplanted construct (US5530101;US5585089;US5693761;US5693762;US6180370;US5859205;US5821337;US6054297;US6407213, where all are incorporated holistically by reference). Humanized antibodies also optimally contain at least some immunoglobulin constant regions (typically those of human immunoglobulins), and therefore typically contain human Fc regions. Humanized antibodies can also be generated using mice with genetically engineered immune systems, for example, as described in Roque et al., 2004, Biotechnol. Prog. 20:639-654, which are incorporated entirely by reference.Various techniques and methods for humanizing and reshaping non-human antibodies are well known in this field (see Tsurushita & Vasquez, 2004, Humanization of Monoclonal Antibodies, Molecular Biology of B Cells, 533-545, Elsevier Science (USA), and the references cited therein, where all of these are incorporated as a whole). Humanization methods are not limited to these, but all are incorporated holistically by reference, including Jones et al., 1986, Nature 321:522-525; Riechmann et al., 1988; Nature 332:323-329; Verhoeyen et al., 1988, Science, 239:1534-1536; Queen et al., 1989, Proc Natl Acad Sci, USA 86:10029-33; He et al., 1998, J. Immunol. 160: 1029-1035; Carter et al., 1992, Proc Natl Acad Sci, USA 89:4285-9; Presta et al., 1997, Cancer Res. 57(20):4593-9; Gorman et al., 1991, This includes methods described in Proc. Natl. Acad. Sci. USA 88:4181-4185; O'Connor et al., 1998, Protein Eng 11:321-8. Other methods for reducing the immunogenicity of humanized or non-human antibody variable regions may include, for example, resurfacing methods, as described in Roguska et al., 1994, Proc. Natl. Acad. Sci. USA 91:969-973, which are incorporated entirely by reference.Other humanization methods may include, but are not limited to, partial transplantation of the CDR, including the methods described in Tan et al., 2002, J. Immunol. 169:1119-1125; De Pascalis et al., 2002, J. Immunol. 169:3076-3084, all of which are incorporated holistically by reference.

[0138] In some embodiments, the antibodies of the present invention include a heavy chain variable region derived from a specific human germline heavy chain immunoglobulin gene and / or a light chain variable region derived from a specific human germline light chain immunoglobulin gene. Such antibodies may contain amino acid differences compared to human germline sequences, for example, by the introduction of innate somatic mutations or intentional site-directed mutations. However, humanized antibodies typically have an amino acid sequence that is at least 80% identical to the amino acid sequence encoded by the human germline immunoglobulin gene and contain amino acid residues that distinguish antibodies derived from human sequences when compared to germline immunoglobulin amino acid sequences of other species (e.g., mouse germline sequences). In certain cases, humanized antibodies may have an amino acid sequence that is at least 95, 96, 97, 98, or 99% identical to the amino acid sequence encoded by the human germline immunoglobulin gene, or even more at least 96%, 97%, 98, or 99% identical. Typically, humanized antibodies derived from a specific human germline sequence exhibit a difference of 10-20 amino acids or less from the amino acid sequence encoded by the human germline immunoglobulin gene. In certain cases, humanized antibodies may exhibit differences of 5 or fewer amino acids from the amino acid sequence encoded by germline immunoglobulin genes, or even 4, 3, 2, or 1 amino acid or less.

[0139] In some embodiments, the antibodies of this disclosure are humanized and affinity-matured as known in the art. Structure-based methods may be used for humanization and affinity-maturation, for example, as described in U.S. Patent No. 7,657,380. Selection-based methods may, but are not limited to, be used to humanize and / or affinity mature antibody variable regions, including methods described in Wu et al., 1999, J. Mol. Biol. 294:151-162; Baca et al., 1997, J. Biol. Chem. 272(16):10678-10684; Rosok et al., 1996, J. Biol. Chem. 271(37): 22611-22618; Rader et al., 1998, Proc. Natl. Acad. Sci. USA 95: 8910-8915; Krauss et al., 2003, Protein Engineering 16(10):753-759, all of which are incorporated holistically by reference.

[0140] II. Characteristics of Antibodies In some embodiments, the anti-BTLA antibodies described herein bind to human and / or cynomolgus monkey BTLA. In some embodiments, the binding of the anti-BTLA antibody to human and / or cynomolgus monkey BTLA is measured by ELISA, e.g., by the exemplary assay described in Example 1. In such embodiments, the antibodies described herein exhibit an EC50 that can range from 0.1 - 20 nM as measured by such an assay. In some embodiments, the binding of the anti-BTLA antibody to human and / or cynomolgus monkey BTLA is measured by FACS, e.g., by the exemplary assay described in Example 2. In such embodiments, the antibodies described herein exhibit an EC50 that ranges from 0.1 - 20 nM as measured by FACS. In further embodiments, the EC50 of the antibodies described herein ranges from about 0.1 - 30, 1 - 28, 2 - 26, 3 - 24, 4 - 22, 5 - 20, 6 - 18, 7 - 16, 8 - 14, or 9 - 12 nM as measured by either ELISA or FACS.

[0141] In some embodiments, the anti-BTLA antibodies described herein bind to human and / or cynomolgus monkey BTLA with high affinity. The K D value can be measured with immobilized antigen or immobilized antibody. The K D value can also be measured in a monovalent or bivalent binding mode. For example, when measured by Bio-Layer interferometry, the K D value between the antibody and human BTLA is 1×10 -6 M or less, 5×10 -7 M or less, 2.5×10 -7 M or less, 1×10 -7 M or less, 5×10 -8 M or less, 1×10 -8 M or less, 1×10 -9 M or less, or 1×10 -10 M or less. The K D value between the antibody and cynomolgus monkey BTLA is 1×10 -6 M or less, 5×10 -7M or less, 2.5×10 -7 M or less, 1×10 -7 M or less, 5×10 -8 M or less, 2.5×10 -8 M or less, 1×10 -8 M or less, 5×10 -9 M or less, 1×10 -9 M or less, 5×10 -10 M or less, or 1 × 10 -10 M may be less than or equal to M. In some embodiments, the K between the antibody and human BTLA is D The values ​​are in the ranges of 0.1nM-1μM, 0.25nM-500nM, 0.5nM-250nM, 1nM-100nM, or 2nM-50nM. In some embodiments, the K between the antibody and cynomolgus monkey BTLA is measured. D The values ​​are in the ranges of 0.1nM-1μM, 0.25nM-500nM, 0.5nM-250nM, 1nM-100nM, or 2nM-50nM.

[0142] The binding affinity of the anti-BTLA antibodies described herein is compared to that of the BTLA monoclonal antibody 4C7 described in U.S. Patent No. 8,563,694. In some embodiments, the anti-BTLA antibodies described herein have a higher binding affinity to human BTLA than 4C7. In some embodiments, the anti-BTLA antibodies described herein have a higher binding affinity to cynomolgus monkey BTLA than 4C7. Table 7 shows some antibody clones and an exemplary K of 4C7. D The following are listed. One advantage of having a higher binding affinity than 4C7 is that the antibodies described herein may be more effective in modulating the immune response and / or participating in the antitumor immune response. Another advantage of having a higher binding affinity than 4C7 for cynomolgus monkey BTLA is that preclinical monkey studies (e.g., pharmacokinetic, pharmacodynamic, safety, and toxicity studies) derived from these antibodies may have better predictive power than studies using 4C7.

[0143] In some embodiments, anti-BTLA antibodies exhibit low immunogenicity when administered to human subjects. These antibodies may contain Fc domains derived from human IgG1, human IgG2, or human IgG3. In some embodiments, these antibodies are humanized using framework regions derived from human immunoglobulins.

[0144] The effects of anti-BTLA antibodies on T cell function can be assayed using various methods known in the art and described herein, including, for example, the method described in Example 5. Therefore, anti-BTLA antibodies can act as BTLA antagonists or BTLA agonists.

[0145] In some embodiments, the described anti-BTLA antibodies act as BTLA antagonists, blocking the interaction between BTLA and HVEM, as well as HVEM-mediated repression of T cell function. As a result, such anti-BTLA antibodies stimulate an immune response. Examples of such anti-BTLA antibodies include a heavy chain variable region containing an amino acid sequence that is at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 21, and at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%) identical to SEQ ID NO: 22, The light chain variable region contains an amino acid sequence that is identical to (88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%); and / or antibodies containing vhCDR1 containing SEQ ID NO: 107, vhCDR2 containing SEQ ID NO: 108, vhCDR3 containing SEQ ID NO: 109, vlCDR1 containing SEQ ID NO: 110, vlCDR2 containing SEQ ID NO: 111, and vlCDR3 containing SEQ ID NO: 112. Alternatively, such an anti-BTLA antibody acting as an antagonist may include a heavy chain variable region containing an amino acid sequence that is at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 31, and at least 80% (e.g., 80%, 81%, 82%, 83%, 84%) identical to SEQ ID NO: 32. Light chain variable regions containing identical amino acid sequences (85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%); and / or may include vhCDR1 containing SEQ ID NO: 137, vhCDR2 containing SEQ ID NO: 138, vhCDR3 containing SEQ ID NO: 139, vlCDR1 containing SEQ ID NO: 140, vlCDR2 containing SEQ ID NO: 141, and vlCDR3 containing SEQ ID NO: 142.Alternatively, such an anti-BTLA antibody acting as an antagonist may include a heavy chain variable region containing an amino acid sequence that is at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 35, and at least 80% (e.g., 80%, 81%, 82%, 83%, 84%) identical to SEQ ID NO: 36. Light chain variable regions containing identical amino acid sequences (85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%); and / or may include vhCDR1 containing SEQ ID NO: 149, vhCDR2 containing SEQ ID NO: 150, vhCDR3 containing SEQ ID NO: 151, vlCDR1 containing SEQ ID NO: 152, vlCDR2 containing SEQ ID NO: 153, and vlCDR3 containing SEQ ID NO: 154.

