BTLA-binding (CD272) antibodies for modulating immune response and

Abstract

The present invention provides novel anti-BTLA antibodies, pharmaceutical compositions comprising such antibodies, and therapeutic methods of using such antibodies and pharmaceutical compositions in the treatment of diseases, such as cancer or autoimmune diseases.

Description

[0001] This application is a divisional application with application number 202080040939.4, application date March 31, 2020, and invention name “BTLA-binding (CD272) antibodies for regulating immune responses and treating diseases”.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims priority to U.S. Provisional Patent Application No. 62 / 827,744, filed on April 1, 2019, which is incorporated herein by reference in its entirety. Technical Field

[0004] The present disclosure relates to novel anti-BTLA antibodies and pharmaceutical compositions comprising such antibodies for use in modulating immune responses and treating diseases, such as cancer or autoimmune diseases. Background Art

[0005] Treating diseases by inducing, enhancing or suppressing immune responses is called immunotherapy. Immunotherapy has shown increasing effectiveness in treating cancer. Generally, the tumor microenvironment enables cancer cells to evade detection by the immune system, for example, by upregulating immunosuppressive proteins (e.g., PDL1, HVEM) expressed by the tumor cells themselves. Leukocytes express specific receptors (also known as immune checkpoint receptors) (e.g., PD1 of PDL1 and BTLA of HVEM) to these inhibitory proteins, so tumor cells can bind to immune checkpoint receptors to suppress immune responses against themselves.

[0006] Many successful immunotherapies for cancer are based on the use of immunomodulatory antibodies that target the immune checkpoints CTLA-4 and PD-1 / PD-L1. However, many FDA-approved antibodies that block immune checkpoint pairs are only effective in about 20% of cancer patients. For responders, treatment is highly effective and durable. However, for the remaining 80% of cancer patients who are non-responders, new immunotherapies targeting other immune checkpoints are needed.

[0007] BTLA (B and T lymphocyte attenuator) is a transmembrane protein that is induced to express during T cell activation. BTLA is also expressed on B cells and dendritic cells. Similar to PD1 and CTLA4, BTLA interacts with B7 homolog BTH4. However, unlike PD-1 and CTLA-4, BTLA also interacts with the tumor necrosis factor receptor family (tumor necrosis familyreceptors). One such receptor is tumor necrosis factor (receptor) superfamily member 14 (TNFRSF14), also known as herpes virus invasion mediator (HVEM). HVEM is a receptor for HSV glycoprotein D involved in viral entry, expressed in hematopoietic cells (including B and T cells) and non-hematopoietic cells (parenchymal cells). In addition, HVEM is expressed in various blood tumors and solid tumors. BTLA-HVEM interactions downregulate T cell responses, such as CD8+T cell responses. Upregulation of BTLA and / or HVEM is a mechanism imposed 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; and upregulation of BTLA in cytotoxic CD8+T cells in the peripheral blood of patients with Hodgkin's lymphoma, B-cell non-Hodgkin's lymphoma, some T-cell non-Hodgkin's lymphoma, and hepatocellular carcinoma. In addition, the lack of HVEM-BTLS signaling will amplify the immune response, leading to dysregulated inflammation and autoimmune diseases. Therefore, regulation of BTLA signaling can target cancer and autoimmune diseases at the same time. Summary of the invention

[0008] In one aspect, the present invention relates to a novel anti-BTLA antibody. In some embodiments, the anti-BTLA antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 1 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 2. In some embodiments, the anti-BTLA antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 3 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 4. In some embodiments, the anti-BTLA antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 5 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 6. In some embodiments, the anti-BTLA antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 7 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 8. In some embodiments, the anti-BTLA antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 9 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 10. In some embodiments, the anti-BTLA antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 11 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 12. In some embodiments, the anti-BTLA antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 13 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 14. In some embodiments, the anti-BTLA antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 15 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 16. In some embodiments, the anti-BTLA antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 17 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 18. In some embodiments, the anti-BTLA antibody comprises 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. In some embodiments, the anti-BTLA antibody comprises 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. In some embodiments, the anti-BTLA antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 23 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 24. In some embodiments, the anti-BTLA antibody comprises 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.In some embodiments, the anti-BTLA antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 27 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 28. In some embodiments, the anti-BTLA antibody comprises 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. In some embodiments, the anti-BTLA antibody comprises 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. In some embodiments, the anti-BTLA antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 33 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 34. In some embodiments, the anti-BTLA antibody comprises 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. In some embodiments, the anti-BTLA antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 37 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 38. In some embodiments, the anti-BTLA antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 39 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 40. In some embodiments, the anti-BTLA antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 41 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 42.

[0009] In some embodiments, the anti-BTLA antibody comprises: a vhCDR1 comprising SEQ ID NO: 47, a vhCDR2 comprising SEQ ID NO: 48, a vhCDR3 comprising SEQ ID NO: 49, a vlCDR1 comprising SEQ ID NO: 50, a vlCDR2 comprising SEQ ID NO: 51, and a vlCDR3 comprising SEQ ID NO: 52. In some embodiments, the anti-BTLA antibody comprises: a vhCDR1 comprising SEQ ID NO: 53, a vhCDR2 comprising SEQ ID NO: 54, a vhCDR3 comprising SEQ ID NO: 55, a vlCDR1 comprising SEQ ID NO: 56, a vlCDR2 comprising SEQ ID NO: 57, and a vlCDR3 comprising SEQ ID NO: 58. In some embodiments, the anti-BTLA antibody comprises: a vhCDR1 comprising SEQ ID NO: 59, a vhCDR2 comprising SEQ ID NO: 60, a vhCDR3 comprising SEQ ID NO: 61, a vlCDR1 comprising SEQ ID NO: 62, a vlCDR2 comprising SEQ ID NO: 63, and a vlCDR3 comprising SEQ ID NO: 64. In some embodiments, the anti-BTLA antibody comprises: a vhCDR1 comprising SEQ ID NO: 65, a vhCDR2 comprising SEQ ID NO: 66, a vhCDR3 comprising SEQ ID NO: 67, a vlCDR1 comprising SEQ ID NO: 68, a vlCDR2 comprising SEQ ID NO: 69, and a vlCDR3 comprising SEQ ID NO: 70. In some embodiments, the anti-BTLA antibody comprises: a vhCDR1 comprising SEQ ID NO: 71, a vhCDR2 comprising SEQ ID NO: 72, a vhCDR3 comprising SEQ ID NO: 73, a vlCDR1 comprising SEQ ID NO: 74, a vlCDR2 comprising SEQ ID NO: 75, and a vlCDR3 comprising SEQ ID NO: 76. In some embodiments, the anti-BTLA antibody comprises: a vhCDR1 comprising SEQ ID NO: 77, a vhCDR2 comprising SEQ ID NO: 78, a vhCDR3 comprising SEQ ID NO: 79, a vlCDR1 comprising SEQ ID NO: 80, a vlCDR2 comprising SEQ ID NO: 81, and a vlCDR3 comprising SEQ ID NO: 82.In some embodiments, the anti-BTLA antibody comprises: a vhCDR1 comprising SEQ ID NO: 83, a vhCDR2 comprising SEQ ID NO: 84, a vhCDR3 comprising SEQ ID NO: 85, a vlCDR1 comprising SEQ ID NO: 86, a vlCDR2 comprising SEQ ID NO: 87, and a vlCDR3 comprising SEQ ID NO: 88. In some embodiments, the anti-BTLA antibody comprises a vhCDR1 comprising SEQ ID NO: 89, a vhCDR2 comprising SEQ ID NO: 90, a vhCDR3 comprising SEQ ID NO: 91, a vlCDR1 comprising SEQ ID NO: 92, a vlCDR2 comprising SEQ ID NO: 93, and a vlCDR3 comprising SEQ ID NO: 94. In some embodiments, the anti-BTLA antibody comprises: a vhCDR1 comprising SEQ ID NO: 95, a vhCDR2 comprising SEQ ID NO: 96, a vhCDR3 comprising SEQ ID NO: 97, a vlCDR1 comprising SEQ ID NO: 98, a vlCDR2 comprising SEQ ID NO: 99, and a vlCDR3 comprising SEQ ID NO: 100. In some embodiments, the anti-BTLA antibody comprises: a vhCDR1 comprising SEQ ID NO: 101, a vhCDR2 comprising SEQ ID NO: 102, a vhCDR3 comprising SEQ ID NO: 103, a vlCDR1 comprising SEQ ID NO: 104, a vlCDR2 comprising SEQ ID NO: 105, and a vlCDR3 comprising SEQ ID NO: 106. In some embodiments, the anti-BTLA antibody comprises: a vhCDR1 comprising SEQ ID NO: 107, a vhCDR2 comprising SEQ ID NO: 108, a vhCDR3 comprising SEQ ID NO: 109, a vlCDR1 comprising SEQ ID NO: 110, a vlCDR2 comprising SEQ ID NO: 111, and a vlCDR3 comprising SEQ ID NO: 112. In some embodiments, the anti-BTLA antibody comprises: a vhCDR1 comprising SEQ ID NO: 113, a vhCDR2 comprising SEQ ID NO: 114, a vhCDR3 comprising SEQ ID NO: 115, a vlCDR1 comprising SEQ ID NO: 116, a vlCDR2 comprising SEQ ID NO: 117, and a vlCDR3 comprising SEQ ID NO: 118.In some embodiments, the anti-BTLA antibody comprises: a vhCDR1 comprising SEQ ID NO: 119, a vhCDR2 comprising SEQ ID NO: 120, a vhCDR3 comprising SEQ ID NO: 121, a vlCDR1 comprising SEQ ID NO: 122, a vlCDR2 comprising SEQ ID NO: 123, and a vlCDR3 comprising SEQ ID NO: 124. In some embodiments, the anti-BTLA antibody comprises: a vhCDR1 comprising SEQ ID NO: 125, a vhCDR2 comprising SEQ ID NO: 126, a vhCDR3 comprising SEQ ID NO: 127, a vlCDR1 comprising SEQ ID NO: 128, a vlCDR2 comprising SEQ ID NO: 129, and a vlCDR3 comprising SEQ ID NO: 130. In some embodiments, the anti-BTLA antibody comprises: a vhCDR1 comprising SEQ ID NO: 131, a vhCDR2 comprising SEQ ID NO: 132, a vhCDR3 comprising SEQ ID NO: 133, a vlCDR1 comprising SEQ ID NO: 134, a vlCDR2 comprising SEQ ID NO: 135, and a vlCDR3 comprising SEQ ID NO: 136. In some embodiments, the anti-BTLA antibody comprises: a vhCDR1 comprising SEQ ID NO: 137, a vhCDR2 comprising SEQ ID NO: 138, a vhCDR3 comprising SEQ ID NO: 139, a vlCDR1 comprising SEQ ID NO: 140, a vlCDR2 comprising SEQ ID NO: 141, and a vlCDR3 comprising SEQ ID NO: 142. In some embodiments, the anti-BTLA antibody comprises: a vhCDR1 comprising SEQ ID NO: 143, a vhCDR2 comprising SEQ ID NO: 144, a vhCDR3 comprising SEQ ID NO: 145, a vlCDR1 comprising SEQ ID NO: 146, a vlCDR2 comprising SEQ ID NO: 147, and a vlCDR3 comprising SEQ ID NO: 148. In some embodiments, the anti-BTLA antibody comprises: a vhCDR1 comprising SEQ ID NO: 149, a vhCDR2 comprising SEQ ID NO: 150, a vhCDR3 comprising SEQ ID NO: 151, a vlCDR1 comprising SEQ ID NO: 152, a vlCDR2 comprising SEQ ID NO: 153, and a vlCDR3 comprising SEQ ID NO: 154.In some embodiments, the anti-BTLA antibody comprises: a vhCDR1 comprising SEQ ID NO: 155, a vhCDR2 comprising SEQ ID NO: 156, a vhCDR3 comprising SEQ ID NO: 157, a vlCDR1 comprising SEQ ID NO: 158, a vlCDR2 comprising SEQ ID NO: 159, and a vlCDR3 comprising SEQ ID NO: 160. In some embodiments, the anti-BTLA antibody comprises: a vhCDR1 comprising SEQ ID NO: 161, a vhCDR2 comprising SEQ ID NO: 162, a vhCDR3 comprising SEQ ID NO: 163, a vlCDR1 comprising SEQ ID NO: 164, a vlCDR2 comprising SEQ ID NO: 165, and a vlCDR3 comprising SEQ ID NO: 166. In some embodiments, the anti-BTLA antibody comprises: vhCDR1 comprising SEQ ID NO: 167, vhCDR2 comprising SEQ ID NO: 168, vhCDR3 comprising SEQ ID NO: 169, vlCDR1 comprising SEQ ID NO: 170, vlCDR2 comprising SEQ ID NO: 171, and vlCDR3 comprising SEQ ID NO: 172.

[0010] In some embodiments, the anti-BTLA antibodies described herein bind to human and / or cynomolgus monkey BTLA.

[0011] In some embodiments, anti-BTLA antibodies act as BTLA antagonists, preventing the binding of BTLA to HVEM and HVEM-mediated inhibition of T cell function. Examples of such antibodies include the following: 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; 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; 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; a vhCDR1 comprising SEQ ID NO: 101, a vhCDR2 comprising SEQ ID NO: 102, a vhCDR3 comprising SEQ ID NO: 103, a vlCDR1 comprising SEQ ID NO: 104, a vlCDR2 comprising SEQ ID NO: 105, and a vlCDR3 comprising SEQ ID NO: 106; a vhCDR1 comprising SEQ ID NO: 107, a vhCDR2 comprising SEQ ID NO: 108, a vhCDR3 comprising SEQ ID NO: 109, a vhCDR4 comprising SEQ ID NO: 110 NO: 110, vlCDR1 comprising SEQ ID NO: 111, and vlCDR3 comprising SEQ ID NO: 112; 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.

[0012] In some embodiments, anti-BTLA antibodies act as BTLA agonists, inhibiting the function of pro-inflammatory immune cells. Examples of such antibodies include the following: 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; 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; 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; a vhCDR1 comprising SEQ ID NO: 131, a vhCDR2 comprising SEQ ID NO: 132, a vhCDR3 comprising SEQ ID NO: 133, a vlCDR1 comprising SEQ ID NO: 134, a vlCDR2 comprising SEQ ID NO: 135, and a vlCDR3 comprising SEQ ID NO: 136; a vhCDR1 comprising SEQ ID NO: 137, a vhCDR2 comprising SEQ ID NO: 138, a vhCDR3 comprising SEQ ID NO: 139, a vhCDR4 comprising SEQ ID NO: 140 NO: 140, vlCDR1 comprising SEQ ID NO: 141, and vlCDR3 comprising SEQ ID NO: 142; 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.

[0013] In some embodiments, the anti-BTLA antibodies described herein include a constant region with an amino acid sequence at least 90% identical to that of human IgG. In some embodiments, the IgG is selected from IgG1, IgG2, IgG3, or IgG4. In some embodiments, the IgG is IgG2.

