Antibody for specifically recognizing B and T lymphocyte attenuators (BTLAs) and application thereof

Abstract

The present application provides antibodies and antigen-binding fragments thereof that specifically recognize B and T lymphocyte attenuators. The invention also provides a preparation method and a use method of the antibody.

Description

[0001] Cross - reference to related applications

[0002] This application claims the priority of U.S. Application No. US63 / 368,870, filed on July 19, 2022, the content of which is incorporated herein by reference in its entirety.

[0003] Submission of the Sequence Listing as an XML FILE text file

[0004] The content of the following submitted XML text file is incorporated herein by reference in its entirety: Text Name: 710262000940SEQLIST.xml, Size: 49713 bytes; Date of Record: 2023.7.14). Technical Field

[0005] This application relates to antibodies that specifically recognize B and T lymphocyte attenuator (BTLA), and methods for their preparation and use, including methods for treating cancer or infectious diseases. Background Art

[0006] BTLA (CD272), a member of the CD28 family, induces immunosuppression by inhibiting the functions and proliferation of B cells and T cells. Structurally, BTLA is a type I transmembrane glycoprotein containing 289 amino acid residues and belongs to the immunoglobulin family (Ning, et al. 2021). Notably, herpesvirus entry mediator (HVEM, also known as tumor necrosis factor receptor superfamily 14, TNFRSF14) has been identified as the specific ligand for BTLA, which belongs to the TNFR family (Compaan, D.M, et al, 2005; Gonzalez, L.C, et al. 2005; Sedy, J.R, et al. 2005). In addition to BTLA, HVEM also binds to LIGHT (also known as TNFSF14) and CD160. Most literature reports that BTLA and CD160 binding to the CRD1 / CRD2 region on HVEM mediate inhibitory signals, while LIGHT transduces stimulatory signals by binding to the CRD2 / CRD3 region (Cai G, et al. 2008: Rodriguez - Barbosa JI, et al. 2019; Sedy, J.R, et al. 2005). However, there are also reports indicating that BTLA and CD160 can induce bidirectional signals in some cases (Gavrieli M and Murphy KM, 2006; Tan CL, et al. 2018).

[0007] HVEM is highly expressed in resting T cells, naive B cells, and memory B cells, but is downregulated in activated T cells and B cells. It is also widely expressed in all other immune cells, certain types of cancer cells, intestinal epithelial cells, etc. Interestingly, HVEM can act as a ligand or receptor and play multiple different roles under different physiological and pathological conditions (Demerlé C, et al. 2021). Although the HVEM / BTLA / CD160 / LIGHT mediates complex signaling pathways, HVEM knockout mice show a stronger CD4+ T cell response to stimulation, indicating that the overall function mediated by HVEM is inhibitory (Wang Y, et al. 2005). In addition, HVEM and BTLA in naive T cells form a cis-heterodimer complex, blocking the binding of external CD160 and other co-signaling molecules to HVEM, thus maintaining T cell tolerance (Cheung TC, et al. 2009).

[0008] In contrast to the broad expression profile of HVEM, the expression of BTLA is restricted to lymphoid organs and is rarely or not expressed in other organs such as the heart, kidney, brain, and liver, etc. (Yu, et al. 2019). Among immune cells, BTLA is mainly expressed in B cells and T cells, but its expression can also be detected in innate immune cells such as dendritic cells (DCs) and monocytes (Ning, et al. 2021). BTLA consists of an Ig-like extracellular domain, a single transmembrane domain (TMD), and an intracellular tail. The cytoplasmic domain of BTLA contains an immunoreceptor tyrosine-based inhibitory motif (ITIM), an immunoreceptor tyrosine-based switch motif (ITSM), and a growth factor receptor-bound protein 2 (Grb-2) associated motif. Activation of the BTLA signaling pathway by HVEM leads to tyrosine phosphorylation of ITIM, enabling the binding of protein tyrosine phosphatases SHP-1 and SHP-2 containing Src homology domain 2 (SH2), thereby inhibiting activation signals in immune cells (Gavrieli M and Murphy KM, 2006; Gavrieli M, et al. 2003; Watanabe N, et al. 2003). Recent studies have shown that, different from PD-1 inhibiting CD28 signaling through SHP-2, BTLA mainly inhibits TCR and CD28 signaling through SHP-1 (Celis-Gutierrez J, et al. 2019; Xu X, et al. 2020). The study also found that the binding of the Grb-2 associated motif to Grb-2 leads to the recruitment of the PI3K protein subunit p85 and T cell activation. Therefore, the BTLA molecule can play a bidirectional regulatory role (Gavrieli M and Murphy KM, 2006).

[0009] BTLA plays a key role in inducing and maintaining T cell immune tolerance. Krieg et al. reported that BTLA is involved in regulating CD8+ T cell homeostasis and memory cell generation (Krieg C, et al. 2007). Studies have also shown that CD8+ T cells lacking BTLA are more prone to induce type I diabetes. In addition, in BTLA knockout mice, even high doses of ovalbumin cannot induce immune tolerance (Liu X, et al. 2009). Moreover, BTLA inhibits follicular helper T cells from producing IL-21, thereby reducing IgG serum levels (Kashiwakuma D, et al. 2010). In addition to T cell functions, BTLA may also affect B cell, dendritic cell, and NKT cell functions (Vendel AC, etal. 2009; Jones A, et al. 2016; Miller ML, etal. 2009).

[0010] BTLA is upregulated in tumor-infiltrating lymphocytes (TILs), while HVEM can be expressed on tumor cells. A large amount of data indicates that the HVEM / BTLA interaction impairs anti-tumor immunity (Demerlé C, et al. 2021). In some reports, the expression level of BTLA is elevated in circulating CD4+ cells, while the expression level remains unchanged in CD8+ T cells. Blocking the BTLA / HVEM interaction can enhance the production of IFN-γ by circulating CD4+ and CD8+ T cells in patients with liver cancer (Liu J, et al. 2018; Zhao Q, et al. 2016). In other publications, it was found that the upregulation of BTLA in CD8+ TILs is associated with poor prognosis in patients with gallbladder cancer (Oguro S, et al. 2015). BTLA is also upregulated in tumor antigen-specific T cells (Fourcade J, et al. 2012; Derre L, et al. 2010). BTLA+ tumor antigen-specific T cells exhibit elevated levels of other inhibitory molecules, such as PD-1, lymphocyte activation gene-3 (LAG3), T cell immunoglobulin and mucin domain-containing molecule 3 (TIM-3), etc. (Quan L, et al. 2018; Fourcade J, et al. 2012). In addition, compared with blocking PD-1 alone, blocking BTLA and PD-1 with antibodies can synergistically enhance the expansion, proliferation, and cytokine production of NY-ESO-1-specific CD8+ T cells in patients with melanoma (Fourcade J, et al. 2012).

[0011] Therefore, there is still a need in the art for therapeutic antibodies that can effectively inhibit or antagonize BTLA and treat BTLA-mediated diseases or disorders (e.g., cancer or infectious diseases).

[0012] All publications, patents, patent applications, and disclosures of published patent applications mentioned herein are incorporated herein by reference in their entirety.

[0013] Application Overview

[0014] In some embodiments, there is provided an isolated anti-BTLA antibody comprising a heavy chain variable domain (V H ) which V H comprises: heavy chain complementarity determining region (HC-CDR) 1 which comprises TFGMGVS (SEQ ID NO:1); HC-CDR2 which comprises HIYWDDDKRFNPSLKS (SEQ ID NO:4); and HC-CDR3 which comprises GNWDGETYFDY (SEQ ID NO:7); and a light chain variable domain (V L ) which V L comprises: light chain complementarity determining region (LC-CDR) 1 which comprises KSTQSLLDSDGKTYLN (SEQ ID NO:10); LC-CDR2 which comprises LVSKLDS (SEQ ID NO:13); and LC-CDR3 which comprises WQGTHFPWT (SEQ ID NO:15).

[0015] In some embodiments, there is provided an isolated anti-BTLA antibody which comprises V H which V H comprises: HC-CDR1 which comprises the amino acid sequence shown in SEQ ID NO:1 or a variant thereof which variant comprises up to about 3 amino acid substitutions; HC-CDR2 which comprises the amino acid sequence shown in SEQ ID NO:4 or a variant thereof which variant comprises up to about 3 amino acid substitutions; and HC-CDR3 which comprises the amino acid sequence shown in SEQ ID NO:7 or a variant thereof which variant comprises up to about 3 amino acid substitutions; and V L which V L comprises: LC-CDR1 which comprises the amino acid sequence shown in SEQ ID NO:10 or a variant thereof which variant comprises up to about 3 amino acid substitutions; LC-CDR2 which comprises the amino acid sequence shown in SEQ ID NO:13 or a variant thereof which variant comprises up to about 3 amino acid substitutions; and LC-CDR3 which comprises the amino acid sequence shown in SEQ ID NO:15 or a variant thereof which variant comprises up to about 3 amino acid substitutions.

[0016] In some embodiments, there is provided an isolated anti-BTLA antibody which comprises: V H which V Hcomprising a V as shown in any of the amino acid sequences of SEQ ID NOs: 18 - 22 H comprising the HC - CDR1, HC - CDR2, and HC - CDR3, and the V L , said V L comprising a V as shown in any of the amino acid sequences of SEQ ID NOs: 25 - 29 L comprising the LC - CDR1, LC - CDR2, and LC - CDR3.

[0017] In some embodiments, there is provided an isolated anti - BTLA antibody comprising: (i) a V H which comprises a V as shown in the amino acid sequence SEQ ID NO: 18 H comprising the HC - CDR1, HC - CDR2, and HC - CDR3; and a V L which comprises a V as shown in the amino acid sequence SEQ ID NO: 25 L comprising the LC - CDR1, LC - CDR2, and LC - CDR3; (ii) a V H which comprises a V as shown in the amino acid sequence SEQ ID NO: 19 H comprising the HC - CDR1, HC - CDR2, and HC - CDR3; and a V L which comprises a V as shown in the amino acid sequence SEQ ID NO: 26 L comprising the LC - CDR1, LC - CDR2, and LC - CDR3; (iii) a V H which comprises a V as shown in the amino acid sequence SEQ ID NO: 19 H comprising the HC - CDR1, HC - CDR2, and HC - CDR3; and a V L which comprises a V as shown in the amino acid sequence SEQ ID NO: 27 L comprising the LC - CDR1, LC - CDR2, and LC - CDR3; (iv) a V H which comprises a V as shown in the amino acid sequence SEQ ID NO: 19 H comprising the HC - CDR1, HC - CDR2, and HC - CDR3; and a V L which comprises a V as shown in the amino acid sequence SEQ ID NO: 28 L comprising the LC - CDR1, LC - CDR2, and LC - CDR3; (v) a V H which comprises a V as shown in the amino acid sequence SEQ ID NO: 19 H comprising the HC - CDR1, HC - CDR2, and HC - CDR3; and a V L, which contains V as shown in the amino acid sequence SEQ ID NO: 29 L containing LC-CDR1, LC-CDR2, and LC-CDR3; (vi) V H , which contains V as shown in the amino acid sequence SEQ ID NO: 20 H containing HC-CDR1, HC-CDR2, and HC-CDR3; and V L , which contains V as shown in the amino acid sequence SEQ ID NO: 26 L containing LC-CDR1, LC-CDR2, and LC-CDR3; (vii) V H , which contains V as shown in the amino acid sequence SEQ ID NO: 20 H containing HC-CDR1, HC-CDR2, and HC-CDR3; and V L , which contains V as shown in the amino acid sequence SEQ ID NO: 27 L containing LC-CDR1, LC-CDR2, and LC-CDR3; (viii) V H , which contains V as shown in the amino acid sequence SEQ ID NO: 20 H containing HC-CDR1, HC-CDR2, and HC-CDR3; and V L , which contains V as shown in the amino acid sequence SEQ ID NO: 28 L containing LC-CDR1, LC-CDR2, and LC-CDR3; (ix) V H , which contains V as shown in the amino acid sequence SEQ ID NO: 20 H containing HC-CDR1, HC-CDR2, and HC-CDR3; and V L , which contains V as shown in the amino acid sequence SEQ ID NO: 29 L containing LC-CDR1, LC-CDR2, and LC-CDR3; (x) V H , which contains V as shown in the amino acid sequence SEQ ID NO: 21 H containing HC-CDR1, HC-CDR2, and HC-CDR3; and V L , which contains V as shown in the amino acid sequence SEQ ID NO: 28 L containing LC-CDR1, LC-CDR2, and LC-CDR3; (xi) V H , which contains V as shown in the amino acid sequence SEQ ID NO: 21 H containing HC-CDR1, HC-CDR2, and HC-CDR3; and V L, which comprises a V as shown in amino acid sequence SEQ ID NO: 29 L comprising LC-CDR1, LC-CDR2 and LC-CDR3; (xii) V H , which comprises a V as shown in amino acid sequence SEQ ID NO: 22 H comprising HC-CDR1, HC-CDR2 and HC-CDR3; and V L , which comprises a V as shown in amino acid sequence SEQ ID NO: 28 L comprising LC-CDR1, LC-CDR2 and LC-CDR3; (xiii) V H , which comprises a V as shown in amino acid sequence SEQ ID NO: 22 H comprising HC-CDR1, HC-CDR2 and HC-CDR3; and V L , which comprises a V as shown in amino acid sequence SEQ ID NO: 29 L comprising LC-CDR1, LC-CDR2 and LC-CDR3.

[0018] In some embodiments, there is provided an isolated anti-BTLA antibody, which comprises: (i) V H , said V H comprising: HC-CDR1, which comprises amino acid sequence SEQ ID NO: 1, HC-CDR2, which comprises amino acid sequence SEQ ID NO: 4, and HC-CDR3, which comprises amino acid sequence SEQ ID NO: 7, or a variant of said V H having at most about 5 amino acid substitutions in its HC-CDRs; and V L , said V L comprising: LC-CDR1, which comprises amino acid sequence SEQ ID NO: 10, LC-CDR2, which comprises amino acid sequence SEQ ID NO: 13, and LC-CDR3, which comprises amino acid sequence SEQ ID NO: 15, or a variant of said V L having at most about 5 amino acid substitutions in its LC-CDRs.

[0019] In some embodiments, any of the isolated anti-BTLA antibodies as described above, which comprises: V H which comprises an amino acid sequence shown in any of SEQ ID NOs: 18-22 or a variant thereof, said variant having at least about 80% sequence identity with an amino acid sequence shown in any of SEQ ID NOs: 18-22; and V L, which comprises an amino acid sequence shown in any of SEQ ID NOs: 25-29 or a variant thereof, said variant having at least about 80% sequence identity with the amino acid sequence shown in any of SEQ ID NOs: 25-29.

[0020] In some embodiments, there is provided an isolated anti-BTLA antibody comprising: (i) V H , which comprises the amino acid sequence SEQ ID NO: 18 or a variant thereof, said variant having at least about 80% sequence identity with the amino acid sequence SEQ ID NO: 18; and V L , which comprises the amino acid sequence SEQ ID NO: 25 or a variant thereof, said variant having at least about 80% sequence identity with the amino acid sequence SEQ ID NO: 25; (ii) V H , which comprises the amino acid sequence SEQ ID NO: 19 or a variant thereof, said variant having at least about 80% sequence identity with the amino acid sequence SEQ ID NO: 19; and V L , which comprises the amino acid sequence SEQ ID NO: 26 or a variant thereof, said variant having at least about 80% sequence identity with the amino acid sequence SEQ ID NO: 26; (iii) V H , which comprises the amino acid sequence SEQ ID NO: 19 or a variant thereof, said variant having at least about 80% sequence identity with the amino acid sequence SEQ ID NO: 19; and V L , which comprises the amino acid sequence SEQ ID NO: 27 or a variant thereof, said variant having at least about 80% sequence identity with the amino acid sequence SEQ ID NO: 27; (iv) V H , which comprises the amino acid sequence SEQ ID NO: 19 or a variant thereof, said variant having at least about 80% sequence identity with the amino acid sequence SEQ ID NO: 19; and V L , which comprises the amino acid sequence SEQ ID NO: 28 or a variant thereof, said variant having at least about 80% sequence identity with the amino acid sequence SEQ ID NO: 28; (v) V H , which comprises the amino acid sequence SEQ ID NO: 19 or a variant thereof, said variant having at least about 80% sequence identity with the amino acid sequence SEQ ID NO: 19; and V L , which comprises the amino acid sequence SEQ ID NO: 29 or a variant thereof, said variant having at least about 80% sequence identity with the amino acid sequence SEQ ID NO: 29; (vi) V H , which comprises the amino acid sequence SEQ ID NO: 20 or a variant thereof, said variant having at least about 80% sequence identity with the amino acid sequence SEQ ID NO: 20; and VL which comprises the amino acid sequence SEQ ID NO:26 or a variant thereof, said variant having at least about 80% sequence identity with the amino acid sequence SEQ ID NO:26; (vii) V H which comprises the amino acid sequence SEQ ID NO:20 or a variant thereof, said variant having at least about 80% sequence identity with the amino acid sequence SEQ ID NO:20; and V L which comprises the amino acid sequence SEQ ID NO:27 or a variant thereof, said variant having at least about 80% sequence identity with the amino acid sequence SEQ ID NO:27; (viii) V H which comprises the amino acid sequence SEQ ID NO:20 or a variant thereof, said variant having at least about 80% sequence identity with the amino acid sequence SEQ ID NO:20; and V L which comprises the amino acid sequence SEQ ID NO:28 or a variant thereof, said variant having at least about 80% sequence identity with the amino acid sequence SEQ ID NO:28; (ix) V H which comprises the amino acid sequence SEQ ID NO:20 or a variant thereof, said variant having at least about 80% sequence identity with the amino acid sequence SEQ ID NO:20; and V L which comprises the amino acid sequence SEQ ID NO:29 or a variant thereof, said variant having at least about 80% sequence identity with the amino acid sequence SEQ ID NO:29; (x) V H which comprises the amino acid sequence SEQ ID NO:21 or a variant thereof, said variant having at least about 80% sequence identity with the amino acid sequence SEQ ID NO:21; and V L which comprises the amino acid sequence SEQ ID NO:28 or a variant thereof, said variant having at least about 80% sequence identity with the amino acid sequence SEQ ID NO:28; (xi) V H which comprises the amino acid sequence SEQ ID NO:21 or a variant thereof, said variant having at least about 80% sequence identity with the amino acid sequence SEQ ID NO:21; and V L which comprises the amino acid sequence SEQ ID NO:29 or a variant thereof, said variant having at least about 80% sequence identity with the amino acid sequence SEQ ID NO:29; (xii) V H which comprises the amino acid sequence SEQ ID NO:22 or a variant thereof, said variant having at least about 80% sequence identity with the amino acid sequence SEQ ID NO:22; and V L, which comprises the amino acid sequence SEQ ID NO:28 or a variant thereof that has at least about 80% sequence identity with the amino acid sequence SEQ ID NO:28; (xiii) V H , which comprises the amino acid sequence SEQ ID NO:22 or a variant thereof that has at least about 80% sequence identity with the amino acid sequence SEQ ID NO:22; and V L , which comprises the amino acid sequence SEQ ID NO:29 or a variant thereof that has at least about 80% sequence identity with the amino acid sequence SEQ ID NO:29.

[0021] In some embodiments, there is provided an isolated anti-BTLA antibody comprising: (i) V H , which comprises the V H comprising HC-CDR1, HC-CDR2, and HC-CDR3 as shown in the amino acid sequence SEQ ID NO:23; and V L , which comprises the V L comprising LC-CDR1, LC-CDR2, and LC-CDR3 as shown in the amino acid sequence SEQ ID NO:30.

[0022] In some embodiments, there is provided an isolated anti-BTLA antibody comprising: (i) V H , said V H comprising: HC-CDR1, which comprises the amino acid sequence SEQ ID NO:2, HC-CDR2, which comprises the amino acid sequence SEQ ID NO:5, and HC-CDR3, which comprises the amino acid sequence SEQ ID NO:8, or a variant of said V H comprising at most about 5 amino acid substitutions in its HC-CDRs; and V L , said V L comprising: LC-CDR1, which comprises the amino acid sequence SEQ ID NO:11, LC-CDR2, which comprises the amino acid sequence SEQ ID NO:14, and LC-CDR3, which comprises the amino acid sequence SEQ ID NO:16, or a variant of said V L comprising at most about 5 amino acid substitutions in its LC-CDRs.

[0023] In some embodiments, any of the isolated anti-BTLA antibodies as described above comprises: V H , which comprises the amino acid sequence shown in SEQ IDNO:23 or a variant thereof that has at least about 80% sequence identity with the amino acid sequence shown in SEQ ID NO:23; and V L, which comprises the amino acid sequence shown in SEQ ID NO:30 or a variant thereof, said variant having at least about 80% sequence identity with the amino acid sequence shown in SEQ ID NO:30.

[0024] In some embodiments, there is provided an isolated anti-BTLA antibody, which comprises: (i) a V H , which comprises a V as shown in the amino acid sequence SEQ ID NO:24 H comprising HC-CDR1, HC-CDR2 and HC-CDR3; and a V L , which comprises a V as shown in the amino acid sequence SEQ ID NO:31 L comprising LC-CDR1, LC-CDR2 and LC-CDR3.

[0025] In some embodiments, there is provided an isolated anti-BTLA antibody, which comprises: (i) a V H , said V H comprising: HC-CDR1, which comprises the amino acid sequence SEQ ID NO:3, HC-CDR2, which comprises the amino acid sequence SEQ ID NO:6, and HC-CDR3, which comprises the amino acid sequence SEQ ID NO:9, or a variant of said V H having at most about 5 amino acid substitutions in its HC-CDRs; and a V L , said V L comprising: LC-CDR1, which comprises the amino acid sequence SEQ ID NO:12, LC-CDR2, which comprises the amino acid sequence SEQ ID NO:13, and LC-CDR3, which comprises the amino acid sequence SEQ ID NO:17, or a variant of said V L having at most about 5 amino acid substitutions in its LC-CDRs.

[0026] In some embodiments, any of the isolated anti-BTLA antibodies as described above comprises: a V H , which comprises the amino acid sequence shown in SEQ IDNO:24 or a variant thereof, said variant having at least about 80% sequence identity with the amino acid sequence shown in SEQ ID NO:24; and a V L , which comprises the amino acid sequence shown in SEQ ID NO:31 or a variant thereof, said variant having at least about 80% sequence identity with the amino acid sequence shown in SEQ ID NO:31.

[0027] In some embodiments, there is provided an isolated anti-BTLA antibody, which has a Kd value for specific binding to human BTLA of from 0.1 pM to 10 nM.

[0028] In some embodiments, a separated anti-BTLA antibody is provided, which competes with any of the above-mentioned separated anti-BTLA antibodies for specific binding to BTLA. In some embodiments, a separated anti-BTLA antibody is provided, which specifically binds to the same epitope as any of the above-mentioned separated anti-BTLA antibodies.

[0029] In some embodiments, any of the above-mentioned separated anti-BTLA antibodies, the separated anti-BTLA antibody comprises an Fc fragment. In some embodiments, the separated anti-BTLA antibody is a full-length IgG antibody. In some embodiments, the separated anti-BTLA antibody is a full-length IgG1, IgG2, IgG3 or IgG4 antibody. In some embodiments, the anti-BTLA antibody is a chimeric, fully human or humanized antibody. In some embodiments, the anti-BTLA antibody is an antigen-binding fragment, and the antigen-binding fragment is selected from the group consisting of Fab, Fab’, F(ab)’2, Fab’-SH, single-chain Fv (scFv), Fv fragment, dAb, Fd, nanobody, diabody and linear antibody.

[0030] In some embodiments, a separated nucleic acid molecule is provided, the nucleic acid molecule encodes any of the above-mentioned anti-BTLA antibodies. In some embodiments, a vector is provided, the vector comprises any of the above-mentioned nucleic acid molecules. In some embodiments, a host cell is provided, the host cell comprises any of the above-mentioned anti-BTLA antibodies, any of the above-mentioned nucleic acid molecules or any of the above-mentioned vectors. In some embodiments, a method for preparing an anti-BTLA antibody is provided, which comprises: a) culturing any of the above-mentioned host cells under conditions capable of effectively expressing the anti-BTLA antibody; and b) obtaining the expressed anti-BTLA antibody from the host cell.

[0031] In some embodiments, a method for treating a disease or disorder in an individual in need thereof is provided, comprising administering to the individual an effective amount of any one of the anti-BTLA antibodies as described above. In some embodiments, use of any one of the anti-BTLA antibodies as described above in the preparation of a pharmaceutical composition for treating a disease or disorder in an individual in need thereof is provided. In some embodiments, use of any one of the anti-BTLA antibodies or a pharmaceutical composition comprising an anti-BTLA antibody in the preparation of a medicament for treating a disease or disorder is provided. In some embodiments, the disease or disorder is related to the BTLA signaling pathway, including cancer or infectious diseases or disorders. In some embodiments, the disease or disorder is selected from, for example, non-small cell lung cancer, adrenal cancer, bladder cancer, brain cancer, pancreatic cancer, breast cancer, colorectal cancer, melanoma, esophageal cancer, gastric cancer, cervical cancer, head and neck cancer, hepatocellular carcinoma, kidney cancer, liver cancer, ovarian cancer, pancreatic cancer, prostate cancer, small cell lung cancer, testicular cancer, thyroid cancer, uterine cancer, and any type of leukemia, lymphoma, and myeloma, and infectious diseases, including but not limited to Human Papilloma Virus (HPV), Human Immunodeficiency Virus (HIV), Herpes Simplex Virus (HSV), Varicella Zoster Virus (VSV), Cytomegalovirus (CMV), Epstein Barr Virus (EBV), Chlamydia, Rickettsia bacteria, Mycobacterium, Staphylococcus, Streptococcus, Pneumococcus, Neisseria meningitidis, and Neisseria gonorrhoeae, Klebsiella, Proteus, Serratia, Pseudomonas, Legionella, Diphtheria, Salmonella, Bacillus, Cholera, Tetanus, Clostridium botulinum, Anthrax, Plague, Leptospirosis, and Lyme disease bacteria.

[0032] Also provided are pharmaceutical compositions, kits, and articles of manufacture comprising any one of the anti-BTLA antibodies as described above. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1A-1B Shown are the binding affinities of the chimeric anti-BTLA antibodies 83F2, 86B7, and 96F11 to human BTLA or rhesus monkey BTLA analyzed by ELISA. Figure 1A Shown are the binding affinities of 83F2, 86B7, and 96F11 to human BTLA. Figure 1B Shown are the binding affinities of 83F2, 86B7, and 96F11 to rhesus monkey BTLA.

[0034] Figure 2 Shown are the blocking activities of chimeric anti-BTLA antibodies 83F2, 86B7, and 96F11 in blocking the binding of human HVEM to human BTLA analyzed by ELISA.

[0035] Figure 3A Shown are Expi293 cells stably overexpressing huBTLA analyzed by FACS. Figure 3B Shown are the binding affinities of exemplary chimeric anti-BTLA antibodies 83F2 and 86B7 to huBTLA-overexpressing Expi293 cells analyzed by FACS. Figure 3C Shown are the blocking activities of exemplary chimeric anti-BTLA antibodies 83F2 and 86B7 in inhibiting the binding of soluble HVEM to huBTLA-overexpressing Expi293 cells analyzed by FACS.

[0036] Figure 4A Shown is a schematic diagram of a Raji-HVEM / Jurkat-BTLA co-culture system stimulated with an anti-αCD3 / CD19 bispecific antibody in the presence or absence of an anti-BTLA antibody, which includes co-culturing HVEM-overexpressing Raji cells with BTLA-overexpressing Jurkat cells. Figure 4B Shown is that in the Raji-HVEM / Jurkat-BTLA co-culture system, chimeric anti-BTLA antibodies 83F2, 86B7, and 96F11 restore T cell IL-2 production by inhibiting the HVEM-BTLA signaling pathway.

[0037] Figure 5A Shown are the binding affinities of exemplary humanized anti-BTLA antibodies SB2003-3, SB2003-4, SB2003-11, and SB2003-12 to huBTLA-overexpressing Expi293 cells analyzed by FACS. Figure 5B Shown are the blocking activities of exemplary humanized anti-BTLA antibodies SB2003-3, SB2003-4, SB2003-11, and SB2003-12 in inhibiting the binding of soluble HVEM to huBTLA-overexpressing Expi293 cells analyzed by FACS.

[0038] Figure 6 Shown is that in the Jurkat-BTLA-FAS cell system, exemplary human anti-BTLA antibodies SB2003-11 and SB2003-12 inhibit huHVEM-Fc-induced apoptosis.

[0039] Figure 7AShown is that in the Raji-HVEM / Jurkat-BTLA co-culture system, the exemplary humanized anti-BTLA antibody SB2003-12 restores T cell IL-2 production by inhibiting the HVEM-BTLA signaling pathway. Figure 7B Shown is that in the Raji-HVEM / primary human T cell co-culture system, the exemplary humanized anti-BTLA antibodies SB2003-11 and SB2003-12 restore T cell IL-2 production by inhibiting the HVEM-BTLA signaling pathway.

[0040] Figure 8A Shown is that murine HVEM binds to human BTLA, while the exemplary humanized anti-BTLA antibody SB2003-12 blocks this interaction. Figure 8B Shown is the average tumor volume of each mouse after treatment with the exemplary humanized anti-BTLA antibody SB2003-12 and the isotype control antibody.

[0041] Detailed description of the present application

[0042] On the one hand, the present application provides anti-BTLA antibody molecules. Through a combination of hybridoma technology, humanization of antibodies, affinity maturation, and appropriately designed biochemical and biological experiments, highly effective antibody molecules that can bind to human BTLA and inhibit the interaction of human BTLA with its receptor have been identified. The results presented herein show that the antibodies in the present application bind to BTLA. And surprisingly, compared with anti-BTLA antibodies known in the art, for example, Icatolimab (also known as tifcemalimab, developed by Shanghai Junshi), the antibodies in the present application have been proven to be more effective in various biological experiments.

[0043] The anti-BTLA antibodies provided by the present application include, for example, full-length anti-BTLA antibodies, anti-BTLA single-chain antibodies (scFvs), anti-BTLA Fc fusion proteins, multispecific (such as bispecific) anti-BTLA antibodies, anti-BTLA immunoconjugates, and the like.

[0044] In some embodiments, the isolated anti-BTLA antibody comprises: a heavy chain variable domain (V H ), the V H comprises: a heavy chain complementarity-determining region (HC-CDR) 1, which comprises TFGMGVS (SEQ ID NO:1); HC-CDR2, which comprises HIYWDDDKRFNPSLKS (SEQ ID NO:4); and HC-CDR3, which comprises GNWDGETYFDY (SEQ ID NO:7); and a light chain variable domain (V L ), the V LComprising: light chain complementarity determining region (LC-CDR) 1, which comprises KSTQSLLDSDGKTYLN (SEQ ID NO:10); LC-CDR2, which comprises LVSKLDS (SEQ ID NO:13); and LC-CDR3, which comprises WQGTHFPWT (SEQ ID NO:15).