[0146] In some other embodiments, the anti-BTLA antibodies described herein act as BTLA agonists and suppress immune cell functions, including pro-inflammatory T cell function. As a result, such anti-BTLA antibodies suppress the immune response. For example, such anti-BTLA antibodies include a heavy chain variable region containing an amino acid sequence that is at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 19, and at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%) identical to SEQ ID NO: 20. Light chain variable regions containing identical amino acid sequences (87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%); and / or may include vhCDR1 containing SEQ ID NO: 101, vhCDR2 containing SEQ ID NO: 102, vhCDR3 containing SEQ ID NO: 103, vlCDR1 containing SEQ ID NO: 104, vlCDR2 containing SEQ ID NO: 105, and vlCDR3 containing SEQ ID NO: 106. Alternatively, such an anti-BTLA antibody may have a heavy chain variable region containing an amino acid sequence that is at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 25, and at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%) identical to SEQ ID NO: 26, Light chain variable regions containing identical amino acid sequences (87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%); and / or may include vhCDR1 containing SEQ ID NO: 119, vhCDR2 containing SEQ ID NO: 120, vhCDR3 containing SEQ ID NO: 121, vlCDR1 containing SEQ ID NO: 122, vlCDR2 containing SEQ ID NO: 123, and vlCDR3 containing SEQ ID NO: 124.Alternatively, such an anti-BTLA antibody acting as an agonist may include a heavy chain variable region containing an amino acid sequence that is at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 29, and at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 8) identical to SEQ ID NO: 30. Light chain variable regions containing identical amino acid sequences (5%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%); and / or may include vhCDR1 containing SEQ ID NO: 131, vhCDR2 containing SEQ ID NO: 132, vhCDR3 containing SEQ ID NO: 133, vlCDR1 containing SEQ ID NO: 134, vlCDR2 containing SEQ ID NO: 135, and vlCDR3 containing SEQ ID NO: 136.

[0147] III. Nucleic Acids of the Present Nucleic acids encoding the anti-BTLA antibodies described herein, as well as expression vectors containing such nucleic acids and host cells transformed with such nucleic acids and / or expression vectors, are also provided. As will be understood by those skilled in the art, the protein sequences shown herein may be encoded by any number of nucleic acid sequences possible by the degenerate of the gene code. Table 3 provides exemplary nucleic acids encoding the heavy chain variable region and light chain variable region of the antibodies described herein.

[0148] [Table 3-1] [Table 3-2] [Table 3-3] [Table 3-4] [Table 3-5] [Table 3-6] [Table 3-7] [Table 3-8] [Table 3-9] [Table 3-10] [Table 3-11]

[0149] Nucleic acid compositions encoding anti-BTLA antibodies and / or BTLA-binding domains are also provided. As will be understood by those skilled in the art, in the case of antigen-binding domains, the nucleic acid composition generally comprises a first nucleic acid encoding the heavy chain variable region and a second nucleic acid encoding the light chain variable region. In the case of scFv, single nucleic acids encoding the heavy chain variable region and the light chain variable region can be made separated by the linker described herein. In the case of traditional antibodies, the nucleic acid composition generally comprises a first nucleic acid encoding the heavy chain and a second nucleic acid encoding the light chain, which spontaneously assemble into a “traditional” tetrameric format of two heavy chains and two light chains upon expression in cells.

[0150] As is known in the art, the nucleic acids encoding the components of the present invention can be incorporated into an expression vector and used to produce the antibody of the present invention depending on the host cell. These two nucleic acids can be incorporated into a single expression vector or into two different expression vectors. Generally, the nucleic acids can be operably linked to any number of regulatory elements (promoters, origins of replication, selectable markers, ribosome binding sites, inducers, etc.) in the expression vector. The expression vector may be extrachromosomal or embedded.

[0151] The nucleic acids and / or expression vectors of the present invention can be introduced into any type of host cell known in the art, including mammalian, bacterial, yeast, insect, and fungal cells. After transfection, single-cell clones can be isolated for cell bank generation using methods known in the art, such as limiting dilution, ELISA, FACS, microscopy, or Clonepix. The clones can be cultured under conditions suitable for scale-up in a bioreactor and maintenance of antibody expression. Antibodies can be isolated and purified using methods known in the art, including centrifugation, deep filtration, cell lysis, homogenization, freeze-thaw cycles, affinity purification, gel filtration, ion exchange chromatography, hydrophobic interaction exchange chromatography, and mixed-mode chromatography.

[0152] IV. Therapeutic application This disclosure provides a method for modulating an immune response in a subject, comprising administering to the subject an effective amount of an anti-BTLA antibody described herein, or a pharmaceutical composition containing an anti-BTLA antibody.

[0153] In some embodiments, methods for modulating an immune response as encompassed by this disclosure include stimulating an immune response in a subject, and in further embodiments, such methods include administering to a subject an effective amount of an anti-BTLA antibody acting as a BTLA antagonist, or by administering a pharmaceutical composition containing an antagonistic anti-BTLA antibody.

[0154] In some embodiments, the methods encompassed in this disclosure include, for example, a heavy chain variable region comprising an amino acid sequence that is at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 21, and a heavy chain variable region comprising an amino acid sequence that is at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, or 90%) identical to SEQ ID NO: 22. The method includes administering an anti-BTLA antibody comprising a light chain variable region containing an amino acid sequence that is identical (in percentages of 1%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%), and / or an anti-BTLA antibody containing vhCDR1 containing SEQ ID NO: 107, vhCDR2 containing SEQ ID NO: 108, vhCDR3 containing SEQ ID NO: 109, vlCDR1 containing SEQ ID NO: 110, vlCDR2 containing SEQ ID NO: 111, and vlCDR3 containing SEQ ID NO: 112.

[0155] In some embodiments, the methods described herein include, for example, a heavy chain variable region comprising an amino acid sequence that is at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 31, and a heavy chain variable region comprising an amino acid sequence that is at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%) identical to SEQ ID NO: 32. The immune response in the subject is stimulated by administering an anti-BTLA antibody containing an amino acid sequence that is identical (in percentages of 1%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%), as well as an anti-BTLA antibody containing vhCDR1 containing SEQ ID NO: 137, vhCDR2 containing SEQ ID NO: 138, vhCDR3 containing SEQ ID NO: 139, vlCDR1 containing SEQ ID NO: 140, vlCDR2 containing SEQ ID NO: 141, and vlCDR3 containing SEQ ID NO: 142.

[0156] In some embodiments, the methods described herein include, for example, a heavy chain variable region comprising an amino acid sequence that is at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 35, and a heavy chain variable region comprising an amino acid sequence that is at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%) identical to SEQ ID NO: 36. The immune response in the subject is stimulated by administering an anti-BTLA antibody containing an amino acid sequence that is identical (in percentages of 1%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%), as well as an anti-BTLA antibody containing vhCDR1 containing SEQ ID NO: 149, vhCDR2 containing SEQ ID NO: 150, vhCDR3 containing SEQ ID NO: 151, vlCDR1 containing SEQ ID NO: 152, vlCDR2 containing SEQ ID NO: 153, and vlCDR3 containing SEQ ID NO: 154.

[0157] In some embodiments, the present disclosure provides methods for suppressing an immune response in a subject, for example, by administering to the subject an effective amount of an anti-BTLA antibody acting as a BTLA agonist, or by administering to the subject a pharmaceutical composition containing such an agonistic anti-BTLA antibody.

[0158] In some other embodiments, the methods described herein include, for example, a heavy chain variable region comprising an amino acid sequence that is at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 19, and a heavy chain variable region comprising an amino acid sequence that is at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, or 8%) identical to SEQ ID NO: 20. The immune response in the subject is suppressed by administering an anti-BTLA antibody containing an amino acid sequence that is identical to (9%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) a light chain variable region; and / or an anti-BTLA antibody containing vhCDR1 containing SEQ ID NO: 101, vhCDR2 containing SEQ ID NO: 102, vhCDR3 containing SEQ ID NO: 103, vlCDR1 containing SEQ ID NO: 104, vlCDR2 containing SEQ ID NO: 105, and vlCDR3 containing SEQ ID NO: 106.