[0014] In another aspect, the invention relates to a nucleic acid composition encoding any of the anti-BTLA antibodies described herein. In some embodiments, the nucleic acid composition comprises a first nucleic acid comprising SEQ ID NO: 185 and a second nucleic acid comprising SEQ ID NO: 186. In some embodiments, the nucleic acid composition comprises a first nucleic acid comprising SEQ ID NO: 187 and a second nucleic acid comprising SEQ ID NO: 188. In some embodiments, the nucleic acid composition comprises a first nucleic acid comprising SEQ ID NO: 189 and a second nucleic acid comprising SEQ ID NO: 190. In some embodiments, the nucleic acid composition comprises a first nucleic acid comprising SEQ ID NO: 191 and a second nucleic acid comprising SEQ ID NO: 192. In some embodiments, the nucleic acid composition comprises a first nucleic acid comprising SEQ ID NO: 193 and a second nucleic acid comprising SEQ ID NO: 194. In some embodiments, the nucleic acid composition comprises a first nucleic acid comprising SEQ ID NO: 195 and a second nucleic acid comprising SEQ ID NO: 196. In some embodiments, the nucleic acid composition comprises a first nucleic acid comprising SEQ ID NO: 197 and a second nucleic acid comprising SEQ ID NO: 198. In some embodiments, the nucleic acid composition comprises a first nucleic acid comprising SEQ ID NO: 199 and a second nucleic acid comprising SEQ ID NO: 200. In some embodiments, the nucleic acid composition comprises a first nucleic acid comprising SEQ ID NO: 201 and a second nucleic acid comprising SEQ ID NO: 202. In some embodiments, the nucleic acid composition comprises a first nucleic acid comprising SEQ ID NO: 203 and a second nucleic acid comprising SEQ ID NO: 204. In some embodiments, the nucleic acid composition comprises a first nucleic acid comprising SEQ ID NO: 205 and a second nucleic acid comprising SEQ ID NO: 206. In some embodiments, the nucleic acid composition comprises a first nucleic acid comprising SEQ ID NO: 207 and a second nucleic acid comprising SEQ ID NO: 208. In some embodiments, the nucleic acid composition comprises a first nucleic acid comprising SEQ ID NO: 209 and a second nucleic acid comprising SEQ ID NO: 210. In some embodiments, the nucleic acid composition comprises a first nucleic acid comprising SEQ ID NO: 211 and a second nucleic acid comprising SEQ ID NO: 212. In some embodiments, the nucleic acid composition comprises a first nucleic acid comprising SEQ ID NO: 213 and a second nucleic acid comprising SEQ ID NO: 214. In some embodiments, the nucleic acid composition comprises a first nucleic acid comprising SEQ ID NO: 215 and a second nucleic acid comprising 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.

[0015] Another aspect of the invention relates to an expression vector composition comprising any 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 embodiments, the first nucleic acid and the second nucleic acid are contained in a single expression vector.

[0016] Another aspect of the invention relates to a host cell comprising any of the expression vectors described herein. Also provided is a method for preparing an anti-BTLA antibody, the method comprising culturing the host cell under conditions wherein the antibody is expressed and recovering the antibody.

[0017] In another aspect, the invention relates to a composition comprising any of the anti-BTLA antibodies described herein and a pharmaceutically acceptable carrier or diluent.

[0018] Also described is a method of modulating an immune response in a subject, the method comprising administering to the subject an effective amount of any of the anti-BTLA antibodies described herein or any of the compositions described herein. 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 or a pharmaceutical composition thereof that acts as a BTLA antagonist. In some embodiments, the method inhibits an immune response in the subject, and the method comprises administering to the subject an effective amount of an anti-BTLA antibody or a pharmaceutical composition thereof that acts as a BTLA agonist.

[0019] In some embodiments, the method stimulates an immune response in a subject, and the method comprises administering to the subject an effective amount of an anti-BTLA antibody, wherein the anti-BTLA antibody comprises: 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 a vhCDR1 comprising SEQ ID NO: 107, a vhCDR2 comprising SEQ ID NO: 108, a vhCDR3 comprising SEQ ID NO: 109, a vlCDR1 comprising SEQ ID NO: 110, a vlCDR2 comprising SEQ ID NO: 111, and a vlCDR3 comprising SEQ ID NO: 112.

[0020] In some embodiments, the method stimulates an immune response in a subject, and the method comprises administering to the subject an effective amount of an anti-BTLA antibody, wherein the anti-BTLA antibody comprises: 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 a vhCDR1 comprising SEQ ID NO: 101, a vhCDR2 comprising SEQ ID NO: 102, a vhCDR3 comprising SEQ ID NO: 103, a vlCDR1 comprising SEQ ID NO: 104, a vlCDR2 comprising SEQ ID NO: 105, and a vlCDR3 comprising SEQ ID NO: 106.

[0021] In some embodiments, the method stimulates an immune response in a subject, and the method comprises administering to the subject an effective amount of an anti-BTLA antibody, wherein the anti-BTLA antibody comprises: 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 a vhCDR1 comprising SEQ ID NO: 119, a vhCDR2 comprising SEQ ID NO: 120, a vhCDR3 comprising SEQ ID NO: 121, a vlCDR1 comprising SEQ ID NO: 122, a vlCDR2 comprising SEQ ID NO: 123, and a vlCDR3 comprising SEQ ID NO: 124.

[0022] In some embodiments, the method inhibits an immune response in a subject, and the method comprises administering to the subject an effective amount of an anti-BTLA antibody, wherein the anti-BTLA antibody comprises: 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 a vhCDR1 comprising SEQ ID NO: 137, a vhCDR2 comprising SEQ ID NO: 138, a vhCDR3 comprising SEQ ID NO: 139, a vlCDR1 comprising SEQ ID NO: 140, a vlCDR2 comprising SEQ ID NO: 141, and a vlCDR3 comprising SEQ ID NO: 142.

[0023] In some embodiments, the method inhibits an immune response in a subject, and the method comprises administering to the subject an effective amount of an anti-BTLA antibody, wherein the anti-BTLA antibody comprises: 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 a vhCDR1 comprising SEQ ID NO: 149, a vhCDR2 comprising SEQ ID NO: 150, a vhCDR3 comprising SEQ ID NO: 151, a vlCDR1 comprising SEQ ID NO: 152, a vlCDR2 comprising SEQ ID NO: 153, and a vlCDR3 comprising SEQ ID NO: 154.

[0024] In some embodiments, the method inhibits an immune response in a subject, and the method comprises administering to the subject an effective amount of an anti-BTLA antibody, wherein the anti-BTLA antibody comprises: 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 a vhCDR1 comprising SEQ ID NO: 131, a vhCDR2 comprising SEQ ID NO: 132, a vhCDR3 comprising SEQ ID NO: 133, a vlCDR1 comprising SEQ ID NO: 134, a vlCDR2 comprising SEQ ID NO: 135, and a vlCDR3 comprising SEQ ID NO: 136.

[0025] On the other hand, the present invention relates to a method for treating cancer in a subject, and the method comprises 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 antagonist. In some embodiments, the cancer to be treated upregulates HVEM compared to the corresponding non-cancerous tissue. In some embodiments, the subject to be treated expresses high levels of BTLA on T cells. The cancer to be treated may be gastric cancer. In some embodiments, anti-BTLA antibodies are used in combination with one or more additional therapeutic agents to treat cancer. In some embodiments, such anti-cancer therapeutic agents are other immune checkpoint inhibitors, such as Ipilimumab, Nivolumab, Pembrolizumab, Avelumab, Durvalumab, and Atezolizumab.

[0026] In another aspect, the invention relates to a method of treating an autoimmune disease in a subject, and the method comprises administering to the subject an effective amount of an anti-BTLA antibody or composition thereof as described herein, wherein the anti-BTLA antibody acts as a BTLA agonist. In some embodiments, the subject to be treated has low levels of HVEM on autoreactive T cells residing at the site of development of the autoimmune disease. In some embodiments, the autoimmune disease is multiple sclerosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] The present invention is best understood from the following detailed description when read in conjunction with the accompanying drawings, which include the following:

[0028] Figure 1 Binding of anti-BTLA antibody 13-F7A to human BTLA was shown 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.

[0029] Figure 2A-2C The figure is a bar graph showing the binding of the anti-BTLA antibody 13-F7A to BTLA by FACS analysis. Figure 2A Shown is the binding of 13-F7A to human BTLA expressed on HEK-293 cells. Figure 2B Shown is the binding of 13-F7A to cynomolgus monkey BTLA expressed on HEK-293 cells. Figure 2C is a negative control showing binding of 13-F7A to HEK-293 cells. Antibodies at 10 μg / ml and 1 μg / ml were tested along with a secondary antibody only control.

[0030] Figure 3 The anti-BTLA antibody 13-F7A was shown to inhibit human BTLA / HVEM interaction by ELISA. Anti-BTLA antibodies 4C7 and 8D5 and human IgG were used as controls.

[0031] Figure 4 The binding affinity of the anti-BTLA antibody 13-F7A to human BTLA was measured by biofilm interferometry. 13-F7A was immobilized on the biosensor tip and tested at 166.7, 55.6, 18.5, and 6.17 nM of recombinant human BTLA. The measured dissociation constant K D It is 5.4×10E-9M.

[0032] Figures 5A-5F Shown is a dose-response curve of EBs stimulating 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 Anti-BTLA antibodies 16-I20A, 15-C19A and 16-H16A and their antagonist effects on T cell function are shown. Figure 5D-5F Anti-BTLA antibodies 12-I8A, 8-M23A and 13-F7A and their agonist effects on T cell function are shown. DETAILED DESCRIPTION

[0033] The present disclosure provides novel anti-BTLA antibodies. The anti-BTLA antibodies described herein bind to human and / or cynomolgus macaque BTLA. In some embodiments, the anti-BTLA antibodies bind to human and / or cynomolgus macaque BTLA with high affinity. In some embodiments, the anti-BTLA antibodies act as functional BTLA antagonists, and when bound to BTLA, the anti-BTLA antibodies prevent the interaction of BTLA with HVEM and prevent HVEM-mediated inhibition of T cell function. In some embodiments, the anti-BTLA antibodies act as functional BTLA agonists, and when bound to BTLA, the anti-BTLA antibodies inhibit T cell function. The present disclosure also provides methods of using such antibodies to modulate the immune response of a subject, and, for example, for the treatment of cancer or autoimmune diseases. In addition, the present disclosure describes nucleic acids encoding these antibodies and host cells comprising such nucleic acids.

[0034] To facilitate understanding of the present invention, a number of terms and expressions are defined below.

[0035] As used herein, each of the following terms has the meaning associated with it in this section.

[0036] As used herein, the articles "a" and "an" refer to one or more (eg, at least one) of the grammatical objects of the article. For example, "an element" refers to one element or a plurality of elements.

[0037] As used herein, "about" refers to a measurable variable, such as an amount, duration, etc., and is meant to include variations of ±20% or ±10% from the specified value, more preferably ±5%, even more preferably ±1%, and still more preferably ±0.1%, which variations are suitable for performing the methods of the present disclosure.

[0038] As used herein, "ablation" means reducing or removing an activity. Thus, for example, "ablation of FcγR binding" means that the Fc region amino acid variant has less than 50% of the initial binding, preferably less than 70-80-90-95-98% loss of activity compared to the Fc region without the specific variant, and generally, the activity is below the level of binding detectable in a Biacore analysis.

[0039] As used herein, "ADCC" or "antibody-dependent cell-mediated cytotoxicity" refers to a cell-mediated reaction in which nonspecific cytotoxic cells expressing FcγRs recognize bound antibody on a target cell and subsequently cause lysis of the target cell. ADCC is associated with binding to FcγRIIIa; increased binding to FcγRIIIa results in increased ADCC activity. As discussed herein, many embodiments of the present invention completely eliminate ADCC activity.

[0040] As used herein, "ADCP" or antibody dependent cell-mediated phagocytosis refers to a cell-mediated reaction in which nonspecific cytotoxic cells expressing FcγRs recognize bound antibody on a target cell and subsequently induce phagocytosis of the target cell.

[0041] Herein, "antigen binding domain" or "ABD" refers to a set of six complementary determining regions (CDRs) that, when present as part of a polypeptide sequence, specifically bind to a target antigen as discussed herein. Thus, an "antigen binding domain" binds to a target antigen as described herein. As known in the art, these CDRs are generally present as a first set of variable heavy CDRs (vhCDR or VHCDR or CDR-HC) and a second set of variable light CDRs (vlCDR or VLCDR or CDR-LC), each comprising three CDRs: vhCDR1, vhCDR2, vhCDR3 of the heavy chain and vlCDR1, vlCDR2 and vlCDR3 of the light chain. The CDRs are present in the variable heavy domain and the variable light domain, respectively, and together form an Fv region. Thus, in some cases, the six CDRs of the antigen binding domain are composed of a variable heavy chain and a variable light chain. In the "Fab" format, a set of 6 CDRs consists of two different polypeptide sequences, a variable heavy domain (vh or VH; comprising vhCDR1, vhCDR2 and vhCDR3) and a variable light domain (vl or VL; comprising vlCDR1, vlCDR2 and vlCDR3), the C-terminus of the vh domain being connected to the N-terminus of the CH1 domain of the heavy chain, and the C-terminus of the vl domain being connected to the N-terminus of the constant light domain (and thus forming a light chain). In the scFv format, the VH and VL domains are covalently linked to a single polypeptide sequence, generally by using a linker as described herein, which can be (starting from the N-terminus) vh-linker-vl or vl-linker-vh, generally preferably the former (including optional domain linkers on each side), depending on the format used. As is known in the art, the CDRs are separated by framework regions in each of the variable heavy and light domains: for the light variable region, these are FR1-vlCDR1-FR2-vlCDR2-FR3-vlCDR3-FR4, for the heavy variable region, these are FR1-vlCDR1-FR2-vlCDR2-FR3-vlCDR3-FR4, the framework regions showing high identity to human germline sequences. Antigen binding domains of the invention include Fab, Fv and scFv.

[0042] "Joint" herein refers to the joint used in scFv and / or other antibody structures. Generally, there are a lot of suitable scFv joints that can be used, including traditional peptide bonds produced by recombinant technology. The joint peptide can mainly include the following amino acid residues: Gly, Ser, Ala or Thr. The joint peptide should have a length sufficient to connect two molecules so that they present the correct conformation relative to each other to retain the required activity. In one embodiment, the length of the joint is about 1 to about 50 amino acids, preferably about 1 to about 30 amino acids in length. In one embodiment, a joint with a length of 1 to 20 amino acids can be used, and in some embodiments, it can be found that the use of a joint with a length of about 5 to about 10 amino acids. Useful joints include glycine-serine polymers, including, for example (GS) n, (GSGGS) n, (GGGGS) n and (GGGS) n (wherein n is an integer of at least 1 (and generally from 3 to 4)), glycine-alanine polymers, alanine-serine polymers and other flexible joints. Alternatively, various non-protein polymers, including but not limited to polyethylene glycol (PEG), polypropylene glycol, polyoxyalkylenes or copolymers of polyethylene glycol and polypropylene glycol, can find use as linkers, i.e. can find use as linkers. Other linker sequences can include any sequence of any length of the CL / CH1 domain but not all residues of the CL / CH1 domain; for example, the first 5-12 amino acid residues of the CL / CH1 domain. The linker can be derived from an immunoglobulin light chain, such as Cκ or Cλ. The linker can be derived from an immunoglobulin heavy chain of any isotype antigen, including, for example, Cγ1, Cγ2, Cγ3, Cγ4, Cα1, Cα2, Cδ, Cε and Cμ. The linker sequence can also be derived from other proteins (e.g., Ig-like proteins (e.g., TCR, FcR, KIR)), hinge region derived sequences and other natural sequences from other proteins. In some embodiments, the linker is a "domain linker" for connecting any two domains as described herein together. While any suitable linker can be used, many embodiments utilize glycine-serine polymers, including, for example, (GS)n, (GSGGS)n, (GGGGS)n, and (GGGS)n, wherein n is an integer of at least 1 (and generally from 3 to 4 to 5), as well as any peptide sequence that allows recombinant attachment of two domains of sufficient length and flexibility to allow each domain to retain its biological function.