[0045] In some embodiments, the isolated anti-BTLA antibody comprises: a heavy chain variable domain (V H ), the V H Comprising: heavy chain complementarity determining region (HC-CDR) 1, which comprises DFWIQ (SEQ ID NO:2); HC-CDR2, which comprises TIYPGDGDTRENQKFKG (SEQ ID NO:5); and HC-CDR3, which comprises GNGNSWFAY (SEQ ID NO:8); and a light chain variable domain (V L ), the V L Comprising: light chain complementarity determining region (LC-CDR) 1, which comprises RASESVDDYGISFIN (SEQ ID NO:11); LC-CDR2, which comprises AASNQGS (SEQ ID NO:14); and LC-CDR3, which comprises LQSREIPYT (SEQ ID NO:16).

[0046] In some embodiments, the isolated anti-BTLA antibody comprises: a heavy chain variable domain (V H ), the V H Comprising: heavy chain complementarity determining region (HC-CDR) 1, which comprises DTYIY (SEQ ID NO:3); HC-CDR2, which comprises RIDPANGHTKYDPRFQD (SEQ ID NO:6); and HC-CDR3, which comprises GGDHPYYVMDW (SEQ ID NO:9); and a light chain variable domain (V L ), the V L Comprising: light chain complementarity determining region (LC-CDR) 1, which comprises KSSQNLLDSDGKTYLI (SEQ ID NO:12); LC-CDR2, which comprises LVSKLDS (SEQ ID NO:13); and LC-CDR3, which comprises WQGTHFPRT (SEQ ID NO:17).

[0047] Also provided are nucleic acids encoding the anti-BTLA antibody, compositions comprising the anti-BTLA antibody, and methods of making and using the anti-BTLA antibody.

[0048] Definitions

[0049] As used herein, "treatment" or "treating" is a method of obtaining a beneficial or desired result, including a clinical result. For the purposes of this application, the beneficial or desired clinical result includes, but is not limited to, one or more of the following: alleviating one or more symptoms caused by a disease, reducing the severity of a disease, stabilizing a disease (e.g., preventing or delaying disease progression), preventing or delaying the spread of a disease (e.g., metastasis), preventing or delaying disease recurrence, delaying or slowing disease progression, improving the disease state, alleviating the disease (partially or completely), reducing the dosage of one or more other medications required to treat the disease, delaying disease progression, improving or enhancing quality of life, increasing body weight, and / or extending survival. At the same time, "treatment" also includes a reduction in disease pathological outcomes (e.g.,

[0050] for cancer, tumor volume). The methods of this application contemplate any one or more aspects of these treatments.

[0051] The term "antibody" includes full-length antibodies and their antigen-binding fragments. A full-length antibody includes two heavy chains and two light chains. The variable regions of the light and heavy chains are responsible for antigen binding. The variable regions in both chains typically include 3 hypervariable loops, known as complementarity-determining regions (CDRs) (light chain (LC) CDRs include LC-CDR1, LC-CDR2, and LC-CDR3, and heavy chain (HC) CDRs include HC-CDR1, HC-CDR2, and HC-CDR3). The CDR boundaries of the antibodies or antigen-binding fragments disclosed herein can be defined or identified by the Kabat, Chothia, or Al-Lazikani conventions (Al-Lazikani 1997; Chothia 1985; Chothia 1987; Chothia 1989; Kabat 1987; Kabat 1991). The 3 CDR regions of the heavy or light chain are inserted between flanking segments called framework regions (FRs), which are more conserved than the CDR regions and form a scaffold to support the hypervariable loops. The constant regions of the heavy and light chains do not participate in antigen binding but exhibit various effector functions. Antibodies are classified based on the amino acid sequence of their heavy-chain constant regions. The five main classes or isotypes of antibodies are IgA, IgD, IgE, IgG, and IgM, which are characterized by having α, δ, ε, γ, and μ heavy chains, respectively. Several of the main antibody classes are divided into subclasses, such as IgG1 (γ1 heavy chain), IgG2 (γ2 heavy chain), IgG3 (γ3 heavy chain), IgG4 (γ4 heavy chain), IgA1 (α1 heavy chain), or IgA2 (α2 heavy chain).

[0052] As used herein, the term "antigen-binding fragment" includes antibody fragments such as diabodies, Fab, Fab', F(ab')2, Fv fragments, disulfide-stabilized Fv fragments (dsFv), (dsFv)2, bispecific dsFv (dsFv-dsFv'), disulfide-stabilized diabodies (ds diabodies), single-chain Fv (scFv), scFv dimers (bivalent diabodies), multispecific antibodies composed of antibody fragments containing one or more CDRs, single-domain antibodies, nanobodies, domain antibodies, bivalent domain antibodies, or any other antibody fragment that is capable of binding an antigen but does not contain the complete antibody structure. An antigen-binding fragment is capable of binding the same antigen as the parental antibody or parental antibody fragment (such as a parental scFv). Antigen-binding fragments also include fusion proteins comprising the above-described antibody fragments. In some embodiments, an antigen-binding fragment may include one or more CDRs from a particular human antibody that are grafted into the framework region from one or more different human antibodies.

[0053] As used herein, the term "epitope" refers to a specific group of atoms or amino acids on an antigen to which an antibody or antibody portion binds. If two antibodies or antibody portions exhibit competitive binding to an antigen, they may bind to the same epitope on the antigen.

[0054] As used herein, a first antibody "competes" with a second antibody for binding to a BTLA target when the first antibody inhibits the binding of the second antibody to the BTLA target by at least 50% (e.g., at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%) at equimolar concentrations, and vice versa. PCT Publication WO

[0055] 03 / 48731 describes a high-throughput antibody "epitope binning" method based on cross-competition.

[0056] As used herein, the terms "specifically binds", "specifically recognizes", or "is specific for" refer to a measurable and reproducible interaction, such as the binding of an antibody to a target that can be determined to be present in a heterogeneous population of molecules, including biomolecules. For example, an antibody being able to specifically recognize a target (which can be an epitope) means that the antibody has a higher affinity, avidity, binds more readily and / or more persistently to that target compared to binding to other targets. In some embodiments, an antibody that specifically recognizes an antigen reacts with one or more antigenic determinants of the antigen, and its binding affinity is at least 10-fold greater than its binding affinity for other targets.

[0057] As described herein, a "separated" anti-BTLA antibody refers to an anti-BTLA antibody that (1) is not related to a naturally occurring protein, (2) does not contain other proteins from the same source, (3) is expressed by cells of different species, or (4) does not exist in nature.

[0058] As described herein, the term "separated nucleic acid" refers to a nucleic acid from a genomic, cDNA, or synthetic source, or a combination thereof. Depending on its source, the "separated nucleic acid" refers to (1) nucleic acid that is not related to all or part of the polynucleotide in the "separated nucleic acid" found in nature, (2) can be operably linked to a polynucleotide that is not linked to it in its natural state, or (3) does not exist as part of a longer sequence in nature.

[0059] As used herein, the term "CDR" or "complementary determining region" means the non - contiguous antigen - binding sites found within the variable domains of heavy - and light - chain polypeptides. These particular regions have been described in Kabat et al., J. Biol. Chem. 252:6609 - 6616 (1977); Kabat et al., U.S. Dept. of Health and Human Services, "Sequences of proteins of immunological interest" (1991); Chothia et al., J. Mol. Biol. 196:901 - 917 (1987); Al - Lazikani B. et al., J. Mol. Biol., 273:927 - 948 (1997); MacCallum et al., J. Mol. Biol. 262:732 - 745 (1996); Abhinandan and Martin, Mol. Immunol., 45:3832 - 3839 (2008); Lefranc M.P. et al., Dev. Comp. Immunol., 27:55 - 77 (2003); and Honegger and Plückthun, J. Mol. Biol., 309:657 - 670 (2001), where, when compared to each other, these definitions include the overlap or subsets of amino acid residues. However, the use of any of these definition methods to indicate the CDRs of an antibody or a graft - antibody or its variants is included within the scope of the term as defined and used herein. The positions of the amino acid residues included in the CDRs defined by each of the above - cited references are listed in Table 1 for comparison. Algorithms for CDR prediction and binding interfaces are known in the art and are described, for example, in Abhinandan and Martin, Mol. Immunol., 45:3832 - 3839 (2008); Ehrenmann F. et al., Nucleic Acids Res., 38:D301 - D307 (2010); and Adolf - Bryfogle J. et al., Nucleic Acids Res., 43:D432 - D438 (2015). The content of the references cited in this paragraph is incorporated herein by reference in its entirety for use in this application and in one or more claims that may be included herein.

[0060] Table 1: CDR Definitions

[0061] <![CDATA[Kabat 1 > <![CDATA[Chothia 2 > <![CDATA[MacCallum 3 > <![CDATA[IMGT 4 > <![CDATA[AHo 5 > <![CDATA[V H CDR1]]> 31-35 26-32 30-35 27-38 25-40 <![CDATA[V H CDR2]]> 50-65 53-55 47-58 56-65 58-77 <![CDATA[V H CDR3]]> 95-102 96-101 93-101 105-117 109-137 <![CDATA[V L CDR1]]> 24-34 26-32 30-36 27-38 25-40 <![CDATA[V L CDR2]]> 50-56 50-52 46-55 56-65 58-77 <![CDATA[V L CDR3]]> 89-97 91-96 89-96 105-117 109-137

[0062] 1 The amino acid residue numbering refers to the nomenclature method in Kabat et al. mentioned above.

[0063] 2 The amino acid residue numbering refers to the nomenclature method in Chothia et al. mentioned above.

[0064] 3 The amino acid residue numbering refers to the nomenclature method in MacCallum et al. mentioned above.

[0065] 4 The amino acid residue numbering refers to the nomenclature method in Lefranc et al. mentioned above.

[0066] 5 The amino acid residue numbering refers to the nomenclature method in Honegger and Plückthun mentioned above.

[0067] The term "chimeric antibody" refers to an antibody in which a portion of the heavy and / or light chain is identical or homologous to the corresponding sequence in an antibody from a particular species or belonging to a particular antibody class or subclass, while the remaining portion of this (these) chain(s) is identical or homologous to the corresponding sequence in an antibody from another species or belonging to another antibody class or subclass, and fragments of such antibodies, provided that they have the biological activity in the present application (see U.S. Patent No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA, 81: 6851-6855 (1984)).

[0068] "Fv" is the smallest antibody fragment that contains the complete antigen recognition and binding site. This fragment is a dimer formed by the tight non-covalent linkage of a heavy chain variable domain and a light chain variable domain. Six hypervariable loops (3 loops in each of the light and heavy chains) are derived from the folding of these two domains. These hypervariable loops provide the amino acid residues for the antibody to bind to the antigen and confer the specificity of the antibody for antigen binding. However, even a single variable domain (or half of the Fv fragment, which only contains the 3 CDRs specific for the antigen) has the ability to recognize and bind the antigen, although its affinity is lower than that of the complete binding site.

[0069] "Single-chain Fv", also abbreviated as "sFv" or "scFv", is an antibody fragment that contains V H and V L antibody domains that are linked into a single polypeptide chain. In some embodiments, the scFv polypeptide further includes V H and V LA linking polypeptide between domains, which enables the scFv to form an ideal structure for antigen binding. For an overview of scFv, see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenberg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).

[0070] The term "diabody" refers to a small antibody fragment prepared by constructing an scFv fragment (see the above paragraph) with a short linker (e.g., 5 to 10 residues) between the V H and V L domains, such that the variable domains pair between chains rather than within a chain, resulting in a bivalent fragment, i.e., a fragment with two antigen-binding sites. A bispecific diabody is a heterodimer of two "crossed" scFv fragments, where the V H and V L domains of the two antibodies are on different polypeptide chains. Diabodies are described in full in EP 404,097; WO 93 / 11161; Hollinger et al., Proc. Natl. Acad. Sci. USA, 90: 6444-6448 (1993).

[0071] "Humanized" forms of non-human (e.g., rodent) antibodies are chimeric antibodies that include minimal sequences from the non-human antibody. In most cases, a humanized antibody is a human immunoglobulin (recipient antibody) in which the hypervariable region (HVR) residues of the recipient antibody are replaced with hypervariable region residues from a non-human species such as mouse, rat, rabbit, or non-human mammal that have the desired antibody specificity, affinity, and properties (donor antibody). In some cases, residues in the human immunoglobulin framework region are replaced with the corresponding non-human residues. Additionally, a humanized antibody can include residues that are not present in either the recipient antibody or the donor antibody. These modifications can further improve the properties of the antibody. Typically, a humanized antibody will contain substantially all, at least one, and usually two variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin, and all or substantially all of the framework regions are human immunoglobulin sequences. A human antibody optionally also includes at least a portion of the immunoglobulin constant region (Fc), typically the constant region of a human immunoglobulin. Specific details can be found in Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992).

[0072] The "percent amino acid sequence identity (%)" or "homology" of the polypeptide and antibody sequences identified herein is defined as the percentage of identical amino acid residues in a candidate sequence compared to a polypeptide sequence of interest, with conservative substitutions being considered part of sequence identity, when sequences are aligned. The percent amino acid sequence identity can be determined by a variety of alignment methods within the skill in the art, e.g., using publicly available computer software such as BLAST, BLAST-2, ALIGN, Megalign (DNASTAR), or MUSCLE software. One of ordinary skill in the art can determine the appropriate parameters for measuring alignment, including any algorithms needed to maximize alignment over the full length of the sequences being compared. However, for the purposes of this application, the percent amino acid sequence identity values are generated using the sequence alignment computer program MUSCLE (Edgar, R.C., Nucleic Acids Research 32(5):1792-1797, 2004; Edgar, R.C., BMC Bioinformatics 5(1):113, 2004).

[0073] The term "Fc receptor" or "FcR" is used to describe a receptor that binds to the Fc region of an antibody. In some embodiments, the FcRs described herein are FcRs that bind IgG antibodies (a γ receptor), including receptors of the FcγRI, FcγRII, and FcγRIII subclasses, including allelic variants and alternatively spliced forms of these receptors. The FcγRII receptor includes FcγRIIA ("activating receptor") and FcγRIIB ("inhibitory receptor"), which have similar amino acid sequences and differ primarily in their cytoplasmic domains. The cytoplasmic domain of the activating receptor FcγRIIA contains an immunoreceptor tyrosine-based activation motif (ITAM). The cytoplasmic domain of the inhibitory receptor FcγRIIB contains an immunoreceptor tyrosine-based inhibitory motif (ITIM) (see M.in Annu.Rev.Immunol. 15:203-234 (1997)). The term also includes allotypes, such as the FcγRIIIA allotypes: FcγRIIIA-Phe158, FcγRIIIA-Val158, FcγRIIA-R131, and / or FcγRIIA-H131. FcRs are described in Ravetch and Kinet, Annu.Rev.Immunol 9:457-92 (1991) and Capel et al., Immunomethods 4:25-34 (1994); and de Haas et al., J.Lab.Clin.Med. 126:330-41 (1995). The term FcR in the present application encompasses other types of FcRs, including FcRs to be identified in the future. The term FcR also includes the neonatal receptor FcRn, which is responsible for the transfer of maternal IgGs to the neonate (Guyer et al., J.Immunol. 117:587 (1976) and Kim et al., J.Immunol. 24:249

[0074] (1994)).

[0075] The term "FcRn" refers to the neonatal Fc receptor (FcRn). FcRn is structurally similar to the major histocompatibility complex (MHC) and is composed of an α chain non-covalently bound to β2-microglobulin. The multiple functions of the neonatal Fc receptor FcRn are reviewed in Ghetie and Ward (2000) Annu.Rev.Immunol. 18, 739-766. FcRn plays an important role in the passive transfer of immunoglobulin IgGs from the mother to the neonate and in the regulation of serum IgG levels. As a salvage receptor, FcRn can bind and transport endocytosed IgGs in an intact form within and between cells and protect them from undergoing the default degradation pathway.

[0076] The "CH1 domain" of the human IgG heavy chain constant region generally extends from amino acid position 118 to amino acid position 215 (EU numbering system).

[0077] The "hinge region" is generally defined as extending from Glu at position 216 to Pro at position 230 of human IgG1 (Burton, Molec. Immunol. 22:161 - 206 (1985)). By placing the first and last cysteine residues that form inter - heavy chain disulfide bonds in the same positions as in IgG1, the hinge regions of other IgG isotypes can be aligned with the IgG1 sequence.

[0078] The "CH2 domain" of the human IgG Fc region generally extends from amino acid position 231 to amino acid position 340. The CH2 domain is unique in that it does not pair tightly with another region, but rather two N - linked branched oligosaccharide chains are inserted between the two CH2 domains of the intact native IgG molecule. It is speculated that the carbohydrates may help to keep the CH2 domain stable as an alternative to domain - to - domain pairing. Burton, Molec. Immunol. 22:161 - 206 (1985).

[0079] The "CH3" domain includes the region in the Fc region extending from the C - terminal residue to the CH2 domain (from amino acid position 341 to the C - terminal of the antibody sequence, usually amino acid residue 446 or 447 of IgG).

[0080] A "functional Fc fragment" has the "effector functions" possessed by the native Fc region sequence. Exemplary "effector functions" include C1q binding; complement - dependent cytotoxicity (CDC); Fc receptor binding; antibody - dependent cell - mediated cytotoxicity (ADCC); phagocytosis; down - regulation of cell surface receptors (such as B - cell receptor; BCR), etc. Such effector functions generally require the Fc region to bind to a binding domain (such as the antibody variable region) and can be evaluated using a variety of experimental methods well - known in the art.

[0081] Antibodies of IgG Fc variants with "altered" FcR - binding affinity or ADCC activity have enhanced or reduced FcR - binding activity and / or ADCC activity compared to the parental polypeptide or a polypeptide containing the native Fc sequence. Fc variants that exhibit "enhanced binding" to FcR have a higher binding affinity for at least one FcR (e.g., lower apparent Kd or IC 50value). In some embodiments, compared to the parental polypeptide, the binding ability is enhanced by 3-fold, such as 5, 10, 25, 50, 60, 100, 150, 200, and even up to 500-fold, or the binding force is increased by 25% to 1000%. An Fc variant that exhibits "reduced binding" to FcR has a lower affinity for at least one FcR compared to the parental polypeptide (e.g., a higher apparent Kd or IC 50 value). Compared to the parental polypeptide, its binding ability is decreased by 40% or more.

[0082] "Antibody-dependent cell-mediated cytotoxicity" or "ADCC" is a form of cytotoxicity in which secreted Ig binds to Fc receptors (FcRs) present on certain cytotoxic cells (e.g., natural killer cells (NK), neutrophils, and macrophages), enabling these cytotoxic effector cells to specifically bind to antigen-bearing target cells and subsequently kill the target cells using cytotoxins. The antibody "arms" the cytotoxic cells and is essential for this killing. Among the major cell types mediating ADCC, NK cells only express FcγRIII, while monocytes express FcγRI, FcγRII, and FcγRIII. The expression of FcRs on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991). To evaluate the ADCC activity of a target molecule, an in vitro ADCC assay can be performed, as described in U.S. Patent No. 5,500,362 or 5,821,337. Effector cells suitable for such assays include peripheral blood mononuclear cells (PBMC) and natural killer cells (NK). Optionally, or in addition, the ADCC activity of the target molecule can also be evaluated in vivo, for example, in an animal model as disclosed in Clynes et al. PNAS (USA) 95:652-656 (1998).

[0083] A polypeptide comprising an Fc region variant exhibits "enhanced ADCC activity" or is capable of more effectively mediating the ADCC effect in the presence of human effector cells compared to a polypeptide comprising a wild-type IgG Fc polypeptide or a parental polypeptide. The polypeptide comprising the Fc region variant can more effectively mediate ADCC both in vitro and in vivo when the amount of the polypeptide comprising the Fc region variant is substantially the same as that of the polypeptide comprising the wild-type IgG Fc polypeptide (or parental polypeptide) during the experiment. Any in vitro ADCC assay method known in the art is generally used to identify such variants, such as an assay or method for identifying ADCC activity, for example, in an animal model. In some embodiments, such variants mediate ADCC with 5 to 100-fold, such as 25 to 50-fold, higher efficiency compared to wild-type Fc (or parental polypeptide).

[0084] "Complement-dependent cytotoxicity" or "CDC" refers to the lysis of target cells in the presence of complement. Activation of the classical complement pathway is initiated by the binding of the first component of the complement system (C1q) to an antibody (subclass of appropriate structure) that has bound homologous antigen. To assess complement activation, a CDC assay can be performed as described in Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996). Polypeptide variants having altered Fc region amino acid sequences and increased or decreased C1q binding ability are described in U.S. Patent No. 6,194,551 B1 and WO 99 / 51642. The contents of these patent publications are hereby incorporated by reference in their entirety. See also Idusogie et al. J. Immunol. 164:4178-4184 (2000).

[0085] Unless otherwise specified, a "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate to each other and encode the same amino acid sequence. A nucleotide sequence encoding a protein or RNA may also include introns, for example, a nucleotide sequence encoding a protein may contain introns in certain forms.

[0086] The term "operably linked" refers to a functional linkage between a regulatory sequence and a heterologous nucleotide sequence such that the latter is expressed. For example, a first nucleotide sequence is operably linked to a second nucleotide sequence when the first nucleotide sequence is in a functional relationship with the second nucleotide sequence. For example, if a promoter affects the transcription or expression of a coding sequence, the promoter is operably linked to the coding sequence. Generally, operably linked DNA sequences are contiguous and, where necessary, two protein-coding regions can be joined in the same reading frame.

[0087] "Homologous" refers to sequence similarity or sequence identity between two polypeptides or between two nucleic acid molecules. Two DNA molecules are homologous at a given position if the same base or amino acid monomer subunit is present at that position in both of the two compared sequences, for example, if both DNA molecules have adenine at the same position. The percentage of homology between two sequences is a function obtained by multiplying the ratio of the number of matching or homologous positions shared between the two sequences to the total number of positions by 100. For example, if 6 out of 10 positions in two sequences are matching or homologous, the homology between the two sequences is 60%. For example, the DNA sequences ATTGCC and TATGGC have 50% homology. Generally, when comparing two sequences, the comparison is made to obtain the maximum homology.

[0088] The "effective amount" of the anti-BTLA antibody or composition disclosed herein refers to an amount sufficient to achieve a particular purpose. The "effective amount" can be determined empirically and by methods known in relation to that purpose.

[0089] The term "therapeutically effective amount" refers to the amount of the anti-BTLA antibody or its composition disclosed herein that is capable of effectively treating a disease or condition in an individual. For example, in the case of cancer, a therapeutically effective amount of the anti-BTLA antibody or its composition is an amount capable of reducing the number of cancer cells; reducing the size or weight of a tumor; inhibiting (i.e., slowing to some extent or preferably stopping) the invasion of tumor cells into surrounding organs; inhibiting (i.e., slowing to some extent or preferably stopping) tumor metastasis; inhibiting tumor growth to some extent; and / or alleviating one or more symptoms associated with cancer to some extent. The anti-BTLA antibody or its composition disclosed herein is capable of blocking and / or killing existing tumor cells to some extent and can be cytostatic or cytotoxic. In some embodiments, the therapeutically effective amount is an amount capable of prolonging the survival of a patient. In some embodiments, the therapeutically effective amount is an amount capable of improving the progression-free survival of a patient.

[0090] As used herein, "pharmaceutically acceptable" or "pharmacologically compatible" refers to a material that is biologically inactive or has no other undesirable properties, e.g., a material that can be incorporated into a pharmaceutical composition administered to a patient without causing a significant adverse biological reaction or, alternatively, does not interact in a harmful manner with any other components contained in the composition. Pharmaceutically acceptable carriers or excipients preferably meet the required standards of toxicological or manufacturing tests and / or are included in the Inactive Ingredients Guides compiled by the U.S. Food and Drug Administration.

[0091] The embodiments of the present application described herein should be understood to include embodiments of "consisting of" and / or "consisting essentially of".

[0092] As used herein, reference to "about" a numerical value or parameter includes (and describes) variations that are specific to that value or parameter itself. For example, a description of "about X" includes a description of "X".

[0093] As used herein, reference to "not" a numerical value or parameter generally means and describes "other than" a particular numerical value or parameter. For example, the method cannot be used to treat cancer type X, meaning that the method is generally used to treat other types of cancer other than cancer type X.

[0094] Unless the context clearly dictates otherwise, the singular forms "a", "an", and "the" as used herein and in the appended claims include plural referents.

[0095] Anti-BTLA antibody

[0096] In one aspect, the present application provides anti-BTLA antibodies that specifically bind to human and / or rhesus macaque BTLA. The anti-BTLA antibodies include, but are not limited to, humanized antibodies, chimeric antibodies, murine antibodies, human antibodies, and antibody molecules comprising heavy and / or light chain CDRs as described herein. In one aspect, the present application provides isolated antibodies that bind to BTLA. Exemplary anti-BTLA antibodies include, for example, full-length anti-BTLA antibodies (such as full-length IgG1 or IgG4), anti-BTLA single-chain antibodies, anti-BTLA Fc fusion proteins, multispecific (such as bispecific) anti-BTLA antibodies, anti-BTLA immunoconjugates, and the like. In some embodiments, the anti-BTLA antibody is a full-length antibody (such as full-length IgG1 or IgG4) or an antigen-binding fragment thereof that specifically binds to BTLA. In some embodiments, the anti-BTLA antibody is Fab, Fab’, F(ab’)2, Fab’-SH, single-chain Fv (scFv), Fv fragment, dAb, Fd, nanobody, diabody, or linear antibody. In some embodiments, an antibody that specifically binds to BTLA means that the affinity of the antibody for binding to BTLA is at least 10 times (including, for example, 10, 10 2 、10 3 、10 4 、10 5 、10 6 、or 10 7 -fold) higher than the binding affinity for a non-target. In some embodiments, the non-target refers to an antigen that is not BTLA. The binding affinity can be determined by methods known in the art, such as ELISA, fluorescence-activated cell sorting (FACS) analysis, or radioimmunoprecipitation assay (RIA). The Kd value can be determined by methods known in the art, such as surface plasmon resonance (SPR) technology or biolayer interferometry (BLI).

[0097] Although anti-BTLA antibodies comprising human sequences are discussed extensively herein (e.g., human heavy and light chain variable domains comprising human CDR sequences), non-human anti-BTLA antibodies are also contemplated. In some embodiments, the non-human anti-BTLA antibodies comprise the human CDR sequences of the anti-BTLA antibodies described herein and non-human framework region sequences. In some embodiments, the non-human framework region sequences include any sequences useful for generating heavy and / or light chain variable domains using one or more of the human CDR sequences described herein, including, for example, mammals such as mice, rats, rabbits, pigs, cattle (e.g., cows, bulls, water buffalo), deer, sheep, goats, chickens, cats, dogs, ferrets, primates (e.g., lesser apes, rhesus monkeys), and the like. In some embodiments, the non-human anti-BTLA antibodies comprise anti-BTLA antibodies generated by grafting one or more of the human CDR sequences described herein into a non-human framework region (e.g., a murine or avian framework region sequence).

[0098] The complete amino acid sequence of an exemplary human BTLA comprises or consists of the amino acid sequence shown in SEQ ID NO: 36. The amino acid sequence of an exemplary murine BTLA extracellular region comprises or consists of the amino acid sequence shown in SEQ ID NO: 37.

[0099] In some embodiments, the anti-BTLA antibodies described herein specifically recognize an epitope in human BTLA. In some embodiments, the anti-BTLA antibodies cross-react with BTLA from species other than human. In some embodiments, the anti-BTLA antibodies are fully specific for human BTLA and do not cross-react with other non-human species.

[0100] In some embodiments, the anti-BTLA antibodies cross-react with at least one allelic variant of the BTLA protein (or a fragment thereof). In some embodiments, the allelic variant has up to 30 (such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, or 30) amino acid substitutions (e.g., conservative substitutions) compared to the naturally occurring BTLA protein (or a fragment thereof). In some embodiments, the anti-BTLA antibodies do not cross-react with any allelic variants of the BTLA protein (or a fragment thereof).

[0101] In some embodiments, the anti-BTLA antibodies cross-react with at least one interspecies variant of the BTLA protein. In some embodiments, for example, the BTLA protein (or a fragment thereof) is human BTLA, and the interspecies variant of the BTLA protein (or a fragment thereof) is a variant in rhesus monkey. In some embodiments, the anti-BTLA antibodies do not cross-react with any interspecies variants of the BTLA protein.

[0102] In some embodiments, any anti-BTLA antibody as described herein, the anti-BTLA antibody comprises an antibody heavy chain constant region and an antibody light chain constant region. In some embodiments, the anti-BTLA antibody comprises a heavy chain constant region of IgG1 type. In some embodiments, the anti-BTLA antibody comprises a heavy chain constant region of IgG2 type. In some embodiments, the anti-BTLA antibody comprises a heavy chain constant region of IgG3 type. In some embodiments, the anti-BTLA antibody comprises a heavy chain constant region of IgG4 type. In some embodiments, the heavy chain constant region comprises (consists of or consists essentially of) the amino acid sequence SEQ ID NO:32. In some embodiments, the heavy chain constant region comprises (consists of or consists essentially of) the amino acid sequence SEQ ID NO:33. In some embodiments, the anti-BTLA antibody comprises a κ light chain constant region. In some embodiments, the light chain constant region comprises (consists of or consists essentially of) the amino acid sequence SEQ ID NO:34. In some embodiments, the anti-BTLA antibody comprises a λ light chain constant region. In some embodiments, the light chain constant region comprises (consists of or consists essentially of) the amino acid sequence SEQ ID NO:35. In some embodiments, the anti-BTLA antibody comprises an antibody heavy chain variable domain and an antibody light chain variable domain.

[0103] In some embodiments, the isolated anti-BTLA antibody comprises a heavy chain variable domain (V H ), the V H comprising: heavy chain complementarity determining region (HC-CDR) 1, which comprises TFGMGVS (SEQ ID NO:1); HC-CDR2, which comprises HIYWDDDKRFNPSLKS (SEQ ID NO:4); and HC-CDR3, which comprises GNWDGETYFDY (SEQ ID NO:7); and a light chain variable domain (V L ), the V L comprising: light chain complementarity determining region (LC-CDR) 1, which comprises KSTQSLLDSDGKTYLN (SEQID NO:10); LC-CDR2, which comprises LVSKLDS (SEQ ID NO:13); and LC-CDR3, which comprises WQGTHFPWT (SEQID NO:15).

[0104] In some embodiments, the anti-BTLA antibody comprises V H , the V HComprising: HC-CDR1, which comprises the amino acid sequence shown in SEQ ID NO: 1 or a variant thereof, said variant comprising up to about 3 (such as 1, 2 or 3) amino acid substitutions; HC-CDR2, which comprises the amino acid sequence shown in SEQ ID NO: 4 or a variant thereof, said variant comprising up to about 3 (such as 1, 2 or 3) amino acid substitutions; and HC-CDR3, which comprises the amino acid sequence shown in SEQ ID NO: 7 or a variant thereof, said variant comprising up to about 3 (such as 1, 2 or 3) amino acid substitutions.

[0105] In some embodiments, the anti-BTLA antibody comprises V H , said V H Comprising: HC-CDR1, which comprises the amino acid sequence shown in SEQ ID NO: 1, HC-CDR2, which comprises the amino acid sequence shown in SEQ ID NO: 4, HC-CDR3, which comprises the amino acid sequence shown in SEQ ID NO: 7.