[0159] In some other embodiments, the methods described herein include, for example, a heavy chain variable region comprising an amino acid sequence that is at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 25, and a heavy chain variable region comprising an amino acid sequence that is at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, or 8%) identical to SEQ ID NO: 26. The immune response in the subject is suppressed by administering an anti-BTLA antibody containing an amino acid sequence that is identical in percentages of 9%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%; and / or vhCDR1 containing SEQ ID NO: 119, vhCDR2 containing SEQ ID NO: 120, vhCDR3 containing SEQ ID NO: 121, vlCDR1 containing SEQ ID NO: 122, vlCDR2 containing SEQ ID NO: 123, and vlCDR3 containing SEQ ID NO: 124.

[0160] In some other embodiments, the methods described herein include, for example, a heavy chain variable region comprising an amino acid sequence that is at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 29, and a heavy chain variable region comprising an amino acid sequence that is at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, or 8%) identical to SEQ ID NO: 30. The immune response in the subject is suppressed by administering an anti-BTLA antibody containing an amino acid sequence that is identical in percentages of 9%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%; and / or vhCDR1 containing SEQ ID NO: 131, vhCDR2 containing SEQ ID NO: 132, vhCDR3 containing SEQ ID NO: 133, vlCDR1 containing SEQ ID NO: 134, vlCDR2 containing SEQ ID NO: 135, and vlCDR3 containing SEQ ID NO: 136.

[0161] This disclosure also provides a method for treating cancer in a subject, such method comprising administering to the subject an effective amount of an anti-BTLA antibody acting as a BTLA antagonist, or a pharmaceutical composition containing such an anti-BTLA antibody. In some embodiments, the cancer to be treated expresses HVEM and / or BTLA on the surface of cancer cells. In some embodiments, the cancer to be treated upregulates HVEM and / or BTLA compared to the corresponding non-cancerous tissue. In some embodiments, the subject to be treated expresses HVEM and / or BTLA on T cells, e.g., CD8+ and / or CD4+ T cells. In some embodiments, the subject to be treated expresses high levels of HVEM and / or BTLA on one or more types of immune cells, including CD4+ T cells, CD8+ T cells, B cells, natural killer T cells, natural killer cells, macrophages, and dendritic cells. In some embodiments, the cancer to be treated uses the BTLA-HVEM pathway to downregulate the T cell response and / or evade immune recognition and destruction. In some embodiments, the cancer being treated is unresponsive to existing immunomodulatory antibodies that target other immune checkpoints, such as CTLA-4, PD-1, or PD-L1.

[0162] In some embodiments, the cancer is a solid tumor such as gastric cancer, colorectal cancer, hepatocellular carcinoma, melanoma, or esophageal squamous cell carcinoma. In some embodiments, the cancer is B-cell chronic lymphocytic leukemia, Hodgkin lymphoma, B-cell non-Hodgkin lymphoma, or T-cell non-Hodgkin lymphoma.

[0163] In some other embodiments, cancer is brain cancer, bladder cancer, breast cancer, cervical cancer, endometrial cancer, esophageal cancer, leukemia, lung cancer, liver cancer, melanoma, ovarian cancer, pancreatic cancer, prostate cancer, rectal cancer, kidney cancer, testicular cancer, or uterine cancer. In yet another embodiment, cancer is vascularized tumor, squamous cell carcinoma, adenocarcinoma, small cell carcinoma, neuroblastoma, sarcoma (e.g., angiosarcoma or chondrosarcoma), laryngeal cancer, parotid gland cancer, biliary tract cancer, thyroid cancer, acral lentiginous melanoma, actinic keratosis, acute lymphoblastic leukemia, acute myeloid leukemia, adenoid cystic carcinoma, adenoma, adenosarcoma, adenosquamous cell carcinoma, anal canal cancer, anal cancer, anorectal cancer (anorectum cancer) Cancer, astrocytic cell tumor, Bartholin's gland carcinoma, basal cell carcinoma, biliary tract cancer, bone cancer, bone marrow cancer, bronchial cancer, bronchial adenocarcinoma, carcinoid, cholangiocarcinoma, chondosarcoma, choroid plexus papilloma / carcinoma, chronic lymphocytic leukemia, chronic myeloid leukemia, clear cell carcinoma, connective tissue carcinoma, cyst adenoma, gastrointestinal cancer, duodenal cancer, endocrine cancer, endoderm sinus tumor, endometrial hyperplasia, endometrial stromal sarcoma, endometrioid adenocarcinoma, endothelial cell carcinoma, ependymal carcinoma, epithelial cell carcinoma, Ewing's sarcoma, eye and orbital cancer, female genital cancer, focal nodular hyperplasia, gallbladder cancer, gastric sinus cancer Cancer, gastric fundus cancer, gastrinoma, gliablastoma, glucagonoma, cardiac cancer, hemangiblastomas, hemangioendothelioma, hemangioma, hepatocellular adenomatosis, hepatobiliary tract cancer, hepatocellular carcinoma, Hodgkin's disease, ileal cancer, insulinoma, intraepithelial neoplasia, interepithelial squamous cell neoplasia, intrahepatic cholangiocarcinoma, invasive squamous cell carcinoma, jejunal cancer, joint cancer, Kaposi's sarcoma, pelvic cancer, large cell carcinoma, colorectal cancer, leiomyosarcoma, lentigo malignant melanoma, lymphoma, male genital cancer, malignant melanoma, malignant mesothelioma, medulloblastoma, medullary epithelioma, meningeal cancer, mesothelial carcinoma, metastatic cancer, oral cancer, mucoepidermoid carcinomaCarcinoma, multiple myeloma, muscle cancer, nasal passage cancer, nervous system cancer, neuroepithelial adenocarcinoma, nodular melanoma, non-epithelial skin cancer, oat cell carcinoma, oligodendroglia, oral cavity cancer, osteosarcoma, papillary serous adenocarcinoma, penile cancer, pharyngeal cancer, pituitary tumor, plasma cell tumor, pseudosarcoma, pulmonary blastoma, rectal cancer, renal cell carcinoma, respiratory cancer, retinoblastoma, rhabdomyosarcoma, sarcoma, serous carcinoma, sinus cancer, skin cancer, small cell carcinoma, small intestine cancer, leiomyocyte cancer, soft tissue cancer, somatostatin-secreting tumor, spinal cancer, squamous cell carcinoma, rhabdomyosarcoma, submesothelial This includes cancer, superficial spreading melanoma, T-cell leukemia, tongue cancer, undifferentiated carcinoma, ureteral cancer, urethral cancer, urinary bladder cancer, urinary tract cancer, cervical cancer, uterine body cancer, uveal melanoma, vaginal cancer, verrucous carcinoma, vipomas, vulva cancer, well-differentiated carcinoma, or Wilms' tumor.

[0164] In some other embodiments, the cancer treated is a non-Hodgkin lymphoma, such as a B-cell lymphoma or a T-cell lymphoma. In certain embodiments, the non-Hodgkin lymphoma is a B-cell lymphoma, such as diffuse large B-cell lymphoma, primary mediastinal B-cell lymphoma, follicular lymphoma, microlymphocytic lymphoma, mantle cell lymphoma, marginal zone B-cell lymphoma, extranodal marginal zone B-cell lymphoma, nodal marginal zone B-cell lymphoma, splenic marginal zone B-cell lymphoma, Burkitt lymphoma, lymphoplasmacytic lymphoma, hairy cell leukemia, or primary central nervous system (CNS) lymphoma. In certain other embodiments, non-Hodgkin lymphoma is T-cell lymphoma, such as precursor T-lymphoblastic lymphoma, peripheral T-cell lymphoma, cutaneous T-cell lymphoma, angioimmunoblastic T-cell lymphoma, extranodal natural killer / T-cell lymphoma, enteropathy-type T-cell lymphoma, subcutaneous panniculitis-like T-cell lymphoma, anaplastic large cell lymphoma, or peripheral T-cell lymphoma.

[0165] This disclosure also provides a method for treating an autoimmune or inflammatory disorder in a subject, comprising administering to the subject an anti-BTLA antibody acting as an effective amount of a BTLA agonist, or a pharmaceutical composition containing such an anti-BTLA antibody. In some embodiments, HVEM and / or BTLA are expressed at low levels in one or more types of immune cells in the subject to be treated, including T cells, B cells, natural killer cells, dendritic cells, endothelial cells, and macrophages. In some embodiments, HVEM and / or BTLA are expressed at low levels in autoreactive immune cells (e.g., T cells, B cells, natural killer cells, dendritic cells, endothelial cells, and macrophages) in the subject at sites where autoimmune diseases occur, e.g., lymph nodes and the central nervous system in a subject with multiple sclerosis, joints in a subject with rheumatoid arthritis, and the gastrointestinal tract in a subject with celiac disease. Administering anti-BTLA antibodies that act as BTLA agonists can suppress pro-inflammatory immune responses, including pro-inflammatory T-cell responses, and modulate immune responses in subjects suffering from autoimmune or inflammatory disorders.