[0043] The term "antibody" is used in the broadest sense and includes, for example, a complete immunoglobulin or an antigen binding portion. The antigen binding portion can be produced by recombinant DNA technology or enzymatic or chemical cleavage of a complete antibody. Thus, the term antibody includes traditional tetrameric antibodies of 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 as described herein.

[0044] "Modification" herein refers to amino acid substitutions, insertions and / or deletions in a polypeptide sequence or changes in a portion chemically attached to a protein. For example, a modification can be a change in a carbohydrate or PEG structure attached to a protein. "Amino acid modification" herein refers to amino acid substitutions, insertions and / or deletions in a polypeptide sequence. For clarity, unless otherwise indicated, amino acid modifications are typically amino acids encoded by DNA, such as the 20 amino acids with codons in DNA and RNA.

[0045] As used herein, "amino acid substitution" or "replacement" refers to the substitution of an amino acid at a specific position in a parent polypeptide sequence having a different amino acid. In particular, in some embodiments, the replacement of an amino acid does not occur naturally at a specific position, nor does it occur naturally in an organism or in any organism. For example, an M252Y substitution refers to a variant polypeptide, in this case, an Fc variant, in which the methionine at position 252 is replaced by tyrosine. For clarity, a protein that is designed into a nucleic acid coding sequence but does not change the starting amino acid (e.g., replacing CGG (encoding arginine) with CGA (still encoding arginine) to increase expression levels in the host organism) is not an "amino acid substitution"; that is, although a new gene encoding the same protein is created, if the protein has the same amino acid at the start at a specific position, it is not an amino acid substitution.

[0046] As used herein, "variant protein" or "protein variant" or "variant" refers to a protein that is distinguished from a parent protein by the modification of at least one amino acid. A protein variant may refer to the protein itself, a composition comprising the protein, or an amino acid sequence encoding the protein. Preferably, the protein variant has at least one amino acid modification compared to the parent protein, for example, from about 1 to 70 amino acid modifications, preferably from about 1 to about 5 amino acid modifications compared to the parent protein. As described below, in some embodiments, the parent polypeptide, such as an Fc parent polypeptide, is a human wild-type sequence, such as an Fc region from IgG1, IgG2, IgG3 or IgG4. The protein variant sequence herein will 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 variant protein may refer to the variant protein itself, a composition comprising the protein variant, or a DNA sequence encoding the protein.

[0047] Thus, as used herein, "antibody variant" or "variant antigen" refers to an antibody that is distinguished from a parent antibody by at least one amino acid modification, as used herein, "IgG variant" or "variant IgG" refers to an antibody that is distinguished from a parent IgG (also, in many cases, from a human IgG sequence) by at least one amino acid modification, as used herein, "immunoglobulin variant" or "variant immunoglobulin" refers to an immunoglobulin sequence that is distinguished from a parent immunoglobulin sequence by at least one amino acid modification. As used herein, "Fc variant" or "variant Fc" refers to a protein that contains amino acid modifications within the Fc domain. The Fc variants in the present invention are defined according to the amino acid modifications that constitute the Fc variants. Thus, for example, M252Y or 252Y is an Fc variant having a tyrosine substituted at position 252 relative to a parent Fc polypeptide, wherein numbering is according to the EU index. Similarly, M252Y / S254T / T256E defines an Fc variant having substitutions M252Y, S254T, and T256E relative to a parent Fc polypeptide. The identity of wild-type amino acid may not be specified, wherein the aforementioned variant is referred to as 252Y / 254T / 256E. It should be noted that the order of the substitution provided is arbitrary, that is, for example, 252Y / 254T / 256E is the same Fc variant as 254T / 252Y / 256E, etc. All positions related to antibodies discussed in the present invention, unless otherwise indicated, the position numbering of amino acids is according to Kabat to number the variable region, and according to the EU index to number the constant region (including the Fc region). The EU index or the EU index or EU numbering scheme as in Kabat 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). Modification can be increase, deletion or replacement. Replacement can include natural amino acids, and in some cases, synthetic amino acids.

[0048] As used herein, "protein" herein refers to at least two covalently linked amino acids, including proteins, polypeptides, oligopeptides and peptides. The peptidyl group may include natural amino acids and peptide bonds.

[0049] As used herein, "Fab" or "Fab region" refers to a polypeptide comprising VH, CH1, VL and CL immunoglobulin domains. Fab can refer to this region in isolation, or in the context of a full-length antibody, antibody fragment or Fab fusion protein.

[0050] As used herein, "Fv" or "Fv fragment" or "Fv region" refers to a polypeptide comprising the VL and VH domains of a single antigen binding domain (ABD). As will be appreciated by those skilled in the art, these are typically composed of two chains, or may be joined (generally using a linker as discussed herein) to form an scFv.

[0051] As used herein, "amino acid" and "amino acid identity" refer to one of the 20 natural amino acids encoded by DNA and RNA.

[0052] As used herein, "effector function" refers to the biochemical events resulting from the interaction of an antibody Fc region and an Fc receptor or ligand. Effector functions include, but are not limited to, ADCC, ADCP, and CDC.

[0053] As used herein, "Fcγ receptor," "FcγR," or "FcgammaR" refers to any member of the family of proteins that bind the IgG antibody Fc region and are encoded by the FcγR gene. In humans, this family includes, but is not limited to, FcγRI (CD64), including subtypes FcγRIa, FcγRIb, and FcγRIc; FcγRII (CD32), including subtypes FcγRIIa (including allotypes H131 and R131), FcγRIIb (including FcγRIIb-1 and FcγRIIb-2), and FcγRIIc; and FcγRIII (CD16), including subtypes FcγRIIIa (including allotypes V158 and F158) and FcγRIIIb (including allotypes FcγRIIb-NA1 and FcγRIIb-NA2) (Jefferis et al., 2002, Immunol Lett 82:57-65, incorporated by reference in its entirety), as well as any undiscovered human FcγR or FcγR subtype or allotype. In some cases, as described herein, binding to one or more FcγR receptors is reduced or eliminated. For example, reduced binding to FcγRIIIa reduces ADCC, and in some cases, reduced binding to both FcγRIIIa and FcγRIIb is desirable.

[0054] As used herein, "FcRn" or "neonatal Fc receptor" refers to a protein that binds IgG antibodies and is at least partially encoded by the FcRn gene. FcRn can be from any organism, including but not limited to humans, mice, rats, rabbits, and monkeys. As is known in the art, a functional FcRn protein includes two polypeptides, commonly 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 specified herein, FcRn or FcRn protein refers to the complex of the FcRn heavy chain and beta-2-microglobulin.

[0055] As used herein, "parent polypeptide" refers to a starting polypeptide that is subsequently modified to produce a variant. A parent polypeptide can be a native polypeptide or a variant or an engineered version of a native polypeptide. A parent polypeptide can refer to the polypeptide itself, a composition comprising a parent polypeptide, or an amino acid sequence encoding the polypeptide. Thus, as used herein, a "parent immunoglobulin" refers to an unmodified immunoglobulin polypeptide that is modified to produce a variant, and as used herein, a "parent antibody" refers to an unmodified antibody that is modified to produce a variant antibody. It should be understood that "parent antibodies" include known commercial recombinantly produced antibodies as described below.

[0056] The "heavy chain constant region" herein refers to the CH1-hinge-CH2-CH3 portion of an antibody, typically from human IgG1, IgG2 or IgG4.

[0057] As used herein, "target antigen" refers to a molecule that is specifically bound by the variable region of a given antibody. In this case, the target antigen is the BTLA protein.

[0058] As used herein, "target cell" refers to a cell that expresses an antigen of interest.

[0059] As used herein, "variable region" refers to the region of an immunoglobulin comprising one or more Ig domains, generally encoded by any of the V.κ, Vλ and / or VH genes that constitute the kappa, lambda and heavy chain immunoglobulin genetic loci, respectively.

[0060] As used herein, "wild type" or "WT" refers to an amino acid sequence or nucleotide sequence found in nature, including allelic variations. A WT protein has an amino acid sequence or nucleotide sequence that has not been intentionally modified.

[0061] As used herein, "position" refers to a position in the sequence of a protein. Positions may be numbered sequentially, or according to an established format, such as the EU index for antibody numbering.

[0062] As used herein, "residue" refers to a position in a protein or its associated amino acid identity. For example, asparagine 297 (also known as Asn297 or N297) is the residue at position 297 in the human antibody IgG1.

[0063] The antibodies of the present invention are generally recombinant. "Recombinant" means that the antibodies are produced in exogenous host cells using recombinant nucleic acid technology.

[0064] "Percentage (%) of amino acid sequence identity" with respect to a protein sequence is defined as the percentage of amino acid residues in a candidate sequence identical to the amino acid residues of a particular (parental) sequence, after aligning the amino acid sequences and introducing intervals, if necessary, to achieve the maximum percentage of sequence identity, without considering any conservative substitutions as part of the sequence identity. Alignment for determining percentage of amino acid sequence identity can be implemented in various ways within the art, for example, using public computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine suitable parameters for measuring alignment, including algorithms required for achieving maximum alignment over the full length of the compared sequences. A specific program is the ALIGN-2 program described in paragraphs

[0279] -

[0280] in US Publication No. 20160244525, which is incorporated herein by reference. Another suitable alignment for nucleic acid sequences is provided by the local homology algorithm of Smith and Waterman, 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, Atlas of Protein Sequences and Structure, MO Dayhoff ed., 5 suppl. 3: 353-358, National Biomedical Research Foundation, Washington, DC, USA and normalized by Gribskov, Nucl. Acids Res. 14 (6): 6745-6763 (1986).

[0065] The example of executing this algorithm to determine the percentage identity of a sequence is provided by the Genetics Computer Group (Genetics Computer Group, Madison, WI) in the "BestFit" utility. The default value parameters for this method are described in the Wisconsin Sequence Analysis Package Program Manual, Version 8 (1995) (available from the Genetics Computer Group, Madison, WI). Another method for setting up percentage identity in the context of the present invention is to use the MPSRCH program package of the University of Edinburgh, which is developed by John F.Collins and Shane S.Sturrok and issued by IntelliGenetics, Inc. (Mountain View, CA). From this set of program packages, the Smith-Waterman algorithm can be adopted, wherein the default value parameters are used for the score table (for example, 12 points for the open penalty of the gap, 1 point for the extension penalty of the gap, and 6 for the gap). From the data generated, the "match (Match)" numerical response "sequence identity (sequence identity)". Other suitable percentage identities or similarities between the calculated sequences are generally known in the art, for example, another comparison program is the BLAST used with the default value parameters. For example, BLASTN and BLASTP can use the following default parameters: genetic code = standard; filter = none; strand = both; cut off = 60; expect = 10; matrix = BLOSUM62; descriptions = 50 sequences; sort by = HIGH SCORRE; databases = non-redundant, GenBank + EMBL + DDBJ + PDB + GenBank CDS translation + Swiss protein + Spupdate + PIR. Details of these programs can be found on the Internet at http: / / blast.ncbi.nlm.nih.gov / Blast.cgi.

[0066] The degree of identity between an amino acid sequence of the invention ("sequence of the invention") and a parental amino acid sequence is calculated as the number of exact matches of the two sequences aligned divided by the length of the "sequence of the invention" or the length of the parental sequence, whichever is the shortest. The result is expressed as a percentage identity.

[0067] In some embodiments, two or more amino acid sequences are at least 50%, 60%, 70%, 80% or 90% identical. In some embodiments, two or more amino acid sequences are at least 95%, 97%, 98%, 99% or even 100% identical.

[0068] "Specific binding" or "specifically binds to" or "specific for" a particular antigen or epitope refers to binding that is significantly different from non-specific interactions. Specific binding can be measured, for example, by determining the binding of a molecule compared to the binding of a control molecule, which is typically of similar molecular structure and does not have the binding affinity. For example, specific binding can be determined by competition with a control molecule similar to the target.

[0069] The terms "kassoc" or "Ka" as used herein are intended to refer to the association rate of a particular antibody-antigen interaction, while the terms "Kdis" or "Kd" as used herein are intended to refer to the dissociation rate of a particular antibody-antigen interaction. D ” is intended to refer to the dissociation constant, obtained from the ratio of Kd to Ka (Kd / Ka) and expressed as a molar concentration (M). The K of an antibody D The K value can be determined using established methods in the art. In some embodiments, the K value for determining an antibody is D The most important method is to use surface plasmon resonance technology, for example, by using a biosensor system, such as In some embodiments, the K of an antibody is determined by biofilm surface interferometry. D In some embodiments, K D The value is measured using an immobilized substance. In other embodiments, K D The K value is measured using an immobilized antibody (e.g., a parental mouse antibody, a chimeric antibody, or a humanized antibody variant). In certain embodiments, K D The value is measured using a bivalent binding mode. In other embodiments, K D Values ​​were measured using the monovalent binding mode.

[0070] "Disease" includes a health condition in an animal, including a human, in which the animal is unable to maintain homeostasis and in which the animal's health condition would continue to deteriorate if the disease condition was not ameliorated.

[0071] In contrast, a "disorder" in an animal (including a human) includes a healthy state in which the animal is able to maintain homeostasis, but in which the animal's health is not as good as it would be without the disorder. Without treatment, the disorder does not necessarily lead to a further deterioration of the animal's health.

[0072] The terms "treatment", "treating", "treat" and the like refer to obtaining a desired pharmacological and / or physiological effect. The effect may be preventive in terms of completely or partially preventing a disease or its symptoms or reducing the likelihood of a disease or its symptoms and / or may be therapeutic in terms of partially or completely curing a disease and / or side effects caused by the disease. "Treatment" as used herein covers the treatment of any disease in mammals, especially humans, including: (a) preventing the disease from occurring in subjects who may be susceptible to the disease but have not been diagnosed with the disease; (b) inhibiting the disease, i.e., preventing its development or progression; and (c) alleviating the disease, i.e., causing regression of the disease and / or alleviation of one or more symptoms of the disease. "Treatment" also refers to the delivery of an agent to provide a pharmacological effect, even in the absence of a disease or condition. For example, "treatment" includes the delivery of a composition that can elicit an immune response or confer immunity in the absence of a disease condition, for example, in the case of a vaccine.

[0073] As used herein, the term "mammal" refers to any mammal, including but not limited to rodent mammals, such as mice and hamsters, and lagomorpha mammals, such as rabbits. In certain embodiments, the mammal is a carnivorous animal, including felines (cats) and canines (dogs). In certain embodiments, the mammal is an artiodactyl, including bovines (cows) and suids (pigs), or a perissodactyl, including equines (horses). Most preferably, the mammal is a primate, a New World monkey (Ceboids) or a Simoids (monkeys) or anthropoids (humans and apes). In certain embodiments, the mammal is a human. In certain embodiments, the mammal is a cynomoglus monkey.

[0074] The term "regression" as used herein and words derived from the term do not necessarily mean 100% or complete regression. On the contrary, there are different degrees of regression, where those of ordinary skill in the art recognize that there are potential benefits or therapeutic effects. In this regard, the disclosed methods can provide regression of any level of any amount of cancer in a mammal. Moreover, the regression provided by the methods of the present invention can include the regression of one or more conditions or symptoms of a disease (e.g., cancer). Also, for the purposes of this article, "regression" can include delaying the onset of the disease, delaying the onset of symptoms, and / or delaying the onset of its condition. With respect to progressive diseases or disorders, "regression" can include slowing the progression of the disease or disorder, slowing the progression of the symptoms of the disease or disorder, and / or slowing the progression of its condition.