[0106] In some embodiments, the anti-BTLA antibody comprises V L , said V L Comprising: LC-CDR1, which comprises the amino acid sequence shown in SEQ ID NO: 10 or a variant thereof, said variant comprising up to about 3 (such as 1, 2 or 3) amino acid substitutions; LC-CDR2, which comprises the amino acid sequence shown in SEQ ID NO: 13 or a variant thereof, said variant comprising up to about 3 (such as 1, 2 or 3) amino acid substitutions; and LC-CDR3, which comprises the amino acid sequence shown in SEQ ID NO: 15 or a variant thereof, said variant comprising up to about 3 (such as 1, 2 or 3) amino acid substitutions.

[0107] In some embodiments, the anti-BTLA antibody comprises V L , said V L Comprising: LC-CDR1, which comprises the amino acid sequence shown in SEQ ID NO: 10, LC-CDR2, which comprises the amino acid sequence shown in SEQ ID NO: 13, LC-CDR3, which comprises the amino acid sequence shown in SEQ ID NO: 15.

[0108] In some embodiments, the anti-BTLA antibody comprises V H , said V HComprising: HC-CDR1, which comprises the amino acid sequence shown in SEQ ID NO: 1 or a variant thereof, said variant comprising up to about 3 (e.g., 1, 2, or 3) amino acid substitutions; HC-CDR2, which comprises the amino acid sequence shown in SEQ ID NO: 4 or a variant thereof, said variant comprising up to about 3 (e.g., 1, 2, or 3) amino acid substitutions; and HC-CDR3, which comprises the amino acid sequence shown in SEQ ID NO: 7 or a variant thereof, said variant comprising up to about 3 (e.g., 1, 2, or 3) amino acid substitutions; and V L , said V L Comprising: LC-CDR1, which comprises the amino acid sequence shown in SEQ ID NO: 10 or a variant thereof, said variant comprising up to about 3 (e.g., 1, 2, or 3) amino acid substitutions; LC-CDR2, which comprises the amino acid sequence shown in SEQ ID NO: 13 or a variant thereof, said variant comprising up to about 3 (e.g., 1, 2, or 3) amino acid substitutions; and LC-CDR3, which comprises the amino acid sequence shown in SEQ ID NO: 15 or a variant thereof, said variant comprising up to about 3 (e.g., 1, 2, or 3) amino acid substitutions.

[0109] In some embodiments, the anti-BTLA antibody comprises V H , said V H Comprising: HC-CDR1, which comprises the amino acid sequence shown in SEQ ID NO: 1, HC-CDR2, which comprises the amino acid sequence shown in SEQ ID NO: 4, and HC-CDR3, which comprises the amino acid sequence shown in SEQ ID NO: 7; and V L , said V L Comprising: LC-CDR1, which comprises the amino acid sequence shown in SEQ ID NO: 10, LC-CDR2, which comprises the amino acid sequence shown in SEQ ID NO: 13, and LC-CDR3, which comprises the amino acid sequence shown in SEQ ID NO: 15.

[0110] In some embodiments, the anti-BTLA antibody comprises V H , said V H Comprising: HC-CDR1, which comprises the amino acid sequence SEQ ID NO: 1, HC-CDR2, which comprises the amino acid sequence SEQ ID NO: 4, HC-CDR3, which comprises the amino acid sequence SEQ ID NO: 7, or a variant of said V H comprising up to about 5 amino acid substitutions in its HC-CDRs; and V L , said V LComprising: LC-CDR1, which comprises the amino acid sequence SEQ ID NO:10, LC-CDR2, which comprises the amino acid sequence SEQ ID NO:13, LC-CDR3, which comprises the amino acid sequence SEQ ID NO:15, or a variant of said V L with up to about 5 amino acid substitutions in its LC-CDRs.

[0111] In some embodiments, the anti-BTLA antibody comprises V H wherein said V H Comprising: HC-CDR1, which comprises the amino acid sequence SEQ ID NO:1, HC-CDR2, which comprises the amino acid sequence SEQ ID NO:4, and HC-CDR3, which comprises the amino acid sequence SEQ ID NO:7; and V L wherein said V L Comprising: LC-CDR1, which comprises the amino acid sequence SEQ ID NO:10, LC-CDR2, which comprises the amino acid sequence SEQ ID NO:13, and LC-CDR3, which comprises the amino acid sequence SEQ ID NO:15.

[0112] In some embodiments, the anti-BTLA antibody comprises V H wherein said V H Comprises V as shown by the amino acid sequence shown in any of SEQ ID NOs: 18-22 H Comprising HC-CDR1, HC-CDR2 and HC-CDR3; and V L wherein said V L Comprises LC-CDR1, LC-CDR2 and LC-CDR3 as shown by the amino acid sequence shown in any of SEQID NOs: 25-29 L Comprising.

[0113] In some embodiments, the anti-BTLA antibody comprises V H which comprises V as shown by the amino acid sequence SEQ ID NO:18 H Comprising HC-CDR1, HC-CDR2 and HC-CDR3; and V L which comprises V as shown by the amino acid sequence SEQ ID NO:25 L Comprising LC-CDR1, LC-CDR2 and LC-CDR3.

[0114] In some embodiments, the anti-BTLA antibody comprises V H which comprises V as shown by the amino acid sequence SEQ ID NO:19 HThe included HC-CDR1, HC-CDR2, and HC-CDR3; and V L which includes a V as shown in amino acid sequence SEQ ID NO:26 L The included LC-CDR1, LC-CDR2, and LC-CDR3.

[0115] In some embodiments, the anti-BTLA antibody includes a V H which includes a V as shown in amino acid sequence SEQ ID NO:19 H The included HC-CDR1, HC-CDR2, and HC-CDR3; and V L which includes a V as shown in amino acid sequence SEQ ID NO:27 L The included LC-CDR1, LC-CDR2, and LC-CDR3.

[0116] In some embodiments, the anti-BTLA antibody includes a V H which includes a V as shown in amino acid sequence SEQ ID NO:19 H The included HC-CDR1, HC-CDR2, and HC-CDR3; and V L which includes a V as shown in amino acid sequence SEQ ID NO:28 L The included LC-CDR1, LC-CDR2, and LC-CDR3.

[0117] In some embodiments, the anti-BTLA antibody includes a V H which includes a V as shown in amino acid sequence SEQ ID NO:19 H The included HC-CDR1, HC-CDR2, and HC-CDR3; and V L which includes a V as shown in amino acid sequence SEQ ID NO:29 L The included LC-CDR1, LC-CDR2, and LC-CDR3.

[0118] In some embodiments, the anti-BTLA antibody includes a V H which includes a V as shown in amino acid sequence SEQ ID NO:20 H The included HC-CDR1, HC-CDR2, and HC-CDR3; and V L which includes a V as shown in amino acid sequence SEQ ID NO:26 L The included LC-CDR1, LC-CDR2, and LC-CDR3.

[0119] In some embodiments, the anti-BTLA antibody includes a V H, which comprises a V as shown in amino acid sequence SEQ ID NO:20 H comprising HC-CDR1, HC-CDR2 and HC-CDR3; and V L , which comprises a V as shown in amino acid sequence SEQ ID NO:27 L comprising LC-CDR1, LC-CDR2 and LC-CDR3.

[0120] In some embodiments, the anti-BTLA antibody comprises a V H , which comprises a V as shown in amino acid sequence SEQ ID NO:20 H comprising HC-CDR1, HC-CDR2 and HC-CDR3; and V L , which comprises a V as shown in amino acid sequence SEQ ID NO:28 L comprising LC-CDR1, LC-CDR2 and LC-CDR3.

[0121] In some embodiments, the anti-BTLA antibody comprises a V H , which comprises a V as shown in amino acid sequence SEQ ID NO:20 H comprising HC-CDR1, HC-CDR2 and HC-CDR3; and V L , which comprises a V as shown in amino acid sequence SEQ ID NO:29 L comprising LC-CDR1, LC-CDR2 and LC-CDR3.

[0122] In some embodiments, the anti-BTLA antibody comprises a V H , which comprises a V as shown in amino acid sequence SEQ ID NO:21 H comprising HC-CDR1, HC-CDR2 and HC-CDR3; and V L , which comprises a V as shown in amino acid sequence SEQ ID NO:28 L comprising LC-CDR1, LC-CDR2 and LC-CDR3.

[0123] In some embodiments, the anti-BTLA antibody comprises a V H , which comprises a V as shown in amino acid sequence SEQ ID NO:21 H comprising HC-CDR1, HC-CDR2 and HC-CDR3; and V L , which comprises a V as shown in amino acid sequence SEQ ID NO:29 L comprising LC-CDR1, LC-CDR2 and LC-CDR3.

[0124] In some embodiments, the anti-BTLA antibody comprises V H , which comprises V as shown in the amino acid sequence SEQ ID NO:22 H HC-CDR1, HC-CDR2 and HC-CDR3; and V L , which comprises V as shown in the amino acid sequence SEQ ID NO:28 L Contains LC-CDR1, LC-CDR2 and LC-CDR3.

[0125] In some embodiments, the anti-BTLA antibody comprises V H , which comprises V as shown in the amino acid sequence SEQ ID NO:22 H HC-CDR1, HC-CDR2 and HC-CDR3; and V L , which comprises V as shown in the amino acid sequence SEQ ID NO:29 L Contains LC-CDR1, LC-CDR2 and LC-CDR3.

[0126] In some embodiments, the anti-BTLA antibody comprises: V H , the V H comprising an amino acid sequence as shown in any one of SEQ ID NOs: 18-22 or a variant thereof, wherein the variant has at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity with the amino acid sequence as shown in any one of SEQ ID NOs: 18-22; and V L , the V L The invention relates to an anti-BTLA antibody comprising an amino acid sequence as set forth in any one of SEQ ID NOs: 25-29 or a variant thereof, wherein the variant has at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity with the amino acid sequence as set forth in any one of SEQ ID NOs: 25-29. In some embodiments, the anti-BTLA antibody comprises V H , the V H comprising an amino acid sequence as shown in any one of SEQ ID NOs: 18-22, and V L , the V L It comprises the amino acid sequence shown in any one of SEQ ID NOs: 25-29.

[0127] In some embodiments, the anti-BTLA antibody comprises: V H , the V Hcomprising the amino acid sequence SEQ ID NO:18 or a variant thereof, said variant having at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence SEQ ID NO:18; and V L , said V L comprising the amino acid sequence SEQ ID NO:25 or a variant thereof, said variant having at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence SEQ ID NO:25. In some embodiments, the anti-BTLA antibody comprises V H , said V H comprising the amino acid sequence SEQ ID NO:18, and V L , said V L comprising the amino acid sequence SEQ ID NO:25.

[0128] In some embodiments, the anti-BTLA antibody comprises: V H , said V H comprising the amino acid sequence SEQ ID NO:19 or a variant thereof, said variant having at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence SEQ ID NO:19; and V L , said V L comprising the amino acid sequence SEQ ID NO:26 or a variant thereof, said variant having at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence SEQ ID NO:26. In some embodiments, the anti-BTLA antibody comprises V H , said V H comprising the amino acid sequence SEQ ID NO:19, and V L , said V L comprising the amino acid sequence SEQ ID NO:26.

[0129] In some embodiments, the anti-BTLA antibody comprises: V H , said V H comprising the amino acid sequence SEQ ID NO:19 or a variant thereof, said variant having at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence SEQ ID NO:19; and V L , said V LComprising the amino acid sequence SEQ ID NO: 27 or a variant thereof, said variant having at least about 80% (such as at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity with the amino acid sequence SEQ ID NO: 27. In some embodiments, the anti-BTLA antibody comprises V H , said V H comprising the amino acid sequence SEQ ID NO: 19, and V L , said V L comprising the amino acid sequence SEQ ID NO: 27.

[0130] In some embodiments, the anti-BTLA antibody comprises: V H , said V H comprising the amino acid sequence SEQ ID NO: 19 or a variant thereof, said variant having at least about 80% (such as at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity with the amino acid sequence SEQ ID NO: 19; and V L , said V L comprising the amino acid sequence SEQ ID NO: 28 or a variant thereof, said variant having at least about 80% (such as at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity with the amino acid sequence SEQ ID NO: 28. In some embodiments, the anti-BTLA antibody comprises V H , said V H comprising the amino acid sequence SEQ ID NO: 19, and V L , said V L comprising the amino acid sequence SEQ ID NO: 28.

[0131] In some embodiments, the anti-BTLA antibody comprises: V H , said V H comprising the amino acid sequence SEQ ID NO: 19 or a variant thereof, said variant having at least about 80% (such as at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity with the amino acid sequence SEQ ID NO: 19; and V L , said V L comprising the amino acid sequence SEQ ID NO: 29 or a variant thereof, said variant having at least about 80% (such as at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity with the amino acid sequence SEQ ID NO: 29. In some embodiments, the anti-BTLA antibody comprises V H , said V Hcomprises the amino acid sequence SEQ ID NO: 19, and V L , said V L comprises the amino acid sequence SEQ ID NO: 29.

[0132] In some embodiments, the anti-BTLA antibody comprises: V H , said V H comprises the amino acid sequence SEQ ID NO: 20 or a variant thereof, the variant having at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence SEQ ID NO: 20; and V L , said V L comprises the amino acid sequence SEQ ID NO: 26 or a variant thereof, the variant having at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence SEQ ID NO: 26. In some embodiments, the anti-BTLA antibody comprises V H , said V H comprises the amino acid sequence SEQ ID NO: 20, and V L , said V L comprises the amino acid sequence SEQ ID NO: 26.

[0133] In some embodiments, the anti-BTLA antibody comprises: V H , said V H comprises the amino acid sequence SEQ ID NO: 20 or a variant thereof, the variant having at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence SEQ ID NO: 20; and V L , said V L comprises the amino acid sequence SEQ ID NO: 27 or a variant thereof, the variant having at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence SEQ ID NO: 27. In some embodiments, the anti-BTLA antibody comprises V H , said V H comprises the amino acid sequence SEQ ID NO: 20, and V L , said V L comprises the amino acid sequence SEQ ID NO: 27.

[0134] In some embodiments, the anti-BTLA antibody comprises: V H , said V HComprising the amino acid sequence SEQ ID NO:20 or a variant thereof, said variant having at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence SEQ ID NO:20; and V L , said V L Comprising the amino acid sequence SEQ ID NO:28 or a variant thereof, said variant having at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence SEQ ID NO:28. In some embodiments, the anti-BTLA antibody comprises V H , said V H Comprising the amino acid sequence SEQ ID NO:20, and V L , said V L Comprising the amino acid sequence SEQ ID NO:28.

[0135] In some embodiments, the anti-BTLA antibody comprises: V H , said V H Comprising the amino acid sequence SEQ ID NO:20 or a variant thereof, said variant having at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence SEQ ID NO:20; and V L , said V L Comprising the amino acid sequence SEQ ID NO:29 or a variant thereof, said variant having at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence SEQ ID NO:29. In some embodiments, the anti-BTLA antibody comprises V H , said V H Comprising the amino acid sequence SEQ ID NO:20, and V L , said V L Comprising the amino acid sequence SEQ ID NO:29.

[0136] In some embodiments, the anti-BTLA antibody comprises: V H , said V H Comprising the amino acid sequence SEQ ID NO:21 or a variant thereof, said variant having at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence SEQ ID NO:21; and V L , said V LComprising the amino acid sequence SEQ ID NO: 28 or a variant thereof, said variant having at least about 80% (such as at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity with the amino acid sequence SEQ ID NO: 28. In some embodiments, the anti-BTLA antibody comprises V H , said V H comprising the amino acid sequence SEQ ID NO: 21, and V L , said V L comprising the amino acid sequence SEQ ID NO: 28.

[0137] In some embodiments, the anti-BTLA antibody comprises: V H , said V H comprising the amino acid sequence SEQ ID NO: 21 or a variant thereof, said variant having at least about 80% (such as at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity with the amino acid sequence SEQ ID NO: 21; and V L , said V L comprising the amino acid sequence SEQ ID NO: 29 or a variant thereof, said variant having at least about 80% (such as at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity with the amino acid sequence SEQ ID NO: 29. In some embodiments, the anti-BTLA antibody comprises V H , said V H comprising the amino acid sequence SEQ ID NO: 21, and V L , said V L comprising the amino acid sequence SEQ ID NO: 29.

[0138] In some embodiments, the anti-BTLA antibody comprises: V H , said V H comprising the amino acid sequence SEQ ID NO: 22 or a variant thereof, said variant having at least about 80% (such as at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity with the amino acid sequence SEQ ID NO: 22; and V L , said V L comprising the amino acid sequence SEQ ID NO: 28 or a variant thereof, said variant having at least about 80% (such as at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity with the amino acid sequence SEQ ID NO: 28. In some embodiments, the anti-BTLA antibody comprises V H , said V Hcomprising the amino acid sequence SEQ ID NO:22, and V L , wherein said V L comprises the amino acid sequence SEQ ID NO:28.

[0139] In some embodiments, the anti-BTLA antibody comprises: V H , wherein said V H comprises the amino acid sequence SEQ ID NO:22 or a variant thereof, said variant having at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence SEQ ID NO:22; and V L , wherein said V L comprises the amino acid sequence SEQ ID NO:29 or a variant thereof, said variant having at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence SEQ ID NO:29. In some embodiments, the anti-BTLA antibody comprises V H , wherein said V H comprises the amino acid sequence SEQ ID NO:22, and V L , wherein said V L comprises the amino acid sequence SEQ ID NO:29.

[0140] In some embodiments, the anti-BTLA antibody comprises V H , wherein said V H comprises: HC-CDR1, which comprises the amino acid sequence SEQ ID NO:2, HC-CDR2, which comprises the amino acid sequence SEQ ID NO:5, HC-CDR3, which comprises the amino acid sequence SEQ ID NO:8, or a variant of said V H that contains up to about 5 amino acid substitutions in its HC-CDRs; and V L , wherein said V L comprises: LC-CDR1, which comprises the amino acid sequence SEQ ID NO:11, LC-CDR2, which comprises the amino acid sequence SEQ ID NO:14, LC-CDR3, which comprises the amino acid sequence SEQ ID NO:16, or a variant of said V L that contains up to about 5 amino acid substitutions in its LC-CDRs.

[0141] In some embodiments, the anti-BTLA antibody comprises V H , wherein said V HComprising: HC-CDR1, which comprises the amino acid sequence SEQ ID NO:2, HC-CDR2, which comprises the amino acid sequence SEQ ID NO:5, and HC-CDR3, which comprises the amino acid sequence SEQ ID NO:8; and V L , said V L Comprising: LC-CDR1, which comprises the amino acid sequence SEQ ID NO:11, LC-CDR2, which comprises the amino acid sequence SEQ ID NO:14, and LC-CDR3, which comprises the amino acid sequence SEQ ID NO:16.

[0142] In some embodiments, the anti-BTLA antibody comprises V H , which comprises HC-CDR1, HC-CDR2 and HC-CDR3 contained in the V H comprised as shown by the amino acid sequence SEQ ID NO:23; and V L , which comprises LC-CDR1, LC-CDR2 and LC-CDR3 contained in the V L comprised as shown by the amino acid sequence SEQ ID NO:30.

[0143] In some embodiments, the anti-BTLA antibody comprises: V H , said V H comprises the amino acid sequence SEQ ID NO:23 or a variant thereof, which variant has at least about 80% (such as at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity with the amino acid sequence SEQ ID NO:23; and V L , said V L comprises the amino acid sequence SEQ IDNO:30 or a variant thereof, which variant has at least about 80% (such as at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity with the amino acid sequence SEQ ID NO:30. In some embodiments, the anti-BTLA antibody comprises V H , said V H comprises the amino acid sequence SEQ ID NO:23, and V L , said V L comprises the amino acid sequence SEQ ID NO:30.

[0144] In some embodiments, the anti-BTLA antibody comprises V H , said V HComprising: HC-CDR1, which comprises the amino acid sequence SEQ ID NO:3, HC-CDR2, which comprises the amino acid sequence SEQ ID NO:6, HC-CDR3, which comprises the amino acid sequence SEQ ID NO:9, or a variant of said V H having up to about 5 amino acid substitutions in its HC-CDRs; and V L wherein said V L Comprising: LC-CDR1, which comprises the amino acid sequence SEQ ID NO:12, LC-CDR2, which comprises the amino acid sequence SEQ ID NO:13, LC-CDR3, which comprises the amino acid sequence SEQ ID NO:17, or a variant of said V L having up to about 5 amino acid substitutions in its LC-CDRs.

[0145] In some embodiments, the anti-BTLA antibody comprises V H wherein said V H Comprising: HC-CDR1, which comprises the amino acid sequence SEQ ID NO:3, HC-CDR2, which comprises the amino acid sequence SEQ ID NO:6, and HC-CDR3, which comprises the amino acid sequence SEQ ID NO:9; and V L wherein said V L Comprising: LC-CDR1, which comprises the amino acid sequence SEQ ID NO:12, LC-CDR2, which comprises the amino acid sequence SEQ ID NO:13, and LC-CDR3, which comprises the amino acid sequence SEQ ID NO:17.

[0146] In some embodiments, the anti-BTLA antibody comprises V H which comprises HC-CDR1, HC-CDR2 and HC-CDR3 as contained in V H shown in the amino acid sequence SEQ ID NO:24; and V L which comprises LC-CDR1, LC-CDR2 and LC-CDR3 as contained in V L shown in the amino acid sequence SEQ ID NO:31.

[0147] In some embodiments, the anti-BTLA antibody comprises: V H wherein said V H comprises the amino acid sequence SEQ ID NO:24 or a variant thereof having at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence SEQ ID NO:24; and V L wherein said V Lcomprising the amino acid sequence SEQ ID NO: 31 or a variant thereof, said variant having at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence SEQ ID NO: 31. In some embodiments, the anti-BTLA antibody comprises V H , said V H comprising the amino acid sequence SEQ ID NO: 24, and V L , said V L comprising the amino acid sequence SEQ ID NO: 31.

[0148] In some embodiments, the above amino acid substitutions are limited to the "exemplary substitutions" shown in Table 4 herein. In some embodiments, the amino acid substitutions are limited to the "preferred substitutions" shown in Table 4 herein.

[0149] In some embodiments, the functional epitope can be resolved by combinatorial alanine scanning. In this process, combinatorial alanine scanning techniques can be used to identify the amino acids in the BTLA protein that are necessary for interaction with the anti-BTLA antibody. In some embodiments, the epitope is conformational, and the crystal structure of the anti-BTLA antibody bound to the BTLA protein can be used to identify the epitope.

[0150] In some embodiments, the present application provides an antibody that competitively binds to BTLA with any of the anti-BTLA antibodies described herein. In some embodiments, an antibody is provided that can competitively bind to the epitope on BTLA with any of the anti-BTLA antibodies described herein. In some embodiments, an anti-BTLA antibody is provided that binds to the same epitope as an anti-BTLA antibody molecule comprising V H and V L , wherein said V H comprises the amino acid sequence shown in any of SEQ ID NOs: 18-24, and said V L comprises the amino acid sequence shown in any of SEQ ID NOs: 25-31. In some embodiments, an anti-BTLA antibody is provided that competitively binds to BTLA with an anti-BTLA antibody comprising V H and V L , wherein said V H comprises the amino acid sequence shown in any of SEQ ID NOs: 18-24, and said V L comprises the amino acid sequence shown in any of SEQ ID NOs: 25-31.

[0151] In some embodiments, competitive assays can be utilized to identify monoclonal antibodies that bind BTLA competitively with the anti-BTLA antibodies described herein. Competitive assays can determine whether two antibodies bind to the same epitope by recognizing the same or spatially overlapping epitopes or by one antibody competitively inhibiting the binding of another antibody to the antigen. In certain embodiments, such competitive antibodies bind to the same epitope as the antibodies described herein. Some exemplary competitive assays include, but are not limited to, conventional assays as described in Harlow and Lane (1988) Antibodies: A Laboratory Manual ch.14 (Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.). Detailed exemplary methods for resolving the epitopes to which antibodies bind are as described in Morris (1996) "Epitope Mapping Protocols," in Methods in Molecular Biology vol.66 (Humana Press, Totowa, N.J.). In some embodiments, if each antibody blocks 50% or more of the binding of the other antibody, they are said to bind to the same epitope. In some embodiments, antibodies that compete with the anti-BTLA antibodies described herein are chimeric antibodies, humanized antibodies, or fully human antibodies.

[0152] Exemplary anti-BTLA antibody sequences are shown in Tables 2 and 3, where CDR numbering is defined according to the EU numbering system Kabat. Those skilled in the art will recognize that there are multiple known algorithms (Kabat definition) for predicting the positions of CDRs and defining the variable regions of the antibody light and heavy chains. Antibodies containing the CDRs, V H and / or V L sequences as described herein, but based on prediction algorithms rather than the antibodies exemplified in the tables below are also within the scope of this application.

[0153] Table 2 Exemplary anti-BTLA antibody CDR sequences

[0154]

[0155]

[0156] Table 3 Exemplary sequences

[0157]

[0158]

[0159]

[0160] BTLA

[0161] BTLA (B and T lymphocyte attenuator) is a member of the receptor CD28 family, which also includes CD28, CTLA-4, ICOS, and PD-1. The initial members of this family, CD28 and ICOS, were discovered through the functional effect of enhancing T cell proliferation after adding monoclonal antibodies (Hutloff et al., Nature, 1999). BTLA was discovered by screening for differential expression in TH1 cells. In addition, BTLA has been described as providing a negative inhibitory signal, similar to CTLA-4. In the presence of agonistic anti-BTLA antibodies, anti-CD3 and anti-CD28-activated T cells exhibit reduced IL-2 production and cell proliferation (Kreig et al., J. Immunol., 2005). Mice lacking the intact BTLA gene have higher titers of anti-DNP-KLH after immunization and increased sensitivity to EAEA (Watanabe et al., Nat. Immunol, 2003).

[0162] The protein structure of BTLA is similar to that of programmed cell death 1 (PD-1) and cytotoxic T lymphocyte-associated antigen 4 (CTLA-4), including an extracellular domain, a transmembrane domain, and a cytoplasmic domain (Sedy JR et al., Nat Immunol., 2005; Gonzalez et al., Proc Natl Acad., 2005). The cytoplasmic domain contains a growth factor receptor-bound protein 2 (Grb-2)-associated motif, an immunoreceptor tyrosine-based switch motif (ITSM), and an immunoreceptor tyrosine-based inhibitory motif (ITIM). Binding of HVEM activates tyrosine phosphorylation of ITIM in BTLA and recruits the protein tyrosine phosphatases SHP-1 and SHP-2 containing Src homology domain 2 (SH2), which usually mediate immunosuppressive effects (Gavrieli et al., Biochem Biophys Res Commmun, 2003; Watanabe N et al., Nat Immunol, 2003). BTLA is widely expressed in lymph nodes, thymus, and spleen, but is rarely or not expressed in other organs such as the heart, kidney, brain, and liver, etc. (Yu, et al. 2019). Among immune cells, BTLA is mainly expressed in B cells and T cells. In the mouse spleen, the expression of BTLA in B cells is higher than that in T cells. Regarding T cells, the expression of BTLA can be detected on CD4+ and CD8+ T cells, and CD4+ T cells express more BTLA than CD8+ T cells (Rio ML et al., Immunobiology. 2010). Additionally, its expression can also be detected in innate immune cells such as dendritic cells (DCs) and monocytes (De Sousa LA et al., Front Immunol, 2018). Binding of HVEM to BTLA has a direct negative impact on the proliferation and activation of B cells and T cells (Cai et al., Immuno Rev, 2009).

[0163] HVEM

[0164] HVEM (TNFRSF14) is a member of the TNFR superfamily and is a shared ligand for co-stimulatory and co-inhibitory receptors. Human and murine HVEM are type I cell surface proteins composed of 283 and 276 amino acids, respectively, with an extracellular domain consisting of four cysteine-rich domains (CRDs), namely CRD1, CRD2, CRD3, and CRD4 (Hsu et al. J. Biol. Chem, 1997). The CRD2 and CRD3 domains of HVEM interact with LIGHT (Rooney et al., J. Biol. Chem, 2000.), and BTLA and CD160 bind to the CRD1 and CRD2 of HVEM (Gonzalez et al., Proc Natl. Acad. Sci, 2005. Cheung et al. Proc. Natl. Acad. 2005.). CDR1 is important for inhibiting the signaling pathway induced by recombinant HVEM-Ig fusion protein because the deletion of this domain leads to the co-stimulation of HVEM-Ig (Admas, et al. Am. J. Transplant, 2002.). HVEM has bifunctional activity, binding to co-inhibitory receptors (such as BTLA or CD160) and attenuating TCR-mediated signal transduction, or acting as a receptor for LIGHT to co-stimulate T cells (Admas, et al. Am. J. Transplant, 2002; Watanabe et al. 2003, Nat. Immunol.).

[0165] HVEM is widely expressed in hematopoietic and non-hematopoietic cells. Interestingly, on the same cell, the expression of LIGHT and HVEM is reciprocally regulated (Tamada et al., J. Immunol., 2000.). HVEM is highly expressed in naive B cells and memory B cells, but not expressed on germinal center-activated B cells (Duhen et al., J. Immunol., 2004). Similar to T cells, HVEM acts on naive B cells through LIGHT expressed on DCs and T cells to co-stimulate B cell proliferation and Ig secretion, thereby enhancing the humoral immune response. In addition to T cells and B cells, HVEM is also widely expressed in other hematopoietic cells (DCs, Tregs, monocytes, neutrophils, and NK cells) and non-hematopoietic cells (parenchymal cells). Triggering HVEM on these cell types can activate its effector functions, increasing bactericidal activity and promoting NK cell activation (Marsters et al. J. Biol. Chem, 1997; Garrieli et al. Adv. Immunol. 2006.; Fan et al. Blood, 2006.).

[0166] Full-length anti-BTLA antibody

[0167] In some embodiments, the anti-BTLA antibody is a full-length anti-BTLA antibody. In some embodiments, the full-length anti-BTLA antibody is IgA, IgD, IgE, IgG or IgM. In some embodiments, the full-length anti-BTLA antibody comprises an IgG constant region, such as the constant region of IgG1, IgG2, IgG3, IgG4 or a variant thereof. In some embodiments, the full-length anti-BTLA antibody comprises a lambda light chain constant region. In some embodiments, the full-length anti-BTLA antibody comprises a kappa light chain constant region. In some embodiments, the full-length anti-BTLA antibody is a full-length human anti-BTLA antibody. In some embodiments, the full-length anti-BTLA antibody comprises a murine immunoglobulin Fc sequence. In some embodiments, the full-length anti-BTLA antibody comprises an Fc sequence that has been altered or otherwise modified such that it has enhanced effector functions of antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC).

[0168] Thus, for example, in some embodiments, there is provided a full-length anti-BTLA antibody comprising an IgG1 constant region, wherein the anti-BTLA antibody specifically binds to BTLA. In some embodiments, the IgG1 is human IgG1. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO:32. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:34. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:35. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO:32 and the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:34. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO:32 and the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:35.

[0169] In some embodiments, there is provided a full-length anti-BTLA antibody comprising an IgG2 constant region, wherein the anti-BTLA antibody specifically binds to BTLA. In some embodiments, the IgG2 is human IgG2. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:34. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:35.

[0170] In some embodiments, provided is a full-length anti-BTLA antibody comprising an IgG3 constant region, wherein the anti-BTLA antibody specifically binds to BTLA. In some embodiments, the IgG3 is human IgG3. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:34. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:35.