[0166] In some embodiments, the autoimmune or inflammatory disorders to be treated are multiple sclerosis, Addison's disease, amyotrophic lateral sclerosis, Crohn's disease, Cushing's syndrome, diabetes mellitus type 1, graft-versus-host disease, Graves' disease, Guillain-Barré syndrome, lupus erythematosus, psoriasis, psoriatic arthritis, rheumatoid arthritis, sarcoidosis, scleroderma, systemic lupus erythematosus, transplant rejection, or vasculitis.

[0167] In some other embodiments, the autoimmune disorders treated include, but are not limited to, acute disseminated encephalomyelitis (ADEM), agammaglobulinemia, alopecia areata, ankylosing spondylitis, antiphospholipid syndrome, antisynthetase syndrome, atopic allergy, atopic dermatitis, autoimmune aplastic anemia, autoimmune cardiomyopathy, autoimmune enteropathy, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune inner ear disease, autoimmune lymphoproliferative syndrome, autoimmune pancreatitis, autoimmune peripheral neuropathy, autoimmune polyendocrine syndrome, autoimmune progesterone dermatitis, autoimmune thrombocytopenic purpura, autoimmune urticaria, autoimmune uveitis, Barlow's disease / Barlow concentric sclerosis, Behçet's disease, Buerger's disease, Vickerstaff-type brainstem encephalitis, Blau syndrome, bullous pemphigoid, cancer, Castleman disease, celiac disease, Chagas disease, and chronic inflammation. Demyelinating polyneuropathy, chronic inflammatory demyelinating polyneuropathy, chronic obstructive pulmonary disease, chronic relapsing multifocal osteomyelitis, Churg-Strauss syndrome, scarring pemphigoid, Cogan syndrome, cold agglutinin disease, complement component 2 deficiency, contact dermatitis, cranial arteritis, Crest syndrome, cutaneous leukocytoclastic vasculitis, Dogo's disease, Darkham's disease, herpetic dermatitis, dermatomyositis, diffuse systemic cutaneous sclerosis, discoid lupus erythematosus, Dressler's syndrome Group, drug-induced lupus, eczema, endometriosis, eosinophilic fasciitis, eosinophilic gastroenteritis, eosinophilic pneumonia, acquired epidermolysis bullosa, erythema nodosum, erythroblastosis fetus, essential mixed cryoglobulinemia, Evans syndrome, progressive ossifying fibrosis, fibrous alveolitis (or idiopathic pulmonary fibrosis), gastritis, gastroenteritis, glomerulonephritis, Goodpasture syndrome, Hashimoto's encephalopathy, Hashimoto's thyroiditis, Henoch-Schönlein purpura, herpes zoster of pregnancy Also known as: gestational pemphigoid, hidradenitis suppurativa, Hughes-Stovin syndrome, hypogammaglobulinemia, idiopathic inflammatory demyelinating disease, idiopathic pulmonary fibrosis, idiopathic thrombocytopenic purpura, IgA nephropathy, inclusion body myositis, interstitial cystitis, juvenile idiopathic arthritis Also known as: juvenile rheumatoid arthritis, Kawasaki disease, Lambert-Eaton myasthenic syndrome, leukocytoclastic vasculitis, lichen planus, lichen sclerosing, linear IgA disease, lupoid hepatitis Also known asAutoimmune hepatitis, Magid syndrome, microscopic colitis, microscopic polyangiitis, Miller-Fischer syndrome, mixed connective tissue disease, plaque scleroderma, Mucher-Habermann disease (also known as) Acute pityriasis lichenoides, multiple sclerosis, myasthenia gravis, myositis, Meniere's disease, narcolepsy, neuromyelitis optica, neurogenic myotonia, ocular pemphigoid, opsoclonus myoclonus syndrome, thyroiditis, relapsing rheumatoid arthritis, PANDAS (pediatric autoimmune streptococcal infection-associated neuropsychiatric disorder), paraneoplastic cerebellar degeneration, paroxysmal nocturnal hemoglobinuria (PNH), Parry-Romberg syndrome, squamous cellulitis, personage-Turner syndrome, pemphigus vulgaris, perivenous encephalomyelitis, pernicious anemia, POEMS syndrome, polyarteritis nodosa, polymyalgia rheumatica, polymyositis, primary biliary cirrhosis, primary sclerosing cholangitis, progressive This includes inflammatory neuropathy, pure red cell aplasia, pyoderma gangrenosum, Rasmussen's encephalitis, Raynaud's phenomenon, Reiter's syndrome, relapsing polychondritis, restless legs syndrome, retroperitoneal fibrosis, rheumatic fever, schizophrenia, Schmidt's syndrome, Schnitzler's syndrome, scleritis, serum sickness, Sjögren's syndrome, spondyloarthritis, stiff person syndrome, Still's disease, subacute bacterial endocarditis (SBE), Suzak's syndrome, Sweet's syndrome, Sydenham's chorea, sympathetic ophthalmitis, Takayasu's arteritis, temporal arteritis, thrombocytopenia, Tolosa-Hunt syndrome, transverse myelitis, ulcerative colitis, undifferentiated spondyloarthritis, urticarial vasculitis, vitiligo, and Wegener's granulomatosis.

[0168] V. Combination Therapy The anti-BTLA antibodies described herein may be used in combination with additional therapeutic agents to treat cancer or autoimmune disorders.

[0169] Examples of therapeutic agents that may be used as part of combination therapy to treat cancer include, for example, radiation, mitomycin, tretinoin, ribomustine, gemcitabine, vincristine, etoposide, cladribine, mitobronitol, methotrexate, doxorubicin, carbocon, pentostatin, nitracrine, dinostatin, cetrorelix, letrozole, raltitrexed, daunorubicin, fadrozol, hotemustine, simalfacin, sobuzoxane, nedaplatin, cytarabine, bicalutamide, vinorelbine, vesnarinone, aminoglutethimide, amsacrine, and proglum. D, eriptinium acetate, ketanserin, doxifluridine, etretinate, isotretinoin, streptozocin, nimustine, vindesine, flutamide, drogenil, butocin, carmofur, razoxane, schizophyllan, carboplatin, mitractol, tegafur, ifosfamide, prednimustine, picibanil, levamisole, teniposide, improsulfan, enocitabine, lislide, oxymetholone, tamoxifen, progesterone, mepitiostane, epithiostanol, formestan, interferon-alpha, interferon-2 This includes variations of the aforementioned drugs that may exhibit different binding and increased or decreased serum half-lives to alpha, interferon-beta, interferon-gamma, colony-stimulating factor-1, colony-stimulating factor-2, denileukin difuticox, interleukin-2, luteinizing hormone-releasing factor and its related receptors.

[0170] An additional class of drugs that may be used as part of combination therapy to treat cancer is immune checkpoint inhibitors. Exemplary immune checkpoint inhibitors include drugs that inhibit one or more of the following: (i) cytotoxic T lymphocyte-associated antigen 4 (CTLA4), (ii) programmed cell death protein 1 (PD1), (iii) PDL1, (iv) LAG3, (v) B7-H3, (vi) B7-H4, and (vii) TIM3, such as ipilimumab, nivolumab, pembrolizumab, avelumab, durvalumab, and atezolizumab.

[0171] Other drugs that may be used as part of combination therapy to treat cancer include monoclonal antibodies that target non-checkpoint targets (e.g., herceptin) and non-cytotoxic drugs (e.g., tyrosine kinase inhibitors).

[0172] Further categories of anticancer drugs include, for example: (i) ALK inhibitors, ATR inhibitors, A2A antagonists, base excision repair inhibitors, Bcr-Abl tyrosine kinase inhibitors, Bruton's tyrosine kinase inhibitors, CDC7 inhibitors, CHK1 inhibitors, cyclin-dependent kinase inhibitors, DNA-PK inhibitors, inhibitors of both DNA-PK and mTOR, DNMT1 inhibitors, DNMT1 inhibitors plus 2-chlorodeoxyadenosine, HDAC inhibitors, Hedgehog signaling pathway inhibitors, IDO inhibitors, JAK inhibitors, mTOR inhibitors, MEK inhibitors (ii) inhibitors selected from harmful agents, MELK inhibitors, MTH1 inhibitors, PARP inhibitors, phosphoinositide 3-kinase inhibitors, inhibitors of both PARP1 and DHODH, proteasome inhibitors, topoisomerase-II inhibitors, tyrosine kinase inhibitors, VEGFR inhibitors, and WEE1 inhibitors; (ii) agonists of OX40, CD137, CD40, GITR, CD27, HVEM, TNFRSF25, or ICOS; and (iii) cytokines selected from IL-12, IL-15, GM-CSF, and G-CSF.