[0075] An "effective amount" or "therapeutically effective amount" of a composition includes an amount of the composition sufficient to provide a beneficial effect to a subject to which the composition has been administered. An "effective amount" of a delivery vehicle includes an amount sufficient to effectively bind or deliver the composition.

[0076] "Individual" or "host" or "subject" or "patient" is any mammalian subject, especially a human, in need of diagnosis, treatment or therapy. Other subjects may include cynomolgus monkeys, cows, dogs, cats, guinea pigs, rabbits, rats, mice, horses, etc.

[0077] As used herein, the term "in combination with" refers to the following uses, for example, wherein the first therapy is administered throughout the administration of the second therapy; wherein the time period for administering the first therapy overlaps with the administration of the second therapy, for example, wherein the administration of the first therapy begins before the administration of the second therapy and ends before the administration of the second therapy ends; wherein the administration of the second therapy begins before the administration of the first therapy begins and ends before the administration of the first therapy ends; wherein the administration of the first therapy begins before the administration of the second therapy begins and ends before the administration of the first therapy ends; wherein the administration of the second therapy begins before the administration of the first therapy begins and ends before the administration of the first therapy ends. Thus, "in combination with" may also refer to a regimen involving the administration of two or more therapies. As used herein, "in combination with" may also refer to the administration of two or more therapies, which may be administered with the same or different formulations, by the same or different routes, and with the same or different dosage types.

[0078] "Encoding" includes the inherent properties of a specific sequence of nucleotides in a polynucleotide (e.g., a gene, cDNA, or mRNA) that serves as a template for the synthesis of other polymers and macromolecules in biological processes, having a defined nucleotide sequence (i.e., rRNA, tRNA, and mRNA) or a defined amino acid sequence and the resulting biological properties. Thus, a gene encodes a protein, and, for example, the transcription and translation of an mRNA corresponds to the production of a protein by a gene in a cell or other biological system. The coding strand, which has the same nucleotide sequence as the mRNA sequence and is often provided in a sequence listing, and the non-coding strand used as a template for transcription of a gene or cDNA can refer to a protein or other product that encodes the gene or cDNA.

[0079] The term "nucleic acid" includes RNA or DNA molecules having more than one nucleotide in any form, including single-stranded, double-stranded, oligonucleotide or polynucleotide. The term "nucleotide sequence" includes the order of nucleotides in an oligonucleotide or polynucleotide in a single-stranded form of a nucleic acid.

[0080] "Nucleic acid construct" refers to a nucleic acid sequence that is constructed to contain one or more functional units that are not found together in nature. Examples include circular, linear, double-stranded, extrachromosomal DNA molecules (plasmids), cosmids (plasmids containing COD sequences from lambda phage), viral genomes containing non-natural nucleic acid sequences, etc.

[0081] The term "operably linked" as used herein includes a polynucleotide having a functional relationship with a second polynucleotide, such as a single-stranded or double-stranded nucleic acid portion comprising two polynucleotides arranged in a nucleic acid portion in such a manner that at least one of the two polynucleotides is able to exert a physiological effect on the other polynucleotide by its characteristics. By way of example, a promoter operably linked to a gene coding region is able to initiate transcription of the coding region. When indicating an operably linked sequence, the order of designation is unimportant. For example, the phrases: "a promoter is operably linked to a nucleotide sequence" and "a nucleotide sequence is operably linked to a promoter" are used interchangeably herein and are considered to be equivalent. In some cases, when the nucleic acid encoding a desired protein also includes a promoter / regulatory sequence, the promoter / regulatory sequence is located at the 5' end of the desired protein coding sequence so that it drives the expression of the desired protein in the cell.

[0082] The terms "oligonucleotide", "polynucleotide" and "nucleic acid molecule" used interchangeably herein refer to a polymeric form of nucleotides of any length (ribonucleotides or deoxyribonucleotides). Thus, the term includes, but is not limited to, single-stranded, double-stranded or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids or polymers comprising purine bases and pyrimidine bases or other natural, chemically or biochemically modified, non-natural or derived nucleotide bases. The backbone of the polynucleotide may contain sugar or phosphate groups (commonly found in RNA or DNA) or modified or substituted sugar or phosphate groups.

[0083] The term "recombinant" as applied to polynucleotides means that the polynucleotide is the product of a combination of various cloning, restriction or ligation steps and other steps, resulting in a construct that is different and / or distinct from a polynucleotide found in nature. The term includes duplications of the original nucleotide construct and progeny of the original viral construct, respectively.

[0084] The term "promoter" as used herein includes a DNA sequence that is operably connected to a nucleic acid sequence to be transcribed (e.g., a nucleic acid sequence encoding a desired molecule). Typically, a promoter is located upstream of a nucleic acid sequence to be transcribed and provides a specific binding site for RNA polymerase and other transcription factors.

[0085] A "vector" is capable of transporting a gene sequence to a target cell. In general, "vector construct," "expression vector," and "gene transfer vector" refer to a nucleic acid construct capable of directing the expression of a gene of interest, and which is capable of transporting a gene sequence to a target cell, either by genomic integration of all or part of the vector or by transient or genetic maintenance of the vector as an extrachromosomal element. Thus, the term includes cloning and expression vectors, as well as integrating vectors.

[0086] The term "regulatory element" as used herein includes nucleotide sequences that control some aspects of the expression of a nucleic acid sequence. Examples of regulatory elements illustratively include enhancers, internal ribosome entry sites (IRES), introns, replication origins, polyadenylation signals (pA), promoters, enhancers, transcription termination sequences, and upstream regulatory regions that contribute to the replication, transcription, and / or post-transcriptional processing of nucleic acid sequences. In some cases, regulatory elements may also include cis-regulatory elements and transposable elements (TEs). Those of ordinary skill in the art can select and use these and other regulatory elements in expression constructs without exceeding routine experiments. Expression constructs may be produced using genetic recombination methods or using known methods to synthesize.

[0087] "Control elements" or "control sequences" are nucleotide sequences involved in molecular interactions that contribute to the regulation of the function of a polynucleotide, including replication, splicing, translation, or degradation of the polynucleotide. Regulation may affect the frequency, speed, or specificity of the process, and may be enhancing or inhibitory in nature. Control elements known in the art include, for example, transcriptional regulatory sequences, such as promoters and enhancers. A promoter is a region of DNA that is capable of binding RNA polymerase under certain conditions and initiates transcription of a coding region that is often located downstream (in the 3' direction) of the promoter.

[0088] As used herein, the statement that an amino acid residue is "phosphorylated" refers to a phosphate group ester-linked 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).

[0089] As used herein, the term "pharmaceutical composition" refers to the combination of an active agent with an inert or active carrier, making the composition particularly suitable for diagnostic or therapeutic use, either in vivo or ex vivo.

[0090] As used herein, the term "pharmaceutically acceptable carrier" refers to any standard pharmaceutical carrier, such as phosphate buffered saline, water, emulsions (e.g., oil-in-water or water-in-oil emulsions), and respective types of wetting agents. The composition may also include stabilizers and preservatives. For examples of carriers, stabilizers, and adjuvants, see, e.g., Martin, Remington's Pharmaceutical Sciences, 15th Ed., Mack Publ. Co., Easton, PA

[1975] .

[0091] In the specification, when compositions are described as having, including or comprising a particular component, or when processes and methods are described as having, including or comprising a particular step, it is contemplated that, in addition, there are compositions of the invention that consist essentially of or consist of that component, and there are processes and methods of the invention that consist essentially of or consist of that process step.

[0092] In general, compositions specifying percentages are by weight unless otherwise indicated. Also, if a variable is not defined, it is defined as indicated by the preceding variable.

[0093] Various aspects of the invention are set forth in the following sections; however, an aspect of the invention described in a particular section is not limited to any particular section.

[0094] I. Antibodies

[0095] The present disclosure provides a novel anti-BTLA antibody. Such antibodies can bind to human and / or cynomolgus monkey BTLA. Table 1 lists peptide sequences of heavy chain variable regions and light chain variable regions (and as specified in Table 1) that can bind to human and / or cynomolgus monkey BTLA. In some embodiments, the heavy chain variable region and the light chain variable region are arranged in a Fab format. In some embodiments, the heavy chain variable region and the light chain variable region are fused together to form an scFv.

[0096]

[0097]

[0098]

[0099]

[0100]

[0101]

[0102]

[0103]

[0104]

[0105] In some embodiments, an anti-BTLA antibody of the present disclosure comprises a heavy chain variable region having 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: 1, and a light chain variable region having 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: 2.

[0106] In some embodiments, the anti-BTLA antibody comprises a vhCDR1 comprising SEQ ID NO: 47, a vhCDR2 comprising SEQ ID NO: 48, a vhCDR3 comprising SEQ ID NO: 49, a vlCDR1 comprising SEQ ID NO: 50, a vlCDR2 comprising SEQ ID NO: 51, and a vlCDR3 comprising SEQ ID NO: 52. In some embodiments, one or more of the six CDRs have 1, 2, 3, 4, or 5 amino acid modifications. In other embodiments, a single CDR contains 1 or 2 amino acid substitutions, and the modified anti-BTLA antibody still binds to human and / or cynomolgus monkey BTLA.

[0107] In some embodiments, an anti-BTLA antibody of the present disclosure comprises a heavy chain variable region having 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:3, and a light chain variable region having 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:4.

[0108] In some embodiments, the anti-BTLA antibody comprises a vhCDR1 comprising SEQ ID NO: 53, a vhCDR2 comprising SEQ ID NO: 54, a vhCDR3 comprising SEQ ID NO: 55, a vlCDR1 comprising SEQ ID NO: 56, a vlCDR2 comprising SEQ ID NO: 57, and a vlCDR3 comprising SEQ ID NO: 58. In other embodiments, a single CDR contains 1 or 2 amino acid substitutions, and the modified anti-BTLA antibody still binds to human and / or cynomolgus monkey BTLA.

[0109] In some embodiments, an anti-BTLA antibody of the present disclosure comprises a heavy chain variable region having 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:5, and a light chain variable region having 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:6.

[0110] In some embodiments, the anti-BTLA antibody comprises a vhCDR1 comprising SEQ ID NO: 59, a vhCDR2 comprising SEQ ID NO: 60, a vhCDR3 comprising SEQ ID NO: 61, a vlCDR1 comprising SEQ ID NO: 62, a vlCDR2 comprising SEQ ID NO: 63, and a vlCDR3 comprising SEQ ID NO: 64. In some embodiments, one or more of the six CDRs have 1, 2, 3, 4, or 5 amino acid modifications. In other embodiments, a single CDR contains 1 or 2 amino acid substitutions, and the modified anti-BTLA antibody still binds to human and / or cynomolgus monkey BTLA.

[0111] In some embodiments, an anti-BTLA antibody of the present disclosure comprises a heavy chain variable region having 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:7, and a light chain variable region having 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:8.

[0112] In some embodiments, the anti-BTLA antibody comprises a vhCDR1 comprising SEQ ID NO: 65, a vhCDR2 comprising SEQ ID NO: 66, a vhCDR3 comprising SEQ ID NO: 67, a vlCDR1 comprising SEQ ID NO: 68, a vlCDR2 comprising SEQ ID NO: 69, and a vlCDR3 comprising SEQ ID NO: 70. In some embodiments, one or more of the six CDRs have 1, 2, 3, 4, or 5 amino acid modifications. In other embodiments, a single CDR contains 1 or 2 amino acid substitutions, and the modified anti-BTLA antibody still binds to human and / or cynomolgus monkey BTLA.

[0113] In some embodiments, an anti-BTLA antibody of the present disclosure comprises a heavy chain variable region having 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:9, and a light chain variable region having 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:10.

[0114] In some embodiments, the anti-BTLA antibody comprises a vhCDR1 comprising SEQ ID NO: 71, a vhCDR2 comprising SEQ ID NO: 72, a vhCDR3 comprising SEQ ID NO: 73, a vlCDR1 comprising SEQ ID NO: 74, a vlCDR2 comprising SEQ ID NO: 75, and a vlCDR3 comprising SEQ ID NO: 76. In some embodiments, one or more of the six CDRs have 1, 2, 3, 4, or 5 amino acid modifications. In other embodiments, a single CDR contains 1 or 2 amino acid substitutions, and the modified anti-BTLA antibody still binds to human and / or cynomolgus monkey BTLA.

[0115] In some embodiments, an anti-BTLA antibody of the present disclosure comprises a heavy chain variable region having 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: 11, and a light chain variable region having 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: 12.

[0116] In some embodiments, the anti-BTLA antibody comprises a vhCDR1 comprising SEQ ID NO: 77, a vhCDR2 comprising SEQ ID NO: 78, a vhCDR3 comprising SEQ ID NO: 79, a vlCDR1 comprising SEQ ID NO: 80, a vlCDR2 comprising SEQ ID NO: 81, and a vlCDR3 comprising SEQ ID NO: 82. In some embodiments, one or more of the six CDRs have 1, 2, 3, 4, or 5 amino acid modifications. In other embodiments, a single CDR contains 1 or 2 amino acid substitutions, and the modified anti-BTLA antibody still binds to human and / or cynomolgus monkey BTLA.

[0117] In some embodiments, an anti-BTLA antibody of the present disclosure comprises a heavy chain variable region having 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: 13, and a light chain variable region having 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: 14.

[0118] In some embodiments, the anti-BTLA antibody comprises a vhCDR1 comprising SEQ ID NO: 83, a vhCDR2 comprising SEQ ID NO: 84, a vhCDR3 comprising SEQ ID NO: 85, a vlCDR1 comprising SEQ ID NO: 86, a vlCDR2 comprising SEQ ID NO: 87, and a vlCDR3 comprising SEQ ID NO: 88. In some embodiments, one or more of the six CDRs have 1, 2, 3, 4, or 5 amino acid modifications. In other embodiments, a single CDR contains 1 or 2 amino acid substitutions, and the modified anti-BTLA antibody still binds to human and / or cynomolgus monkey BTLA.

[0119] In some embodiments, an anti-BTLA antibody of the present disclosure comprises a heavy chain variable region having 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: 15, and a light chain variable region having 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: 16.

[0120] In some embodiments, the anti-BTLA antibody comprises a vhCDR1 comprising SEQ ID NO: 89, a vhCDR2 comprising SEQ ID NO: 90, a vhCDR3 comprising SEQ ID NO: 91, a vlCDR1 comprising SEQ ID NO: 92, a vlCDR2 comprising SEQ ID NO: 93, and a vlCDR3 comprising SEQ ID NO: 94. In some embodiments, one or more of the six CDRs have 1, 2, 3, 4, or 5 amino acid modifications. In other embodiments, a single CDR contains 1 or 2 amino acid substitutions, and the modified anti-BTLA antibody still binds to human and / or cynomolgus monkey BTLA.

[0121] In some embodiments, an anti-BTLA antibody of the present disclosure comprises a heavy chain variable region having 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: 17, and a light chain variable region having 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: 18.

[0122] In some embodiments, the anti-BTLA antibody comprises a vhCDR1 comprising SEQ ID NO: 95, a vhCDR2 comprising SEQ ID NO: 96, a vhCDR3 comprising SEQ ID NO: 97, a vlCDR1 comprising SEQ ID NO: 98, a vlCDR2 comprising SEQ ID NO: 99, and a vlCDR3 comprising SEQ ID NO: 100. In some embodiments, one or more of the six CDRs have 1, 2, 3, 4, or 5 amino acid modifications. In other embodiments, a single CDR contains 1 or 2 amino acid substitutions, and the modified anti-BTLA antibody still binds to human and / or cynomolgus monkey BTLA.