[0171] In some embodiments, provided is a full-length anti-BTLA antibody comprising an IgG4 constant region, wherein the anti-BTLA antibody specifically binds to BTLA. In some embodiments, the IgG4 is human IgG4. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO:33. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:34. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:35. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO:33 and the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:34. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO:33 and the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:35.

[0172] In some embodiments, there is provided a full-length anti-BTLA antibody comprising an IgG1 constant region, wherein the anti-BTLA antibody comprises: a) a heavy chain variable domain comprising: HC-CDR1, which comprises the amino acid sequence shown in any one of SEQ ID NOs: 1-3 or a variant thereof, the variant comprising up to about 3 (e.g., 1, 2, or 3) amino acid substitutions; HC-CDR2, which comprises the amino acid sequence shown in any one of SEQ ID NOs: 4-6 or a variant thereof, the variant comprising up to about 3 (e.g., 1, 2, or 3) amino acid substitutions; and HC-CDR3, which comprises the amino acid sequence shown in any one of SEQ ID NOs: 7-9 or a variant thereof, the variant comprising up to about 3 (e.g., 1, 2, or 3) amino acid substitutions; and b) a light chain variable domain comprising: LC-CDR1, which comprises the amino acid sequence shown in any one of SEQ ID NOs: 10-12 or a variant thereof, the variant comprising up to about 3 (e.g., 1, 2, or 3) amino acid substitutions, LC-CDR2, which comprises the amino acid sequence shown in any one of SEQ ID NOs: 13-14 or a variant thereof, the variant comprising up to about 3 (e.g., 1, 2, or 3) amino acid substitutions; and LC-CDR3, which comprises the amino acid sequence shown in any one of SEQ ID NOs: 15-17 or a variant thereof, the variant comprising up to about 3 (e.g., 1, 2, or 3) amino acid substitutions. In some embodiments, the IgG1 is human IgG1. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO: 32. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO: 34. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO: 35. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO: 32 and the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO: 34. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO: 32 and the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO: 35.

[0173] In some embodiments, there is provided a full-length anti-BTLA antibody comprising an IgG2 constant region, wherein the anti-BTLA antibody comprises: a) a heavy chain variable domain comprising: HC-CDR1, which comprises the amino acid sequence shown in any one of SEQ ID NOs: 1-3 or a variant thereof, the variant comprising up to about 3 (e.g., 1, 2, or 3) amino acid substitutions; HC-CDR2, which comprises the amino acid sequence shown in any one of SEQ ID NOs: 4-6 or a variant thereof, the variant comprising up to about 3 (e.g., 1, 2, or 3) amino acid substitutions; and HC-CDR3, which comprises the amino acid sequence shown in any one of SEQ ID NOs: 7-9 or a variant thereof, the variant comprising up to about 3 (e.g., 1, 2, or 3) amino acid substitutions; and b) a light chain variable domain comprising: LC-CDR1, which comprises the amino acid sequence shown in any one of SEQ ID NOs: 10-12 or a variant thereof, the variant comprising up to about 3 (e.g., 1, 2, or 3) amino acid substitutions; LC-CDR2, which comprises the amino acid sequence shown in any one of SEQ ID NOs: 13-14 or a variant thereof, the variant comprising up to about 3 (e.g., 1, 2, or 3) amino acid substitutions; and LC-CDR3, which comprises the amino acid sequence shown in any one of SEQ ID NOs: 15-17 or a variant thereof, the variant comprising up to about 3 (e.g., 1, 2, or 3) amino acid substitutions. In some embodiments, the IgG2 is human IgG2. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 34. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 35.

[0174] In some embodiments, there is provided a full-length anti-BTLA antibody comprising an IgG3 constant region, wherein the anti-BTLA antibody comprises: a) a heavy chain variable domain, the heavy chain variable domain comprising: HC-CDR1, which comprises the amino acid sequence shown in any one of SEQ ID NOs: 1-3 or a variant thereof, the variant comprising up to about 3 (e.g., 1, 2, or 3) amino acid substitutions; HC-CDR2, which comprises the amino acid sequence shown in any one of SEQ ID NOs: 4-6 or a variant thereof, the variant comprising up to about 3 (e.g., 1, 2, or 3) amino acid substitutions; and HC-CDR3, which comprises the amino acid sequence shown in any one of SEQ ID NOs: 7-9 or a variant thereof, the variant comprising up to about 3 (e.g., 1, 2, or 3) amino acid substitutions; and b) a light chain variable domain, the light chain variable domain comprising: LC-CDR1, which comprises the amino acid sequence shown in any one of SEQ ID NOs: 10-12 or a variant thereof, the variant comprising up to about 3 (e.g., 1, 2, or 3) amino acid substitutions; LC-CDR2, which comprises the amino acid sequence shown in any one of SEQ ID NOs: 13-14 or a variant thereof, the variant comprising up to about 3 (e.g., 1, 2, or 3) amino acid substitutions; and LC-CDR3, which comprises the amino acid sequence shown in any one of SEQ ID NOs: 15-17 or a variant thereof, the variant comprising up to about 3 (e.g., 1, 2, or 3) amino acid substitutions. In some embodiments, the IgG3 is human IgG3. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO: 34. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO: 35.

[0175] In some embodiments, there is provided a full-length anti-BTLA antibody comprising an IgG4 constant region, wherein the anti-BTLA antibody comprises: a) a heavy chain variable domain comprising: HC-CDR1, which comprises the amino acid sequence shown in any one of SEQ ID NOs: 1-3 or a variant thereof, the variant comprising up to about 3 (e.g., 1, 2, or 3) amino acid substitutions; HC-CDR2, which comprises the amino acid sequence shown in any one of SEQ ID NOs: 4-6 or a variant thereof, the variant comprising up to about 3 (e.g., 1, 2, or 3) amino acid substitutions; and HC-CDR3, which comprises the amino acid sequence shown in any one of SEQ ID NOs: 7-9 or a variant thereof, the variant comprising up to about 3 (e.g., 1, 2, or 3) amino acid substitutions; and b) a light chain variable domain comprising: LC-CDR1, which comprises the amino acid sequence shown in any one of SEQ ID NOs: 10-12 or a variant thereof, the variant comprising up to about 3 (e.g., 1, 2, or 3) amino acid substitutions, LC-CDR2, which comprises the amino acid sequence shown in any one of SEQ ID NOs: 13-14 or a variant thereof, the variant comprising up to about 3 (e.g., 1, 2, or 3) amino acid substitutions, and LC-CDR3, which comprises the amino acid sequence shown in any one of SEQ ID NOs: 15-17 or a variant thereof, the variant comprising up to about 3 (e.g., 1, 2, or 3) amino acid substitutions. In some embodiments, the IgG4 is human IgG4. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO: 33. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO: 34. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO: 35. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO: 33 and the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO: 34. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO: 33 and the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO: 35.

[0176] In some embodiments, there is provided a full-length anti-BTLA antibody comprising an IgG1 constant region, wherein the anti-BTLA antibody comprises: a) a heavy chain variable domain comprising: HC-CDR1, which comprises the amino acid sequence shown in any of SEQ ID NOs: 1-3, HC-CDR2, which comprises the amino acid sequence shown in any of SEQ ID NOs: 4-6, and HC-CDR3, which comprises the amino acid sequence shown in any of SEQ ID NOs: 7-9, or a variant of the heavy chain variable domain, wherein the HC-CDR sequence contains up to about 5 (e.g., 1, 2, 3, 4, or 5) amino acid substitutions; and b) a light chain variable domain comprising: LC-CDR1, which comprises the amino acid sequence shown in any of SEQ ID NOs: 10-12, LC-CDR2, which comprises the amino acid sequence shown in any of SEQ ID NOs: 13-14, and LC-CDR3, which comprises the amino acid sequence shown in any of SEQ ID NOs: 15-17, or a variant of the light chain variable domain, wherein the LC-CDR sequence contains up to about 5 (e.g., 1, 2, 3, 4, or 5) amino acid substitutions. In some embodiments, the IgG1 is human IgG1. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO: 32. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO: 34. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO: 35. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO: 32 and the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO: 34. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO: 32 and the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO: 35.

[0177] In some embodiments, a full-length anti-BTLA antibody comprising an IgG4 constant region is provided, wherein the anti-BTLA antibody comprises a) a heavy chain variable domain comprising: HC-CDR1, which comprises an amino acid sequence shown in any of SEQ ID NOs: 1-3, HC-CDR2, which comprises an amino acid sequence shown in any of SEQ ID NOs: 4-6, and HC-CDR3, which comprises an amino acid sequence shown in any of SEQ ID NOs: 7-9, or a variant of the heavy chain variable domain, wherein the HC-CDR sequence contains up to about 5 (e.g., 1, 2, 3, 4, or 5) amino acid substitutions; and b) a light chain variable domain comprising: LC-CDR1, which comprises an amino acid sequence shown in any of SEQ ID NOs: 10-12, LC-CDR2, which comprises an amino acid sequence shown in any of SEQ ID NOs: 13-14, and LC-CDR3, which comprises an amino acid sequence shown in any of SEQ ID NOs: 15-17, or a variant of the light chain variable domain, wherein the LC-CDR sequence contains up to about 5 (e.g., 1, 2, 3, 4, or 5) amino acid substitutions. In some embodiments, the IgG4 is human IgG4. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO: 33. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO: 34. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO: 35. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO: 33 and the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO: 34. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO: 33 and the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO: 35.

[0178] In some embodiments, there is provided a full-length anti-BTLA antibody comprising an IgG1 constant region, wherein the anti-BTLA antibody comprises: a) a heavy chain variable domain comprising: HC-CDR1, which comprises the amino acid sequence shown in any one of SEQ ID NOs: 1-3; HC-CDR2, which comprises the amino acid sequence shown in any one of SEQ ID NOs: 4-6; and HC-CDR3, which comprises the amino acid sequence shown in any one of SEQ ID NOs: 7-9; and b) a light chain variable domain comprising: LC-CDR1, which comprises the amino acid sequence shown in any one of SEQ ID NOs: 10-12; LC-CDR2, which comprises the amino acid sequence shown in any one of SEQ ID NOs: 13-14; and LC-CDR3, which comprises the amino acid sequence shown in any one of SEQ ID NOs: 15-17. In some embodiments, the IgG1 is human IgG1. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO: 32. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO: 34. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO: 35. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO: 32 and the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO: 34. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO: 32 and the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO: 35.

[0179] In some embodiments, provided is a full-length anti-BTLA antibody comprising an IgG4 constant region, wherein the anti-BTLA antibody comprises: a) a heavy chain variable domain, the heavy chain variable domain comprising: HC-CDR1, which comprises an amino acid sequence shown in any of SEQ ID NOs: 1-3, HC-CDR2, which comprises an amino acid sequence shown in any of SEQ ID NOs: 4-6, and HC-CDR3, which comprises an amino acid sequence shown in any of SEQ ID NOs: 7-9; and b) a light chain variable domain, the light chain variable domain comprising: LC-CDR1, which comprises an amino acid sequence shown in any of SEQ ID NOs: 10-12, LC-CDR2, which comprises an amino acid sequence shown in any of SEQ ID NOs: 13-14, and LC-CDR3, which comprises an amino acid sequence shown in any of SEQ ID NOs: 15-17. In some embodiments, the IgG4 is human IgG4. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO: 33. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO: 34. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO: 35. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO: 33 and the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO: 34. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO: 33 and the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO: 35.

[0180] In some embodiments, there is provided a full-length anti-BTLA antibody comprising an IgG1 constant region, wherein the anti-BTLA antibody comprises: a) a heavy chain variable domain comprising: HC-CDR1, which comprises the amino acid sequence SEQ ID NO:1, HC-CDR2, which comprises the amino acid sequence SEQ ID NO:4, and HC-CDR3, which comprises the amino acid sequence SEQ ID NO:7; and b) a light chain variable domain comprising: LC-CDR1, which comprises the amino acid sequence SEQ ID NO:10, LC-CDR2, which comprises the amino acid sequence SEQ ID NO:13, and LC-CDR3, which comprises the amino acid sequence SEQ ID NO:15. In some embodiments, the IgG1 is human IgG1. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO:32. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:34. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:35. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO:32 and the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:34. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO:32 and the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:35.

[0181] In some embodiments, there is provided a full-length anti-BTLA antibody comprising an IgG1 constant region, wherein the anti-BTLA antibody comprises: a) a heavy chain variable domain comprising: HC-CDR1, which comprises the amino acid sequence SEQ ID NO:2, HC-CDR2, which comprises the amino acid sequence SEQ ID NO:5, and HC-CDR3, which comprises the amino acid sequence SEQ ID NO:8; and b) a light chain variable domain comprising: LC-CDR1, which comprises the amino acid sequence SEQ ID NO:11, LC-CDR2, which comprises the amino acid sequence SEQ ID NO:14, and LC-CDR3, which comprises the amino acid sequence SEQ ID NO:16. In some embodiments, the IgG1 is human IgG1. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO:32. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:34. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:35. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO:32 and the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:34. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO:32 and the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:35.

[0182] In some embodiments, a full-length anti-BTLA antibody comprising an IgG1 constant region is provided, wherein the anti-BTLA antibody comprises: a) a heavy chain variable domain, the heavy chain variable domain comprising: HC-CDR1, which comprises the amino acid sequence SEQ ID NO:3, HC-CDR2, which comprises the amino acid sequence SEQ ID NO:6, and HC-CDR3, which comprises the amino acid sequence SEQ ID NO:9; and b) a light chain variable domain, the light chain variable domain comprising: LC-CDR1, which comprises the amino acid sequence SEQ ID NO:12, LC-CDR2, which comprises the amino acid sequence SEQ ID NO:13, and LC-CDR3, which comprises the amino acid sequence SEQ ID NO:17. In some embodiments, the IgG1 is human IgG1. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO:32. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:34. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:35. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO:32 and the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:34. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO:32 and the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:35.

[0183] In some embodiments, a full-length anti-BTLA antibody comprising an IgG4 constant region is provided, wherein the anti-BTLA antibody comprises: a) a heavy chain variable domain comprising: HC-CDR1, which comprises the amino acid sequence SEQ ID NO:1, HC-CDR2, which comprises the amino acid sequence SEQ ID NO:4, and HC-CDR3, which comprises the amino acid sequence SEQ ID NO:7; and b) a light chain variable domain comprising: LC-CDR1, which comprises the amino acid sequence SEQ ID NO:10, LC-CDR2, which comprises the amino acid sequence SEQ ID NO:13, and LC-CDR3, which comprises the amino acid sequence SEQ ID NO:15. In some embodiments, the IgG4 is human IgG4. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO:33. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:34. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:35. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO:33 and the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:34. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO:33 and the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:35.

[0184] In some embodiments, a full-length anti-BTLA antibody comprising an IgG4 constant region is provided, wherein the anti-BTLA antibody comprises: a) a heavy chain variable domain comprising: HC-CDR1, which comprises the amino acid sequence SEQ ID NO:2, HC-CDR2, which comprises the amino acid sequence SEQ ID NO:5, and HC-CDR3, which comprises the amino acid sequence SEQ ID NO:8; and b) a light chain variable domain comprising: LC-CDR1, which comprises the amino acid sequence SEQ ID NO:11, LC-CDR2, which comprises the amino acid sequence SEQ ID NO:14, and LC-CDR3, which comprises the amino acid sequence SEQ ID NO:16. In some embodiments

[0185] In the example, the IgG4 is human IgG4. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO:33. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:34. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:35. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO:33 and the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:34. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO:33 and the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:35.

[0186] In some embodiments, there is provided a full-length anti-BTLA antibody comprising an IgG4 constant region, wherein the anti-BTLA antibody comprises: a) a heavy chain variable domain comprising: HC-CDR1, which comprises the amino acid sequence SEQ ID NO:3, HC-CDR2, which comprises the amino acid sequence SEQ ID NO:6, and HC-CDR3, which comprises the amino acid sequence SEQ ID NO:9; and b) a light chain variable domain comprising: LC-CDR1, which comprises the amino acid sequence SEQ ID NO:12, LC-CDR2, which comprises the amino acid sequence SEQ ID NO:13, and LC-CDR3, which comprises the amino acid sequence SEQ ID NO:17. In some embodiments, the IgG4 is human IgG4. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO:33. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:34. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:35. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO:33 and the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:34. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO:33 and the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:35.

[0187] In some embodiments, there is provided a full-length anti-BTLA antibody comprising an IgG1 constant region, wherein the anti-BTLA antibody comprises: a heavy chain variable domain (V H )), the V Hcomprising an amino acid sequence shown in any of SEQ ID NOs: 18 - 24 or a variant thereof, said variant having at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity with the amino acid sequence shown in any of SEQ ID NOs: 18 - 24; and a light chain variable domain (V L ), said V L comprising an amino acid sequence shown in any of SEQ ID NOs: 25 - 31 or a variant thereof, said variant having at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity with the amino acid sequence shown in any of SEQ ID NOs: 25 - 31. In some embodiments, the IgG1 is human IgG1. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO: 32. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO: 34. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO: 35. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO: 32 and the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO: 34. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO: 32 and the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO: 35.

[0188] In some embodiments, there is provided a full - length anti - BTLA antibody comprising an IgG2 constant region, wherein the anti - BTLA antibody comprises: a heavy chain variable domain (V H ), said V H comprising an amino acid sequence shown in any of SEQ ID NOs: 18 - 24 or a variant thereof, said variant having at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity with the amino acid sequence shown in any of SEQ ID NOs: 18 - 24; and a light chain variable domain (V L ), said V Lcomprising an amino acid sequence shown in any of SEQ ID NOs: 25 - 31 or a variant thereof, said variant having at least about 80% (such as at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity with the amino acid sequence shown in any of SEQ ID NOs: 25 - 31. In some embodiments, the IgG2 is human IgG2. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO: 34. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO: 35.

[0189] In some embodiments, there is provided a full - length anti - BTLA antibody comprising an IgG3 constant region, wherein the anti - BTLA antibody comprises: a heavy chain variable domain (V H ), said V H comprising an amino acid sequence shown in any of SEQ ID NOs: 18 - 24 or a variant thereof, said variant having at least about 80% (such as at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity with the amino acid sequence shown in any of SEQ ID NOs: 18 - 24; and a light chain variable domain (V L ), said V L comprising an amino acid sequence shown in any of SEQ ID NOs: 25 - 31 or a variant thereof, said variant having at least about 80% (such as at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity with the amino acid sequence shown in any of SEQ ID NOs: 25 - 31. In some embodiments, the IgG3 is human IgG3. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO: 34. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO: 35.

[0190] In some embodiments, there is provided a full - length anti - BTLA antibody comprising an IgG4 constant region, wherein the anti - BTLA antibody comprises: a heavy chain variable domain (V H ), said V H comprising an amino acid sequence shown in any of SEQ ID NOs: 18 - 24 or a variant thereof, said variant having at least about 80% (such as at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity with the amino acid sequence shown in any of SEQ ID NOs: 18 - 24; and a light chain variable domain (V L ), said V LComprising an amino acid sequence shown in any of SEQ ID NOs: 25-31 or a variant thereof, said variant having at least about 80% (such as at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity with the amino acid sequence shown in any of SEQ ID NOs: 25-31. In some embodiments, the IgG4 is human IgG4. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO: 33. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO: 34. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO: 35. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO: 33 and the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO: 34. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO: 33 and the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO: 35.

[0191] In some embodiments, there is provided a full-length anti-BTLA antibody comprising an IgG1 constant region, wherein the anti-BTLA antibody comprises: a heavy chain variable domain (V H ), said (V H ) comprising an amino acid sequence shown in any of SEQ ID NOs: 18-24, and a light chain variable domain (V L ), said (V L ) comprising an amino acid sequence shown in any of SEQ ID NOs: 25-31. In some embodiments, the IgG1 is human IgG1. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO: 32. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO: 34. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO: 35. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO: 32 and the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO: 34. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO: 32 and the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO: 35.

[0192] In some embodiments, there is provided a full-length anti-BTLA antibody comprising an IgG4 constant region, wherein the anti-BTLA antibody comprises: a heavy chain variable domain (V H ), said V Hcomprising an amino acid sequence shown in any of SEQ ID NOs: 18-24, and a light chain variable domain (V L ), wherein the V L comprises an amino acid sequence shown in any of SEQ ID NOs: 25-31. In some embodiments, the IgG4 is human IgG4. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO: 33. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO: 34. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO: 35. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO: 33 and the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO: 34. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO: 33 and the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO: 35.

[0193] In some embodiments, there is provided a full-length anti-BTLA antibody comprising an IgG1 constant region, wherein the anti-BTLA antibody comprises: V H which comprises the amino acid sequence SEQ ID NO: 18 or a variant thereof that has at least about 80% sequence identity with the amino acid sequence SEQ ID NO: 18; and V L which comprises the amino acid sequence SEQ ID NO: 25 or a variant thereof that has at least about 80% sequence identity with the amino acid sequence SEQ ID NO: 25. In some embodiments, the IgG1 is human IgG1. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO: 32. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO: 34. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO: 35. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO: 32 and the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO: 34. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO: 32 and the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO: 35.

[0194] In some embodiments, there is provided a full-length anti-BTLA antibody comprising an IgG1 constant region, wherein the anti-BTLA antibody comprises: V H, which comprises the amino acid sequence SEQ ID NO:19 or a variant thereof, said variant having at least about 80% sequence identity to the amino acid sequence SEQ ID NO:19; and V L , which comprises the amino acid sequence SEQ ID NO:26 or a variant thereof, said variant having at least about 80% sequence identity to the amino acid sequence SEQ ID NO:26. In some embodiments, the IgG1 is human IgG1. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO:32. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:34. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:35. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO:32 and the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:34. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO:32 and the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:35.

[0195] In some embodiments, there is provided a full-length anti-BTLA antibody comprising an IgG1 constant region, wherein the anti-BTLA antibody comprises: V H , which comprises the amino acid sequence SEQ ID NO:19 or a variant thereof, said variant having at least about 80% sequence identity to the amino acid sequence SEQ ID NO:19; and V L , which comprises the amino acid sequence SEQ ID NO:27 or a variant thereof, said variant having at least about 80% sequence identity to the amino acid sequence SEQ ID NO:27. In some embodiments, the IgG1 is human IgG1. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO:32. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:34. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:35. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO:32 and the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:34. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO:32 and the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:35.

[0196] In some embodiments, there is provided a full-length anti-BTLA antibody comprising an IgG1 constant region, wherein the anti-BTLA antibody comprises: V H, which comprises the amino acid sequence SEQ ID NO:19 or a variant thereof, said variant having at least about 80% sequence identity with the amino acid sequence SEQ ID NO:19; and V L , which comprises the amino acid sequence SEQ ID NO:28 or a variant thereof, said variant having at least about 80% sequence identity with the amino acid sequence SEQ ID NO:28. In some embodiments, the IgG1 is human IgG1. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO:32. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:34. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:35. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO:32 and the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:34. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO:32 and the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:35.

[0197] In some embodiments, there is provided a full-length anti-BTLA antibody comprising an IgG1 constant region, wherein the anti-BTLA antibody comprises: V H , which comprises the amino acid sequence SEQ ID NO:19 or a variant thereof, said variant having at least about 80% sequence identity with the amino acid sequence SEQ ID NO:19; and V L , which comprises the amino acid sequence SEQ ID NO:29 or a variant thereof, said variant having at least about 80% sequence identity with the amino acid sequence SEQ ID NO:29. In some embodiments, the IgG1 is human IgG1. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO:32. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:34. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:35. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO:32 and the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:34. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO:32 and the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:35.

[0198] In some embodiments, there is provided a full-length anti-BTLA antibody comprising an IgG1 constant region, wherein the anti-BTLA antibody comprises: V H, which comprises the amino acid sequence SEQ ID NO:20 or a variant thereof, said variant having at least about 80% sequence identity with the amino acid sequence SEQ ID NO:20; and V L , which comprises the amino acid sequence SEQ ID NO:26 or a variant thereof, said variant having at least about 80% sequence identity with the amino acid sequence SEQ ID NO:26. In some embodiments, the IgG1 is human IgG1. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO:32. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:34. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:35. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO:32 and the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:34. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO:32 and the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:35.

[0199] In some embodiments, there is provided a full-length anti-BTLA antibody comprising an IgG1 constant region, wherein the anti-BTLA antibody comprises: V H , which comprises the amino acid sequence SEQ ID NO:20 or a variant thereof, said variant having at least about 80% sequence identity with the amino acid sequence SEQ ID NO:20; and V L , which comprises the amino acid sequence SEQ ID NO:27 or a variant thereof, said variant having at least about 80% sequence identity with the amino acid sequence SEQ ID NO:27. In some embodiments, the IgG1 is human IgG1. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO:32. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:34. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:35. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO:32 and the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:34. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO:32 and the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:35.

[0200] In some embodiments, there is provided a full-length anti-BTLA antibody comprising an IgG1 constant region, wherein the anti-BTLA antibody comprises: V H, which comprises the amino acid sequence SEQ ID NO:20 or a variant thereof, said variant having at least about 80% sequence identity with the amino acid sequence SEQ ID NO:20; and V L , which comprises the amino acid sequence SEQ ID NO:28 or a variant thereof, said variant having at least about 80% sequence identity with the amino acid sequence SEQ ID NO:28. In some embodiments, the IgG1 is human IgG1. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO:32. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:34. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:35. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO:32 and the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:34. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO:32 and the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:35.

[0201] In some embodiments, there is provided a full-length anti-BTLA antibody comprising an IgG1 constant region, wherein the anti-BTLA antibody comprises: V H , which comprises the amino acid sequence SEQ ID NO:20 or a variant thereof, said variant having at least about 80% sequence identity with the amino acid sequence SEQ ID NO:20; and V L , which comprises the amino acid sequence SEQ ID NO:29 or a variant thereof, said variant having at least about 80% sequence identity with the amino acid sequence SEQ ID NO:29. In some embodiments, the IgG1 is human IgG1. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO:32. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:34. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:35. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO:32 and the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:34. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO:32 and the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:35.

[0202] In some embodiments, there is provided a full-length anti-BTLA antibody comprising an IgG1 constant region, wherein the anti-BTLA antibody comprises: V H, which comprises the amino acid sequence SEQ ID NO:21 or a variant thereof, said variant having at least about 80% sequence identity with the amino acid sequence SEQ ID NO:21; and V L , which comprises the amino acid sequence SEQ ID NO:28 or a variant thereof, said variant having at least about 80% sequence identity with the amino acid sequence SEQ ID NO:28. In some embodiments, the IgG1 is human IgG1. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO:32. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:34. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:35. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO:32 and the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:34. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO:32 and the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:35.

[0203] In some embodiments, there is provided a full-length anti-BTLA antibody comprising an IgG1 constant region, wherein the anti-BTLA antibody comprises: V H , which comprises the amino acid sequence SEQ ID NO:21 or a variant thereof, said variant having at least about 80% sequence identity with the amino acid sequence SEQ ID NO:21; and V L , which comprises the amino acid sequence SEQ ID NO:29 or a variant thereof, said variant having at least about 80% sequence identity with the amino acid sequence SEQ ID NO:29. In some embodiments, the IgG1 is human IgG1. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO:32. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:34. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:35. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO:32 and the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:34. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO:32 and the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:35.

[0204] In some embodiments, there is provided a full-length anti-BTLA antibody comprising an IgG1 constant region, wherein the anti-BTLA antibody comprises: V H, which comprises the amino acid sequence SEQ ID NO:22 or a variant thereof, said variant having at least about 80% sequence identity with the amino acid sequence SEQ ID NO:22; and V L , which comprises the amino acid sequence SEQ ID NO:28 or a variant thereof, said variant having at least about 80% sequence identity with the amino acid sequence SEQ ID NO:28. In some embodiments, the IgG1 is human IgG1. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO:32. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:34. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:35. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO:32 and the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:34. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO:32 and the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:35.

[0205] In some embodiments, there is provided a full-length anti-BTLA antibody comprising an IgG1 constant region, wherein the anti-BTLA antibody comprises: V H , which comprises the amino acid sequence SEQ ID NO:22 or a variant thereof, said variant having at least about 80% sequence identity with the amino acid sequence SEQ ID NO:22; and V L , which comprises the amino acid sequence SEQ ID NO:29 or a variant thereof, said variant having at least about 80% sequence identity with the amino acid sequence SEQ ID NO:29. In some embodiments, the IgG1 is human IgG1. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO:32. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:34. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:35. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO:32 and the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:34. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO:32 and the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:35.

[0206] In some embodiments, there is provided a full-length anti-BTLA antibody comprising an IgG1 constant region, wherein the anti-BTLA antibody comprises: V H, which comprises the amino acid sequence SEQ ID NO:23 or a variant thereof, said variant having at least about 80% sequence identity with the amino acid sequence SEQ ID NO:23; and V L , which comprises the amino acid sequence SEQ ID NO:30 or a variant thereof, said variant having at least about 80% sequence identity with the amino acid sequence SEQ ID NO:30. In some embodiments, the IgG1 is human IgG1. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO:32. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:34. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:35. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO:32 and the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:34. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO:32 and the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:35.

[0207] In some embodiments, there is provided a full-length anti-BTLA antibody comprising an IgG1 constant region, wherein the anti-BTLA antibody comprises: V H , which comprises the amino acid sequence SEQ ID NO:24 or a variant thereof, said variant having at least about 80% sequence identity with the amino acid sequence SEQ ID NO:24; and V L , which comprises the amino acid sequence SEQ ID NO:31 or a variant thereof, said variant having at least about 80% sequence identity with the amino acid sequence SEQ ID NO:31. In some embodiments, the IgG1 is human IgG1. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO:32. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:34. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:35. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO:32 and the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:34. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO:32 and the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:35.

[0208] In some embodiments, there is provided a full-length anti-BTLA antibody comprising an IgG4 constant region, wherein the anti-BTLA antibody comprises: V H, which comprises the amino acid sequence SEQ ID NO:18 or a variant thereof, said variant having at least about 80% sequence identity with the amino acid sequence SEQ ID NO:18; and V L , which comprises the amino acid sequence SEQ ID NO:25 or a variant thereof, said variant having at least about 80% sequence identity with the amino acid sequence SEQ ID NO:25. In some embodiments, the IgG4 is human IgG4. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO:33. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:34. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:35. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO:33 and the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:34. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO:33 and the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:35.

[0209] In some embodiments, there is provided a full-length anti-BTLA antibody comprising an IgG4 constant region, wherein the anti-BTLA antibody comprises: V H , which comprises the amino acid sequence SEQ ID NO:19 or a variant thereof, said variant having at least about 80% sequence identity with the amino acid sequence SEQ ID NO:19; and V L , which comprises the amino acid sequence SEQ ID NO:26 or a variant thereof, said variant having at least about 80% sequence identity with the amino acid sequence SEQ ID NO:26. In some embodiments, the IgG4 is human IgG4. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO:33. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:34. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:35. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO:33 and the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:34. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO:33 and the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:35.

[0210] In some embodiments, there is provided a full-length anti-BTLA antibody comprising an IgG4 constant region, wherein the anti-BTLA antibody comprises: V H, which comprises the amino acid sequence SEQ ID NO:19 or a variant thereof, said variant having at least about 80% sequence identity with the amino acid sequence SEQ ID NO:19; and V L , which comprises the amino acid sequence SEQ ID NO:27 or a variant thereof, said variant having at least about 80% sequence identity with the amino acid sequence SEQ ID NO:27. In some embodiments, the IgG4 is human IgG4. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO:33. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:34. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:35. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO:33 and the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:34. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO:33 and the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:35.