[0173] The antibodies of the present invention can also be used as an adjunct to the surgical removal of cancer from a primary lesion.

[0174] Exemplary therapeutic agents that may be used as part of a combination therapy with the anti-BTLA antibodies disclosed herein for treating autoimmune or inflammatory disorders, delaying the progression of autoimmune or inflammatory disorders, preventing recurrence of autoimmune or inflammatory disorders, or alleviating the symptoms of autoimmune or inflammatory disorders include any anti-inflammatory and / or immunosuppressive therapies known in the art and described herein. In some embodiments, anti-inflammatory and / or immunosuppressive therapies include, but are not limited to, methotrexate, cyclosporine A (e.g., including cyclosporine microemulsions), tacrolimus, corticosteroids, statins, interferon beta, nonsteroidal anti-inflammatory drugs, and 6-MP (also known as mercaptopurine, 6-mercaptopurine, or Purinethol).

[0175] In some embodiments, anti-inflammatory and / or immunosuppressive therapies for combination with anti-BTLA antibodies disclosed herein include, but are not limited to, TOPK inhibitors (e.g., OTS964((R)-9-(4-(1-(dimethylamino)propan-2-yl)phenyl)-8-hydroxy-6-methylthieno[2,3-c]quinoline-4(5H)-one) (Oncotherapy) Science)), tyrosine kinase inhibitors (e.g., axitinib, dasatinib, icotinib), topoisomerase inhibitors (e.g., topotecan), sphingosine-1-phosphate receptor agonists (e.g., fingolimod, KRP-203), anti-T cell immunoglobulins (e.g., AtGam), anti-IL-2 receptor antibodies (e.g., daclizumab), amides (CTX), ifosfamide (IFO), adriamycin (ADM), daunorubicin (DNR), vincristine (VCR), vinblastine (VBL), et These include Poside (VP16), Vermeer (Vumon), carboplatin (CBP), tacrolimus, sirolimus, everolimus, azathioprine, Brekinal, leflunomide, LEA-29Y, anti-CD3 antibodies (e.g., OKT3), aspirin, B7-CD28 blocking molecules (e.g., beratacept, abatacept), CD40-CD154 blocking molecules (anti-CD40 antibodies), acetaminophen, ibuprofen, naproxen, piroxicam, and anti-inflammatory steroids (e.g., prednisolone or dexamethasone).

[0176] In some embodiments, anti-inflammatory and / or immunosuppressive therapies for combination with anti-BTLA antibodies disclosed herein include, for example, ablation of autoimmune cells by administration of TNF-alpha, CFA, interleukin-1 (IL-1), proteasome inhibitors, NFκB inhibitors, anti-inflammatory drugs, tissue plasminogen activators (TPAs), lipopolysaccharides, ultraviolet light, and intracellular mediators of the TNF-alpha signaling pathway. Such agents induce apoptosis of autoreactive lymphocytes by disrupting the downstream pathway of TNF-alpha receptor signaling or by acting downstream of TNF-alpha receptor binding (Baldwin et al., Ann. Rev. Immunol. (1996) 12:141; Baltimore, Cell (1996) 87:13).

[0177] In some embodiments, the anti-BTLA antibodies disclosed herein are used in conjunction with surgical methods to treat or otherwise alleviate autoimmune diseases.

[0178] For example, anti-BTLA antibodies acting as BTLA agonists may be used in combination with any existing treatment for multiple sclerosis, such as corticosteroids (e.g., oral prednisone and intravenous methylprednisolone), plasmapheresis, ocrelizumab, beta-interferon, glatiramer acetate, dimethyl fumarate, fingolimod, feriflunomide, natalizumab, alemtuzumab, and / or mitoxantrone, to treat the symptoms of multiple sclerosis, slow the progression of multiple sclerosis, prevent relapses of multiple sclerosis, or alleviate the symptoms of multiple sclerosis.

[0179] The amounts of antibodies and additional therapeutic agents, as well as the relative timing of their administration, may be selected to achieve the desired combined therapeutic effect. For example, when combination therapy is administered to a patient requiring such administration, the therapeutic agents in the combination, or the pharmaceutical composition or composition containing the therapeutic agents, may be administered in any order, such as sequentially, simultaneously, together, or concurrently. Furthermore, for example, a multispecific binding protein may be administered during the time when the additional therapeutic agent exerts its prophylactic or therapeutic effect, and vice versa.

[0180] VI. Pharmaceutical Compositions and Administration This disclosure also features pharmaceutical compositions / formulations containing therapeutically effective amounts of anti-BTLA antibodies as described herein. The compositions may be formulated for use in various drug delivery systems. One or more physiologically acceptable excipients or carriers may also be included in the composition for a suitable formulation. Suitable formulations for use in this disclosure can be found in Remington's Pharmaceutical Sciences, Mack Publishing Company, Philadelphia, Pa., 17th ed., 1985. For a concise review of methods for drug delivery, see, for example, Langer (Science 249:1527-1533, 1990).

[0181] The antibodies of this disclosure may be present in lyophilized formulations or in liquid aqueous pharmaceutical formulations. The aqueous carriers of interest herein are pharmaceutically acceptable (safe and non-toxic with respect to administration to humans) and useful for the preparation of liquid formulations. Exemplary carriers include sterile water for injection (SWFI), bacteriostatic water for injection (BWFI), pH buffer (e.g., phosphate-buffered saline), sterile saline, Ringer's solution, or dextrose solution.

[0182] The antibodies of this disclosure may be present in a lyophilized formulation comprising a protein and a lyoprotectant. The lyoprotectant may be a sugar, such as a disaccharide. In certain embodiments, the lyoprotectant is sucrose or maltose. The lyophilized formulation may also include one or more buffers, surfactants, fillers, and / or preservatives.

[0183] The actual dose level of the active ingredient in the pharmaceutical composition of the present invention may vary to obtain an amount of the active ingredient that is effective in achieving the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient. For adults, the dose may be in the range of 0.1 mg to 1 g and preferably 0.5 mg to 500 mg of active antibody per dose. Alternatively, the patient dose may be adjusted relative to the patient's approximate body weight or surface area. Other factors determining the appropriate dose may include the disease or condition being treated or prevented, the severity of the disease, the route of administration, and the patient's age, sex, and medical condition. Further refinements of the calculations required to determine the appropriate dose for treatment are routinely made by those skilled in the art, particularly in light of the dose information and assays disclosed herein. Doses may also be determined through the use of known assays that determine the dose in conjunction with appropriate dose-response data. Individual patient doses may be adjusted while monitoring disease progression. Blood levels of the targetable construct or complex in the patient may be measured to determine whether the dose needs to be adjusted to reach or maintain an effective concentration. Pharmacological genomics may be used to determine which targetable construct and / or complex, and its dose, is most likely to be effective for a given individual (Schmitz et al., Clinica Chimica Acta 308: 43-53, 2001; Steimer et al., Clinica Chimica Acta 308: 33-41, 2001).

[0184] The dosage may be administered one or more times daily, weekly, monthly, or annually, or once every 2 to 20 years. Those skilled in the art can easily estimate the frequency of administration based on measurements of the residence time and concentration of the targetable construct or complex in body fluids or tissues. The administration of the present invention may be intravenous, intra-arterial, intraperitoneal, intramuscular, subcutaneous, intrapleural, subarachnoid, or intracavitary, by perfusion through a catheter or by direct intrafocal injection. This may be administered one or more times daily, one or more times weekly, one or more times monthly, and one or more times annually. [Examples]

[0185] Although the present invention has been described in general terms here, it will be more clearly understood by reference to the following examples, which are included solely for illustrative purposes of certain aspects and embodiments of the invention and are not intended to limit the invention.

[0186] [Example 1] Anti-BTLA antibodies bind to recombinant BTLA. The binding of anti-BTLA antibodies to human or cynomolgus monkey BTLA was assayed by ELISA. A plate (384 wells) was coated overnight at 4°C with 20 μl BTLA(ECD)-HIS (1 μg / ml). The wells were then washed with PBS and blocked at room temperature for 1 hour with 55 μl blocking buffer. The wells were then washed with PBS and the anti-BTLA antibodies of the present invention, as well as anti-BTLA antibodies 4C7 and 8D5 (listed in U.S. Patent No. 8,563,694), and human IgG control were added to the wells at various concentrations (67, 22, 7.4, 2.5, 0.8, 0.3, 0.09, 0.03, 0.01, 0.004, 0.001 nM), and incubated at room temperature for 1 hour. The wells were then washed with PBS and 20 μl of diluted HRP-conjugated secondary anti-human IgG antibody were added to the wells and incubated at room temperature for 45 minutes. After removing excess HRP-conjugated secondary antibody, substrate solution was added to the wells to generate a chemiluminescent signal. Photon emission was determined using an Omegastar plate reader, and relative light units (RLU) were calculated. Figure 1 shows an exemplary binding profile of anti-BTLA antibody 13-F7A to human BTLA, compared to 4C7 and 8D5 antibodies. Human IgG was used as a negative control. Table 4 summarizes the binding EC50 of various anti-BTLA antibodies to human and cynomolgus monkey BTLA as measured by ELISA.