[0123] In some embodiments, an anti-BTLA antibody of the present disclosure comprises a heavy chain variable region having 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 light chain variable region having 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: 20.

[0124] In some embodiments, the anti-BTLA antibody comprises a vhCDR1 comprising SEQ ID NO: 101, a vhCDR2 comprising SEQ ID NO: 102, a vhCDR3 comprising SEQ ID NO: 103, a vlCDR1 comprising SEQ ID NO: 104, a vlCDR2 comprising SEQ ID NO: 105, and a vlCDR3 comprising SEQ ID NO: 106. In some embodiments, one or more of the six CDRs have 1, 2, 3, 4, or 5 amino acid modifications. In other embodiments, a single CDR contains 1 or 2 amino acid substitutions, and the modified anti-BTLA antibody still binds to human and / or cynomolgus monkey BTLA.

[0125] In some embodiments, an anti-BTLA antibody of the present disclosure comprises a heavy chain variable region having 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 light chain variable region having 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: 22.

[0126] In some embodiments, the anti-BTLA antibody comprises a vhCDR1 comprising SEQ ID NO: 107, a vhCDR2 comprising SEQ ID NO: 108, a vhCDR3 comprising SEQ ID NO: 109, a vlCDR1 comprising SEQ ID NO: 110, a vlCDR2 comprising SEQ ID NO: 111, and a vlCDR3 comprising SEQ ID NO: 112. In some embodiments, one or more of the six CDRs have 1, 2, 3, 4, or 5 amino acid modifications. In other embodiments, a single CDR contains 1 or 2 amino acid substitutions, and the modified anti-BTLA antibody still binds to human and / or cynomolgus monkey BTLA.

[0127] In some embodiments, an anti-BTLA antibody of the present disclosure comprises a heavy chain variable region having 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: 23, and a light chain variable region having 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: 24.

[0128] In some embodiments, the anti-BTLA antibody comprises a vhCDR1 comprising SEQ ID NO: 113, a vhCDR2 comprising SEQ ID NO: 114, a vhCDR3 comprising SEQ ID NO: 115, a vlCDR1 comprising SEQ ID NO: 116, a vlCDR2 comprising SEQ ID NO: 117, and a vlCDR3 comprising SEQ ID NO: 118. In some embodiments, one or more of the six CDRs have 1, 2, 3, 4, or 5 amino acid modifications. In other embodiments, a single CDR contains 1 or 2 amino acid substitutions, and the modified anti-BTLA antibody still binds to human and / or cynomolgus monkey BTLA.

[0129] In some embodiments, an anti-BTLA antibody of the present disclosure comprises a heavy chain variable region having 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 light chain variable region having 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: 26.

[0130] In some embodiments, the anti-BTLA antibody comprises a vhCDR1 comprising SEQ ID NO: 119, a vhCDR2 comprising SEQ ID NO: 120, a vhCDR3 comprising SEQ ID NO: 121, a vlCDR1 comprising SEQ ID NO: 122, a vlCDR2 comprising SEQ ID NO: 123, and a vlCDR3 comprising SEQ ID NO: 124. In some embodiments, one or more of the six CDRs have 1, 2, 3, 4, or 5 amino acid modifications. In other embodiments, a single CDR contains 1 or 2 amino acid substitutions, and the modified anti-BTLA antibody still binds to human and / or cynomolgus monkey BTLA.

[0131] In some embodiments, an anti-BTLA antibody of the present disclosure comprises a heavy chain variable region having 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: 27, and a light chain variable region having 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: 28.

[0132] In some embodiments, the anti-BTLA antibody comprises a vhCDR1 comprising SEQ ID NO: 125, a vhCDR2 comprising SEQ ID NO: 126, a vhCDR3 comprising SEQ ID NO: 127, a vlCDR1 comprising SEQ ID NO: 128, a vlCDR2 comprising SEQ ID NO: 129, and a vlCDR3 comprising SEQ ID NO: 130. In some embodiments, one or more of the six CDRs have 1, 2, 3, 4, or 5 amino acid modifications. In other embodiments, a single CDR contains 1 or 2 amino acid substitutions, and the modified anti-BTLA antibody still binds to human and / or cynomolgus monkey BTLA.

[0133] In some embodiments, an anti-BTLA antibody of the present disclosure comprises a heavy chain variable region having 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 light chain variable region having 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: 30.

[0134] In some embodiments, the anti-BTLA antibody comprises a vhCDR1 comprising SEQ ID NO: 131, a vhCDR2 comprising SEQ ID NO: 132, a vhCDR3 comprising SEQ ID NO: 133, a vlCDR1 comprising SEQ ID NO: 134, a vlCDR2 comprising SEQ ID NO: 135, and a vlCDR3 comprising SEQ ID NO: 136. In some embodiments, one or more of the six CDRs have 1, 2, 3, 4, or 5 amino acid modifications. In other embodiments, a single CDR contains 1 or 2 amino acid substitutions, and the modified anti-BTLA antibody still binds to human and / or cynomolgus monkey BTLA.

[0135] In some embodiments, an anti-BTLA antibody of the present disclosure comprises a heavy chain variable region having 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 light chain variable region having 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:32.

[0136] In some embodiments, the anti-BTLA antibody comprises a vhCDR1 comprising SEQ ID NO: 137, a vhCDR2 comprising SEQ ID NO: 138, a vhCDR3 comprising SEQ ID NO: 139, a vlCDR1 comprising SEQ ID NO: 140, a vlCDR2 comprising SEQ ID NO: 141, and a vlCDR3 comprising SEQ ID NO: 142. In some embodiments, one or more of the six CDRs have 1, 2, 3, 4, or 5 amino acid modifications. In other embodiments, a single CDR contains 1 or 2 amino acid substitutions, and the modified anti-BTLA antibody still binds to human and / or cynomolgus monkey BTLA.

[0137] In some embodiments, an anti-BTLA antibody of the present disclosure comprises a heavy chain variable region having 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: 33, and a light chain variable region having 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: 34.

[0138] In some embodiments, the anti-BTLA antibody comprises a vhCDR1 comprising SEQ ID NO: 143, a vhCDR2 comprising SEQ ID NO: 144, a vhCDR3 comprising SEQ ID NO: 145, a vlCDR1 comprising SEQ ID NO: 146, a vlCDR2 comprising SEQ ID NO: 147, and a vlCDR3 comprising SEQ ID NO: 148. In some embodiments, one or more of the six CDRs have 1, 2, 3, 4, or 5 amino acid modifications. In other embodiments, a single CDR contains 1 or 2 amino acid substitutions, and the modified anti-BTLA antibody still binds to human and / or cynomolgus monkey BTLA.

[0139] In some embodiments, an anti-BTLA antibody of the present disclosure comprises a heavy chain variable region having 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 light chain variable region having 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:36.

[0140] In some embodiments, the anti-BTLA antibody comprises a vhCDR1 comprising SEQ ID NO: 149, a vhCDR2 comprising SEQ ID NO: 150, a vhCDR3 comprising SEQ ID NO: 151, a vlCDR1 comprising SEQ ID NO: 152, a vlCDR2 comprising SEQ ID NO: 153, and a vlCDR3 comprising SEQ ID NO: 154. In some embodiments, one or more of the six CDRs have 1, 2, 3, 4, or 5 amino acid modifications. In other embodiments, a single CDR contains 1 or 2 amino acid substitutions, and the modified anti-BTLA antibody still binds to human and / or cynomolgus monkey BTLA.

[0141] In some embodiments, an anti-BTLA antibody of the present disclosure comprises a heavy chain variable region having 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: 37, and a light chain variable region having 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: 38.

[0142] In some embodiments, the anti-BTLA antibody comprises a vhCDR1 comprising SEQ ID NO: 155, a vhCDR2 comprising SEQ ID NO: 156, a vhCDR3 comprising SEQ ID NO: 157, a vlCDR1 comprising SEQ ID NO: 158, a vlCDR2 comprising SEQ ID NO: 159, and a vlCDR3 comprising SEQ ID NO: 160. In some embodiments, one or more of the six CDRs have 1, 2, 3, 4, or 5 amino acid modifications. In other embodiments, a single CDR contains 1 or 2 amino acid substitutions, and the modified anti-BTLA antibody still binds to human and / or cynomolgus monkey BTLA.

[0143] In some embodiments, an anti-BTLA antibody of the present disclosure comprises a heavy chain variable region having 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:39, and a light chain variable region having 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:40.

[0144] In some embodiments, the anti-BTLA antibody comprises a vhCDR1 comprising SEQ ID NO: 161, a vhCDR2 comprising SEQ ID NO: 162, a vhCDR3 comprising SEQ ID NO: 163, a vlCDR1 comprising SEQ ID NO: 164, a vlCDR2 comprising SEQ ID NO: 165, and a vlCDR3 comprising SEQ ID NO: 166. In some embodiments, one or more of the six CDRs have 1, 2, 3, 4, or 5 amino acid modifications. In other embodiments, a single CDR contains 1 or 2 amino acid substitutions, and the modified anti-BTLA antibody still binds to human and / or cynomolgus monkey BTLA.

[0145] In some embodiments, an anti-BTLA antibody of the present disclosure comprises a heavy chain variable region having 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:41, and a light chain variable region having 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:42.

[0146] In some embodiments, the anti-BTLA antibody comprises a vhCDR1 comprising SEQ ID NO: 167, a vhCDR2 comprising SEQ ID NO: 168, a vhCDR3 comprising SEQ ID NO: 169, a vlCDR1 comprising SEQ ID NO: 170, a vlCDR2 comprising SEQ ID NO: 171, and a vlCDR3 comprising SEQ ID NO: 172. In some embodiments, one or more of the six CDRs have 1, 2, 3, 4, or 5 amino acid modifications. In other embodiments, a single CDR contains 1 or 2 amino acid substitutions, and the modified anti-BTLA antibody still binds to human and / or cynomolgus monkey BTLA.

[0147] In addition to the sequence variants in the heavy and light chain variable regions and / or CDRs described herein, the framework regions of the heavy and / or light chain variable regions can also be changed. In some embodiments, the variants in the framework regions (e.g., except for the CDRs) maintain 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 variant can maintain at least about 80, 85, 90, or 95% identity with any one of the light chain V-GENE, light chain J-GENE, heavy chain V-GENE, heavy chain J-GENE, and heavy chain D-GENE alleles.

[0148]

[0149]

[0150]

[0151] In some embodiments, variations are made in the framework regions that maintain at least 80, 85, 90, or 95% identity to the germline gene sequences set forth in Table 2, while keeping the 6 CDRs unchanged.

[0152] In some embodiments, variations are made in the framework regions and 6 CDRs that maintain at least 80, 85, 90, or 95% identity to the germline gene sequence described in Table 2. 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., as long as the total number of changes in a set of 6 CDRs is less than 6 amino acid modifications, the CDRs may be modified to change any combination of CDRs; for example, there may be 1 change in vlCDR1, 2 changes in vlCDR2, no changes in vlCDR3, etc.)).

[0153] By selecting CDRs and / or those amino acid sequences from the heavy and light chain variable regions described herein and appropriately combining them with amino acid sequences of the framework regions and / or constant regions of the heavy and light chains of the antibodies, one skilled in the art will be able to design anti-BTLA antibodies according to the invention. The antibody framework regions and / or constant regions (Fc domains) described in the invention may be derived from antibodies of any species, such as from humans, rabbits, dogs, cats, mice, horses or monkeys.

[0154] In some embodiments, the constant region is derived from humans, including heavy chain constant regions derived from those IgG, IgA, IgM, IgE and IgD subtypes or variants thereof and light chain constant regions derived from κ or λ subtypes or variants thereof. 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 human IgG1, IgG2, IgG3 or IgG4 constant regions. In some other embodiments, the amino acid sequence of the constant region is at least 80%, 85%, 90% or 95% identical to the antibody constant region of 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.

[0155] In some embodiments, the antibodies described herein may be derived from a mixture of different species, for example, to form chimeric antibodies and / or humanized antibodies. Generally, both "chimeric antibodies" and "humanized antibodies" refer to antibodies that bind to regions from more than one species. For example, a "chimeric antibody" typically comprises a variable region from a mouse (or in some cases, a rat) and a constant region from a human. A "humanized antibody" generally refers to a non-human antibody that has had a variable domain framework region that has been exchanged with a sequence found in a human antibody. Generally, in a humanized antibody, the entire antibody except the CDR is encoded by a polynucleotide of human origin, or is identical to such an antibody (except within its CDR). Some or all of the CDRs are encoded by nucleic acids derived from non-human organisms and transplanted into the human antibody variable region β sheet framework region to produce an antibody whose specificity is determined by the transplanted CDRs. The production of such antibodies is described in, for example, WO 92 / 11018; Jones, 1986, Nature 321:522-525, Verhoeyen et al., 1988, Science 239:1534-1536, all of which are incorporated herein by reference. "Back mutation" of selected acceptor framework residues to corresponding donor residues is usually required to restore the affinity lost in the initial graft construct (US 5530101; US ​​5585089; US 5693761; US ​​5693762; US 6180370; US 5859205; US5821337; US 6054297; US 6407213, all of which are incorporated herein by reference). Humanized antibodies also optimally comprise at least a portion of an immunoglobulin constant region, typically a human immunoglobulin constant region, and therefore typically comprise a human Fc region. Humanized antibodies can also be produced using mice with genetically engineered immune systems (such as described in, e.g., Roque et al., 2004, Biotechnol. Prog. 20: 639-654, incorporated herein by reference in its entirety). Various techniques and methods for humanizing and modifying non-human antibodies are known in the art (see, Tsurushita & Vasquez, 2004, Humanization of Monoclonal Antibodies, Molecular Biology of B Cells, 533-545, Elsevier Science (USA)) and references cited therein, all of which are incorporated herein by reference in their entirety).Humanization methods include, but are not limited to, those described in 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., 1998, Proc Natl Acad Sci, USA 89:4285-9; al., 1991, Proc. Natl. Acad. Sci. USA 88:4181-4185; O'Connor et al., 1998, Protein Eng 11:321-8, all of which are incorporated herein by reference in their entirety. Humanization or other methods for reducing the immunogenicity of non-human antibody variable regions may include methods of reshaping the surface, such as described in, for example, Roguska et al., 1994, Proc. Natl. Acad. Sci. USA 91:969-973, which are incorporated herein by reference in their entirety. Other humanization methods may involve the transplantation of only part of the CDR, including but not limited to 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 herein by reference in their entirety.

[0156] 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. Compared with human germline sequences, such antibodies may include amino acid differences, for example, due to naturally occurring somatic mutations or intentionally introduced site-directed mutations. However, compared with germline immunoglobulin amino acid sequences of other species (e.g., mouse germline sequences), humanized antibodies are generally at least 80% identical amino acid sequences to the amino acid sequences encoded by human germline immunoglobulin genes, and include amino acid residues that recognize antibodies derived from human sequences. In some cases, humanized antibodies may be amino acid sequences with at least 95, 96, 97, 98, or 99% (or even at least 96%, 97%, 98%, or 99%) identity to the amino acid sequences encoded by human germline immunoglobulin genes. Typically, humanized antibodies derived from specific human germline sequences will show differences of no more than 10-20 amino acids from the amino acid sequences encoded by human germline immunoglobulins. In certain cases, the humanized antibody may display no more than 5, or even no more than 4, 3, 2, or 1 amino acid difference from the amino acid sequence encoded by a human germline immunoglobulin.

[0157] In some embodiments, the antibodies of the present disclosure are humanized and affinity matured as known in the art.Structure-based approaches can be employed for humanization and affinity maturation, for example, as described in US Patent No. 7,657,380. Selection-based methods can be used to humanize and / or affinity mature antibody variable regions, including but not limited to the 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 herein by reference in their entirety.