[0211] In some embodiments, there is provided a full-length anti-BTLA antibody comprising an IgG4 constant region, wherein the anti-BTLA antibody comprises: V H , which comprises the amino acid sequence SEQ ID NO:19 or a variant thereof, said variant having at least about 80% sequence identity with the amino acid sequence SEQ ID NO:19; and V L , which comprises the amino acid sequence SEQ ID NO:28 or a variant thereof, said variant having at least about 80% sequence identity with the amino acid sequence SEQ ID NO:28. In some embodiments, the IgG4 is human IgG4. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO:33. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:34. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:35. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO:33 and the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:34. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO:33 and the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:35.

[0212] In some embodiments, there is provided a full-length anti-BTLA antibody comprising an IgG4 constant region, wherein the anti-BTLA antibody comprises: V H, which comprises the amino acid sequence SEQ ID NO:19 or a variant thereof, said variant having at least about 80% sequence identity with the amino acid sequence SEQ ID NO:19; and V L , which comprises the amino acid sequence SEQ ID NO:29 or a variant thereof, said variant having at least about 80% sequence identity with the amino acid sequence SEQ ID NO:29. In some embodiments, the IgG4 is human IgG4. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO:33. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:34. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:35. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO:33 and the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:34. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO:33 and the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:35.

[0213] In some embodiments, there is provided a full-length anti-BTLA antibody comprising an IgG4 constant region, wherein the anti-BTLA antibody comprises: V H , which comprises the amino acid sequence SEQ ID NO:20 or a variant thereof, said variant having at least about 80% sequence identity with the amino acid sequence SEQ ID NO:20; and V L , which comprises the amino acid sequence SEQ ID NO:26 or a variant thereof, said variant having at least about 80% sequence identity with the amino acid sequence SEQ ID NO:26. In some embodiments, the IgG4 is human IgG4. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO:33. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:34. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:35. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO:33 and the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:34. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO:33 and the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:35.

[0214] In some embodiments, there is provided a full-length anti-BTLA antibody comprising an IgG4 constant region, wherein the anti-BTLA antibody comprises: V H, which comprises the amino acid sequence SEQ ID NO:20 or a variant thereof, said variant having at least about 80% sequence identity with the amino acid sequence SEQ ID NO:20; and V L , which comprises the amino acid sequence SEQ ID NO:27 or a variant thereof, said variant having at least about 80% sequence identity with the amino acid sequence SEQ ID NO:27. In some embodiments, the IgG4 is human IgG4. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO:33. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:34. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:35. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO:33 and the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:34. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO:33 and the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:35.

[0215] In some embodiments, there is provided a full-length anti-BTLA antibody comprising an IgG4 constant region, wherein the anti-BTLA antibody comprises: V H , which comprises the amino acid sequence SEQ ID NO:20 or a variant thereof, said variant having at least about 80% sequence identity with the amino acid sequence SEQ ID NO:20; and V L , which comprises the amino acid sequence SEQ ID NO:28 or a variant thereof, said variant having at least about 80% sequence identity with the amino acid sequence SEQ ID NO:28. In some embodiments, the IgG4 is human IgG4. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO:33. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:34. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:35. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO:33 and the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:34. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO:33 and the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:35.

[0216] In some embodiments, there is provided a full-length anti-BTLA antibody comprising an IgG4 constant region, wherein the anti-BTLA antibody comprises: V H, which comprises the amino acid sequence SEQ ID NO:20 or a variant thereof, said variant having at least about 80% sequence identity with the amino acid sequence SEQ ID NO:20; and V L , which comprises the amino acid sequence SEQ ID NO:29 or a variant thereof, said variant having at least about 80% sequence identity with the amino acid sequence SEQ ID NO:29. In some embodiments, the IgG4 is human IgG4. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO:33. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:34. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:35. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO:33 and the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:34. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO:33 and the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:35.

[0217] In some embodiments, there is provided a full-length anti-BTLA antibody comprising an IgG4 constant region, wherein the anti-BTLA antibody comprises: V H , which comprises the amino acid sequence SEQ ID NO:21 or a variant thereof, said variant having at least about 80% sequence identity with the amino acid sequence SEQ ID NO:21; and V L , which comprises the amino acid sequence SEQ ID NO:28 or a variant thereof, said variant having at least about 80% sequence identity with the amino acid sequence SEQ ID NO:28. In some embodiments, the IgG4 is human IgG4. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO:33. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:34. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:35. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO:33 and the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:34. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO:33 and the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:35.

[0218] In some embodiments, there is provided a full-length anti-BTLA antibody comprising an IgG4 constant region, wherein the anti-BTLA antibody comprises: V H, which comprises the amino acid sequence SEQ ID NO:21 or a variant thereof, said variant having at least about 80% sequence identity with the amino acid sequence SEQ ID NO:21; and V L , which comprises the amino acid sequence SEQ ID NO:29 or a variant thereof, said variant having at least about 80% sequence identity with the amino acid sequence SEQ ID NO:29. In some embodiments, the IgG4 is human IgG4. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO:33. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:34. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:35. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO:33 and the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:34. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO:33 and the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:35.

[0219] In some embodiments, there is provided a full-length anti-BTLA antibody comprising an IgG4 constant region, wherein the anti-BTLA antibody comprises: V H , which comprises the amino acid sequence SEQ ID NO:22 or a variant thereof, said variant having at least about 80% sequence identity with the amino acid sequence SEQ ID NO:22; and V L , which comprises the amino acid sequence SEQ ID NO:28 or a variant thereof, said variant having at least about 80% sequence identity with the amino acid sequence SEQ ID NO:28. In some embodiments, the IgG4 is human IgG4. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO:33. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:34. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:35. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO:33 and the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:34. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO:33 and the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:35.

[0220] In some embodiments, there is provided a full-length anti-BTLA antibody comprising an IgG4 constant region, wherein the anti-BTLA antibody comprises: V H, which comprises the amino acid sequence SEQ ID NO:22 or a variant thereof, said variant having at least about 80% sequence identity with the amino acid sequence SEQ ID NO:22; and V L , which comprises the amino acid sequence SEQ ID NO:29 or a variant thereof, said variant having at least about 80% sequence identity with the amino acid sequence SEQ ID NO:29. In some embodiments, the IgG4 is human IgG4. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO:33. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:34. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:35. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO:33 and the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:34. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO:33 and the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:35.

[0221] In some embodiments, there is provided a full-length anti-BTLA antibody comprising an IgG4 constant region, wherein the anti-BTLA antibody comprises: V H , which comprises the amino acid sequence SEQ ID NO:23 or a variant thereof, said variant having at least about 80% sequence identity with the amino acid sequence SEQ ID NO:23; and V L , which comprises the amino acid sequence SEQ ID NO:30 or a variant thereof, said variant having at least about 80% sequence identity with the amino acid sequence SEQ ID NO:30. In some embodiments, the IgG4 is human IgG4. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO:33. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:34. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:35. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO:33 and the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:34. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO:33 and the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:35.

[0222] In some embodiments, there is provided a full-length anti-BTLA antibody comprising an IgG4 constant region, wherein the anti-BTLA antibody comprises: V H, which comprises the amino acid sequence SEQ ID NO:24 or a variant thereof, said variant having at least about 80% sequence identity with the amino acid sequence SEQ ID NO:24; and V L , which comprises the amino acid sequence SEQ ID NO:31 or a variant thereof, said variant having at least about 80% sequence identity with the amino acid sequence SEQ ID NO:31. In some embodiments, the IgG4 is human IgG4. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO:33. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:34. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:35. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO:33 and the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:34. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO:33 and the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:35.

[0223] Binding affinity

[0224] Binding affinity is expressed as Kd, Koff, Kon or Ka. As used herein, the term Koff refers to the rate constant of dissociation of an antibody from an antigen / antibody complex, determined by a kinetic selection device. The term Kon refers to the association rate constant of an antibody binding to an antigen to form an antigen / antibody complex. The equilibrium dissociation constant Kd as used herein refers to the dissociation constant for a particular antibody-antigen interaction, which is the antigen concentration required for half of all antibody binding sites to be occupied by antigen and equilibrium to be reached in a solution of antibody molecules, and is equal to Koff / Kon. The determination of Kd assumes that all binding molecules are in solution. In the case of an antibody linked to a cell wall, such as in a yeast expression system, the corresponding equilibrium dissociation rate constant is expressed as EC50, which is a good approximation of Kd. The affinity association constant Ka is the reciprocal of the dissociation constant Kd.

[0225] The dissociation constant (Kd) can be used as an indicator of the affinity of the antibody moiety for the antigen. For example, a simple analysis can be performed using an antibody labeled with various labels and a Biacore instrument (manufactured by Amersham Biosciences) by the Scatchard method, and the interaction between biomolecules can be analyzed by surface plasmon resonance according to the user manual or the accompanying kit. The Kd values obtained using these methods are expressed in units of M. An antibody that specifically binds to a target may have, for example, ≤10 -7 M, ≤10 -8 M, ≤10 -9M, ≤ 10 -10 M, ≤ 10 -11 M, ≤ 10 -12 M or ≤ 10 -13 The Kd value of M.

[0226] The binding specificity of the antibody can be experimentally determined by methods known in the art. These methods include, but are not limited to, Western blots, ELISA-, RIA-, ECL-, IRMA-, EIA-, BIAcore assays, and peptide scanning, etc.

[0227] In some embodiments, the anti-BTLA antibody specifically binds to the BTLA target, and its Kd value is 10 -7 M to 10 -13 M (e.g., 10 -7 M to 10 -13 M, 10 -8 M to 10 -13 M, 10 -9 M to 10 -13 M or 10 -10 M to 10 -12 M). Thus, in some embodiments, the Kd value of the binding between the anti-BTLA antibody and BTLA is 10 -7 M to 10 -13 M, 1×10 -7 M to 5×10 -13 M, 10 -7 M to 10 -12 M, 10 -7 M to 10 -11 M, 10 -7 M to 10 -10 M, 10 -7 M to 10 -9 M, 10 -8 M to 10 -13 M, 1×10 -8 M to 5×10 -13 M, 10 -8 M to 10 - 12 M, 10 -8 M to 10 -11 M, 10 -8 M to 10 -10 M, 10 -8 M to 10 -9 M, 5×10 -9 M to 1×10 -13 M, 5×10 -9 M to 1×10 -12 M, 5×10 -9 M to 1×10-11 M, 5 × 10 -9 M to 1 × 10 -10 M, 10 -9 M to 10 -13 M, 10 -9 M to 10 -12 M, 10 -9 M to 10 -11 M, 10 -9 M to 10 -10 M, 5 × 10 -10 M to 1 × 10 -13 M, 5 × 10 -10 M to 1 × 10 -12 M, 5 × 10 -10 M to 1 × 10 -11 M, 10 -10 M to 10 - 13 M, 1 × 10 -10 M to 5 × 10 -13 M, 1 × 10 -10 M to 1 × 10 -12 M, 1 × 10 -10 M to 5 × 10 -12 M, 1 × 10 -10 M to 1 × 10 - 11 M, 10 -11 M to 10 -13 M, 1 × 10 -11 M to 5 × 10 -13 M, 10 -11 M to 10 -12 M, 10 -12 M to 10 -13 M. In some embodiments, the Kd value of the binding between the anti-BTLA antibody and BTLA is 10 -7 M to 10 -13 M.

[0228] In some embodiments, the Kd value of the binding between the anti-BTLA antibody and a non-target is higher than the Kd value of the binding between the anti-BTLA antibody and the target, and in some of the embodiments cited herein, the binding affinity of the anti-BTLA antibody for the target (e.g., BTLA) is higher than the binding affinity of the anti-BTLA antibody for the non-target. In some embodiments, the non-target refers to an antigen other than BTLA. In some embodiments, the difference in the Kd values of the binding of the anti-BTLA antibody (against BTLA) to non-BTLA targets is at least 10-fold, such as 10 - 100-fold, 100 - 1000-fold, 10 3 -10 4 -fold, 10 4 -10 5 -fold, 105 -10 6 times, 10 6 -10 7 times, 10 7 -10 8 times, 10 8 -10 9 times, 10 9 -10 10 times, 10 10 -10 11 times, 10 11 -10 12 times.

[0229] In some embodiments, the Kd value of the anti-BTLA antibody binding to a non-target is 10 -1 M to 10 -6 M (e.g., 10 -1 M to 10 -6 M, 10 -1 M to 10 -5 M, 10 -2 M to 10 -4 M). In some embodiments, the non-target refers to an antigen other than BTLA. Therefore, in some embodiments, the Kd value of the binding between the anti-BTLA antibody and a non-BTLA target is 10 -1 M to 10 -6 M, 1×10 -1 M to 5×10 -6 M, 10 -1 M to 10 -5 M, 1×10 -1 M to 5×10 -5 M, 10 -1 M to 10 -4 M, 1×10 -1 M to 5×10 -4 M, 10 -1 M to 10 - 3 M,

[0230] 1×10 -1 M to 5×10 -3 M, 10 -1 M to 10 -2 M, 10 -2 M to 10 -6 M, 1×10 -2 M to 5×10 -6 M, 10 -2 M to 10 - 5 M, 1×10 -2 M to 5×10 -5 M, 10-2 M to 10 -4 M, 1×10 -2 M to 5×10 -4 M, 10 -2 M to 10 -3 M, 10 -3 M to 10 -6 M, 1×10 -3 M to 5×10 -6 M, 10 -3 M to 10 -5 M, 1×10 -3 M to 5×10 -5 M, 10 -3 M to 10 -4 M, 10 -4 M to 10 -6 M, 1×10 -4 M to 5×10 -6 M, 10 -4 M to 10 -5 M, 10 -5 M to 10 -6 M.

[0231] In some embodiments, when it is mentioned that an anti-BTLA antibody specifically recognizes a BTLA target with high binding affinity and binds to non-targets with low binding affinity, the Kd value of the anti-BTLA antibody binding to the BTLA target is 10 -7 M to 10 -13 M (e.g., 10 -7 M to 10 -13 M, 10 -8 M to 10 -13 M, 10 -9 M to 10 -13 M, 10 -10 M to 10 -12 M), and the Kd value of binding to non-targets is 10 -1 M to 10 -6 M (e.g., 10 -1 M to 10 -6 M, 10 -1 M to 10 -5 M, 10 -2 M to 10 -4 M).

[0232] In some embodiments, when it is mentioned that an anti-BTLA antibody specifically recognizes BTLA, the binding affinity of the anti-BTLA antibody is compared with the binding affinity of a control anti-BTLA antibody (such as Ref). In some embodiments, the Kd value of the binding between the control anti-BTLA antibody and BTLA can be at least 2 times, such as 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 10 - 100 times, 100 - 1000 times, 10 3 -10 4 times.

[0233] Nucleic acid

[0234] Nucleic acid molecules encoding anti-BTLA antibodies are also contemplated. In some embodiments, there is provided a (or a set of) nucleic acid encoding a full-length anti-BTLA antibody, including any of the full-length anti-BTLA antibodies described herein. In some embodiments, the nucleic acid (or a set of nucleic acids) of the anti-BTLA antibody described herein may further include a nucleic acid sequence encoding a polypeptide tag (such as a protein purification tag, His tag, HA tag).

[0235] At the same time, isolated host cells containing anti-BTLA antibodies, isolated nucleic acids encoding anti-BTLA antibody polypeptide components, or vectors containing nucleic acids encoding anti-BTLA antibody polypeptide components described herein are also contemplated.

[0236] This application also includes variants of these nucleic acid sequences. For example, variants include nucleotide sequences that hybridize to the nucleic acid sequence encoding the anti-BTLA antibody of this application at least under moderately stringent hybridization conditions.

[0237] This application also provides vectors into which the nucleic acid sequences in this application can be inserted.

[0238] In short, a natural or synthetic nucleic acid encoding an anti-BTLA antibody is inserted into a suitable expression vector such that the nucleic acid is operably linked to 5' and 3' end regulatory elements, such as including a promoter (such as a lymphocyte-specific promoter) and a 3' untranslated region (UTR), and an anti-BTLA antibody (such as a full-length anti-BTLA antibody) can be expressed. The vector is suitable for replication and integration in eukaryotic host cells. Typical cloning and expression vectors contain transcriptional and translational terminators, initiation sequences, and promoters that regulate the expression of the target nucleic acid sequence.

[0239] The nucleic acids described in the present application can also be used for nucleic acid immunization and gene therapy by using standard gene delivery protocols. Nucleic acid delivery methods are known in the art. See, for example, U.S. Pat. Nos. 5,399,346, 5,580,859, 5,589,466, the entire contents of which are incorporated herein by reference. In some embodiments, the present application also provides gene therapy vectors.

[0240] The nucleic acids can be cloned into many types of vectors. For example, the nucleic acids can be cloned into vectors including, but not limited to, plasmids, phagemids, phage derivatives, animal viruses, and cosmids. Particularly interesting vectors include expression vectors, replication vectors, probe generation vectors, and sequencing vectors.

[0241] In addition, the expression vectors can be provided to cells in the form of viral vectors. Viral vector technology is well known in the art and is described, for example, in Green and Sambrook (2013, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York), and other virology or molecular biology manuals. Viruses that can be used as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpesviruses, and lentiviruses. Generally, suitable vectors include an origin of replication that functions in at least one organism, a promoter sequence, convenient restriction endonuclease sites, and one or more selectable markers (see, for example, WO 01 / 96584; WO 01 / 29058; and U.S. Pat. No. 6,326,193).

[0242] Many virus-based systems have been developed for gene transfer into mammalian cells. For example, retroviruses provide a convenient platform for gene delivery systems. Techniques known in the art can be applied to insert the selected gene into a vector and package it in retroviral particles. The recombinant virus is then isolated and delivered to the cells of a subject, either in vivo or in vitro. Many retroviral systems are known in the art. In some embodiments, adenoviral vectors are used. Many adenoviral vectors are known in the art. In some embodiments, lentiviral vectors are used. Vectors derived from retroviruses, such as lentiviruses, are suitable tools for achieving long-term gene transfer because they enable long-term stable integration of the transgene and propagation in daughter cells. Lentiviral vectors have additional advantages over retroviruses derived from tumors, such as murine leukemia virus, because they can transduce non-dividing cells, such as hepatocytes. At the same time, they also have the additional advantage of low immunogenicity.

[0243] Other promoter elements, such as enhancers, regulate the frequency of transcriptional initiation. They are usually located 30 - 110 bp upstream of the start site, although recently many promoters have been found to contain functional elements downstream of the start site as well. The spacing between promoter elements is generally flexible, so that the promoter function is maintained when the elements are interchanged or moved relative to each other. In the thymidine kinase (tk) promoter, the spacing between promoter elements can be increased to 50 bp before activity begins to decline.

[0244] One example of a suitable promoter is the immediate early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a strong constitutive promoter sequence that can drive high-level expression of any polynucleotide sequence operably linked thereto. Another example of a suitable promoter is the elongation factor 1α (EF-1α) promoter. However, other constitutive promoters can also be used, including but not limited to the simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus long terminal repeat (HIV-LTR) promoter, MoMuLV promoter, avian leukosis virus promoter, Epstein-Barr virus immediate early promoter, Rous sarcoma virus promoter, and human gene promoters, such as, including but not limited to, actin promoter, myosin promoter, hemoglobin promoter, and creatine kinase promoter. In addition, the present application should not be limited to the use of only constitutive promoters; inducible promoters are also part of what is contemplated in the present application. The use of inducible promoters provides a molecular switch that can initiate the expression of a polynucleotide sequence operably linked thereto when such expression is desired and turn off the expression when it is not. Inducible promoters include, but are not limited to, metallothionein promoter, glucocorticoid promoter, progesterone promoter, and tetracycline promoter.

[0245] In some embodiments, the expression of the anti-BTLA antibody is inducible. In some embodiments, the nucleic acid sequence encoding the anti-BTLA antibody is operably linked to an inducible promoter, including any of the inducible promoters described herein.

[0246] Inducible promoter

[0247] The use of inducible promoters provides a molecular switch that can initiate the expression of a polynucleotide sequence operably linked thereto when expression is desired and turn off the expression when expression is not desired. Exemplary inducible promoters applicable in eukaryotic cells include, but are not limited to, hormone regulatory elements (see, for example, Mader, S. and White, J. H. (1993) Proc. Natl. Acad. Sci. USA 90:5603-5607), synthetic ligand regulatory elements (see Spencer, D. M. et al (1993) Science 262:1019-1024), and ionizing radiation regulatory elements (see Manome, Y. et al. (1993) Biochemistry 32:10607-10613; Datta, R. et al. (1992) Proc. Natl. Acad. Sci. USA 89:1014-10153). Other exemplary inducible promoters applicable in mammalian systems in vivo or in vitro are described in Gingrich et al. (1998) Annual Rev. Neurosci 21:377-405. In some embodiments, the inducible promoter system for expressing the anti-BTLA antibody is the Tet system. In some embodiments, the inducible promoter system for expressing the anti-BTLA antibody is the Escherichia coli lac repressor system.

[0248] An exemplary inducible promoter system employed in the present application is the Tet system. This system is based on the Tet system described by Gossen et al (1993). In an exemplary embodiment, the target polynucleotide is controlled by a promoter containing one or more Tet operator (TetO) sites. In the non-activated state, the Tet repressor (TetR) binds to the TetO site and inhibits transcription of the promoter. In the activated state, for example, in the presence of an inducer such as tetracycline (Tc), anhydrotetracycline, doxycycline (Dox), or an active analog thereof, the inducer causes TetR to be released from TetO, resulting in transcription. Doxycycline is a member of the tetracycline antibiotic family and its chemical name is 1-dimethylamino-2,4a,5,7-pentahydroxy-11-methyl-4,6-dioxo-1,4a,11,11a,12,12a-hexahydrotetracene-3-carboxamide.

[0249] In one embodiment, TetR is codon-optimized for expression in mammalian cells, such as mouse or human cells. Due to the degeneracy of the genetic code, most amino acids are encoded by more than one codon, such that a given nucleic acid sequence has a large number of variants without any change in the encoded amino acid sequence. However, many organisms differ in codon usage, also known as "codon bias" (i.e., the preference for a particular codon for a given amino acid). Codon bias is generally related to the presence of the cognate tRNA species for a particular codon, which in turn enhances the efficiency of mRNA translation. Thus, coding sequences (e.g., from prokaryotes) can be customized by codon optimization to enhance their expression in different species (e.g., eukaryotes).

[0250] Other specific variants of the Tet system include the following "Tet-Off" and "Tet-On" systems. In the Tet-off system, transcription is inactivated in the presence of Tc or Dox. In this system, the tetracycline-regulated transcriptional activator protein (tTA), which consists of a fusion of TetR with the strong transcriptional activation domain of herpes simplex virus VP16, regulates the expression of the target nucleic acid under the transcriptional control of the tetracycline response promoter element (TRE). The TRE element consists of a tandem of TetO sequences fused to a promoter (usually a minimal promoter sequence derived from the immediate early promoter of human cytomegalovirus). In the absence of Tc or Dox, tTA binds to the TRE and activates transcription of the target gene. In the presence of Tc or Dox, tTA cannot bind to the TRE and the target gene is not expressed.

[0251] Conversely, in the Tet-On system, transcription is activated in the presence of Tc or Dox. The Tet-On system is based on the reverse tetracycline-regulated transcriptional activator rtTA. Like tTA, rtTA is a fusion protein consisting of the TetR repressor and the VP16 transcriptional activation domain. However, a change in four amino acids in the DNA-binding region of TetR alters the binding properties of rtTA such that it can only recognize the tetO sequence on the target transgene TRE in the presence of Dox. Thus, in the Tet-On system, rtTA can only activate transcription of the TRE-regulated target gene in the presence of Dox.

[0252] Another inducible promoter system is the lac repressor system of Escherichia coli (see Brown et al., Cell 49:603-612 (1987)). The Lac repressor system functions by regulating the transcription of a target polynucleotide operably linked to a promoter containing the lac operator (lacO). The Lac repressor (lacR) binds to LacO, thereby preventing the transcription of the target polynucleotide. The expression of the target polynucleotide is induced by a suitable inducer, for example, isopropyl-β-D-thiogalactopyranoside (IPTG).

[0253] To assess the expression of a polypeptide or a portion thereof, the expression vector to be introduced into the cell may also contain a selectable marker gene or a reporter gene or both, so as to facilitate the identification and selection of expressing cells from a population of cells transfected or infected with the viral vector. In other aspects, the selectable marker can be carried on a separate DNA fragment and used in co-transfection experiments. Both the selectable marker gene and the reporter gene can be flanked by suitable regulatory sequences to enable their expression in the host cell. Useful selectable markers include, for example, antibiotic resistance genes such as neo and similar genes.

[0254] Reporter genes can be used to identify potential transfected cells and to evaluate the function of regulatory sequences. Generally, a reporter gene is a gene that is not present in or not expressed by the recipient organism or tissue, and encodes a polypeptide whose expression is manifested as some easily detectable property, such as enzyme activity. After the DNA is introduced into the recipient cell, the expression of the reporter gene is detected at an appropriate time. Suitable reporter genes can include genes encoding luciferase, β-galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase or green fluorescent protein (see, Ui-Tel et al., 2000 FEBS Letters 479:79-82). Suitable expression systems are well known and can be prepared by known techniques or obtained commercially. Generally, the construct of the smallest 5' flanking region that can show the highest expression level of the reporter gene is identified as the promoter. Such promoter regions can be linked to the reporter gene and used to evaluate the ability of certain substances to regulate promoter-driven transcription.

[0255] In some embodiments, a nucleic acid encoding any of the full-length anti-BTLA antibodies described herein is provided. In some embodiments, the nucleic acid comprises one or more nucleic acid sequences encoding the heavy and light chains of the full-length anti-BTLA antibody. In some embodiments, each of the one or more nucleic acid sequences is contained in a separate vector. In some embodiments, at least some of the nucleic acid sequences are contained in the same vector. In some embodiments, all of the nucleic acid sequences are contained in the same vector. The vector can be selected from, for example, mammalian expression vectors and viral vectors (such as vectors derived from retroviruses, adenoviruses, adeno-associated viruses, herpesviruses and lentiviruses).

[0256] Methods for introducing genes into cells and expressing them are known in the art. In the context of expression vectors, the vector can be easily introduced into host cells, such as mammalian cells, bacteria, yeast, or insect cells, by any method in the art. For example, the expression vector can be introduced into the host cell by physical, chemical, or biological methods.

[0257] Physical methods for introducing polynucleotides into host cells include calcium phosphate precipitation, liposome transfection, gene gun, microinjection, electroporation, and the like. Methods for preparing cells containing vectors and / or exogenous nucleic acids are well known in the art. See, for example, Green and Sambrook (2013, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York). In some embodiments, the polynucleotide is introduced into the host cell by calcium phosphate transfection.

[0258] Biological methods for introducing a target polynucleotide into a host cell include the use of DNA and RNA vectors. Viral vectors, especially retroviral vectors, have become the most widely used method for inserting genes into mammalian cells, such as human cells. Other viral vectors can be derived from lentiviruses, poxviruses, herpes simplex virus type 1, adenoviruses, and adeno-associated viruses, etc. See, e.g., U.S. Pat. Nos. 5,350,674 and 5,585,362.

[0259] Chemical methods for introducing polynucleotides into host cells include colloidal dispersion systems, such as polymer complexes, nanocapsules, microspheres, magnetic beads, and lipid-based systems, which include water-in-oil emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system used as a delivery vector in vivo and in vitro is a liposome (e.g., an artificial membrane vesicle).

[0260] In the case of using a non-viral delivery system, an exemplary delivery vehicle is a liposome. Consider using lipid formulations to introduce nucleic acids into host cells (in vitro, ex vivo, or in vivo). On the other hand, the nucleic acid can be associated with lipids. The nucleic acid associated with lipids can be encapsulated into the aqueous interior of liposomes, dispersed within the lipid bilayer of liposomes, linked to liposomes via a linking molecule that binds to both the liposome and the oligonucleotide, embedded in liposomes, form a complex with liposomes, dispersed in a lipid-containing solution, mixed with lipids, bound to lipids, suspended in lipids, included in micelles or mixed with micelles, or otherwise associated with lipids. Lipid-, lipid / DNA-, or lipid / expression vector-related compositions in solution are not limited to any particular structure. For example, they may exist in a bilayer structure, in micelles, or in a "collapsed" structure. They can also be simply dispersed in solution, possibly forming aggregates of non-uniform size or shape. Lipids are fatty substances and can be naturally occurring or synthetic lipids. For example, lipids include the fat droplets naturally present in the cytoplasm, and a class of compounds containing long-chain aliphatic hydrocarbons and their derivatives, such as fatty acids, alcohols, amines, amino alcohols, and aldehydes.

[0261] Regardless of the method used to introduce foreign nucleic acids into host cells or otherwise expose the cells to the inhibitors of the present application, in order to confirm the presence of a recombinant DNA sequence in the host cell, a variety of experiments can be performed. Such experiments include, for example, "molecular biology" experiments well-known to those skilled in the art. For example, Southern and Northern blotting, RT-PCR, and PCR; "biochemical" experiments, such as detecting the presence or absence of a particular polypeptide, for example, by immunological methods (ELISAs and Western blots) or by the experiments described herein, are all within the scope of the present application.

[0262] Preparation of anti-BTLA antibody

[0263] In some embodiments, the anti-BTLA antibody is a monoclonal antibody or derived from a monoclonal antibody. In some embodiments, the anti-BTLA antibody comprises the V H and V L , or variants thereof. In some embodiments, the anti-BTLA antibody further comprises the CH1 and CL regions from a monoclonal antibody, or variants thereof. Monoclonal antibodies can be prepared using methods known in the art, including the hybridoma cell method, the phage display method, or the application of recombinant DNA methods. In addition, exemplary phage display methods are described herein and in the following examples.

[0264] In the hybridoma cell method, an immunizing agent is typically used to immunize hamsters, mice, or other suitable host animals to elicit lymphocytes that produce or are capable of producing antibodies that specifically bind to the immunizing agent. Alternatively, lymphocytes can be immunized in vitro. The immunizing agent can include polypeptides or fusion proteins of the target protein. Generally, if human cells are required, peripheral blood lymphocytes (PBLs) are used, while if cells from non-human mammalian sources are needed, splenocytes or lymph node cells are used. Lymphocytes are fused with an immortal cell line using a suitable fusogen, such as polyethylene glycol, to form hybridoma cells. The immortal cell line is typically a transformed mammalian cell, especially myeloma cells of rodent, bovine, and human origin. Rat or mouse myeloma cell lines are commonly used. Hybridoma cells can be cultured in a suitable medium, which preferably contains one or more substances that inhibit the growth or survival of unfused immortal cells. For example, if the parental cells lack hypoxanthine-guanine phosphoribosyltransferase (HGPRT or HPRT), the hybridoma cell medium typically includes hypoxanthine, aminopterin, and thymidine (HAT medium), which can prevent the growth of HGPRT-deficient cells.

[0265] In some embodiments, the immortal cell line fuses efficiently, ensuring high-level stable expression of the antibody by the selected antibody-producing cells and being sensitive to certain media, such as HAT medium. In some embodiments, the immortal cell line is a mouse myeloma cell line, which can be obtained from, for example, the Salk Cell Collection Center in San Diego, California, and the American Type Culture Collection in Manassas, Virginia. Human myeloma and mouse-human hybrid myeloma cell lines are also described for the preparation of human monoclonal antibodies.