[0187] [Table 4]

[0188] [Example 2] Anti-BTLA antibodies bind to BTLA expressed on cells. The binding of anti-BTLA antibodies to BTLA was evaluated using HEK293 cells expressing human or cynomolgus monkey BTLA. Various concentrations of anti-BTLA antibodies (67, 22, 7.4, 2.5, 0.8, 0.3, 0.09, 0.03, 0.01, 0.004, and 0.001 nM) were incubated with cells at room temperature for 1 hour. Fluorophore-conjugated anti-human IgG secondary antibodies were then added, and the cells were analyzed by flow cytometry. Figures 2A–2B show exemplary binding profiles of the anti-BTLA antibody 13-F7A to HEK293 cells expressing human or cynomolgus monkey BTLA. Compared to control HEK293 cells (Figure 2C), 13-F7A binds to both human and cynomolgus monkey BTLA. Table 5 summarizes the binding EC50 of various anti-BTLA antibodies to human and cynomolgus monkey BTLA as measured by FACS.

[0189] [Table 5]

[0190] [Example 3] Anti-BTLA antibodies inhibit BTLA / HVEM interaction. The effect of anti-BTLA antibodies on BTLA / HVEM interactions was assayed using an ELISA-based bioassay. Each well of a 384-well ELISA plate was coated with 20 μl of 5 μl / ml human HVEM-rat IgG2a at 37°C for 1 hour (or overnight at 4°C). After coating, the plates were washed twice with washing solution (PBS containing 0.05% Tween-20) using a Molecular Devices Aquamax2000 plate washer, and then blocked with blocking solution (PBS containing 3% BSA) at room temperature (RT) for 1 hour. During the blocking step, 1 μg / ml human BTLA-human Fc or 0.5 μg / ml cynomolgus monkey BTLA-human Fc was mixed with anti-BTLA antibody, 4C7, 8D5, or human IgG2 control at various concentrations (67, 22, 7.4, 2.5, 0.8, 0.3, 0.09, 0.03, 0.01, 0.004, 0.001 nM). After the blocking step was completed, the plate was washed twice in a plate washer. 20 μl of the protein / antibody mixture was then added to the plate and incubated at RT for 1 hour. Unbound material was discarded, and the plate was washed four times in a plate washer. 20 μl of secondary antibody (anti-human Fc-HRP) was then added to each well and incubated at RT for 45 minutes. Unbound material was discarded, and the plate was washed eight times in a plate washer. 20 μl of chemiluminescent substrate was then added to each well, luminescence was read at a gain of 3600, and relative light units (RLU) were calculated. Figure 3 shows an exemplary profile of the anti-BTLA antibody 13-F7A. 13-F7A inhibited human BTLA interaction with human HVEM to a similar degree as 4C7. However, 8D5 and human IgG controls had only a slight effect on the interaction between human BTLA and HVEM. Table 6 provides a summary of IC50 values ​​for different anti-BTLA antibodies that inhibit the interaction between human BTLA and HVEM, as well as the interaction between cynomolgus monkey BTLA and HVEM.

[0191] [Table 6]

[0192] [Example 4] Anti-BTLA antibodies bind to BTLA with high affinity. The binding affinity of anti-BTLA antibodies to human and cynomolgus monkey BTLA was measured by Bio-Layer Interferometry (Octet® system from ForteBIO). Each anti-BTLA antibody at 10 μg / ml was immobilized on the biosensor chip surface via anti-human-Fc (AHC) capture. Human or cynomolgus monkey BTLA was diluted in PBS and loaded into 3-fold serial dilutions at concentrations ranging from 166.7 nM to 6.17 nM. The binding curves were fitted to a 1:1 interaction model using analytical software provided by the Octet® system. Figure 4 shows exemplary association and dissociation curves between anti-BTLA antibody 13-F7A and human BTLA at different concentrations (166.7 nM, 55.6 nM, 18.5 nM, and 6.17 nM). Table 7 shows the binding and dissociation constants (K) for different anti-BTLA antibodies to human and cynomolgus monkey BTLA as measured by Bio-Layer Interferometry. D This provides an overview of the following:

[0193] [Table 7]

[0194] [Example 5] Anti-BTLA antibodies regulate T cell function. The effect of anti-BTLA antibodies on T cell function was assayed. Peripheral blood mononuclear cells (PBMCs) were isolated from heparinized whole blood of human donors by density gradient centrifugation. PBMCs were injected into complete RPMI1640 (RPMI1640, 10% FCS, l-glutamine, penicillin, and streptomycin) at a dose of 1 × 10⁶. 6The cells were resuspended at 1 / ml and 100 μl of cells were seeded into each well of a 96-well cell culture cluster plate. 1 ng / ml of Staphylococcus enterotoxin B (SEB) was added to each well along with its respective anti-BTLA antibody. The cells were incubated at 37°C under 5% CO2 for 48 hours, and the supernatant was collected. The concentration of IL-2 in the supernatant was measured by ELISA using an anti-IL-2 capture antibody (R&D System MAB602).

[0195] Figures 5A–5F show dose-response curves of IL-2 release from primary T cells in the presence of different anti-BTLA antibodies and SEB. Human IgG2 and IgG4 were used as controls for the assay because, in this example, the anti-BTLA antibodies contain the Fc domain from human IgG2, while 8D5 and 4C7 contain the Fc domain from human IgG4, respectively. The anti-BTLA antibodies 16-I20A, 15-C19A, and 16-H16A increased SEB-induced IL-2 secretion by T cells and therefore acted as BTLA antagonists. In addition, they exhibited higher maximum antagonist activity compared to the 4C7 antibody. The EC50 of each antibody tested was calculated, and 16-I20A showed efficacy in antagonizing T cell function compared to a similar EC50, i.e., the 4C7 antibody. The anti-BTLA antibodies 12-I8A, 8-M23A, and 13-F7A suppressed SEB-induced IL-2 secretion by T cells and therefore acted as BTLA agonists.

[0196] Embedding by reference The full disclosures of each patent document and scientific paper referenced herein are incorporated by reference for all purposes.

[0197] Equivalents The present invention can be embodied in other specific forms, without departing from its spirit or essential features. Therefore, the embodiments described herein are not limiting to the invention as described herein and are considered illustrative in all respects. Accordingly, the scope of the invention is indicated not by the foregoing description but by the appended claims, and all variations falling within the equivalent meaning and scope of the claims are intended to be incorporated into the invention.

Claims

1. a) A heavy chain variable region containing the amino acid sequence of SEQ ID NO: 1 and a light chain variable region containing the amino acid sequence of SEQ ID NO: 2; b) A heavy chain variable region containing the amino acid sequence of SEQ ID NO: 3 and a light chain variable region containing the amino acid sequence of SEQ ID NO: 4; c) A heavy chain variable region containing the amino acid sequence of SEQ ID NO: 5 and a light chain variable region containing the amino acid sequence of SEQ ID NO: 6; d) Heavy chain variable region containing the amino acid sequence of SEQ ID NO: 7 and light chain variable region containing the amino acid sequence of SEQ ID NO: 8; e) A heavy chain variable region containing the amino acid sequence of SEQ ID NO: 9 and a light chain variable region containing the amino acid sequence of SEQ ID NO: 10; f) A heavy chain variable region containing the amino acid sequence of SEQ ID NO: 11 and a light chain variable region containing the amino acid sequence of SEQ ID NO: 12; g) A heavy chain variable region containing the amino acid sequence of SEQ ID NO: 13 and a light chain variable region containing the amino acid sequence of SEQ ID NO: 14; h) A heavy chain variable region containing the amino acid sequence of SEQ ID NO: 15 and a light chain variable region containing the amino acid sequence of SEQ ID NO: 16; i) A heavy chain variable region containing the amino acid sequence of SEQ ID NO: 17 and a light chain variable region containing the amino acid sequence of SEQ ID NO: 18; j) A heavy chain variable region containing the amino acid sequence of SEQ ID NO: 19 and a light chain variable region containing the amino acid sequence of SEQ ID NO: 20; k) A heavy chain variable region containing the amino acid sequence of SEQ ID NO: 21 and a light chain variable region containing the amino acid sequence of SEQ ID NO: 22; l) A heavy chain variable region containing the amino acid sequence of SEQ ID NO: 23 and a light chain variable region containing the amino acid sequence of SEQ ID NO: 24; m) A heavy chain variable region containing the amino acid sequence of SEQ ID NO: 25 and a light chain variable region containing the amino acid sequence of SEQ ID NO: 26; n) A heavy chain variable region containing the amino acid sequence of SEQ ID NO: 29 and a light chain variable region containing the amino acid sequence of SEQ ID NO: 30; o) A heavy chain variable region containing the amino acid sequence of SEQ ID NO: 31 and a light chain variable region containing the amino acid sequence of SEQ ID NO: 32; p) Heavy chain variable region containing the amino acid sequence of SEQ ID NO: 33 and light chain variable region containing the amino acid sequence of SEQ ID NO: 34; q) A heavy chain variable region containing the amino acid sequence of SEQ ID NO: 35 and a light chain variable region containing the amino acid sequence of SEQ ID NO: 36; r) A heavy chain variable region containing the amino acid sequence of SEQ ID NO: 37 and a light chain variable region containing the amino acid sequence of SEQ ID NO: 38; s) A heavy chain variable region containing the amino acid sequence of SEQ ID NO: 39 and a light chain variable region containing the amino acid sequence of SEQ ID NO: 40; or t) Heavy chain variable region containing the amino acid sequence of SEQ ID NO: 41 and light chain variable region containing the amino acid sequence of SEQ ID NO: 42 Antibodies containing this substance.