[0158] II. Characteristics of Antibodies

[0159] In some embodiments, the anti-BTLA antibodies described herein bind to human and / or cynomolgus monkey BTLA antibodies. In some embodiments, the binding of anti-BTLA antibodies to human and / or cynomolgus monkey BTLA is measured by ELISA, such as the exemplary analysis described in Example 1. In these embodiments, the antibodies described herein show an EC50 measured by these analyses, which may range from 0.1-20 nM. In some embodiments, the binding of anti-BTLA antibodies to human and / or cynomolgus monkey BTLA is measured by FACS, such as the exemplary analysis described in Example 2. In these embodiments, the antibodies described herein show an EC50 measured by FACS, which may range from 0.1-20 nM. In other embodiments, the EC50 of the antibodies described herein measured by ELISA or FACS is in the range of about 0.1-30, 1-28, 2-26, 3-24, 4-22, 5-20, 6-18, 7-16, 8-14 or 9-12 nM.

[0160] In some embodiments, the anti-BTLA antibodies described herein bind to human and / or cynomolgus monkey BTLA with high affinity. K can be measured using antigen immobilization or antibody immobilization. D K can also be measured in monovalent binding mode or bivalent binding mode. D For example, when measured by biofilm interferometry, the K between the antibody and human BTLA is D The value can be 1×10 -6 M or smaller, 5×10 -7 M or smaller, 2.5×10 -7 M or smaller, 1×10 -7 M or smaller, 5×10 -8 M or smaller, 1×10 -8 M or smaller, 1×10 -9 M or smaller, or 1×10 -10 M or less. K between antibody and cynomolgus monkey BTLA D The value can be 1×10 -6 M or smaller, 5×10 -7 M or smaller, 2.5×10 -7 M or smaller, 1×10 -7 M or smaller, 5×10 -8 M or smaller, 2.5×10 -8 M, 1×10 -8 M or smaller, 5×10 -9 M or smaller, 1×10 -9 M or smaller, 5×10 -10 M or 1×10 -10 M or less. In some embodiments, the K between the antibody and human BTLA is DThe value range is 0.1 nM–1 μM, 0.25 nM-500 nM, 0.5 nM-250 nM, 1 nM-100 nM, or 2 nM-50 nM. In some embodiments, the K between the antibody and cynomolgus monkey BTLA is D Values ​​range from 0.1 nM–1 μM, 0.25 nM-500 nM, 0.5 nM-250 nM, 1 nM-100 nM, or 2 nM-50 nM.

[0161] The binding affinity of the anti-BTLA antibodies described herein is compared to the BTLA monoclonal antibody 4C7 described in US Patent No. 8,563,694. In some embodiments, the anti-BTLA antibodies described herein have a higher binding affinity to human BTLA antibodies than 4C7. In some embodiments, the anti-BTLA antibodies described herein have a higher binding affinity to cynomolgus macaque BTLA than 4C7. Table 7 lists some antibody clones and exemplary K values ​​of 4C7 D One advantage of having a higher binding affinity than 4C7 is that the antibodies described herein may be more effective in modulating an immune response and / or participating in an anti-tumor immune response. Another advantage of having a higher binding affinity to cynomolgus macaque BTLA than to 4C7 is that monkey clinical studies (e.g., pharmacokinetic, pharmacodynamic, safety, toxicity studies) derived from these antibodies may have better predictive power than studies using 4C7.

[0162] In some embodiments, anti-BTLA antibodies exhibit low immunogenicity when administered to human subjects. These antibodies may comprise an Fc binding domain derived from human IgG1, human IgG2, or human IgG3. In some embodiments, these antibodies are humanized using a framework region derived from human immunoglobulin.

[0163] The effect of an anti-BTLA antibody on T cell function can be determined using a variety of methods known in the art and described herein, including, for example, the method described by Example 5. Thus, an anti-BTLA antibody can act as a BTLA antagonist or a BTLA agonist.

[0164] In some embodiments, the anti-BTLA antibodies act as BTLA antagonists, blocking the interaction between BTLA and HVEM and HVEM-mediated inhibition of T cell function. Therefore, such anti-BTLA antibodies stimulate immune responses. Examples of such anti-BTLA antibodies include antibodies comprising: 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 light 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: 20; and / or an antibody comprising SEQ ID NO: NO: 101, vhCDR1 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. Alternatively, such anti-BTLA antibodies that are antagonists may include: 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 light 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: 22; and / or a light 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: 22; NO:107, vhCDR1 comprising SEQ ID NO:108, vhCDR2 comprising SEQ ID NO:109, vlCDR1 comprising SEQ ID NO:110, vlCDR2 comprising SEQ ID NO:111 and vlCDR3 comprising SEQ ID NO:112.Alternatively, such anti-BTLA antibodies that are antagonists may include: 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 light 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: 26; and / or a light 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: 26; NO:119, vhCDR1 comprising SEQ ID NO:120, vhCDR2 comprising SEQ ID NO:121, vlCDR1 comprising SEQ ID NO:122, vlCDR2 comprising SEQ ID NO:123 and vlCDR3 comprising SEQ ID NO:124.

[0165] In some other embodiments, the anti-BTLA antibodies described herein act as BTLA agonists, inhibiting immune cell function, including pro-inflammatory T cell function. Thus, such anti-BTLA antibodies inhibit immune responses. For example, such anti-BTLA antibodies may include: 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 light 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:30; and / or a light 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; 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. Alternatively, such anti-BTLA antibodies may include: 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 light 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: 32; and / or a light 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: NO:137, vhCDR1 comprising SEQ ID NO:138, vhCDR2 comprising SEQ ID NO:139, vlCDR1 comprising SEQ ID NO:140, vlCDR2 comprising SEQ ID NO:141, and vlCDR3 comprising SEQ ID NO:142.Alternatively, such anti-BTLA antibodies that are agonists may include: 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 light 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: 36; and / or a light 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: NO:149, vhCDR1 comprising SEQ ID NO:150, vhCDR2 comprising SEQ ID NO:151, vlCDR1 comprising SEQ ID NO:152, vlCDR2 comprising SEQ ID NO:153 and vlCDR3 comprising SEQ ID NO:154.

[0166] III. Nucleic Acids of the Invention

[0167] Also provided are 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. Those skilled in the art will appreciate that due to the degeneracy of the gene codon, the protein sequences described herein can be encoded by any number of possible nucleic acid sequences. Table 3 gives exemplary nucleic acids encoding the heavy chain variable regions and light chain variable regions of the antibodies described herein.

[0168]

[0169]

[0170]

[0171]

[0172]

[0173]

[0174]

[0175]

[0176]

[0177]

[0178]

[0179]

[0180] Also provided are nucleic acid compositions encoding anti-BTLA antibodies and / or BTLA binding domains. Those skilled in the art will appreciate that, in the case of an antigen binding domain, the nucleic acid composition generally includes a first nucleic acid encoding a heavy chain variable region and a second nucleic acid encoding a light chain variable region. In the case of an scFv, a single nucleic acid encoding a heavy chain variable region and a light chain variable region can be prepared, separated by a linker as described herein. In the case of a conventional antibody, the nucleic acid composition generally includes a first nucleic acid encoding a heavy chain variable region and a second nucleic acid encoding a light chain variable region, which, when expressed in a cell, spontaneously assembles into a "conventional" tetramer format of two heavy chains and two light chains.

[0181] As known in the art, the nucleic acid encoding the elements of the present invention can be incorporated into an expression vector, and depending on the host cell, is used to produce the antibody of the present invention. The two nucleic acids can be incorporated into a single expression vector or into two different expression vectors. Generally, the nucleic acid can be operably connected to any number of regulatory elements (promoter, replication origin, selectable marker, ribosome binding site, induction factor, etc.) in an expression vector. The expression vector can be an extrachromosomal vector or an integration vector.

[0182] Nucleic acid and / or expression vector of the present invention can be introduced into any type of host cell known in the art, including mammalian cells, bacterial cells, yeast cells, insect cells and fungal cells. After transfection, single cell clones can be isolated using methods known in the art (e.g., limiting dilution, ELISA, FACS, microscope or Clonepix) for cell bank generation. Under conditions suitable for bioreactor scale-up and maintaining antibody expression, clones can be cultivated. Antibodies can be isolated and purified using methods known in the art (including centrifugation, deep filtration, cell lysis, homogenization, freeze-thaw, affinity purification, gel filtration, ion exchange chromatography, hydrophobic interaction exchange chromatography and mixed mode chromatography).

[0183] IV. Therapeutic Applications

[0184] The present disclosure provides a method of modulating an immune response in a subject, the method comprising administering to the subject an effective amount of an anti-BTLA antibody described herein or a pharmaceutical composition comprising an anti-BTLA antibody.

[0185] In some embodiments, the present disclosure encompasses methods of modulating an immune response comprising stimulating an immune response in a subject, and in other embodiments, such methods comprise administering to the subject an effective amount of an anti-BTLA antibody that is a BTLA antagonist, or administering a pharmaceutical composition containing an antagonistic anti-BTLA antibody.

[0186] In some embodiments, the methods encompassed by the present disclosure include methods of stimulating an immune response in a subject, e.g., by administering an anti-BTLA antibody comprising 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 light 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. NO: 22 has 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; and / or comprises a vhCDR1 of SEQ ID NO: 107, a vhCDR2 of SEQ ID NO: 108, a vhCDR3 of SEQ ID NO: 109, a vlCDR1 of SEQ ID NO: 110, a vlCDR2 of SEQ ID NO: 111, and a vlCDR3 of SEQ ID NO: 112.

[0187] In some embodiments, the methods described herein stimulate an immune response in a subject, e.g., by administering an anti-BTLA antibody comprising 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 light 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. NO: 20 has 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; and / or comprises a vhCDR1 of SEQ ID NO: 101, a vhCDR2 of SEQ ID NO: 102, a vhCDR3 of SEQ ID NO: 103, a vlCDR1 of SEQ ID NO: 104, a vlCDR2 of SEQ ID NO: 105, and a vlCDR3 of SEQ ID NO: 106.

[0188] In some embodiments, the methods described herein stimulate an immune response in a subject, e.g., by administering an anti-BTLA antibody comprising 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 light 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. NO:26 has 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; and / or comprises a vhCDR1 of SEQ ID NO: 119, a vhCDR2 of SEQ ID NO: 120, a vhCDR3 of SEQ ID NO: 121, a vlCDR1 of SEQ ID NO: 122, a vlCDR2 of SEQ ID NO: 123, and a vlCDR3 of SEQ ID NO: 124.

[0189] In some embodiments, the disclosure provides a method of inhibiting an immune response in a subject, for example, by administering to the subject an effective amount of an anti-BTLA antibody that is a BTLA agonist, or by administering to the subject a pharmaceutical composition containing such an agonistic anti-BTLA antibody.

[0190] In some other embodiments, the methods described herein inhibit an immune response in a subject, for example, by administering an anti-BTLA antibody comprising 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 light 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. ID NO: 32 has 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; and / or comprises a vhCDR1 of SEQ ID NO: 137, a vhCDR2 of SEQ ID NO: 138, a vhCDR3 of SEQ ID NO: 139, a vlCDR1 of SEQ ID NO: 140, a vlCDR2 of SEQ ID NO: 141, and a vlCDR3 of SEQ ID NO: 142.

[0191] In some other embodiments, the methods described herein inhibit an immune response in a subject, for example, by administering an anti-BTLA antibody comprising 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 light 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. ID NO: 36 has 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; and / or comprises a vhCDR1 of SEQ ID NO: 149, a vhCDR2 of SEQ ID NO: 150, a vhCDR3 of SEQ ID NO: 151, a vlCDR1 of SEQ ID NO: 152, a vlCDR2 of SEQ ID NO: 153, and a vlCDR3 of SEQ ID NO: 154.

[0192] In some other embodiments, the methods described herein inhibit an immune response in a subject, for example, by administering an anti-BTLA antibody comprising 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 light 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. ID NO: 30 having 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; and / or comprises a vhCDR1 of SEQ ID NO: 131, a vhCDR2 of SEQ ID NO: 132, a vhCDR3 of SEQ ID NO: 133, a vlCDR1 of SEQ ID NO: 134, a vlCDR2 of SEQ ID NO: 135, and a vlCDR3 of SEQ ID NO: 136.

[0193] The present disclosure also provides a method for treating cancer in a subject, the method comprising administering to the subject an effective amount of an anti-BTLA antibody 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 HEVM and / or BTLA compared to corresponding non-cancerous tissues. In some embodiments, the subject to be treated expresses HVEM and / or BTLA on T cells (e.g., on 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 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 T cell responses and / or escape immune recognition and destruction. In some embodiments, the cancer to be treated is not responsive to existing immunomodulatory antibodies targeting other immune checkpoints (e.g., CTLA-4, PD-1, or PD-L1).

[0194] 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's lymphoma, B-cell non-Hodgkin's lymphoma, or T-cell non-Hodgkin's lymphoma.

[0195] In some other embodiments, the 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 other embodiments, the cancer is hemangioma, squamous cell carcinoma, adenocarcinoma, small cell carcinoma, neuroblastoma, sarcoma (e.g., angiosarcoma or chondrosarcoma), laryngeal cancer, parotid cancer, biliary cancer, thyroid cancer, acral melanoma, actinic keratosis, acute lymphocytic leukemia, acute myeloid leukemia, adenoid cystic carcinoma, adenoma, adenosarcoma, adenosquamous carcinoma, anal canal cancer, anal cancer, anorectal cancer, astrocytoma, Bartholin's gland carcinoma, basal cell carcinoma, bile duct cancer, bone cancer, bone marrow cancer, bronchial cancer, duct cancer, bronchial gland cancer, carcinoid, bile duct cancer (cholangiocarcinoma), chondrosarcoma, choroid plexus papilloma / carcinoma, chronic lymphocytic leukemia, chronic myeloid leukemia, clear cell carcinoma, connective tissue cancer, cystadenoma, digestive system cancer, duodenal cancer, endocrine system cancer, endodermal sinus tumor, endometrial hyperplasia, endometrial stromal sarcoma, endometrioid adenocarcinoma, endothelial cell carcinoma, ependymal carcinoma, epithelial cell carcinoma, Ewing's sarcoma sarcoma), eye and orbit cancer, female genital cancer, focal nodular hyperplasia of the liver, gallbladder cancer, gastric antral cancer, gastric fundus cancer, gastrinoma, glioblastoma, glucagonoma, heart cancer, hemangioblastoma, hemangioendothelioma, hemangioma, hepatic adenoma, hepatocellular carcinoma, Hodgkin's disease, ileal cancer, insulinoma, intraepithelial neoplasia, intraepithelial squamous cell neoplasia, intrahepatic bile duct cancer, invasive squamous cell carcinoma, jejunum cancer, joint cancer, Kaposi's sarcoma 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, mesothelioma, metastatic cancer, oral cancer, mucoepidermoid carcinoma, multiple myeloma, muscle cancer, nasal passage cancer, nervous system cancer, neuroepithelial adenocarcinoma nodular melanoma, non-epithelial skin cancer, oat cell carcinoma, oligodendroglial carcinoma, oral cancer, osteosarcoma, papillary serous adenocarcinoma, penile cancer, pharynx cancercancer, pituitary tumor, plasmacytoma, pseudosarcoma, pulmonary blastoma, rectal cancer, renal cell cancer, respiratory cancer, eye cancer, rhabdomyosarcoma, sarcoma, serous carcinoma, sinus cancer, skin cancer, small cell carcinoma, small intestine cancer, leiomyosarcoma, soft tissue cancer, somatostatin-secreting tumor, spinal cancer, squamous cell carcinoma, striated muscle cancer, submesothelial cancer, superficial spreading melanoma, T-cell leukemia, tongue cancer, undifferentiated carcinoma, ureteral cancer, urethral cancer, urinary bladder cancer, urinary system cancer, cervical cancer, endometrial cancer, uveal melanoma, vaginal cancer, verrucous carcinoma, vipoma, vulvar cancer, well-differentiated carcinoma, or Wilms tumor.