[0266] Then, it can be determined whether monoclonal antibodies against the polypeptide are present in the medium in which the hybridoma cells are cultured. The binding specificity of the monoclonal antibodies produced by the hybridoma cells can be determined by immunoprecipitation or in vitro binding assays, such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA). Such techniques or analytical methods are known in the art. The binding affinity of the monoclonal antibodies can be determined by Scatchard analysis as described, for example, in Munson and Pollard, Anal. Biochem., 107:220 (1980).

[0267] After the desired hybridoma cells are identified, the target clones can be subcloned by the limiting dilution method and cultured by standard methods. Suitable media for this purpose include, for example, Dulbecco's Modified Eagle Medium (DMEM) and RPMI-1640 medium. Alternatively, hybridoma cells can be grown in the mammalian body in the form of ascites.

[0268] The monoclonal antibodies secreted by subclones can be isolated or purified from the culture medium or ascites by conventional immunoglobulin purification methods, such as protein A-agarose gel, hydroxyapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

[0269] In some embodiments, according to any of the anti-BTLA antibodies described herein, the anti-BTLA antibody comprises a sequence cloned from an antibody library (e.g., a phage library displaying scFv or Fab fragments). The clone can be identified by screening a combinatorial library of antibody fragments for the desired activity. For example, a variety of methods are known in the art for generating phage display libraries and screening these libraries to obtain antibodies with the desired binding properties. These methods are reviewed, for example, in Hoogenboom et al., Methods in Molecular Biology 178:1-37 (O'Brien et al., ed., Human Press, Totowa, N.J., 2001), and are further described, for example, in McCafferty et al., Nature 348:552-554; Clackson et al., Nature 352:624-628 (1991); Marks et al., J. Mol. Biol. 222:581-597 (1992); Marks and Bradbury, Methods in Molecular Biology 248:161-175 (Lo, ed., Human Press, Totowa, N.J., 2003); Sidhu et al., J. Mol. Biol. 338(2):299-310 (2004); Lee et al., J. Mol. Biol. 340(5):1073-1093 (2004); Fellouse, Proc. Natl. Acad. Sci. USA 101(34):12467-12472 (2004); and Lee et al., J. Immunol. Methods 284(1-2):119-132 (2004).

[0270] In certain phage display methods, the V is cloned separately by polymerase chain reaction (PCR). H and V LAll the components of the gene are randomly recombined in a phage library, and then phages that can bind to the antigen are screened, as described in Winter et al., Ann. Rev. Immunol., 12:433-455 (1994). Phages usually display antibody fragments in the form of scFv fragments or Fab fragments. Library phages of immune origin provide high-affinity antibodies against the immunogen without the need to construct hybridoma cells. Alternatively, a naive library (e.g., from humans) can be cloned to provide a single source of antibodies against a variety of non-self and self antigens without any immunization, as described in Griffiths et al., EMBO J, 12:725-734 (1993). Finally, a naive library can also be prepared by cloning non-rearranged V-gene fragments from stem cells and using PCR primers containing random sequences to encode the hypervariable CDR3 region and complete rearrangement in vitro, as described in Hoogenboom and Winter, J. Mol. Biol., 227:381-388 (1992). Patent publications describing human antibody phage libraries include, for example, U.S. Pat. No. 5,750,373 and US Patent Publication Nos. 2005 / 0079574, 2005 / 0119455, 2005 / 0266000, 2007 / 0117126, 2007 / 0160598, 2007 / 0237764, 2007 / 0292936, and 2009 / 0002360.

[0271] The anti-BTLA antibody is prepared by a method of screening an anti-BTLA antibody moiety that can specifically bind to the target BTLA in a library through phage display. The library can be a human scFv phage display library having at least 1×10 9 (e.g., at least 1×10 9 , 2.5×10 9 , 5×10 9 , 7.5×10 9 , 1×10 10 , 2.5×10 10 , 5×10 10 , 7.5×10 10 or 1×10 11)Unique human antibody fragments of species diversity. In some embodiments, the library is a human natural library constructed from DNA extracted from PMBCs and spleens of healthy subjects, and contains all human heavy and light chain subfamilies. In some embodiments, the library is a human natural library constructed from DNA extracted from PMBCs isolated from patients with various diseases, such as patients with autoimmune diseases, cancer patients, and patients with infectious diseases. In some embodiments, the library is a semi-synthetic human library, wherein the heavy chain CDR3 is completely random, and all amino acids (except cysteine) are present at any given position with the same probability. (See, for example, Hoet, R.M. et al., Nat. Biotechnol. 23(3):344-348, 2005). In some embodiments, the length of the heavy chain CDR3 of the semi-synthetic human library is between 5 and 24 (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24) amino acids. In some embodiments, the library is a fully synthetic phage display library. In some embodiments, the library is a non-human phage display library.

[0272] Phage clones with high affinity for the target BTLA can be screened by iterative binding of phage to the target BTLA, which is bound to a solid support (such as beads for solution panning or mammalian cells for cell panning), followed by removal of unbound phage and elution of specifically bound phage. Subsequently, the eluted bound phage clones are used to infect suitable host cells, such as E. coli XL1-Blue, for expression and purification. Specific binding of BTLA can be enriched by multiple rounds of panning (e.g., 2, 3, 4, 5, 6, or more rounds), such as solution panning, cell panning, or a combination of both. Specific binding of the enriched phage clones to the target BTLA can be detected by any method known in the art, including, for example, ELISA and FACS.

[0273] Another method of screening antibody libraries is to display proteins on the surface of yeast cells. Wittrup et al. (U.S. Patents 6,699,658 and 6,696,251) developed a method for yeast cell display libraries. In this yeast display system, one component consists of the yeast lectin protein (Aga1) anchored to the yeast cell wall, and the other component consists of the second subunit of the lectin protein Aga2, which can bind to the Aga1 protein via a disulfide bond and thus be displayed on the surface of yeast cells. The Aga1 protein is expressed by integrating the Aga1 gene into the yeast chromosome. A single-chain variable fragment (scFv) library is fused with the Aga2 gene in the yeast display plasmid, and after transformation, the library can be retained in yeast due to the presence of an additional nutritional marker. Both the Aga1 and Aga2 proteins are expressed under the control of a galactose-inducible promoter.

[0274] Human antibody V gene libraries (V H and V K fragments) were obtained by PCR using a set of degenerate primers (Sblattero, D. and Bradbury, A. Immunotechnology 3, 271-278 1998). The PCR templates were commercially available RNAs or cDNAs, including PBMC, spleen, lymph nodes, bone marrow, and tonsils. After independent V H and V K PCR libraries were combined, they were assembled into scFv form by overlap extension PCR (Sheets, M.D. et al, Proc. Natl. Acad. Sci. USA 95, 6157-6162 1998). To construct a yeast scFv display library, the resulting scFv PCR products were cloned into a yeast display plasmid in yeast by homologous recombination. (Chao, G, et al, Nat Protoc. 2006; 1(2): 755-68. Miller KD, et al. Current Protocols in Cytometry 4.7.1-4.7.30, 2008).

[0275] The mammalian cell display system can be utilized to screen for anti-BTLA antibodies, where the antibody portion is displayed on the cell surface and specific BTLA-targeting antibodies are isolated by antigen-directed screening methods (as described in U.S. patent No. 7,732,195 B2). A Chinese hamster ovary (CHO) cell library displaying a large number of human IgG antibody genes can be established and used to discover clones expressing high-affinity antibody genes. Another display system has been developed that enables the simultaneous surface display and secretion of the same protein through alternative splicing, where the displayed protein phenotype remains genotype-related, allowing the secreted soluble antibody to be characterized in both biophysical and cell function-based assays. This method overcomes many of the limitations of previous mammalian cell displays and enables the direct screening and maturation of antibodies in the form of full-length, glycosylated IgGs (Peter M. Bowers, et al, Methods 2014, 65: 44-56). The transient expression system is suitable for single-round antigen selection prior to antibody gene rescue and is thus most useful for selecting antibodies from smaller libraries. Stable episomal vectors provide an attractive option. Episomal vectors can be efficiently transfected and stably maintained at low copy numbers, allowing for multiple rounds of panning and the dissection of more complex antibody libraries.

[0276] The IgG library was constructed based on the ligation of germline sequence V gene segments isolated from a pool of human donors to rearranged (D)J regions. RNA collected from 2000 human blood samples was reverse transcribed into cDNA, and V H and V K specific primers were used to amplify V H and V K fragments, which were purified by gel extraction. The V H and V K fragments were separately subcloned into display vectors containing IgG1 or κ constant regions and then electroporated or transduced into 293T cells to prepare the IgG library. To prepare the scFv antibody display library, V H and V K were ligated to generate scFv, which was then subcloned into a display vector and electroporated or transduced into 293T cells. It is well known that IgG libraries are constructed based on the ligation of germline sequence V gene segments isolated from a pool of donors, which can be mice, rats, rabbits, or monkeys.

[0277] Monoclonal antibodies can also be prepared by recombinant DNA methods, such as those described in U.S. Patent No. 4,816,567. The DNA encoding the monoclonal antibodies described herein can be readily isolated and sequenced by conventional methods (e.g., by oligonucleotide probes that specifically bind to genes encoding murine antibody light and heavy chains). The hybridoma cells described above or the BTLA-specific phage clones of the present application can serve as a source of such DNA. After isolation, the DNA can be placed in an expression vector, which is then transfected into a host cell, such as simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not produce immunoglobulins, to obtain monoclonal antibodies synthesized in the recombinant host cells. The DNA can also be modified, e.g., by replacing the human heavy and light chain constant regions with coding sequences and / or replacing homologous non-human sequences with framework regions (U.S. Patent No. 4,816,567; Morrison et al., supra), or by covalently linking the coding sequence of all or part of a non-immunoglobulin polypeptide to the coding sequence of the immunoglobulin. Such non-immunoglobulin polypeptides can replace the constant region of the antibody herein or can replace one of the antigen-binding sites in the variable domain of the antibody herein to form a chimeric bivalent antibody.

[0278] The antibodies can be monovalent antibodies. Methods for preparing monovalent antibodies are known in the art. For example, a recombinant expression method involving immunoglobulin light chains and modified heavy chains. The heavy chain is typically truncated at any position in the Fc region to prevent heavy chain cross-linking. Alternatively, the relevant cysteine residues are replaced with other amino acid residues or deleted to prevent cross-linking.

[0279] In vitro methods are also suitable for preparing monovalent antibodies. Digesting the antibody to produce antibody fragments, particularly Fab fragments, can be accomplished using any method known in the art.

[0280] Antibody variable domains having the desired binding specificity (antibody-antigen binding sites) can be fused to immunoglobulin constant regions. Preferably, they are fused to immunoglobulin heavy chain constant regions, which include at least part of the hinge, CH2, and CH3 regions. In some embodiments, a first heavy chain constant region (CH1) containing the necessary sites for light chain binding appears in at least one of the fusions. The DNA encoding the immunoglobulin heavy chain fusion, and if desired, also including the DNA encoding the immunoglobulin light chain, is inserted into a separate expression vector and co-transfected into a suitable host organism.

[0281] Fully human and humanized antibodies

[0282] The anti-BTLA antibody (such as a full-length anti-BTLA antibody) can be a fully human antibody or a humanized antibody. The humanized form of a non-human (such as murine) antibody portion is a chimeric immunoglobulin, immunoglobulin chain, or fragment thereof (e.g., Fv, Fab, Fab’, F(ab’)2, scFv, or other antigen-binding sequences of an antibody), which typically includes a minimal sequence derived from a non-human immunoglobulin. A humanized antibody includes a human immunoglobulin, immunoglobulin chain, or fragment thereof (the acceptor antibody), wherein the residues of the acceptor CDRs are replaced with CDR residues from a non-human source (the donor antibody) having the desired specificity, affinity, and properties, such as murine, rat, or rabbit CDRs. In some embodiments, human immunoglobulin Fv framework region residues are replaced with corresponding non-human residues. A humanized antibody can also contain amino acid residues that are neither part of the acceptor antibody nor in the introduced CDR or framework region sequences. Typically, a humanized antibody contains at least one, and usually two, variable domains, wherein all or substantially all of the CDR regions correspond to the CDR regions of a non-human immunoglobulin and all or substantially all of the framework regions are human immunoglobulin consensus sequences.

[0283] Typically, a humanized antibody contains one or more amino acid residues introduced from a non-human source. Those non-human amino acid residues are often referred to as “grafted” residues, and typically originate from a “grafted” variable domain. According to some embodiments, humanization can generally be performed in accordance with the following method of Winter and his colleagues (Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988)), by replacing the corresponding sequences of a human antibody with rodent CDRs or CDR sequences. Thus, such a “humanized” antibody portion (U.S. Patent No. 4,816,567), which is substantially less than a complete human antibody, has had its variable domains replaced with the corresponding sequences from a non-human source. In practice, a humanized antibody portion is typically a human antibody portion in which some CDR residues and possibly some framework region residues have been replaced with residues from similar sites in a rodent antibody.

[0284] Fully human antibodies are an alternative to humanization. For example, transgenic animals (e.g., mice) can currently be prepared that are capable of generating a complete library of fully human antibodies upon immunization without generating endogenous immunoglobulins. For example, it has been reported that homozygous deletion of the antibody heavy chain joining region (JH) gene in chimeric and germline mutant mice completely inhibits the production of endogenous antibodies. Transfer of the human germline immunoglobulin gene array into such germline mutant mice results in the production of human antibodies upon antigen stimulation, see, e.g., Jakobovits et al., PNAS USA, 90:2551 (1993); Jakobovits et al., Nature, 362:255-258 (1993); Bruggemann et al., Year in Immunol., 7:33 (1993); U.S. Patent Nos. 5,545,806, 5,569,825, 5,591,669, 5,545,807; and WO 97 / 17852. Alternatively, fully human antibodies can be prepared by introducing the human immunoglobulin locus into a transgenic animal (e.g., a mouse in which the endogenous immunoglobulin genes have been partially or completely silenced). Upon antigen stimulation, the production of fully human antibodies can be found to be very similar in all respects to their production in humans, including gene rearrangement, assembly, and antibody repertoire. This method is described, e.g., in U.S. Patent Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; and 5,661,016, and Marks et al., Bio / Technology, 10:779-783 (1992); Lonberg et al., Nature, 368:856-859 (1994); Morrison, Nature, 368:812-813 (1994); Fishwild et al., Nature Biotechnology, 14:845-851 (1996); Neuberger, Nature Biotechnology, 14:826 (1996); Lonberg and Huszar, Intern. Rev. Immunol., 13:65-93 (1995).

[0285] Fully human antibodies can also be generated by in vitro activation of B cells (see U.S. Patents 5,567,610 and 5,229,275) or by using various techniques known in the art, including phage display libraries. Hoogenboom and Winter, J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581 (1991). The techniques of Cole et al. and Boerner et al. can also be used to prepare fully human monoclonal antibodies. See Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985) and Boerner et al., J. Immunol., 147(1):86 - 95 (1991).

[0286] Anti - BTLA antibody variants

[0287] In some embodiments, the amino acid sequences of the anti - BTLA antibody variants provided herein (e.g., full - length anti - BTLA antibodies) are also contemplated. For example, it may be desirable to improve the binding affinity and / or other biological activities of the antibody. The amino acid sequences of the antibody variants can be prepared by introducing appropriate modifications in the nucleotide sequences encoding the antibody or by peptide synthesis. Such modifications include, for example, deletions and / or insertions and / or substitutions of residues in the amino acid sequence of the antibody. The final construct can be accomplished by any combination of amino acid residue deletions, insertions, and substitutions so as to have the desired characteristics. For example, antigen - binding ability.

[0288] In some embodiments, anti - BTLA antibody variants having one or more amino acid substitutions are provided. Target sites for substitution mutations include hypervariable regions (HVRs) and framework regions (FRs). Amino acid substitutions can be introduced into the target antibody and the products screened for the desired activities, such as improved biological activity, maintained / improved antigen - binding ability, reduced immunogenicity, or improved ADCC or CDC.

[0289] Conservative substitutions are shown in Table 4 below.

[0290] Table 4 Conservative substitutions

[0291]

[0292]

[0293] Amino acids are divided into different classes according to the properties of their side chains:

[0294] a. Hydrophobic amino acids: Norleucine, Met, Ala, Val, Leu, Ile;

[0295] b. Neutral hydrophilic amino acids: Cys, Ser, Thr, Asn, Gln;

[0296] c. Acidic amino acids: Asp, Glu;

[0297] d. Basic amino acids: His, Lys, Arg;

[0298] e. Amino acids containing those affecting the chain direction: Gly, Pro;

[0299] f. Aromatic amino acids: Trp, Tyr, Phe.

[0300] Substitution of non-conservative amino acids involves substituting one of the above categories for another.

[0301] An exemplary substitution variant is an affinity matured antibody, which can be conveniently produced by, for example, phage display-based affinity maturation techniques. Briefly, one or more CDR residues are mutated, the variant antibody portion is displayed on the phage, and variants with specific biological activities (e.g., biological activity mediated by T cell activation in the Raji-HVEM / T cell co-culture assay or binding affinity) are screened. Alterations (e.g., substitutions) can be made in the HVRs region to obtain improved biological activity mediated by T cell activation in the Raji-HVEM / T cell co-culture assay or binding affinity. Alterations can be made in the "hot spots" of the HVRs, i.e., residues encoded by codons that undergo high-frequency mutations during somatic maturation (see, e.g., Chowdhury, Methods Mol. Biol. 207:179-196 (2008)), and / or at specific determinant residues (SDRs), and the binding affinities of the resulting variants V H and V L are detected. Methods for constructing and reselecting affinity matured antibodies from secondary libraries have been described in a number of publications, e.g., Hoogenboom et al. in Methods in Molecular Biology 178:1-37 (O'Brien et al., ed., Human Press, Totowa, NJ, (2001)).

[0302] In some embodiments of affinity maturation, diversity is introduced into the selected variable genes for affinity maturation by any of a variety of methods (e.g., error-prone PCR, chain shuffling or oligonucleotide-directed mutagenesis). A secondary library is then created. The library is screened to identify antibody variants with the desired affinity. Another method of introducing diversity involves an HVR-mediated approach, where several HVR residues (e.g., 4-6 residues at a time) are randomized. HVR residues involved in antigen binding are specifically identified, e.g., using alanine-scan mutagenesis or modeling. Typically the CDR-H3 and CDR-L3 regions are particularly targeted.

[0303] In some embodiments, substitutions, insertions or deletions may occur within one or more HVRs, provided that such changes do not substantially reduce the ability of the antibody to bind antigen. For example, conservative changes (e.g., conservative substitutions provided herein) that do not substantially reduce binding affinity may be made in the HVRs. These changes may occur outside of HVR "hot spots" or SDRs regions. In some embodiments the variant V H and V L sequences, each HVR either remains unaltered or contains no more than 1, 2 or 3 amino acid substitutions.

[0304] A useful method for identifying amino acid residues or regions in an antibody that can be targeted for mutagenesis is called "alanine-scan mutagenesis", as described in Cunningham and Wells (1989) Science, 244:1081-1085. In this method, one or a group of target residues (e.g., charged residues such as arginine, aspartic acid, histidine, lysine and glutamic acid) are replaced with a neutral or negatively charged amino acid (e.g., alanine or glutamic acid) to determine whether the interaction of the antibody with antigen is affected. Substitutions can be further introduced at the position of the amino acid to demonstrate functional sensitivity to the initial substitution. Alternatively / additionally, the contact sites between the antibody and antigen are identified by the crystal structure of the antigen-antibody complex. These contact site residues and adjacent residues can be targeted or eliminated as substitution candidates. Variants are screened to determine whether they have the desired properties.

[0305] Insertions of amino acid sequences, including fusions at the amino- and / or carboxy-termini, ranging in length from 1 residue to polypeptides containing 100 or more residues, as well as the insertion of 1 or more amino acid residues within the sequence. Examples of terminal insertions include antibodies having a methionyl residue at the N-terminus. Other insertion variants of the antibody molecule include fusing an enzyme (e.g., ADEPT) or a polypeptide that increases the serum half-life of the antibody at the N- or C-terminus of the antibody molecule.

[0306] Fc region variant

[0307] In some embodiments, one or more amino acid modifications are introduced into the Fc region of an antibody described herein (e.g., a full-length anti-BTLA antibody or an anti-BTLA antibody fusion protein), thereby generating an Fc region variant. In some embodiments, the Fc region variant has enhanced ADCC potency, which is generally associated with binding to Fc-binding receptors (FcRs). In some embodiments, the Fc region variant has reduced ADCC potency. There are many examples of alterations or mutations in the Fc sequence that affect its potency. For example, WO 00 / 42072 and Shields et al. J Biol. Chem. 9(2):6591-6604 (2001) describe antibody variants with enhanced or reduced binding to FcRs. The contents of these publications are incorporated herein by reference.

[0308] Antibody-dependent cell-mediated cytotoxicity (ADCC) is a mechanism of action of therapeutic antibodies against tumor cells. ADCC is a cell-mediated immune defense in which effector cells of the immune system actively lyse target cells (e.g., cancer cells) when an antigen on the surface of the target cell membrane is bound by a specific antibody (e.g., an anti-BTLA antibody). Typically, the ADCC effect involves NK cells activated by the antibody. NK cells express the Fc receptor CD16. This receptor recognizes and binds to the Fc portion of the antibody molecule bound to the surface of the target cell. The most common Fc receptor on the surface of NK cells is CD16 or FcγRIII. Binding of the Fc receptor to the Fc region of the antibody results in activation of the NK cell, release of cytotoxic granules, and subsequent apoptosis of the target cell. The killing effect of ADCC on tumor cells can be measured by a specific assay using NK-92 cells transfected with a high-affinity FcR. The results are compared to wild-type NK-92 cells that do not express the FcR.

[0309] In some embodiments, the present application also provides anti-BTLA antibody variants (e.g., full-length anti-BTLA antibody variants) that comprise an Fc region with partial but not all effector functions, such that they have an extended half-life in vivo, yet specific effector functions (e.g., CDC or ADCC) are non-essential or detrimental, and such anti-BTLA antibodies are desirable candidates for the present application. Reduction / elimination of CDC and / or ADCC activity is confirmed by performing cytotoxicity assays in vitro and / or in vivo. For example, lack of FcγR binding ability (and thus likely lack of ADCC activity) but retention of FcRn binding ability of the antibody is confirmed by an Fc receptor (FcR) binding assay. Among the major cells mediating ADCC, NK cells express only FcγRIII, while monocytes express FcγRI, FcγRII, and FcγRIII. The expression of FcRs on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet Annu. Rev. Immunol. 9:457-492 (1991). Non-limiting examples of assessing the ADCC activity of a target molecule in vitro are described in U.S. Pat. No. 5,500,362 (see, e.g., Hellstrom, I. et al. Proc. Nat'l Acad. Sci. USA 83:7059-7063 (1986) and Hellstrom, I et al., Proc. Nat'l Acad. Sci. USA 82:1499-1502 (1985); U.S. Pat. No. 5,821,337 (see Bruggemann, M. et al., J. Exp. Med. 166:1351-1361 (1987)). Alternatively, non-radioactive assay methods can be employed (see, e.g., ACTI TM Flow cytometry non-radioactive cytotoxicity assay (CellTechnology, Inc., Mountain View, Calif.) and CYTOTOX 96 TMNon-radioactive cytotoxicity assays (Promega, Madison, Wis.). Effector cells used in such assay experiments include peripheral blood mononuclear cells (PBMC) and natural killer cells (NK). Alternatively, additionally, the ADCC activity of the target molecule is assayed in vivo, for example, in an animal model as described in Clynes et al. Proc. Nat'l Acad. Sci. USA 95:652-656 (1998). A C1q binding assay can also be performed to confirm that the antibody does not bind to C1q and thus lacks CDC activity. See, for example, C1q and C3c binding ELISAs in WO 2006 / 029879 and WO 2005 / 100402. To evaluate complement activation, a CDC assay can be performed (see, for example, Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996); Cragg, M. S. et al., Blood 101:1045-1052 (2003); and Cragg, M. S. and M. J. Glennie, Blood 103:2738-2743 (2004)). Methods known in the art are used to determine FcRn binding and in vivo clearance / half-life (see, for example, Petkova, S. B. et al., Int'l. Immunol. 18(12):1759-1769 (2006)).

[0310] Antibodies with reduced effector function, including substitutions of one or more residues at Fc region residues 238, 265, 269, 270, 297, 327, and 329 (U.S. Pat. No. 6,737,056). These Fc variants include Fc variants with substitutions of two or more residues at positions 265, 269, 270, 297, and 327, including the Fc variant referred to as "DANA", which has alanine substitutions at residues 265 and 297 (U.S. Pat. No. 7,332,581).

[0311] Antibody variants with increased or decreased binding to FcRs have been described (see, for example, U.S. Pat. No. 6,737,056; WO 2004 / 056312, and Shields et al., J. Biol. Chem. 9(2):6591-6604 (2001)).

[0312] In some embodiments, there is provided a variant of an anti - BTLA antibody (e.g., a full - length anti - BTLA antibody) that comprises a variant Fc region having one or more amino acid substitutions capable of enhancing ADCC. In some embodiments, the Fc region variant comprises one or more amino substitutions capable of enhancing ADCC, and these substitutions are at positions 298, 333, and / or 334 (EU residue numbering) of the Fc region. In some embodiments, the anti - BTLA antibody (e.g., a full - length anti - BTLA antibody) variant includes amino acid substitutions at positions S298A, E333A, and K334A in the Fc region.

[0313] In some embodiments, the alteration of the Fc region results in an alteration (i.e., enhancement or attenuation) of C1q binding and / or complement - dependent cytotoxicity (CDC), as described in U.S. Pat. No. 6,194,551, WO 99 / 51642, and Idusogie et al., J. Immunol. 164:4178 - 4184 (2000).

[0314] In some embodiments, there is provided a variant of an anti - BTLA antibody (e.g., a full - length anti - BTLA antibody) that comprises a variant Fc region having one or more amino acid substitutions capable of extending the half - life or enhancing binding to the Fc receptor (FcRn). Antibodies with extended half - lives and improved FcRn binding are described in US2005 / 0014934A1 (Hinton et al.). These antibodies have Fc regions that contain one or more amino acid substitutions that enhance the binding of the Fc region to FcRn. These Fc variants contain one or more substitutions at residues at positions 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424, or 434 of the Fc region, such as a substitution at residue 434 of the Fc region (U.S. Pat. No. 7,371,826).

[0315] See also Duncan & Winter, Nature 322:738 - 40 (1988); U.S. Pat. No. 5,648,260;

[0316] Examples of other Fc region variants are provided in U.S. Pat. No. 5,624,821 and WO 94 / 29351.

[0317] This application contemplates anti - BTLA antibodies (e.g., full - length anti - BTLA antibodies) that include any one of the Fc variants described herein or combinations thereof.

[0318] Glycosylation variant

[0319] In some embodiments, the anti-BTLA antibodies provided herein (e.g., full-length anti-BTLA antibodies) are modified to increase or decrease the degree of glycosylation of the anti-NGF antibody. By modifying the amino acid sequence of the anti-NGF antibody or its polypeptide portion to add or remove one or more glycosylation sites, glycosylation sites on the anti-BTLA antibody can be conveniently added or deleted.

[0320] Where the anti-BTLA antibody comprises an Fc region, the sugars linked thereto can be modified. Natural antibodies produced by mammalian cells typically contain branched biantennary oligosaccharides that are generally N-linked to Asn297 in the CH2 domain of the Fc region, see, e.g., Wright et al., TIBTECH 15:26-32 (1997). The oligosaccharide can comprise various sugars such as mannose, N-acetylglucosamine (GlcNAc), galactose, and sialic acid, as well as trehalose linked to the GlcNAc in the "stem" of the biantennary oligosaccharide structure. In some embodiments, the anti-BTLA antibodies of the present application can be modified with oligosaccharides to produce anti-BTLA antibody variants with certain improved properties.

[0321] The N-glycans linked to the CH2 domain of the Fc region are heterogeneous. Antibodies or Fc fusion proteins produced in CHO cells are fucosylated through fucosyltransferase activity, see Shoji-Hosaka et al., J. Biochem. 2006, 140:777-83. Typically, a small fraction of naturally occurring non-fucosylated IgGs can be detected in human serum. N-glycosylation of the Fc region is important for its binding to FcγR; non-fucosylated N-glycans enhance the binding ability of Fc to FcγRIIIa. Enhanced binding ability to FcRIIIa results in enhanced ADCC effect, which is beneficial in certain antibody therapeutic applications that require cytotoxicity.

[0322] In some embodiments, the enhanced effector function can be detrimental when Fc-mediated cytotoxicity is not desired. In some embodiments, the Fc fragment or the CH2 domain is non-glycosylated. In some embodiments, the N-glycosylation site in the CH2 domain is mutated to prevent its glycosylation.

[0323] In some embodiments, anti-BTLA antibody (e.g., full-length anti-BTLA antibody) variants are provided that comprise an Fc region, wherein the carbohydrate structure linked to the Fc region has reduced fucose or lacks fucose, which may enhance ADCC function. Specifically, anti-BTLA antibodies are provided herein that have reduced fucose relative to the same anti-BTLA antibody produced by wild-type CHO cells. That is, they are characterized by having a lower amount of fucose compared to antibodies produced by native CHO cells (e.g., CHO cells that produce the native glycosylated form, CHO cells containing the native FUT8 gene). In some embodiments, the N-linked glycans of the anti-BTLA antibody have less than 50%, 40%, 30%, 20%, 10%, or 5% fucose. For example, the fucose content of the anti-BTLA antibody may be 1%-80%, 1%-65%, 5%-65%, or 20%-40%. In some embodiments, the N-linked glycans of the anti-BTLA antibody do not contain fucose, i.e., wherein the anti-BTLA antibody is completely devoid of fucose, or has no fucose or is defucosylated. The fucose content is determined by calculating the average fucose content within the sugar chains linked to Asn297 relative to the total amount of all sugar structures (such as complex, hybrid, or mannose structures) linked to Asn297 as measured by MALDI-TOF mass spectrometry, as described in WO 2008 / 077546. Asn297 refers to the asparagine residue at position 297 in the Fc region (EU Fc region residue numbering system). However, due to minor sequence variations in the antibody, Asn297 may also be located upstream or downstream of position 297 by ±3 amino acids, i.e., between positions 294 and 300. These fucosylation variants may have enhanced ADCC function. See, for example, US Patent Publication Nos. US2003 / 0157108 (Presta, L.), US2004 / 0093621 (Kyowa Hakko Kogyo Co., Ltd).Examples of publications related to "defucosylated" or "fucose - deficient" antibody variants include US 2003 / 0157108; WO 2000 / 61739; WO 2001 / 29246; US 2003 / 0115614; US 2002 / 0164328; US 2004 / 0093621; US 2004 / 0132140; US 2004 / 0110704; US2004 / 0110282; US 2004 / 0109865; WO 2003 / 085119; WO 2003 / 084570; WO 2005 / 035586; WO2005 / 035778; WO 2005 / 053742; WO 2002 / 031140; Okazaki et al. J. Mol. Biol. 336:1239 - 1249(2004); Yamane - Ohnuki et al. Biotech. Bioeng. 87:614(2004). Cell lines capable of producing defucosylated antibodies include Lec13 CHO cells lacking protein fucosylation function (Ripka et al. Arch. Biochem. Biophys. 249:533 - 545(1986); US Pat Appl No US2003 / 0157108 A1, Presta, L; and WO 2004 / 056312 A1, Adams et al., especially Example 11), and gene - knockout cell lines, such as α - 1,6 - fucosyltransferase gene, FUT8 gene - knockout CHO cells (see Yamane - Ohnuki et al. Biotech. Bioeng. 87:614(2004); Kanda, Y. et al., Biotechnol. Bioeng., 94(4):680 - 688(2006); and WO2003 / 085107).