2. a) vhCDR1 containing sequence number 47, vhCDR2 containing sequence number 48, vhCDR3 containing sequence number 49, vlCDR1 containing sequence number 50, vlCDR2 containing sequence number 51, and vlCDR3 containing sequence number 52; b) vhCDR1 containing SEQ ID NO: 53, vhCDR2 containing SEQ ID NO: 54, vhCDR3 containing SEQ ID NO: 55, vlCDR1 containing SEQ ID NO: 56, vlCDR2 containing SEQ ID NO: 57, and vlCDR3 containing SEQ ID NO: 58; c) vhCDR1 containing SEQ ID NO: 59, vhCDR2 containing SEQ ID NO: 60, vhCDR3 containing SEQ ID NO: 61, vlCDR1 containing SEQ ID NO: 62, vlCDR2 containing SEQ ID NO: 63, and vlCDR3 containing SEQ ID NO: 64; d) vhCDR1 containing SEQ ID NO: 65, vhCDR2 containing SEQ ID NO: 66, vhCDR3 containing SEQ ID NO: 67, vlCDR1 containing SEQ ID NO: 68, vlCDR2 containing SEQ ID NO: 69, and vlCDR3 containing SEQ ID NO: 70; e) vhCDR1 containing SEQ ID NO: 71, vhCDR2 containing SEQ ID NO: 72, vhCDR3 containing SEQ ID NO: 73, vlCDR1 containing SEQ ID NO: 74, vlCDR2 containing SEQ ID NO: 75, and vlCDR3 containing SEQ ID NO: 76; f) vhCDR1 containing SEQ ID NO: 77, vhCDR2 containing SEQ ID NO: 78, vhCDR3 containing SEQ ID NO: 79, vlCDR1 containing SEQ ID NO: 80, vlCDR2 containing SEQ ID NO: 81, and vlCDR3 containing SEQ ID NO: 82; g) vhCDR1 containing SEQ ID NO: 83, vhCDR2 containing SEQ ID NO: 84, vhCDR3 containing SEQ ID NO: 85, vlCDR1 containing SEQ ID NO: 86, vlCDR2 containing SEQ ID NO: 87, and vlCDR3 containing SEQ ID NO: 88; h) vhCDR1 containing SEQ ID NO: 89, vhCDR2 containing SEQ ID NO: 90, vhCDR3 containing SEQ ID NO: 91, vlCDR1 containing SEQ ID NO: 92, vlCDR2 containing SEQ ID NO: 93, and vlCDR3 containing SEQ ID NO: 94; i) vhCDR1 containing sequence number 95, vhCDR2 containing sequence number 96, vhCDR3 containing sequence number 97, vlCDR1 containing sequence number 98, vlCDR2 containing sequence number 99, and vlCDR3 containing sequence number 100; j) vhCDR1 containing sequence number 101, vhCDR2 containing sequence number 102, vhCDR3 containing sequence number 103, vlCDR1 containing sequence number 104, vlCDR2 containing sequence number 105, and vlCDR3 containing sequence number 106; k) vhCDR1 containing sequence number 107, vhCDR2 containing sequence number 108, vhCDR3 containing sequence number 109, vlCDR1 containing sequence number 110, vlCDR2 containing sequence number 111, and vlCDR3 containing sequence number 112; l) vhCDR1 containing sequence number 113, vhCDR2 containing sequence number 114, vhCDR3 containing sequence number 115, vlCDR1 containing sequence number 116, vlCDR2 containing sequence number 117, and vlCDR3 containing sequence number 118; m) vhCDR1 containing sequence number 119, vhCDR2 containing sequence number 120, vhCDR3 containing sequence number 121, vlCDR1 containing sequence number 122, vlCDR2 containing sequence number 123, and vlCDR3 containing sequence number 124; n) vhCDR1 containing sequence number 131, vhCDR2 containing sequence number 132, vhCDR3 containing sequence number 133, vlCDR1 containing sequence number 134, vlCDR2 containing sequence number 135, and vlCDR3 containing sequence number 136; o) vhCDR1 containing sequence number 137, vhCDR2 containing sequence number 138, vhCDR3 containing sequence number 139, vlCDR1 containing sequence number 140, vlCDR2 containing sequence number 141, and vlCDR3 containing sequence number 142; p) vhCDR1 containing sequence number 143, vhCDR2 containing sequence number 144, vhCDR3 containing sequence number 145, vlCDR1 containing sequence number 146, vlCDR2 containing sequence number 147, and vlCDR3 containing sequence number 148; q) vhCDR1 containing sequence number 149, vhCDR2 containing sequence number 150, vhCDR3 containing sequence number 151, vlCDR1 containing sequence number 152, vlCDR2 containing sequence number 153, and vlCDR3 containing sequence number 154; r) vhCDR1 containing sequence number 155, vhCDR2 containing sequence number 156, vhCDR3 containing sequence number 157, vlCDR1 containing sequence number 158, vlCDR2 containing sequence number 159, and vlCDR3 containing sequence number 160; s) vhCDR1 containing sequence number 161, vhCDR2 containing sequence number 162, vhCDR3 containing sequence number 163, vlCDR1 containing sequence number 164, vlCDR2 containing sequence number 165, and vlCDR3 containing sequence number 166; or t) vhCDR1 containing SEQ ID NO: 167, vhCDR2 containing SEQ ID NO: 168, vhCDR3 containing SEQ ID NO: 169, vlCDR1 containing SEQ ID NO: 170, vlCDR2 containing SEQ ID NO: 171, and vlCDR3 containing SEQ ID NO: 172 Antibodies containing this substance.

3. The antibody according to claim 1 or 2, which binds to human and / or cynomolgus monkey BTLA.

4. A heavy chain variable region containing the amino acid sequence of SEQ ID NO: 21 and a light chain variable region containing the amino acid sequence of SEQ ID NO: 22; A heavy chain variable region containing the amino acid sequence of SEQ ID NO: 31 and a light chain variable region containing the amino acid sequence of SEQ ID NO: 32; A heavy chain variable region containing the amino acid sequence of SEQ ID NO: 35 and a light chain variable region containing the amino acid sequence of SEQ ID NO: 36; vhCDR1 containing sequence number 107, vhCDR2 containing sequence number 108, vhCDR3 containing sequence number 109, vlCDR1 containing sequence number 110, vlCDR2 containing sequence number 111, and vlCDR3 containing sequence number 112; vhCDR1 containing SEQ ID NO: 137, vhCDR2 containing SEQ ID NO: 138, vhCDR3 containing SEQ ID NO: 139, vlCDR1 containing SEQ ID NO: 140, vlCDR2 containing SEQ ID NO: 141, and vlCDR3 containing SEQ ID NO: 142; or Includes vhCDR1 containing sequence number 149, vhCDR2 containing sequence number 150, vhCDR3 containing sequence number 151, vlCDR1 containing sequence number 152, vlCDR2 containing sequence number 153, and vlCDR3 containing sequence number 154, The antibody according to claim 1 or 2, which acts as a BTLA antagonist and blocks the binding of BTLA to HVEM and the HVEM-mediated suppression of T cell function.

5. A heavy chain variable region containing the amino acid sequence of SEQ ID NO: 19 and a light chain variable region containing the amino acid sequence of SEQ ID NO: 20; A heavy chain variable region containing the amino acid sequence of SEQ ID NO: 25 and a light chain variable region containing the amino acid sequence of SEQ ID NO: 26; A heavy chain variable region containing the amino acid sequence of SEQ ID NO: 29 and a light chain variable region containing the amino acid sequence of SEQ ID NO: 30; vhCDR1 containing sequence number 101, vhCDR2 containing sequence number 102, vhCDR3 containing sequence number 103, vlCDR1 containing sequence number 104, vlCDR2 containing sequence number 105, and vlCDR3 containing sequence number 106; vhCDR1 containing SEQ ID NO: 119, vhCDR2 containing SEQ ID NO: 120, vhCDR3 containing SEQ ID NO: 121, vlCDR1 containing SEQ ID NO: 122, vlCDR2 containing SEQ ID NO: 123, and vlCDR3 containing SEQ ID NO: 124; or Includes vhCDR1 containing SEQ ID NO: 131, vhCDR2 containing SEQ ID NO: 132, vhCDR3 containing SEQ ID NO: 133, vlCDR1 containing SEQ ID NO: 134, vlCDR2 containing SEQ ID NO: 135, and vlCDR3 containing SEQ ID NO: 136, The antibody according to claim 1 or 2, which acts as a BTLA agonist and suppresses pro-inflammatory T cell function.