[0196] In some other embodiments, the cancer to be 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, small lymphocytic lymphoma, mantle cell lymphoma, marginal zone B-cell lymphoma, extranodal marginal zone B-cell lymphoma, intranodal 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, the non-Hodgkin lymphoma is a T-cell lymphoma, such as precursor T-cell 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.

[0197] The present disclosure also provides a method for treating an autoimmune or inflammatory disorder in a subject, the method comprising administering to the subject an effective amount of an anti-BTLA antibody as 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 (including T cells, B cells, natural killer cells, dendritic cells, endothelial cells, and macrophages) of the subject to be treated. In some embodiments, HVEM and / or BTLA are expressed at low levels on autoimmune cells (e.g., T cells, B cells, natural killer cells, dendritic cells, endothelial cells, and macrophages) residing in the site of occurrence of autoimmune diseases in the subject (e.g., lymph nodes and central nervous system of subjects with multiple sclerosis, joints of subjects with rheumatoid arthritis, gastrointestinal tract of subjects with celiac disease). Administration of an anti-BTLA antibody as a BTLA agonist can inhibit proinflammatory immune responses, including proinflammatory T cell responses, and regulate the immune response of subjects with autoimmune or inflammatory disorders.

[0198] In some embodiments, the autoimmune or inflammatory disorder to be treated is multiple sclerosis, Addison's disease, amyotrophic lateral sclerosis, Crohn's disease, Cushing's Syndrome, type 1 diabetes, 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.

[0199] In some other embodiments, the autoimmune disorder to be treated includes, but is 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's concentric sclerosis, Behcet's syndrome (Behcet's disease), Berger's disease, Bickerstaff brainstem encephalitis, Blau syndrome, bullous pemphigoid, cancer, giant lymphadenopathy (Castleman's disease), celiac disease, Chagas disease, chronic inflammatory demyelinating polyneuropathy, chronic inflammatory demyelinating polyneuropathy, chronic obstructive pulmonary disease, slowly relapsing multifocal osteomyelitis, Churg-Strauss syndrome, cicatricial pemphigoid, Cogan syndrome, cold agglutinin disease, complement component 2 deficiency (Complement component2deficiency), contact dermatitis, cranial arteritis, CREST syndrome, cutaneous leukocytoclastic vasculitis, Dego's disease, Dercum's disease, dermatitis herpetiformis, dermatomyositis, diffuse cutaneous systemic sclerosis, discoid lupus erythematosus, Dressler syndrome, drug-induced lupus erythematosus, eczema, endometriosis, eosinophilic fasciitis, eosinophilic gastroenteritis, eosinophilic pneumonia, epidermolysis bullosa acquisita, erythema nodosum, erythroblastosis fetalis, essential mixed cryoglobulinemia, Evan's syndrome, progressive ossific fibrodysplasia, fibrosing alveolitis (or idiopathic pulmonary fibrosis), gastritis, gastrointestinal pemphigoid, glomerulonephritis, Goodpaster syndrome, Hashimoto's encephalopathy, Hashimoto's thyroiditis thyroiditis), Henoch-Schonlein purpura, Herpes gestationis aka GestationalPemphigoid), 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 asthenic syndrome, leukoproliferative vasculitis, lichen planus, lichen sclerosus, linear IgA disease, lupus hepatitis also known as autoimmune hepatitis, Majeed syndrome, microscopic colitis, microscopic polyangiitis, Miller-Fisher syndrome, mixed connective tissue disease, Morphea, Mucha-Habermann disease also known as pityriasis lichenoides and condyloma acuminatum, multiple sclerosis, myasthenia gravis, myositis, Ménière's disease disease), narcolepsy, neuromyelitis optica, neuromyotonia, ocular cicatricial pemphigoid, opsoclonus-myoclonus syndrome, Ord's thyroiditis, relapsing rheumatic disease, PANDAS (pediatric autoimmune neuropsychiatric disorders associated with streptococci), paraneoplastic cerebellar degeneration, paroxysmal nocturnal hemoglobinuria (PNH), Parry Romberg syndrome, pars planitis, Parsonage-Turner syndrome, pemphigus vulgaris, perivenous encephalomyelitis, pernicious anemia, POEMS syndrome, polyarteritis nodosa, polymyalgia rheumatica, polymyositis, primary biliary cirrhosis, primary sclerosing cholangitis, progressive inflammatory neuropathy, pure red cell aplasia, pyoderma gangrenosum, Rasmussen encephalitis, Raynaud's phenomenon, Reiter syndrome, relapsing polychondritis, restless legs syndrome, retroperitoneal fibrosis, rheumatic fever, schizophrenia, Schmidt syndrome, Schnitzler syndrome, scleritis, serum sickness, syndrome, spondyloarthropathies, stiff person syndrome, Still's disease, subacute bacterial endocarditis (SBE), Susac syndrome, Sweet syndrome, Sydenham chorea, sympathetic ophthalmia, Takayasu's arteritis, temporal arteritis, thrombocytopenia, Tolosa-Hunt syndrome, transverse myelitis, ulcerative colitis, undifferentiated spondyloarthropathies, urticarial vasculitis, vitiligo, Wegener's granulomatosis.

[0200] V. Combination Therapy

[0201] The anti-BTLA antibodies described herein can be used in combination with other therapeutic agents to treat cancer or autoimmune diseases.

[0202] Exemplary therapeutic agents that can be used as part of a combination therapy to treat cancer include, for example, radiation, mitomycin, tretinoin, ribomustin, gemcitabine, vincristine, etoposide, cladribine, mitobronitol, methotrexate, doxorubicin, carboquone, pentostatin, nitracrine, zinostatin, cetrorelix, letrozole, raltitrexed, daunorubicin, fadrozole, formox statins, thymosin, sobuzoxane, nedaplatin, cytarabine, bicalutamide, vinorelbine, vesnarinone, aminoglutethimide, amsacrine, proglumide, elipromide, ketanserin, doxifluridine, acitretin, isotretinoin, streptozotocin, nimustine, vindesine, flutamid, drogenistein l, butocin, carmofur, razoxane, sizofilan, carboplatin, mitolactol, tegafur, ifosfamide, prednimustine, picibanil, levamisole, teniposide, improsulfan, enocitabine, lisuride, oxymetholone, tamoxifen, progesterone, mepitiostane, epitiostanol, formestane, interferon-α, interferon-2α, interferon-β, interferon-γ, colony stimulating factor-1, colony stimulating factor-2, denileukindiftitox, interleukin-2, luteinizing hormone releasing factor and variants of the above agents that may exhibit different binding to their cognate receptors and increase or decrease serum half-life.

[0203] Another class of agents that can be used as part of a combination therapy to treat cancer is immune checkpoint inhibitors. Exemplary immune checkpoint inhibitors include agents 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.

[0204] However, other agents that may be used as part of combination therapy to treat cancer are monoclonal antibody agents directed against non-checkpoint targets (e.g., Herceptin) and non-cytotoxic agents (e.g., tyrosine kinase inhibitors).

[0205] However, other classes of anticancer agents include, for example: (i) inhibitors selected from the group consisting of 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, DNA-PK and mTOR inhibitors, DNMT1 inhibitors, DNMT1 inhibitors plus 2-chlorodeoxyadenosine, HDAC inhibitors, Hedgehog signaling pathway inhibitors, IDO inhibitors, JAK inhibitors, mTOR inhibitors, MEK inhibitors, MELK inhibitors, MTH1 inhibitors, PARP inhibitors, phosphoinositide 3-kinase inhibitors, PARP1 and DHODH inhibitors, 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.

[0206] The antibodies of the invention may also be used as an adjunct to surgical removal of cancer from the primary lesion.

[0207] Exemplary therapeutic agents that can be used as part of a combination therapy with an anti-BTLA antibody disclosed herein to treat, delay progression, prevent relapse, or alleviate symptoms of an autoimmune or inflammatory disease include, for example, any anti-inflammatory and / or immunosuppressive therapy known in the art and described herein. In some embodiments, anti-inflammatory and / or immunosuppressive therapies include, but are not limited to, methotrexate, cyclosporin A (including, for example, cyclosporin microemulsion), tacrolimus, corticosteroids, statins, interferon beta, nonsteroidal anti-inflammatory drugs, and 6-MP (mercaptopurine, also known as 6-mercaptopurine or Purinethol).

[0208] In some embodiments, anti-inflammatory and / or immunosuppressive therapies combined with the 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-methylthienyl[2,3-c]quinolin-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), doxorubicin (ADM), daunorubicin (DNR), vincristine (VCR), vinblastine (VBL), etoposide (VP16) , vermeer (Vumon), carboplatin (CBP), tacrolimus, sirolimus, everolimus, azathioprine, brequinar, leflunomide, LEA-29Y, anti-CD3 antibodies (e.g., OKT3), aspirin, B7-CD28 blocking molecules (e.g., belatacept, abatacept), CD40-CD154 blocking molecules (anti-CD40 antibodies), acetaminophen, ibuprofen, naproxen, piroxicam, and anti-inflammatory steroids (e.g., prednisolone or dexamethasone).

[0209] In some embodiments, the anti-inflammatory and / or immunosuppressive therapy disclosed herein in combination with an anti-BTLA antibody comprises eliminating autoimmune cells, for example, by administering TNF-α, CFA, interleukin-1 (IL-1), proteasome inhibitors, NFκB inhibitors, anti-inflammatory drugs, tissue plasminogen activator (TPA), lipopolysaccharide, ultraviolet light, and intracellular mediators of the TNF-α signaling pathway. Such drugs act by interrupting downstream pathways of TNF-α receptor signaling or downstream of TNF-α receptor binding, inducing apoptosis of autologous lymphocytes. (Baldwin et al., Ann. Rev. Immunol. (1996) 12: 141; Baltimore, Cell (1996) 87: 13).

[0210] In some embodiments, the anti-BTLA antibodies disclosed herein are used in conjunction with surgical procedures to treat or otherwise ameliorate an autoimmune disease.

[0211] For example, to treat, delay the progression of, prevent relapse of, or alleviate symptoms of multiple osteoporosis, anti-BTLA antibodies that are BTLA agonists can be combined with any existing therapy for treating multiple sclerosis, such as corticosteroids (e.g., oral prednisone and intravenous methylprednisolone), plasmapheresis, orelizumab, beta interferon, glatiramer acetate, dimethyl fumarate, fingolimod, feriflunomide, natalizumab, alemtuzumab, and / or mitoxantrone.

[0212] The amount of antibody and additional therapeutic agent and the relative timing of administration can be selected to achieve the desired combined therapeutic effect. For example, when administering a combination therapy to a patient in need of such administration, the therapeutic agents or pharmaceutical compositions or compositions comprising therapeutic agents in the combination can be administered in any order, e.g., sequentially, concurrently, together, simultaneously, etc. In addition, for example, a multispecific binding protein can be administered while the additional therapeutic agent exerts its prophylactic or therapeutic effect, or vice versa.

[0213] VI. Pharmaceutical Compositions and Administration

[0214] The present disclosure also includes pharmaceutical compositions / formulations containing a therapeutically effective amount of an anti-BTLA antibody described herein. The composition can 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 proper formulation. Suitable formulations for the present disclosure can be found in Remington's Pharmaceutical Sciences, Mack Publishing Company, Philadelphia, Pa., 17th ed., 1985. For a brief review of drug delivery methods, see, for example, Langer (Science 249: 1527-1533, 1990).

[0215] The antibodies disclosed herein may be present in a lyophilized formulation or a liquid aqueous pharmaceutical formulation. The aqueous carriers of interest herein are pharmaceutically acceptable (safe and non-toxic to humans) and can be used to prepare liquid formulations. Illustrative carriers include sterile water for injection (SWFI), bacteriostatic water for injection (BWFI), pH buffered solutions (e.g., phosphate buffered saline), sterile saline, Ringer's solution, or glucose solution.

[0216] The antibodies disclosed herein may be present in a lyophilized formulation, including a protein and a lyophilizing agent. A 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 of a buffer, a surfactant, a bulking agent, and / or a preservative.

[0217] The actual dosage level of the active ingredient in the pharmaceutical composition of the present invention can be varied so as to obtain an effective active ingredient content to achieve the therapeutic response required for a specific patient, composition and mode of administration without causing toxicity to the patient. It can be administered in the range of 0.1 mg to 1 g, preferably in the range of 0.5 mg to 500 mg of active antibody per administration to adults. Alternatively, the dosage of the patient can be determined based on the approximate body weight or surface area of ​​the patient. Other factors for determining the appropriate dosage may include the disease or condition to be treated or prevented, the severity of the disease, the route of administration, and the age, sex and physical condition of the patient. Those skilled in the art will generally further refine the calculations required to determine the appropriate dosage for treatment, particularly in light of the dosage information and analytical methods disclosed herein. The dosage can also be determined by using a known detection method for determining the dosage used with appropriate dose-response data. The dosage of individual patients can be adjusted as the disease progresses. The level of the targetable construct or complex in the patient's blood can be measured to understand whether the dosage needs to be adjusted to reach or maintain an effective concentration. Pharmacogenomics can be used to determine which targetable constructs and / or complexes and their dosages are most likely to be effective for a particular individual (Schmitz et al., Clinica Chimica Acta 308:43-53, 2001; Steimer et al., Clinica Chimica Acta 308:33-41, 2001).

[0218] The dosage may be given once or more per day, once or more per week, once or more per month, or once or more per year, or even once every 2 to 20 years. One of ordinary skill in the art can easily estimate the repetition rate of the dosage based on the measured residence time and the concentration of the targetable construct or complex in the body fluid or tissue. The administration of the present invention can be intravenous, intraarterial, intraperitoneal, intramuscular, subcutaneous, intrapleural, intrathecal, intracavitary, by catheter perfusion or direct intralesional injection. This may be administered once or more per day, once or more per week, once or more per month, once or more per year.

[0219] Example

[0220] The invention is now generally described and will be more readily understood by reference to the following examples, which are merely intended to illustrate certain aspects and embodiments of the invention and are not intended to limit the invention.

[0221] Example 1 - Anti-BTLA Antibody Binding to Recombinant BTLA

[0222] The binding of anti-BTLA antibodies to human or cynomolgus monkey BTLA was detected by ELISA. The plate (384 wells) was coated with 20 μl BTLA (ECD)-HIS (1 μg / ml) at 4°C overnight. The wells were then washed with PBS and then blocked with 55 μl blocking buffer for 1 hour at room temperature. The wells were then washed with PBS, and the anti-BTLA antibodies of the present invention and anti-BTLA antibodies 4C7 and 8D5 (described in U.S. Pat. No. 8,563,694) and human IgG controls were added to the wells at different 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 second anti-human IgG antibody was added to the wells and incubated at room temperature for 45 minutes. After removing the excess HRP-conjugated second antibody, a substrate solution was added to the wells to form a chemiluminescent signal. Photon emission was measured using an Omegastar plate reader and relative light units (RLU) were calculated. Figure 1 An exemplary binding curve of anti-BTLA antibody 13-F7A binding to human BTLA and comparison to 4C7 and 8D5 antibodies is shown. Human IgG was used as a negative control. Table 4 summarizes the EC50s of binding of various anti-BTLA antibodies to human and cynomolgus monkey BTLA as determined by ELISA.