[0324] Variants of anti-BTLA antibodies (such as full-length anti-BTLA antibodies) further provide bisected oligosaccharides, e.g., wherein the biantennary oligosaccharide linked to the Fc region of the anti-BTLA antibody is bisected by GlcNAc. Such variants of anti-BTLA antibodies (such as full-length anti-BTLA antibodies) may have reduced fucosylation and / or enhanced ADCC function. Examples of such antibody variants are described in WO 2003 / 011878 (Jean-Mairet et al.); U.S. Pat. No. 6,602,684 (Umana et al.); US2005 / 0123546 (Umana et al.), and Ferrara et al., Biotechnology and Bioengineering, 93(5):851-861 (2006). Also provided are variants of anti-BTLA antibodies (such as full-length anti-BTLA antibodies) that have at least one galactose residue in the oligosaccharide linked to the Fc region. Such variants of anti-BTLA antibodies may have enhanced CDC function. Such variants are described, for example, in WO 1997 / 30087 (Patel et al.); WO 1998 / 58964 (Raju, S.); and WO 1999 / 22764 (Raju, S).

[0325] In some embodiments, the variant of the anti-BTLA antibody (such as full-length anti-BTLA antibody) comprises an Fc region capable of binding to FcγRIII. In some embodiments, the variant of the anti-BTLA antibody (such as full-length anti-BTLA antibody) comprising the Fc region has ADCC activity in the presence of human effector cells (such as T cells), or has enhanced ADCC activity in the presence of human effector cells compared to other identical anti-BTLA antibodies (such as full-length anti-BTLA antibodies) having a human wild-type IgG1 Fc region.

[0326] Cysteine engineering variant

[0327] In some embodiments, it is desirable to prepare cysteine-engineered anti-BTLA antibodies (such as full-length anti-BTLA antibodies) in which one or more amino acid residues are replaced with cysteine residues. In some embodiments, the substituted residues occur at accessible sites of the anti-BTLA antibody. By replacing those residues with cysteine, reactive thiol groups are located at accessible sites of the anti-BTLA antibody and can be used to conjugate the anti-BTLA antibody to other moieties, such as a drug moiety or a linker-drug moiety, to prepare an anti-BTLA immunoconjugate as further described herein. Cysteine-engineered anti-BTLA antibodies (such as, full-length anti-BTLA antibodies) can be prepared as described, for example, in U.S. Pat. No. 7,521,541.

[0328] Derivative

[0329] In some embodiments, the anti-BTLA antibodies provided herein (e.g., full-length anti-BTLA antibodies) can be further modified to include other non-protein moieties known in the art and readily available. Moieties suitable for derivatizing anti-BTLA antibodies include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol / propanediol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymers, polyamino acids (homopolymers or random copolymers), dextran or poly(n-vinylpyrrolidone) polyethylene glycol, propylene glycol homopolymers, propylene oxide / ethylene oxide copolymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde has advantages in manufacturing due to its stability in water. The polymer can have any molecular weight and can be branched or unbranched. The number of polymers attached to the anti-BTLA antibody can vary, and if more than one polymer is attached, they can be the same or different molecules. Generally, the number and / or type of polymer used for derivatization can be determined based on considerations including, but not limited to, the need to improve the properties or functions of the anti-BTLA antibody, whether the anti-BTLA antibody derivative is used for treatment under specific conditions, etc.

[0330] Pharmaceutical composition

[0331] Also provided herein are compositions (e.g., pharmaceutical compositions, also referred to herein as formulations) comprising any one of the anti-BTLA antibodies (e.g., full-length anti-BTLA antibodies), nucleic acids encoding the antibodies, vectors comprising the nucleic acids encoding the antibodies, or host cells comprising the nucleic acids or vectors described herein. In some embodiments, provided is a pharmaceutical composition comprising any one of the anti-BTLA antibodies described herein and a pharmaceutically acceptable carrier.

[0332] Suitable anti-BTLA antibody formulations can be obtained by mixing an anti-BTLA antibody of the desired purity with an optional pharmaceutically acceptable carrier, excipient, or stabilizer (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)), and formulated into a lyophilized preparation or a liquid preparation. The acceptable carrier, excipient, or stabilizer is non-toxic to the recipient at the dosages and concentrations employed, and includes buffers such as phosphates, citric acid, and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (e.g., cetyltrimethylammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol; butanol or benzyl alcohol; alkyl esters of p-hydroxybenzoic acid, such as methyl p-hydroxybenzoate or propyl p-hydroxybenzoate; catechol; resorcinol; cyclohexanol; 3-pentanol, and m-cresol); low molecular weight (less than 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone; amino acids, such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrin; chelating agents such as EDTA; sugars, such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (such as zinc-protein complexes); and / or nonionic surfactants such as TWEEN TM , PLURONICS TM or polyethylene glycol (PEG); Exemplary formulations are described in WO98 / 56418 and are hereby incorporated by reference expressly. Lyophilized preparations suitable for subcutaneous administration are described in WO97 / 04801. Such lyophilized preparations can be reconstituted with a suitable diluent into a formulation with a high protein concentration, and the reconstituted formulation can be administered to an individual to be treated herein by subcutaneous administration. Cationic liposomes or liposomes can be used to deliver the anti-BTLA antibody in the present application to cells.

[0333] The formulations described herein, in addition to containing an anti-BTLA antibody (e.g., a full-length anti-BTLA antibody), can further contain one or more other active substances necessary for treating a specific condition, preferably substances having complementary activities and no adverse reactions to each other. For example, in addition to the anti-BTLA antibody, it may be necessary to further contain an anti-tumor agent, a growth inhibitor, a cytotoxic agent, or a chemotherapeutic agent. These molecules are present in a combination in an amount effective for the intended purpose. The effective amount of the other substances depends on the content of the anti-BTLA antibody in the formulation, the type of disease or condition or treatment, and other factors as described above. These drugs are generally used at the same dosages and administration routes as described herein, or at 1% to 99% of the currently applied dosages.

[0334] The anti-BTLA antibody (e.g., full-length anti-BTLA antibody) can also be encapsulated in microcapsules prepared, for example, by coacervation techniques and interfacial polymerization, such as hydroxymethylcellulose or gelatin-microcapsules and poly(methyl methacrylate) microcapsules in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules) or in a coarse emulsion, respectively. Sustained-release formulations can be prepared.

[0335] Sustained-release formulations of the anti-BTLA antibody (e.g., full-length anti-BTLA antibody) can be prepared. Suitable examples of sustained-release formulations include solid hydrophobic polymer semipermeable matrices containing the antibody (or its fragment), and these matrices are in the form of shaped articles, e.g., films or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (e.g., poly(2-hydroxyethyl methacrylate) or poly(vinyl alcohol)), polylactic acid (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and L-glutamic acid ethyl ester, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as LUPRON DEPOTTM (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D(-)-3-hydroxybutyric acid. Although polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid can release molecules for more than 100 days, certain hydrogels can release proteins in a shorter time. When the encapsulated antibody stays in the body for a long time, they may denature or aggregate due to exposure to a humid environment at 37°C, which may lead to loss of biological activity or alteration of immunogenicity. Rational strategies can be designed to stabilize the anti-BTLA antibody according to the corresponding mechanisms. For example, if it is found that the aggregation mechanism is the formation of intermolecular S-S bonds through thiol-disulfide exchange, stabilization can be achieved by modifying sulfhydryl residues, lyophilizing in an acidic solution, controlling the water content, using appropriate additives, and developing specific polymer matrix compositions.

[0336] In some embodiments, the anti-BTLA antibody (e.g., full-length anti-BTLA antibody) is formulated in a buffer containing citrate, sodium chloride, acetate, succinate, glycine, polysorbate 80 (Tween 80), or any combination thereof.

[0337] Formulations for in vivo administration must be sterile. This can be easily achieved, for example, by filtering through a sterile filtration membrane.

[0338] Therapeutic methods using anti-BTLA antibodies

[0339] An anti-BTLA antibody (e.g., a full-length anti-BTLA antibody) and / or the compositions described herein can be administered to an individual (e.g., a mammal such as a human) to treat diseases and / or disorders caused by dysregulation of the BTLA signaling pathway (e.g., cancer or infectious diseases), including but not limited to non-small cell lung cancer, adrenal cancer, bladder cancer, brain cancer, pancreatic cancer, breast cancer, colorectal cancer, melanoma, esophageal cancer, gastric cancer, cervical cancer, head and neck cancer, hepatocellular carcinoma, kidney cancer, liver cancer, ovarian cancer, pancreatic cancer, prostate cancer, small cell lung cancer, testicular cancer, thyroid cancer, uterine cancer, and any type of leukemia, lymphoma, and myeloma, as well as infectious diseases, including but not limited to Human Papilloma Virus (HPV), Human Immunodeficiency Virus (HIV), Herpes Simplex Virus (HSV), Varicella Zoster Virus (VSV), Cytomegalovirus (CMV), Epstein Barr Virus (EBV), Chlamydia, Rickettsia bacteria, Mycobacterium, Staphylococcus, Streptococcus, Pneumococcus, Meningococcus, and Gonococcus (Conococci), Klebsiella, Proteus, Serratia, Pseudomonas, Legionella, Diphtheria, Salmonella, Bacillus, Cholera, Tetanus, Botulism, Anthrax, Plague, Leptospirosis, and Lymes disease bacteria. Accordingly, in some embodiments, the present application provides a method for treating diseases and / or disorders caused by dysregulation of the BTLA signaling pathway (e.g., cancer or infectious diseases), comprising administering to an individual an effective amount of a composition (e.g., a pharmaceutical composition) comprising an anti-BTLA antibody (e.g., a full-length anti-BTLA antibody), such as any of the anti-BTLA antibodies described herein (e.g., a full-length anti-BTLA antibody), and in some embodiments, the individual is a human.

[0340] For example, in some embodiments, a method for treating an individual having a disease associated with the BTLA signaling pathway (e.g., cancer or an infectious disease) is provided, comprising administering to the individual an effective amount of a pharmaceutical composition comprising a BTLA antibody (e.g., a full-length anti-BTLA antibody) that specifically binds to an epitope on human BTLA, wherein the epitope comprises the amino acid residues of human BTLA. In some embodiments, the anti-BTLA antibody is a full-length antibody. In some embodiments, the full-length anti-BTLA antibody is an IgG1 or IgG4 antibody. In some embodiments, the disease or disorder is selected from, for example, non-small cell lung cancer, adrenal cancer, bladder cancer, brain cancer, pancreatic cancer, breast cancer, colorectal cancer, melanoma, esophageal cancer, gastric cancer, cervical cancer, head and neck cancer, hepatocellular carcinoma, kidney cancer, liver cancer, ovarian cancer, pancreatic cancer, prostate cancer, small cell lung cancer, testicular cancer, thyroid cancer, uterine cancer, and any type of leukemia, lymphoma, and myeloma, and infectious diseases, including but not limited to Human Papilloma Virus (HPV), Human Immunodeficiency Virus (HIV), Herpes Simplex Virus (HSV), Varicella Zoster Virus (VSV), Cytomegalovirus (CMV), Epstein Barr Virus (EBV), chlamydia, rickettsial bacteria, mycobacteria, staphylococcus, streptococcus, pneumococcus, meningococcus, and gonococcus (conococci), klebsiella, proteus, serratia, pseudomonas, legionella, diphtheria, salmonella, bacillus, cholera, tetanus, botulism, anthrax, plague, leptospirosis, and Lymes disease bacteria. In some embodiments, the individual is human.

[0341] For example, in some embodiments, a method for treating an individual having a disease associated with the BTLA signaling pathway (e.g., cancer or an infectious disease) is provided, comprising administering to the individual an effective amount of a pharmaceutical composition comprising an anti-BTLA antibody (e.g., a full-length anti-BTLA antibody), > heavy chain variable domain (V H ) of which the V HComprising: heavy chain complementarity determining region (HC-CDR) 1, which comprises TFGMGVS (SEQ ID NO:1); HC-CDR2, which comprises HIYWDDDKRFNPSLKS (SEQ ID NO:4); and HC-CDR3, which comprises GNWDGETYFDY (SEQ ID NO:7); and a light chain variable domain (V L ), said V L Comprising: light chain complementarity determining region (LC-CDR) 1, which comprises KSTQSLLDSDGKTYLN (SEQ ID NO:10);

[0342] LC-CDR2, which comprises LVSKLDS (SEQ ID NO:13); and LC-CDR3, which comprises WQGTHFPWT (SEQ ID NO:15). In some embodiments, the anti-BTLA antibody is a full-length antibody. In some embodiments, the full-length anti-BTLA antibody is an IgG1 or IgG4 antibody. In some embodiments, the disease or disorder is selected from, for example, non-small cell lung cancer, adrenal cancer, bladder cancer, brain cancer, pancreatic cancer, breast cancer, colorectal cancer, melanoma, esophageal cancer, gastric cancer, cervical cancer, head and neck cancer, hepatocellular carcinoma, kidney cancer, liver cancer, ovarian cancer, pancreatic cancer, prostate cancer, small cell lung cancer, testicular cancer, thyroid cancer, uterine cancer, and any type of leukemia, lymphoma, and myeloma, and infectious diseases, including but not limited to Human Papilloma Virus (HPV), Human Immunodeficiency Virus (HIV), Herpes Simplex Virus (HSV), Varicella Zoster Virus (VSV), Cytomegalovirus (CMV), Epstein Barr Virus (EBV), chlamydia, rickettsial bacteria, mycobacteria, staphylococcus, streptococcus, pneumococcus, meningococcus, and gonococcus (conococci), klebsiella, proteus, serratia, pseudomonas, legionella, diphtheria, salmonella, bacillus, cholera, tetanus, botulinum, anthrax, plague, leptospirosis, and Lyme disease bacteria. In some embodiments, the individual is human.

[0343] In some embodiments, there is provided a method for treating an individual having a disease associated with the BTLA signaling pathway (e.g., cancer or infectious disease), comprising administering to the individual an effective amount of a composition comprising an anti-BTLA antibody, wherein the antibody comprises VH , said V H comprises: HC-CDR1, which comprises the amino acid sequence shown in SEQ ID NO:1, HC-CDR2, which comprises the amino acid sequence shown in SEQ ID NO:4, and HC-CDR3, which comprises the amino acid sequence shown in SEQ ID NO:7, or a variant of said V H with up to about 5 amino acid substitutions in its HC-CDRs; and V L , said V L comprises: LC-CDR1, which comprises the amino acid sequence shown in SEQ ID NO:10, LC-CDR2, which comprises the amino acid sequence shown in SEQ ID NO:13, and LC-CDR3, which comprises the amino acid sequence shown in SEQ ID NO:15, or a variant of said V L with up to about 5 amino acid substitutions in its LC-CDRs.

[0344] In some embodiments, provided is a method for treating an individual suffering from a disease associated with the BTLA signaling pathway (e.g., cancer or an infectious disease), comprising administering to the individual an effective amount of a composition comprising an anti-BTLA antibody, wherein the antibody comprises: V H , said V H comprises an amino acid sequence shown in any of SEQ ID NOs: 18-22 or a variant thereof, said variant having at least about 80% sequence identity with an amino acid sequence shown in any of SEQ ID NOs: 18-22; and V L , said V L comprises an amino acid sequence shown in any of SEQ ID NOs: 25-29 or a variant thereof, said variant having at least about 80% sequence identity with an amino acid sequence shown in any of SEQ ID NOs: 25-29.

[0345] In some embodiments, the anti-BTLA antibody described herein is a full-length anti-BTLA antibody comprising an IgG1 or IgG4 constant region. In some embodiments, the IgG1 is human IgG1. In some embodiments, the IgG4 is human IgG4. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:32. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:33. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:34. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:35.

[0346] In some embodiments, provided is a method for treating an individual having a disease associated with the BTLA signaling pathway (e.g., cancer or an infectious disease), comprising administering to the individual an effective amount of a composition comprising an anti-BTLA antibody, wherein the antibody comprises: V H wherein the V H comprises: HC-CDR1, which comprises the amino acid sequence SEQ ID NO:1, HC-CDR2, which comprises the amino acid sequence SEQ ID NO:4, and HC-CDR3, which comprises the amino acid sequence SEQ ID NO:7, or a variant of the V H having up to about 5 amino acid substitutions in its HC-CDRs; and V L wherein the V L comprises: LC-CDR1, which comprises the amino acid sequence SEQ ID NO:10, LC-CDR2, which comprises the amino acid sequence SEQ ID NO:13, and LC-CDR3, which comprises the amino acid sequence SEQ ID NO:15, or a variant of the V L having up to about 5 amino acid substitutions in its LC-CDRs.

[0347] In some embodiments, the anti-BTLA antibody described herein comprises: V H wherein the V H comprises the amino acid sequence SEQ ID NO:18 or a variant thereof having at least about 80% sequence identity to the amino acid sequence SEQ ID NO:18; and V L wherein the V L comprises the amino acid sequence SEQ ID NO:25 or a variant thereof having at least about 80% sequence identity to the amino acid sequence SEQ ID NO:25. In some embodiments, the anti-BTLA antibody described herein is a full-length anti-BTLA antibody comprising an IgG1 or IgG4 constant region. In some embodiments, the IgG1 is human IgG1. In some embodiments, the IgG4 is human IgG4. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO:32. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO:33. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:34. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:35.

[0348] In some embodiments, the anti-BTLA antibody described herein comprises: V H wherein the V Hcomprising the amino acid sequence SEQ ID NO: 19 or a variant thereof, said variant having at least about 80% sequence identity with the amino acid sequence SEQ ID NO: 19; and V L , said V L comprising the amino acid sequence SEQ ID NO: 26 or a variant thereof, said variant having at least about 80% sequence identity with the amino acid sequence SEQ ID NO: 26. In some embodiments, the anti-BTLA antibodies described herein are full-length anti-BTLA antibodies comprising an IgG1 or IgG4 constant region. In some embodiments, the IgG1 is human IgG1. In some embodiments, the IgG4 is human IgG4. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO: 32. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO: 33. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO: 34. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO: 35.

[0349] In some embodiments, the anti-BTLA antibodies described herein comprise: V H , said V H comprising the amino acid sequence SEQ ID NO: 19 or a variant thereof, said variant having at least about 80% sequence identity with the amino acid sequence SEQ ID NO: 19; and V L , said V L comprising the amino acid sequence SEQ ID NO: 27 or a variant thereof, said variant having at least about 80% sequence identity with the amino acid sequence SEQ ID NO: 27. In some embodiments, the anti-BTLA antibodies described herein are full-length anti-BTLA antibodies comprising an IgG1 or IgG4 constant region. In some embodiments, the IgG1 is human IgG1. In some embodiments, the IgG4 is human IgG4. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO: 32. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO: 33. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO: 34. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO: 35.

[0350] In some embodiments, the anti-BTLA antibodies described herein comprise: V H , said V H comprising the amino acid sequence SEQ ID NO: 19 or a variant thereof, said variant having at least about 80% sequence identity with the amino acid sequence SEQ ID NO: 19; and VL wherein the V L comprises the amino acid sequence SEQ ID NO:28 or a variant thereof, said variant having at least about 80% sequence identity with the amino acid sequence SEQ ID NO:28. In some embodiments, the anti-BTLA antibody described herein is a full-length anti-BTLA antibody comprising an IgG1 or IgG4 constant region. In some embodiments, the IgG1 is human IgG1. In some embodiments, the IgG4 is human IgG4. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO:32. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO:33. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:34. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:35.

[0351] In some embodiments, the anti-BTLA antibody described herein comprises: V H wherein the V H comprises the amino acid sequence SEQ IDNO:19 or a variant thereof, said variant having at least about 80% sequence identity with the amino acid sequence SEQ ID NO:19; and V L wherein the V L comprises the amino acid sequence SEQ ID NO:29 or a variant thereof, said variant having at least about 80% sequence identity with the amino acid sequence SEQ ID NO:29. In some embodiments, the anti-BTLA antibody described herein is a full-length anti-BTLA antibody comprising an IgG1 or IgG4 constant region. In some embodiments, the IgG1 is human IgG1. In some embodiments, the IgG4 is human IgG4. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO:32. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO:33. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:34. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:35.

[0352] In some embodiments, the anti-BTLA antibody described herein comprises: V H wherein the V H comprises the amino acid sequence SEQ IDNO:20 or a variant thereof, said variant having at least about 80% sequence identity with the amino acid sequence SEQ ID NO:20; and V L wherein the V LComprising the amino acid sequence SEQ ID NO:26 or a variant thereof, said variant having at least about 80% sequence identity with the amino acid sequence SEQ ID NO:26. In some embodiments, the anti-BTLA antibodies described herein are full-length anti-BTLA antibodies comprising an IgG1 or IgG4 constant region. In some embodiments, the IgG1 is human IgG1. In some embodiments, the IgG4 is human IgG4. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO:32. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO:33. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:34. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:35.

[0353] In some embodiments, the anti-BTLA antibodies described herein comprise: V H , said V H comprising the amino acid sequence SEQ ID NO:20 or a variant thereof, said variant having at least about 80% sequence identity with the amino acid sequence SEQ ID NO:20; and V L , said V L comprising the amino acid sequence SEQ ID NO:27 or a variant thereof, said variant having at least about 80% sequence identity with the amino acid sequence SEQ ID NO:27. In some embodiments, the anti-BTLA antibodies described herein are full-length anti-BTLA antibodies comprising an IgG1 or IgG4 constant region. In some embodiments, the IgG1 is human IgG1. In some embodiments, the IgG4 is human IgG4. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO:32. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO:33. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:34. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:35.

[0354] In some embodiments, the anti-BTLA antibodies described herein comprise: V H , said V H comprising the amino acid sequence SEQ ID NO:20 or a variant thereof, said variant having at least about 80% sequence identity with the amino acid sequence SEQ ID NO:20; and V L , said V LComprising the amino acid sequence SEQ ID NO:28 or a variant thereof, said variant having at least about 80% sequence identity with the amino acid sequence SEQ ID NO:28. In some embodiments, the anti-BTLA antibody described herein is a full-length anti-BTLA antibody comprising an IgG1 or IgG4 constant region. In some embodiments, the IgG1 is human IgG1. In some embodiments, the IgG4 is human IgG4. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO:32. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO:33. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:34. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:35.

[0355] In some embodiments, the anti-BTLA antibody described herein comprises: V H , said V H comprising the amino acid sequence SEQ ID NO:20 or a variant thereof, said variant having at least about 80% sequence identity with the amino acid sequence SEQ ID NO:20; and V L , said V L comprising the amino acid sequence SEQ ID NO:29 or a variant thereof, said variant having at least about 80% sequence identity with the amino acid sequence SEQ ID NO:29. In some embodiments, the anti-BTLA antibody described herein is a full-length anti-BTLA antibody comprising an IgG1 or IgG4 constant region. In some embodiments, the IgG1 is human IgG1. In some embodiments, the IgG4 is human IgG4. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO:32. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO:33. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:34. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:35.

[0356] In some embodiments, the anti-BTLA antibody described herein comprises: V H , said V H comprising the amino acid sequence SEQ ID NO:21 or a variant thereof, said variant having at least about 80% sequence identity with the amino acid sequence SEQ ID NO:21; and V L , said V LComprising the amino acid sequence SEQ ID NO:28 or a variant thereof, said variant having at least about 80% sequence identity with the amino acid sequence SEQ ID NO:28. In some embodiments, the anti-BTLA antibody described herein is a full-length anti-BTLA antibody comprising an IgG1 or IgG4 constant region. In some embodiments, the IgG1 is human IgG1. In some embodiments, the IgG4 is human IgG4. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO:32. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO:33. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:34. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:35.

[0357] In some embodiments, the anti-BTLA antibody described herein comprises: V H , said V H comprising the amino acid sequence SEQ ID NO:21 or a variant thereof, said variant having at least about 80% sequence identity with the amino acid sequence SEQ ID NO:21; and V L , said V L comprising the amino acid sequence SEQ ID NO:29 or a variant thereof, said variant having at least about 80% sequence identity with the amino acid sequence SEQ ID NO:29. In some embodiments, the anti-BTLA antibody described herein is a full-length anti-BTLA antibody comprising an IgG1 or IgG4 constant region. In some embodiments, the IgG1 is human IgG1. In some embodiments, the IgG4 is human IgG4. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO:32. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO:33. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:34. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:35.

[0358] In some embodiments, the anti-BTLA antibody described herein comprises: V H , said V H comprising the amino acid sequence SEQ ID NO:22 or a variant thereof, said variant having at least about 80% sequence identity with the amino acid sequence SEQ ID NO:22; and V L , said V LComprising the amino acid sequence SEQ ID NO:28 or a variant thereof, said variant having at least about 80% sequence identity with the amino acid sequence SEQ ID NO:28. In some embodiments, the anti-BTLA antibody described herein is a full-length anti-BTLA antibody comprising an IgG1 or IgG4 constant region. In some embodiments, the IgG1 is human IgG1. In some embodiments, the IgG4 is human IgG4. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO:32. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO:33. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:34. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:35.

[0359] In some embodiments, the anti-BTLA antibody described herein comprises: V H , said V H Comprising the amino acid sequence SEQ IDNO:22 or a variant thereof, said variant having at least about 80% sequence identity with the amino acid sequence SEQ ID NO:22; and V L , said V L Comprising the amino acid sequence SEQ ID NO:29 or a variant thereof, said variant having at least about 80% sequence identity with the amino acid sequence SEQ ID NO:29. In some embodiments, the anti-BTLA antibody described herein is a full-length anti-BTLA antibody comprising an IgG1 or IgG4 constant region. In some embodiments, the IgG1 is human IgG1. In some embodiments, the IgG4 is human IgG4. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO:32. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO:33. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:34. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:35.

[0360] For example, in some embodiments, provided is a method for treating an individual suffering from a disease associated with the BTLA signaling pathway (e.g., cancer or an infectious disease), comprising administering to the individual an effective amount of a pharmaceutical composition comprising an anti-BTLA antibody (e.g., a full-length anti-BTLA antibody), a heavy chain variable domain (V H ), said V HComprising: HC-CDR1, which comprises the amino acid sequence SEQ ID NO:2, HC-CDR2, which comprises the amino acid sequence SEQ ID NO:5, and HC-CDR3, which comprises the amino acid sequence SEQ ID NO:8, or a variant of said V H in which there are at most about 5 amino acid substitutions in its HC-CDRs; and a light chain variable domain (V L ), said V L Comprising: LC-CDR1, which comprises the amino acid sequence SEQ ID NO:11, LC-CDR2, which comprises the amino acid sequence SEQ ID NO:14, and LC-CDR3, which comprises the amino acid sequence SEQ ID NO:16, or a variant of said V L in which there are at most about 5 amino acid substitutions in its LC-CDRs. In some embodiments, the anti-BTLA antibody is a full-length antibody. In some embodiments, the full-length anti-BTLA antibody is an IgG1 or IgG4 antibody. In some embodiments, the disease or disorder is selected from, for example, non-small cell lung cancer, adrenal cancer, bladder cancer, brain cancer, pancreatic cancer, breast cancer, colorectal cancer, melanoma, esophageal cancer, gastric cancer, cervical cancer, head and neck cancer, hepatocellular carcinoma, kidney cancer, liver cancer, ovarian cancer, pancreatic cancer, prostate cancer, small cell lung cancer, testicular cancer, thyroid cancer, uterine cancer, and any type of leukemia, lymphoma, and myeloma, and infectious diseases, including but not limited to Human Papilloma Virus (HPV), Human Immunodeficiency Virus (HIV), Herpes Simplex Virus (HSV), Varicella Zoster Virus (VSV), Cytomegalovirus (CMV), Epstein Barr Virus (EBV), Chlamydia, Rickettsia bacteria, Mycobacteria, Staphylococcus, Streptococcus, Pneumococcus, Meningococcus, and Gonococci, Klebsiella, Proteus, Serratia, Pseudomonas, Legionella, Diphtheria, Salmonella, Bacillus, Cholera, Tetanus, Botulinum, Anthrax, Plague, Leptospirosis, and Lymes disease bacteria. In some embodiments, the individual is human.

[0361] In some embodiments, there is provided a method for treating an individual having a disease (e.g., cancer or infectious disease) associated with the BTLA signaling pathway, comprising administering to the individual an effective amount of a composition comprising an anti-BTLA antibody, wherein the antibody comprises: VH , said V H comprises the amino acid sequence shown in SEQ ID NO:23 or a variant thereof, said variant having at least about 80% sequence identity with the amino acid sequence shown in SEQ ID NO:23; and V L , said V L comprises the amino acid sequence shown in SEQ ID NO:30 or a variant thereof, said variant having at least about 80% sequence identity with the amino acid sequence shown in SEQ ID NO:30.

[0362] In some embodiments, the anti-BTLA antibodies described herein are full-length anti-BTLA antibodies comprising an IgG1 or IgG4 constant region. In some embodiments, the IgG1 is human IgG1. In some embodiments, the IgG4 is human IgG4. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO:32. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO:33. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:34. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:35.

[0363] For example, in some embodiments, provided is a method for treating an individual suffering from a disease associated with the BTLA signaling pathway (e.g., cancer or an infectious disease), comprising administering to the individual an effective amount of a pharmaceutical composition comprising an anti-BTLA antibody (e.g., a full-length anti-BTLA antibody), a heavy chain variable domain (V H ), said V H comprising: HC-CDR1, which comprises the amino acid sequence SEQ ID NO:3, HC-CDR2, which comprises the amino acid sequence SEQ ID NO:6, and HC-CDR3, which comprises the amino acid sequence SEQ ID NO:9, or a variant of said V H having at most about 5 amino acid substitutions in its HC-CDRs; and a light chain variable domain (V L ), said V L comprising: LC-CDR1, which comprises the amino acid sequence SEQ ID NO:12, LC-CDR2, which comprises the amino acid sequence SEQ ID NO:13, and LC-CDR3, which comprises the amino acid sequence SEQ ID NO:17, or said V LVariants thereof with substitutions of up to about 5 amino acids in the LC-CDRs. In some embodiments, the anti-BTLA antibody is a full-length antibody. In some embodiments, the full-length anti-BTLA antibody is an IgG1 or IgG4 antibody. In some embodiments, the disease or disorder is selected from, for example, non-small cell lung cancer, adrenal cancer, bladder cancer, brain cancer, pancreatic cancer, breast cancer, colorectal cancer, melanoma, esophageal cancer, gastric cancer, cervical cancer, head and neck cancer, hepatocellular carcinoma, kidney cancer, liver cancer, ovarian cancer, pancreatic cancer, prostate cancer, small cell lung cancer, testicular cancer, thyroid cancer, uterine cancer, and any type of leukemia, lymphoma, and myeloma, and infectious diseases including but not limited to Human Papilloma Virus (HPV), Human Immunodeficiency Virus (HIV), Herpes Simplex Virus (HSV), Varicella Zoster Virus (VSV), Cytomegalovirus (CMV), Epstein Barr Virus (EBV), Chlamydia, Rickettsia bacteria, Mycobacteria, Staphylococcus, Streptococcus, Pneumococcus, Neisseria meningitidis, and Neisseria gonorrhoeae, Klebsiella, Proteus, Serratia, Pseudomonas, Legionella, Diphtheria, Salmonella, Bacillus, Cholera, Tetanus, Clostridium botulinum, Anthrax, Plague, Leptospirosis, and Lyme disease bacteria. In some embodiments, the individual is human.