6. The antibody according to any one of claims 1 to 5, comprising a constant region having an amino acid sequence that is at least 90% identical to that of human IgG.

7. The antibody according to claim 6, wherein human IgG is selected from the group consisting of IgG1, IgG2, IgG3, and IgG4.

8. The antibody according to claim 7, wherein IgG is IgG2.

9. A nucleic acid composition encoding an antibody according to any one of claims 1 to 8.

10. a) A first nucleic acid containing SEQ ID NO: 185 and a second nucleic acid containing SEQ ID NO: 186; b) A first nucleic acid containing SEQ ID NO: 187 and a second nucleic acid containing SEQ ID NO: 188; c) A first nucleic acid containing SEQ ID NO: 189 and a second nucleic acid containing SEQ ID NO: 190; d) A first nucleic acid containing SEQ ID NO: 191 and a second nucleic acid containing SEQ ID NO: 192; e) A first nucleic acid containing SEQ ID NO: 193 and a second nucleic acid containing SEQ ID NO: 194; f) A first nucleic acid containing SEQ ID NO: 195 and a second nucleic acid containing SEQ ID NO: 196; g) A first nucleic acid containing SEQ ID NO: 197 and a second nucleic acid containing SEQ ID NO: 198; h) A first nucleic acid containing SEQ ID NO: 199 and a second nucleic acid containing SEQ ID NO: 200; i) A first nucleic acid containing SEQ ID NO: 201 and a second nucleic acid containing SEQ ID NO: 202; j) A first nucleic acid containing SEQ ID NO: 203 and a second nucleic acid containing SEQ ID NO: 204; k) A first nucleic acid containing SEQ ID NO: 205 and a second nucleic acid containing SEQ ID NO: 206; l) A first nucleic acid containing SEQ ID NO: 207 and a second nucleic acid containing SEQ ID NO: 208; m) A first nucleic acid containing sequence number 209 and a second nucleic acid containing sequence number 210; n) A first nucleic acid containing SEQ ID NO: 211 and a second nucleic acid containing SEQ ID NO: 212; o) A first nucleic acid containing SEQ ID NO: 213 and a second nucleic acid containing SEQ ID NO: 214; p) A first nucleic acid containing SEQ ID NO: 215 and a second nucleic acid containing SEQ ID NO: 216; q) A first nucleic acid containing SEQ ID NO: 217 and a second nucleic acid containing SEQ ID NO: 218; r) A first nucleic acid containing SEQ ID NO: 219 and a second nucleic acid containing SEQ ID NO: 220; s) A first nucleic acid containing SEQ ID NO: 221 and a second nucleic acid containing SEQ ID NO: 222; t) A first nucleic acid containing SEQ ID NO: 223 and a second nucleic acid containing SEQ ID NO: 224; or u) A first nucleic acid containing SEQ ID NO: 225 and a second nucleic acid containing SEQ ID NO: 226 The nucleic acid composition according to claim 9, comprising:

11. An expression vector composition comprising the nucleic acid composition according to claim 10, wherein a first nucleic acid is contained in a first expression vector, and a second nucleic acid is contained in a second expression vector.

12. An expression vector composition comprising the nucleic acid composition according to claim 10, wherein the first nucleic acid and the second nucleic acid are contained in a single expression vector.

13. A host cell comprising the expression vector composition according to claim 11 or 12.

14. A method for producing an antibody, comprising culturing the host cell described in claim 13 under conditions in which the antibody is expressed, and recovering the antibody.

15. A composition comprising the antibody according to any one of claims 1 to 8, and a pharmaceutically acceptable carrier or diluent.

16. A method for modulating an immune response in a subject, comprising administering to the subject an effective amount of an antibody according to any one of claims 1 to 8, or a composition according to claim 15.

17. The method according to claim 16, comprising stimulating an immune response in a subject and administering to the subject an effective amount of an antibody according to any one of claims 1 to 8 or the composition according to claim 15, wherein the antibody acts as a BTLA antagonist.

18. The method according to claim 17, wherein the antibody comprises a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 21 and a light chain variable region containing the amino acid sequence of SEQ ID NO: 22; and / or vhCDR1 containing SEQ ID NO: 107, vhCDR2 containing SEQ ID NO: 108, vhCDR3 containing SEQ ID NO: 109, vlCDR1 containing SEQ ID NO: 110, vlCDR2 containing SEQ ID NO: 111, and vlCDR3 containing SEQ ID NO:

112.

19. The method according to claim 17, wherein the antibody comprises a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 31 and a light chain variable region containing the amino acid sequence of SEQ ID NO: 32; and / or vhCDR1 containing SEQ ID NO: 137, vhCDR2 containing SEQ ID NO: 138, vhCDR3 containing SEQ ID NO: 139, vlCDR1 containing SEQ ID NO: 140, vlCDR2 containing SEQ ID NO: 141, and vlCDR3 containing SEQ ID NO:

142.

20. The method according to claim 17, wherein the antibody comprises a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 35 and a light chain variable region containing the amino acid sequence of SEQ ID NO: 36; and / or vhCDR1 containing SEQ ID NO: 149, vhCDR2 containing SEQ ID NO: 150, vhCDR3 containing SEQ ID NO: 151, vlCDR1 containing SEQ ID NO: 152, vlCDR2 containing SEQ ID NO: 153, and vlCDR3 containing SEQ ID NO:

154.

21. The method according to claim 16, comprising suppressing the immune response in a subject and administering to the subject an effective amount of an antibody according to any one of claims 1 to 8 or the composition according to claim 15, wherein the antibody acts as a BTLA agonist.

22. The method according to claim 21, wherein the antibody comprises a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 19 and a light chain variable region containing the amino acid sequence of SEQ ID NO: 20; and / or vhCDR1 containing SEQ ID NO: 101, vhCDR2 containing SEQ ID NO: 102, vhCDR3 containing SEQ ID NO: 103, vlCDR1 containing SEQ ID NO: 104, vlCDR2 containing SEQ ID NO: 105, and vlCDR3 containing SEQ ID NO:

106.

23. The method according to claim 21, wherein the antibody comprises a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 25 and a light chain variable region containing the amino acid sequence of SEQ ID NO: 26; and / or vhCDR1 containing SEQ ID NO: 119, vhCDR2 containing SEQ ID NO: 120, vhCDR3 containing SEQ ID NO: 121, vlCDR1 containing SEQ ID NO: 122, vlCDR2 containing SEQ ID NO: 123, and vlCDR3 containing SEQ ID NO:

124.

24. The method according to claim 21, wherein the antibody comprises a heavy chain variable region having an amino acid sequence at least 90% identical to SEQ ID NO: 29 and a light chain variable region having an amino acid sequence at least 90% identical to SEQ ID NO: 30; and / or vhCDR1 having SEQ ID NO: 131, vhCDR2 having SEQ ID NO: 132, vhCDR3 having SEQ ID NO: 133, vlCDR1 having SEQ ID NO: 134, vlCDR2 having SEQ ID NO: 135, and vlCDR3 having SEQ ID NO:

136.

25. A method for treating cancer in a subject, comprising administering to the subject an effective amount of an antibody according to any one of claims 1 to 8, or a composition according to claim 15, wherein the antibody acts as a BTLA antagonist.

26. The method according to claim 25, wherein cancer is upregulated by HVEM.

27. The method according to claim 25 or 26, wherein the T cells have a high level of BTLA.

28. The method according to any one of claims 25 to 27, wherein the cancer is stomach cancer.

29. The method according to any one of claims 25 to 28, wherein the antibody is combined with one or more additional therapeutic agents for treating cancer.

30. The method according to claim 29, wherein the additional therapeutic agent is another immune checkpoint inhibitor.

31. The method according to claim 30, wherein the other immune checkpoint inhibitor is selected from the group consisting of ipilimumab, nivolumab, pembrolizumab, avelumab, durvalumab, and atezolizumab.

32. A method for treating an autoimmune disease in a subject, comprising administering to the subject an effective amount of an antibody according to any one of claims 1 to 8, or a composition according to claim 15, wherein the antibody acts as a BTLA agonist.

33. The method according to claim 32, wherein low levels of HVEM are expressed in autoreactive T cells located in the site where an autoimmune disease occurs.

34. The method according to claim 32 or 33, wherein the autoimmune disease is multiple sclerosis.

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