[0223] Table 4

[0224]

[0225] Example 2 - Anti-BTLA Antibodies Binding to BTLA Expressed on Cells

[0226] HEK 293 cells expressing human or cynomolgus macaque BTLA were used to evaluate the binding of anti-BTLA antibodies to BTLA. Different 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. Figure 2A-2B The binding curves of anti-BTLA antibody 13-F7A to HEK293 cells expressing human or cynomolgus monkey BTLA are shown. Compared with the control group HEK 293 cells ( Figure 2C ), 13-F7A binds to both human BTLA and cynomolgus monkey BTLA. Table 5 summarizes the EC50s of binding of various anti-BTLA antibodies to human and cynomolgus monkey BTLA as measured by FACS.

[0227] Table 5

[0228]

[0229]

[0230] Example 3 - Anti-BTLA Antibodies Inhibit BTLA / HVEM Interactions

[0231] ELISA-based biofunctional analysis was used to detect the effect of anti-BTLA antibodies on BTLA / HVEM interaction. Each well of a 384-well ELISA plate was coated with 20 μl of 5 μg / ml human HVEM-rat IgG2a for 1 hour at 37°C (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 Aquamax 2000 plate washer, and then blocked with blocking solution (PBS containing 3% BSA) for 1 hour at room temperature (RT). In the blocking step, 1 μg / ml of human BTLA-human Fc or 0.5 μg / ml of cynomolgus monkey BTLA-human Fc was mixed with different concentrations (67, 22, 7.4, 2.5, 0.8, 0.3, 0.09, 0.03, 0.01, 0.004, 0.001 nM) of anti-BTLA antibodies, 4C7, 8D5 or human IgG2 control. After the blocking step, the plate was washed twice with a plate washer. Then 20 μl of protein / antibody mixture was added to the plate and incubated for 1 hour at RT. Unbound material was discarded and the plate was washed four times with a plate washer. Then 20 μl of secondary antibody (anti-human Fc-HRP) was added to each well and incubated for 45 minutes at RT. Unbound material was discarded and the plate was washed 8 times with a plate washer. Then 20 μl of chemiluminescent substrate was added to each well, the luminescence value was read at a gain of 3600, and the relative light units (RLU) were calculated. Figure 3 An exemplary curve for the anti-BTLA antibody 13-F7A is shown. 13-F7A inhibits the interaction of human BTLA with human HVEM to a similar extent as 4C7. However, 8D5 and human IgG controls have little effect on the interaction between human BTLA and HVEM. Table 6 shows a summary of the IC50s of different anti-BTLA antibodies for inhibiting the interaction between human BTLA and HVEM and the interaction between cynomolgus monkey BTLA and HVEM.

[0232] Table 6

[0233]

[0234] Example 4 - Anti-BTLA Antibodies Bind to BTLA with High Affinity

[0235] Through biofilm interferometry (from FortéBIO The binding affinity of anti-BTLA antibodies to human and cynomolgus macaque BTLA was measured using a 3-fold serial dilution system. 10 μg / ml of each anti-BTLA antibody was immobilized on the biosensor tip surface via anti-human Fc (AHC) capture. Human or cynomolgus macaque BTLA was diluted in PBS and loaded at concentrations ranging from 166.7 nM to 6.17 nM. The analysis software provided by the system fits the binding curve to a 1:1 interaction model. Figure 4 Exemplary binding 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) are shown. Table 7 shows the binding dissociation constants (K) of different anti-BTLA antibodies to human and cynomolgus monkey BTLA measured by biofilm interferometry. D )’s summary.

[0236] Table 7

[0237]

[0238] Example 5 - Anti-BTLA antibodies modulate T cell function

[0239] The effect of anti-BTLA antibodies on T cell function was examined. Peripheral blood mononuclear cells (PBMCs) were isolated from heparinized whole blood of human donors by density gradient centrifugation. PBMCs were plated at 1×10 6 The cells were resuspended in complete RPMI 1640 (RPMI 1640, 10% FCS, l-glutamine, penicillin and streptomycin) at 400 μl / ml and 100 μl cells were seeded per well in a 96-well cell culture plate. 1 ng / ml Staphylococcal enterotoxin B (SEB) was added to the wells along with each anti-BTLA antibody. The cells were cultured for 48 hours at 37°C and 5% carbon dioxide, and the supernatant was collected. The concentration of IL-2 in the supernatant was detected by ELISA using an anti-IL2 capture antibody (R&D Systems MAB602).

[0240] Figures 5A-5FThe dose-response curves of IL-2 released by primary T cells in the presence of different anti-BTLA antibodies and SEB are shown. Human IgG2 and IgG4 were used as controls for the assay because, in this example, the anti-BTLA antibodies include an Fc domain from human IgG2, and 8D5 and 4C7 each include an Fc domain from human IgG4. Anti-BTLA antibodies 16-I20A, 15-C19A, and 16-H16A enhanced SEB-induced IL-2 secretion by T cells and thus can be used as BTLA antagonists. In addition, they showed higher maximum antagonistic activity compared to the 4C7 antibody. The EC50 of each tested antibody was calculated, and 16-I20A showed a similar EC50, i.e., the potency of antagonizing T cell function, compared to the 4C7 antibody. Anti-BTLA antibodies 12-I8A, 8-M23A, and 13-F7A inhibited SEB-induced IL-2 secretion by T cells and thus can be used as BTLA agonists.

[0241] Incorporated by Reference

[0242] The entire disclosure of each patent document and scientific article mentioned herein is incorporated by reference for all purposes.

[0243] Equivalent

[0244] The present invention may be embodied in other specific forms without departing from its spirit or essential characteristics. Therefore, the above embodiments should be considered in all respects as illustrative rather than limiting the invention described herein. Therefore, the scope of the present invention is indicated by the appended claims rather than by the foregoing description, and all changes within the meaning and equivalent range of the claims are intended to be included herein.

Claims

1. An antibody or antigen-binding fragment thereof that binds to human and / or cynomolgus macaque BTLA, the antibody or antigen-binding fragment thereof comprising: a) a vhCDR1 comprising the amino acid sequence of SEQ ID NO: 149, a vhCDR2 comprising the amino acid sequence of SEQ ID NO: 150, a vhCDR3 comprising the amino acid sequence of SEQ ID NO: 151, a vlCDR1 comprising the amino acid sequence of SEQ ID NO: 152, a vlCDR2 comprising the amino acid sequence of SEQ ID NO: 153, and a vlCDR3 comprising the amino acid sequence of SEQ ID NO: 154; b) a vhCDR1 comprising the amino acid sequence of SEQ ID NO: 107, a vhCDR2 comprising the amino acid sequence of SEQ ID NO: 108, a vhCDR3 comprising the amino acid sequence of SEQ ID NO: 109, a vlCDR1 comprising the amino acid sequence of SEQ ID NO: 110, a vlCDR2 comprising the amino acid sequence of SEQ ID NO: 111, and a vlCDR3 comprising the amino acid sequence of SEQ ID NO: 112; c) a vhCDR1 comprising the amino acid sequence of SEQ ID NO: 119, a vhCDR2 comprising the amino acid sequence of SEQ ID NO: 120, a vhCDR3 comprising the amino acid sequence of SEQ ID NO: 121, a vlCDR1 comprising the amino acid sequence of SEQ ID NO: 122, a vlCDR2 comprising the amino acid sequence of SEQ ID NO: 123, and a vlCDR3 comprising the amino acid sequence of SEQ ID NO: 124; d) a vhCDR1 comprising the amino acid sequence of SEQ ID NO: 131, a vhCDR2 comprising the amino acid sequence of SEQ ID NO: 132, a vhCDR3 comprising the amino acid sequence of SEQ ID NO: 133, a vlCDR1 comprising the amino acid sequence of SEQ ID NO: 134, a vlCDR2 comprising the amino acid sequence of SEQ ID NO: 135, and a vlCDR3 comprising the amino acid sequence of SEQ ID NO: 136; or e) a vhCDR1 comprising the amino acid sequence of SEQ ID NO: 137, a vhCDR2 comprising the amino acid sequence of SEQ ID NO: 138, a vhCDR3 comprising the amino acid sequence of SEQ ID NO: 139, a vlCDR1 comprising the amino acid sequence of SEQ ID NO: 140, a vlCDR2 comprising the amino acid sequence of SEQ ID NO: 141, and a vlCDR3 comprising the amino acid sequence of SEQ ID NO:

142.

2. The antibody or antigen-binding fragment thereof according to claim 1, wherein: a) the antibody or antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region: a vhCDR1 comprising the amino acid sequence of SEQ ID NO: 149, a vhCDR2 comprising the amino acid sequence of SEQ ID NO: 150, a vhCDR3 comprising the amino acid sequence of SEQ ID NO: 151, a vlCDR1 comprising the amino acid sequence of SEQ ID NO: 152, a vlCDR2 comprising the amino acid sequence of SEQ ID NO: 153, and a vlCDR3 comprising the amino acid sequence of SEQ ID NO: 154, the heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 35, and the light chain variable region comprising the amino acid sequence of SEQ ID NO: 36; b) the antibody or antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region: a vhCDR1 comprising the amino acid sequence of SEQ ID NO: 107, a vhCDR2 comprising the amino acid sequence of SEQ ID NO: 108, a vhCDR3 comprising the amino acid sequence of SEQ ID NO: 109, a vlCDR1 comprising the amino acid sequence of SEQ ID NO: 110, a vlCDR2 comprising the amino acid sequence of SEQ ID NO: 111, and a vlCDR3 comprising the amino acid sequence of SEQ ID NO: 112, the heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 21, and the light chain variable region comprising the amino acid sequence of SEQ ID NO: 22; c) the antibody or antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region: a vhCDR1 comprising the amino acid sequence of SEQ ID NO: 119, a vhCDR2 comprising the amino acid sequence of SEQ ID NO: 120, a vhCDR3 comprising the amino acid sequence of SEQ ID NO: 121, a vlCDR1 comprising the amino acid sequence of SEQ ID NO: 122, a vlCDR2 comprising the amino acid sequence of SEQ ID NO: 123, and a vlCDR3 comprising the amino acid sequence of SEQ ID NO: 124, the heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 25, and the light chain variable region comprising the amino acid sequence of SEQ ID NO: 26; d) the antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region: a vhCDR1 comprising the amino acid sequence of SEQ ID NO: 131, a vhCDR2 comprising the amino acid sequence of SEQ ID NO: 132, a vhCDR3 comprising the amino acid sequence of SEQ ID NO: 133, a vlCDR1 comprising the amino acid sequence of SEQ ID NO: 134, a vlCDR2 comprising the amino acid sequence of SEQ ID NO: 135, and a vlCDR3 comprising the amino acid sequence of SEQ ID NO: 136, the heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 29, and the light chain variable region comprising the amino acid sequence of SEQ ID NO: 30; or e) the antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region: vhCDR1 comprising the amino acid sequence of SEQ ID NO: 137, vhCDR2 comprising the amino acid sequence of SEQ ID NO: 138, vhCDR3 comprising the amino acid sequence of SEQ ID NO: 139, vlCDR1 comprising the amino acid sequence of SEQ ID NO: 140, vlCDR2 comprising the amino acid sequence of SEQ ID NO: 141 and vlCDR3 comprising the amino acid sequence of SEQ ID NO: 142, the heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 31, and the light chain variable region comprising the amino acid sequence of SEQ ID NO:

32.

3. The antibody or antigen-binding fragment thereof according to claim 1 or claim 2, wherein: The antibody or antigen-binding fragment thereof acts as a BTLA antagonist and promotes IL-2 secretion of activated T cells; or the antibody or antigen-binding fragment thereof acts as a BTLA agonist and inhibits pro-inflammatory T cell function. 4 . The antibody or antigen-binding fragment thereof according to claim 3 , wherein the antibody or antigen-binding fragment thereof inhibits HVEM-mediated inhibition of T cell function.

5. The antibody or antigen-binding fragment thereof according to claim 1 or claim 2, wherein the antibody or antigen-binding fragment thereof comprises a humanized IgG constant region selected from the group consisting of IgG1, IgG2, IgG3 or IgG4.

6. The antibody or antigen-binding fragment thereof according to claim 1 or claim 2, wherein the antigen-binding fragment is selected from the group consisting of Fv, Fab and scFv fragments.

7. A nucleic acid composition encoding the antibody or antigen-binding fragment thereof according to any one of the preceding claims.

8. The nucleic acid composition according to claim 7, comprising: a) a first nucleic acid comprising SEQ ID NO: 219 and a second nucleic acid comprising SEQ ID NO: 220; b) a first nucleic acid comprising SEQ ID NO: 205 and a second nucleic acid comprising SEQ ID NO: 206; c) a first nucleic acid comprising SEQ ID NO: 209 and a second nucleic acid comprising SEQ ID NO: 210; d) a first nucleic acid comprising SEQ ID NO: 213 and a second nucleic acid comprising SEQ ID NO: 214; or e) a first nucleic acid comprising SEQ ID NO: 215 and a second nucleic acid comprising SEQ ID NO:

216.

9. An expression vector composition comprising the nucleic acid composition according to claim 8, wherein the first nucleic acid is contained in a first expression vector and the second nucleic acid is contained in a second expression vector.

10. An expression vector composition comprising the nucleic acid composition according to claim 8, wherein the first nucleic acid and the second nucleic acid are contained in a single expression vector.

11. A host cell comprising an expression vector composition comprising the nucleic acid composition of claim 8, wherein (i) the first nucleic acid is contained in a first expression vector and the second nucleic acid is contained in a second expression vector; or (ii) the first nucleic acid and the second nucleic acid are contained in a single expression vector.

12. A method for preparing an antibody or an antigen-binding fragment thereof, the method comprising culturing the host cell according to claim 11 under conditions in which the antibody or the antigen-binding fragment thereof is expressed and recovering the antibody or the antigen-binding fragment thereof.

13. A composition comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, and a pharmaceutically acceptable carrier or diluent.

14. Use of the antibody or antigen-binding fragment thereof of any one of claims 1 to 6 in the preparation of a medicament for modulating an immune response in a subject.

15. Use of the antibody or antigen-binding fragment thereof according to claim 14, wherein the antibody or antigen-binding fragment is a BTLA antagonist.

16. Use of the antibody or antigen-binding fragment thereof according to claim 14, wherein the antibody or antigen-binding fragment acts as a BTLA agonist.

17. Use of the antibody or antigen-binding fragment thereof of any one of claims 1 to 6 in the preparation of a medicament for treating cancer in a subject.

18. Use of the antibody or antigen-binding fragment thereof according to claim 17, wherein the antibody or antigen-binding fragment is a BTLA antagonist.

19. The use of the antibody or antigen-binding fragment thereof according to claim 17, wherein the antibody or antigen-binding fragment acts as a BTLA agonist.

20. Use of the antibody or antigen-binding fragment thereof of any one of claims 1 to 6 in the preparation of a medicament for treating an autoimmune disease in a subject.

21. Use of the antibody or antigen-binding fragment thereof according to claim 20, wherein the antibody or antigen-binding fragment acts as a BTLA agonist.

22. Use of the antibody or antigen-binding fragment thereof according to claim 21, wherein the subject expresses low levels of HVEM on autoreactive T cells residing at sites of development of autoimmune diseases.

23. The use of the antibody or antigen-binding fragment thereof according to claim 20, wherein the autoimmune disease is multiple sclerosis.

Images