[0364] In some embodiments, there is provided a method of treating an individual having a disease associated with the BTLA signaling pathway (e.g., cancer or an infectious disease), comprising administering to the individual an effective amount of a composition comprising an anti-BTLA antibody, wherein the antibody comprises: V H , said V H comprises the amino acid sequence shown in SEQ ID NO:24 or a variant thereof that has at least about 80% sequence identity to the amino acid sequence shown in SEQ ID NO:24; and V L , said V L comprises the amino acid sequence shown in SEQ ID NO:31 or a variant thereof that has at least about 80% sequence identity to the amino acid sequence shown in SEQ ID NO:31.

[0365] In some embodiments, the anti-BTLA antibodies described herein are full-length anti-BTLA antibodies comprising an IgG1 or IgG4 constant region. In some embodiments, the IgG1 is human IgG1. In some embodiments, the IgG4 is human IgG4. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO:32. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence SEQ ID NO:33. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:34. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:35.

[0366] In some embodiments, the individual is a mammal (such as a human, non-human primate, rat, mouse, cow, horse, pig, sheep, goat, dog, cat, etc.). In some embodiments, the individual is a human. In some embodiments, the individual is a clinical patient, a clinical trial volunteer, an experimental animal, etc. In some embodiments, the individual is less than 60 years old (including, for example, less than 50, 40, 30, 25, 20, 15, or 10 years old). In some embodiments, the individual is greater than 60 years old (including, for example, greater than 70, 80, 90, or 100 years old). In some embodiments, the individual is diagnosed with or genetically predisposed to one or more of the diseases or disorders described herein (such as cancer or infectious diseases). In some embodiments, the individual has one or more risk factors associated with one or more of the diseases or disorders described herein.

[0367] In some embodiments, the present application provides a method of delivering an anti-BTLA antibody (such as any of the anti-BTLA antibodies described herein, such as an isolated anti-BTLA antibody) to cells expressing BTLA on their surface in an individual, the method comprising administering to the individual a composition comprising the anti-BTLA antibody.

[0368] Many diagnostic methods for cancer or infectious diseases or any other diseases showing abnormal BTLA expression and the clinical descriptions of these diseases are known in the art. Such methods include, but are not limited to, for example, immunohistochemistry, PCR, and fluorescence in situ hybridization (FISH).

[0369] In some embodiments, the anti-BTLA antibodies (such as full-length anti-BTLA antibodies) and / or compositions of the present application are used in combination with a second, third, or fourth agent (including, for example, immunogenic drugs such as purified tumor antigens; standard cancer treatments such as chemotherapy regimens; antibodies that can be used to activate the host immune response; cytokine therapy (e.g., interferon, GM-CSF, G-CSF, IL-2); or bispecific antibody therapy that provides enhanced tumor antigen presentation, or combinations thereof) to treat diseases associated with the BTLA signaling pathway.

[0370] In some embodiments, cancer treatment can be evaluated by, for example, tumor regression, shrinkage of tumor weight or size, time to progression, survival time, progression-free survival, overall response rate, response duration, quality of life, protein expression, and / or activity. Methods for determining treatment efficacy can be employed, including, for example, measuring the response by radiological imaging.

[0371] In some embodiments, the treatment effect is measured as the percentage of tumor growth inhibition (%TGI), calculated using the equation 100 - (T / C × 100), where T is the average relative tumor volume of the treated tumor and C is the average relative tumor volume of the untreated tumor. In some embodiments, the %TGI is about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, or greater than 95%. In some embodiments, the treatment effect is measured by a change in granulocyte shape and / or an increase in granulocyte survival rate. In some embodiments, the treatment effect is measured by an increase in cytokines secreted by monocytes.

[0372] Dosage and method of administering anti - BTLA antibody

[0373] The dosage of the anti - BTLA antibody composition (e.g., an isolated anti - BTLA antibody) administered to an individual (e.g., a human) may vary depending on the specific composition, mode of administration, and type of disease being treated. In some embodiments, the amount of the composition (e.g., a composition comprising an anti - BTLA antibody) is effective in producing an objective response (e.g., a partial response or a complete response) in the treatment of cancer or an infectious disease. In some embodiments, the amount of the anti - BTLA antibody composition is sufficient to produce a complete response in an individual. In some embodiments, the amount of the anti - BTLA antibody composition is sufficient to produce a partial response in an individual. In some embodiments, the dosage of the anti - BTLA antibody composition (e.g., when administered alone) is sufficient to produce a total response rate higher than 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 64%, 65%, 70%, 75%, 80%, 85%, or 90% in a population of individuals treated with the anti - BTLA antibody composition. The response of an individual to the treatment methods described herein can be determined, for example, by an ACR score.

[0374] In some embodiments, the amount of the composition (e.g., a composition comprising an isolated anti-BTLA antibody) is sufficient to prolong the progression-free survival of an individual. In some embodiments, the amount of the composition is sufficient to prolong the overall survival of an individual. In some embodiments, in a population of individuals treated with an anti-BTLA antibody composition, the amount of the composition (e.g., when administered alone) is sufficient to produce a clinical benefit of greater than 50%, 60%, 70%, or 77%.

[0375] In some embodiments, the amount of the composition (e.g., a composition comprising an isolated anti-BTLA antibody), when used alone or in combination with a second, third, and / or fourth agent, is an amount sufficient to control symptoms and reduce the risk of disease exacerbation compared to the corresponding activity of other untreated subjects before treatment. Standard methods can be employed to measure the magnitude of this efficacy, such as in vitro assays of purified enzymes, cell-based assays, animal models, or human trials.

[0376] In some embodiments, when the composition is administered to an individual, the amount of the anti-BTLA antibody (e.g., a full-length anti-BTLA antibody) in the composition is below the level that causes a toxic effect (i.e., an effect above the clinically acceptable toxicity level), or at a level where potential side effects can be controlled or tolerated.

[0377] In some embodiments, following the same dosing regimen, the amount of the composition approaches the maximum tolerated dose (MTD) of the composition. In some embodiments, the amount of the composition is greater than 80%, 90%, 95%, or 98% of the MTD.

[0378] In some embodiments, the content of the anti-BTLA antibody (e.g., a full-length anti-BTLA antibody) in the composition ranges from 0.001 μg to 1000 μg.

[0379] In any of the embodiments described above, the effective amount of the anti-BTLA antibody (e.g., a full-length anti-BTLA antibody) in the composition, calculated per body weight, ranges from 0.1 μg / kg to 100 mg / kg.

[0380] An anti-BTLA antibody composition can be administered to an individual (such as a human) by various routes, including, for example, intravenous injection, intraarterial administration, intraperitoneal injection, intralung administration, oral administration, inhalation administration, intravascular administration, intramuscular injection, intratracheal administration, subcutaneous injection, intraocular administration, intrathecal administration, mucosal administration, or transdermal administration. In some embodiments, a sustained-release formulation of the composition is used. In some embodiments, the composition is administered intravenously. In some embodiments, the composition is administered by artery. In some embodiments, the composition is administered intraperitoneally. In some embodiments, the composition is administered intrahepatically. In some embodiments, the composition is administered by hepatic artery infusion. In some embodiments, the composition is administered to a site distant from the first lesion.

[0381] Articles and kits

[0382] In some embodiments of the present application, an article is provided, which comprises a substance capable of being used for treating a disease related to the BTLA signaling pathway (such as cancer or an infectious disease), or for delivering an anti-BTLA antibody (such as a full-length anti-BTLA antibody) to cells expressing BTLA on the surface. The article may include a container and a label or package insert on or attached to the container. Suitable containers include, for example, bottles, vials, syringes, etc. The container can be made of various materials, such as glass or plastic. Generally, the container contains a composition capable of effectively treating the diseases or disorders described herein and has a sterile port (for example, the container can be an intravenous infusion bag or a vial with a lid pierceable by a subcutaneous injection needle). At least one active substance in the composition is the anti-BTLA antibody described in the present application. The label or package insert indicates the specific diseases that the composition can be used to treat. The label or package insert further contains instructions for administering the anti-BTLA antibody composition to a patient. Articles and kits including combination therapies are within the scope of consideration herein.

[0383] A package insert refers to the instructions usually included in the commercial packaging of a therapeutic product, which contain information on indications, usage, dosage, administration, contraindications, and / or warnings related to the use of these therapeutic products. In some embodiments, the package insert indicates that the composition can be used to treat diseases related to the BTLA signaling pathway (such as cancer or infectious diseases). In some embodiments, the package insert indicates that the composition can be used to treat the following diseases, including non-small cell lung cancer, adrenal cancer, bladder cancer, brain cancer, pancreatic cancer, breast cancer, colorectal cancer, melanoma, esophageal cancer, gastric cancer, cervical cancer, head and neck cancer, hepatocellular carcinoma, kidney cancer, liver cancer, ovarian cancer, pancreatic cancer, prostate cancer, small cell lung cancer, testicular cancer, thyroid cancer, uterine cancer, and any type of leukemia, lymphoma, and myeloma, as well as infectious diseases, including but not limited to Human Papilloma Virus (HPV), Human Immunodeficiency Virus (HIV), Herpes Simplex Virus (HSV), Varicella Zoster Virus (VSV), Cytomegalovirus (CMV), Epstein Barr Virus (EBV), Chlamydia, Rickettsia bacteria, Mycobacterium, Staphylococcus, Streptococcus, Pneumococcus, Neisseria meningitidis, and Neisseria gonorrhoeae, Klebsiella, Proteus, Serratia, Pseudomonas, Legionella, Diphtheria, Salmonella, Bacillus, Cholera, Tetanus, Clostridium botulinum, Anthrax, Plague, Leptospirosis, and Lyme disease bacteria.

[0384] In addition, the article may further include a second container containing a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate buffer, Ringer's solution, or glucose solution. Other materials required from a commercial and user perspective may also be included, including other buffers, diluents, filters, needles, and syringes.

[0385] Also provided are kits that can be used for a variety of purposes, such as treating diseases associated with the BTLA signaling pathway (e.g., cancer or infectious diseases), or delivering an anti-BTLA antibody (e.g., a full-length anti-BTLA antibody) to cells expressing BTLA on their surface, optionally in combination with a product. The kits of the present application include one or more containers that contain an anti-BTLA antibody composition (or single-dose form and / or product), and in some embodiments, further include another agent (e.g., an agent described herein) and / or instructions for use consistent with any of the methods described herein. The kit may further include a description for selecting a suitable treatment for an individual. The instructions for use accompanying the kit in the present application are typically written instructions on a label or package insert (e.g., a sheet of paper included in the kit), and machine-readable instructions (e.g., instructions on a magnetic or optical storage disc) are also acceptable.

[0386] For example, in some embodiments, the kit includes a composition comprising an anti-BTLA antibody (e.g., a full-length anti-BTLA antibody). In some embodiments, the kit includes: a) a composition comprising any one of the anti-BTLA antibodies described herein, and b) at least one effective amount of another agent that can enhance the effect of the anti-BTLA antibody (such as a therapeutic effect, a detection effect). In some embodiments, the kit includes: a) a composition comprising any one of the anti-BTLA antibodies described herein, and b) instructions for use for administering the anti-BTLA antibody composition to an individual for treating a disease associated with the BTLA signaling pathway (e.g., cancer or infectious diseases). In some embodiments, the kit includes: a) a composition comprising any one of the anti-BTLA antibodies described herein, and b) at least one effective amount of another agent that can enhance the effect of the anti-BTLA antibody (such as a therapeutic effect, a detection effect), and c) instructions for use for administering the anti-BTLA antibody composition and other substances to an individual for treating a disease associated with the BTLA signaling pathway (e.g., cancer or infectious diseases). The anti-BTLA antibody and other substances may be present in separate containers or the same container. For example, the kit may include a specific composition or two or more compositions, wherein one composition includes the anti-BTLA antibody and another composition includes another agent.

[0387] In some embodiments, the kit comprises a nucleic acid (or a group of nucleic acids) encoding an anti-BTLA antibody (e.g., a full-length anti-BTLA antibody). In some embodiments, the kit comprises: a) a nucleic acid (or a group of nucleic acids) encoding an anti-BTLA antibody (e.g., a full-length anti-BTLA antibody), and b) a host cell for expressing the nucleic acid (or the group of nucleic acids). In some embodiments, the kit comprises: a) a nucleic acid (or a group of nucleic acids) encoding an anti-BTLA antibody (e.g., a full-length anti-BTLA antibody), and b) instructions for use, applicable to: i) expressing the anti-BTLA antibody in a host cell, ii) preparing a composition comprising the anti-BTLA antibody, and iii) administering to an individual a composition comprising the anti-BTLA antibody for treating a disease associated with the BTLA signaling pathway (e.g., cancer or an infectious disease). In some embodiments, the kit includes: a) a nucleic acid (or a group of nucleic acids) encoding an anti-BTLA antibody (e.g., a full-length anti-BTLA antibody), b) a host cell for expressing the nucleic acid (or the group of nucleic acids), and c) instructions for use, applicable to: i) expressing the anti-BTLA antibody in a host cell, ii) preparing a composition comprising the anti-BTLA antibody, and iii) administering to an individual a composition comprising the anti-BTLA antibody for treating a disease associated with the BTLA signaling pathway (e.g., cancer or an infectious disease).

[0388] The kit described in the present application is packaged in a suitable form. Suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packages (such as sealed polyester films or plastic bags), etc. The kit may optionally provide other components, such as buffers and instruction information. Accordingly, the present application also provides an article, which includes vials, bottles, jars, flexible packages (such as sealed polyester films or plastic bags), etc.

[0389] The instructions for use regarding the anti-BTLA antibody composition generally include information such as dosage, dosing cycle, and route of administration, etc. The container can be unit-dose, large-packaged (e.g., multi-dose packaging), or sub-unit-dose. For example, a kit containing a sufficient dose of an anti-BTLA antibody as described herein (e.g., a full-length anti-BTLA antibody) is provided for long-term effective treatment of an individual, such as for one week, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 2 weeks, 3 weeks, 4 weeks, 6 weeks, 8 weeks, 3 months, 4 months, 5 months, 7 months, 8 months, 9 months, or longer. The kit may also contain multiple unit-doses of the anti-BTLA antibody, pharmaceutical compositions, and instructions for use, and is packaged in an amount sufficient for storage and use in a pharmacy, e.g., a hospital pharmacy and a compounding pharmacy.

[0390] Those skilled in the art will recognize several possible embodiments within the scope and spirit of the present application. The present application will now be described in more detail by reference to the following non-limiting examples. The following examples further illustrate the present application, but should not be construed as limiting its scope in any way. Detailed Description of Specific Embodiments

[0391] The following typical examples illustrate various features and embodiments of the present invention, which are intended to illustrate rather than limit. Those skilled in the art can easily understand that the specific examples are only illustrative examples of the present invention and are more fully described in the following claims. Each embodiment and feature described in the present application should be understood as interchangeable and combinable with each embodiment included in the application.

[0392] Example 1: Preparation of BTLA Polypeptides

[0393] This example describes the preparation of various BTLA polypeptide constructs, which are used as antigens to induce the production and screening of anti-BTLA antibodies of the present disclosure.

[0394] The coding sequences of the extracellular domains (ECDs) of human BTLA (huBTLA) and mouse BTLA (musBTLA) were synthesized and subcloned into the expression vector pTTal using the In-Fusion seamless cloning kit (Takara, cat#639691) with the restriction endonucleases HindIII and EcoRI as recognition sites. The amino acid sequences of huBTLA(ECD) and musBTLA(ECD) are shown in Table 5. For purification and detection, all constructs have the following sequences: human IgG1 Fc at the C-terminus, mouse IgG2a Fc at the C-terminus, or a 10×His-tag sequence at the C-terminus. A total of six fusion proteins were expressed and purified.

[0395] Table 5 BTLA Polypeptide Sequences

[0396]

[0397] The fusion proteins were expressed in Expi293 cells (Thermo Fisher Scientific) according to the manufacturer's operating instructions. Briefly, Expi293 cells were transfected with the expression vector, and the above cells were cultured at 37 °C, 8% CO2 and 120 rpm for 5 days.

[0398] For the purification of the Fc fusion protein, after collection, the clarified supernatant medium was mixed with MabSelect protein A resin (GE Healthcare) equilibrated with PBS buffer and incubated with gentle rotation at room temperature for 1.5 h. After incubation, the suspension was loaded into a column, and the resin was washed with 20 column volumes of PBS buffer containing 0.15 M NaCl, and then eluted with 3 column volumes of 50 mM sodium phosphate (pH 3.0). The pH of the eluate was

[0399] quickly adjusted to pH 5.2 with 1 M Tris-HCl (pH 9.0), and the buffer was replaced with PBS buffer using a PD-10 column (GE Healthcare).

[0400] For the purification of the His-tag protein, after collection, the clarified supernatant medium was loaded into a Histrap column (GE

[0401] Healthcare) pre-equilibrated with 20 mM sodium phosphate buffer (pH 7.4) containing 0.25 M NaCl

[0402] and 5 mM imidazole (pH 8.0). The column was washed with 10 column volumes of 20 mM sodium phosphate buffer (pH 7.4) containing 0.25 M NaCl and 15 mM imidazole (pH 8.0), and then eluted with 3 column volumes of 20 mM sodium phosphate buffer (pH 7.4) containing 0.25 M NaCl and 100 mM

[0403] imidazole (pH 8.0). The elution buffer was replaced with PBS buffer using a PD-10 column.

[0404] Example 2: Preparation of anti-BTLA antibody using the hybridoma method and its screening and identification

[0405] This example describes a method for preparing anti-BTLA antibodies using mouse hybridoma technology, as well as methods for screening and selecting antibodies for further characterization.

[0406]

[0407] Immunization and fusion ​: Recombinant human BTLA ECD fused with His- or mouse IgG2a Fc tag produced in Expi293 cells was used to immunize Balb / c mice, and the adjuvants were RIBI (Sigma Aldrich, cat#S6322-1VL), Titermax (Sigma Aldrich, cat#T2684-1ML), Freund's (incomplete Freund's adjuv...

Claims

1. An isolated anti-BTLA antibody, comprising: Heavy chain variable domain (V H ), said V H comprising: Heavy chain complementarity determining region (HC-CDR) 1, comprising TFGMGVS (SEQ ID NO:1) or a variant thereof, said variant comprising up to about 3 amino acid substitutions; HC-CDR2, comprising HIYWDDDKRFNPSLKS (SEQ ID NO:4) or a variant thereof, said variant comprising up to about 3 amino acid substitutions; and HC-CDR3, comprising GNWDGETYFDY (SEQ ID NO:7) or a variant thereof, said variant comprising up to about 3 amino acid substitutions; and a variable light chain domain (V L ), wherein the V L comprises: Light chain complementarity determining region (LC-CDR) 1, comprising KSTQSLLDSDGKTYLN (SEQ ID NO:10) or a variant thereof, said variant comprising up to about 3 amino acid substitutions; LC-CDR2, comprising LVSKLDS (SEQ ID NO:13) or a variant thereof, said variant comprising up to about 3 amino acid substitutions; and LC-CDR3, comprising WQGTHFPWT (SEQ ID NO:15) or a variant thereof, said variant comprising up to about 3 amino acid substitutions.

2. The isolated anti-BTLA antibody according to claim 1, comprising: V H , which comprises an amino acid sequence shown in any of SEQ ID NOs: 18-22 or a variant thereof, said variant being identical to SEQ an amino acid sequence shown in any of ID NOs: 18 - 22 having at least about 80% sequence identity; and V L , which comprises an amino acid sequence shown in any of SEQ ID NOs: 25-29 or a variant thereof, said variant being identical to an amino acid sequence shown in any of SEQ ID NOs: 25 - 29 having at least about 80% sequence identity.

3. The isolated anti-BTLA antibody according to any one of claims 1 - 2, comprising: (i)V H which comprises the amino acid sequence SEQ ID NO:18 or a variant thereof, said variant being identical to the amino acid sequence SEQ ID NO:18 has at least about 80% sequence identity; and V L , which comprises the amino acid sequence SEQ ID NO:25 or a variant thereof, said variant having at least about 80% sequence identity with the amino acid sequence SEQ ID NO:25; (ii)V H , which comprises the amino acid sequence SEQ ID NO:19 or a variant thereof, said variant being related to the amino acid sequence SEQ ID NO:19 has at least about 80% sequence identity; and V L , which comprises an amino acid sequence SEQ ID NO:26 or a variant thereof, said variant having at least about 80% sequence identity with the amino acid sequence SEQ ID NO:26; (iii)V H which comprises the amino acid sequence SEQ ID NO:19 or a variant thereof, said variant having at least about 80% sequence identity with the amino acid sequence SEQ ID NO:19; and V L which comprises the amino acid sequence SEQ ID NO:27 or a variant thereof, said variant having at least about 80% sequence identity with the amino acid sequence SEQ ID NO:27; (iv)V H , which comprises the amino acid sequence SEQ ID NO: 19 or a variant thereof, said variant having at least about 80% sequence identity with the amino acid sequence SEQ ID NO: 19; and V L , which comprises the amino acid sequence SEQ ID NO: 28 or a variant thereof, said variant having at least about 80% sequence identity with the amino acid sequence SEQ ID NO: 28; (v)V H , which comprises the amino acid sequence SEQ ID NO: 19 or a variant thereof, said variant having at least about 80% sequence identity with the amino acid sequence SEQ ID NO: 19; and V L , which comprises the amino acid sequence SEQ ID NO: 29 or a variant thereof, said variant having at least about 80% sequence identity with the amino acid sequence SEQ ID NO: 29; (vi) V H which comprises the amino acid sequence SEQ ID NO:20 or a variant thereof, said variant having at least about 80% sequence identity with the amino acid sequence SEQ ID NO:20; and V L which comprises the amino acid sequence SEQ ID NO:26 or a variant thereof, said variant having at least about 80% sequence identity with the amino acid sequence SEQ ID NO:26; (vii)V H , which comprises the amino acid sequence SEQ ID NO: 20 or a variant thereof, said variant having at least about 80% sequence identity with the amino acid sequence SEQ ID NO: 20; and V L , which comprises the amino acid sequence SEQ ID NO: 27 or a variant thereof, said variant having at least about 80% sequence identity with the amino acid sequence SEQ ID NO: 27; (viii)V H , which comprises the amino acid sequence SEQ ID NO:20 or a variant thereof, said variant having at least about 80% sequence identity with the amino acid sequence SEQ ID NO:20; and V L , which comprises the amino acid sequence SEQ ID NO:28 or a variant thereof, said variant having at least about 80% sequence identity with the amino acid sequence SEQ ID NO:28; (ix)V H which comprises the amino acid sequence SEQ ID NO: 20 or a variant thereof, said variant having at least about 80% sequence identity with the amino acid sequence SEQ ID NO: 20; and V L which comprises the amino acid sequence SEQ ID NO: 29 or a variant thereof, said variant having at least about 80% sequence identity with the amino acid sequence SEQ ID NO: 29; (x)V H , which comprises the amino acid sequence SEQ ID NO: 21 or a variant thereof, said variant having at least about 80% sequence identity with the amino acid sequence SEQ ID NO: 21; and V L , which comprises the amino acid sequence SEQ ID NO: 28 or a variant thereof, said variant having at least about 80% sequence identity with the amino acid sequence SEQ ID NO: 28; (xi)V H which comprises the amino acid sequence SEQ ID NO: 21 or a variant thereof, said variant having at least about 80% sequence identity with the amino acid sequence SEQ ID NO: 21; and V L which comprises the amino acid sequence SEQ ID NO: 29 or a variant thereof, said variant having at least about 80% sequence identity with the amino acid sequence SEQ ID NO: 29; (xii)V H which comprises the amino acid sequence SEQ ID NO:22 or a variant thereof, said variant having at least about 80% sequence identity with the amino acid sequence SEQ ID NO:22; and V L which comprises the amino acid sequence SEQ ID NO:28 or a variant thereof, said variant having at least about 80% sequence identity with the amino acid sequence SEQ ID NO:28; or (xiii)V H which comprises the amino acid sequence SEQ ID NO: 22 or a variant thereof, said variant having at least about 80% sequence identity with the amino acid sequence SEQ ID NO: 22; and V L which comprises the amino acid sequence SEQ ID NO: 29 or a variant thereof, said variant having at least about 80% sequence identity with the amino acid sequence SEQ ID NO:

29.

4. The isolated anti-BTLA antibody according to any one of claims 1-3, wherein the anti-BTLA antibody comprises V H , and the V H comprises a V H comprising the HC- CDR1, HC-CDR2, and HC-CDR3; and V L , said V L comprises a V as shown by any one of the amino acid sequences of SEQ ID NOs: 25-29 L comprising LC-CDR1, LC-CDR2, and LC-CDR3.

5. An isolated anti-BTLA antibody, wherein said anti-BTLA antibody comprises: (i)V H wherein said V H comprises: HC-CDR1, which comprises the amino acid sequence SEQ ID NO:2, HC- CDR2, which comprises the amino acid sequence SEQ ID NO:5, and HC-CDR3, which comprises the amino acid sequence SEQ ID NO:8, or a variant of said V H which contains at most about 5 amino acid substitutions in its HC-CDRs; and V L , said V L comprising: LC-CDR1, which comprises the amino acid sequence SEQ ID NO:11, LC-CDR2, which comprises the amino acid sequence SEQ ID NO:14, and LC-CDR3, which comprises the amino acid sequence SEQ ID NO:16, or a variant of said V L which contains at most about 5 amino acid substitutions in its LC-CDRs; or (ii)V H , said V H comprises: HC-CDR1, which comprises the amino acid sequence SEQ ID NO:3, HC- CDR2, which comprises the amino acid sequence SEQ ID NO:6, and HC-CDR3, which comprises the amino acid sequence SEQ ID NO:9, or a variant of said V H that contains up to about 5 amino acid substitutions in its HC-CDRs; and V L wherein said V L comprises: LC-CDR1, which comprises the amino acid sequence SEQID NO:12, LC-CDR2, which comprises the amino acid sequence SEQ ID NO:13, and LC-CDR3 It comprises the amino acid sequence SEQ ID NO:17, or a variant of said V L with up to about 5 amino acid substitutions in its LC-CDRs.

6. The isolated anti-BTLA antibody according to claim 5, comprising: V H which comprises an amino acid sequence shown in any of SEQ ID NOs: 23-24 or a variant thereof, said variant having at least about 80% sequence identity with the amino acid sequence shown in any of SEQ ID NOs: 23-24; and V L , which comprises an amino acid sequence shown in any of SEQ ID NOs: 30-31 or a variant thereof, said variant having at least about 80% sequence identity with an amino acid sequence shown in any of SEQ ID NOs: 30-31.

7. The isolated anti-BTLA antibody according to any one of claims 5 - 6, comprising: (i)V H which comprises the amino acid sequence SEQ ID NO:23 or a variant thereof, said variant being identical to the amino acid sequence SEQ ID NO: 23 has at least about 80% sequence identity; and V L , which comprises an amino acid sequence SEQ ID NO:30 or a variant thereof, said variant having at least about 80% sequence identity with the amino acid sequence SEQ ID NO:30; or (ii)V H , which comprises the amino acid sequence SEQ ID NO:24 or a variant thereof, said variant being identical to the amino acid sequence SEQ ID NO:24 has at least about 80% sequence identity; and V L , which comprises an amino acid sequence SEQ ID NO:31 or a variant thereof, said variant having at least about 80% sequence identity with the amino acid sequence SEQ ID NO:

31.

8. The isolated anti-BTLA antibody according to any one of claims 5-7, wherein the anti-BTLA antibody comprises V H , said V H comprises V as shown in any one of the amino acid sequences of SEQ ID NOs: 23-24 H comprising HC- CDR1, HC-CDR2, and HC-CDR3; and V L , said V L comprises a V as shown by any of the amino acid sequences in SEQ ID NOs: 30-31 L comprising LC-CDR1, LC-CDR2, and LC-CDR3.

9. The isolated anti-BTLA antibody according to any one of claims 5 - 8, wherein said anti-BTLA antibody comprises: (i)V H which comprises a V as set forth in amino acid sequence SEQ ID NO:23 H comprising HC-CDR1, HC-CDR2 and HC-CDR3; and V L , which comprises as shown in amino acid sequence SEQ ID NO: 30 The V shown L LC-CDR1, LC-CDR2, and LC-CDR3 contained; or (ii)V H , which comprises a V as shown in amino acid sequence SEQ ID NO:24 H comprising HC-CDR1, HC-CDR2 and HC-CDR3; and V L , which comprises as shown in amino acid sequence SEQ ID NO:31 The V shown L including LC-CDR1, LC-CDR2, and LC-CDR3.

10. An isolated anti-BTLA antibody that competes with the isolated anti-BTLA antibody according to any one of claims 1 - 9 for specific binding to BTLA, or specifically binds to the same epitope as the isolated anti-BTLA antibody according to any one of claims 1 - 9.

11. The isolated anti-BTLA antibody according to any one of claims 1-10, wherein the anti-BTLA antibody has a K d value of from about 0.1 pM to about 1 nM for binding to BTLA.

12. The isolated anti-BTLA antibody according to claim 11, wherein the anti-BTLA antibody comprises an Fc fragment.

13. The isolated anti-BTLA antibody according to claim 12, wherein the anti-BTLA antibody is a full-length IgG antibody.

14. The isolated anti-BTLA antibody according to claim 13, wherein the anti-BTLA antibody is the isolated anti-BTLA antibody according to claim 12, wherein the anti-BTLA antibody is an IgG1, IgG2, IgG3 or IgG4 antibody.

15. The isolated anti-BTLA antibody according to any one of claims 1-14, wherein the anti-BTLA antibody is a chimeric, fully human or humanized antibody.

16. The isolated anti-BTLA antibody according to any one of claims 1-11, wherein the anti-BTLA antibody is an antigen-binding fragment, and the antigen-binding fragment is selected from the group consisting of Fab, Fab’, F(ab’)2, Fab’-SH, single-chain antibody (scFv), Fv fragment, dAb, Fd or diabody.

17. An isolated nucleic acid molecule encoding the isolated anti-BTLA antibody according to any one of claims 1-16.

18. A vector comprising the isolated nucleic acid molecule according to claim 17.

19. An isolated host cell comprising the isolated anti-BTLA antibody according to any one of claims 1-16, the isolated nucleic acid molecule according to claim 17 or the vector according to claim 18.

20. A method for preparing an isolated anti-BTLA antibody, comprising: a) culturing the host cell according to claim 19 under conditions effective for expressing the anti-BTLA antibody; and b) obtaining the expressed anti-BTLA antibody from the host cell.

21. A pharmaceutical composition comprising the anti-BTLA antibody according to any one of claims 1-16, the nucleic acid molecule according to claim 17, the vector according to claim 18, the isolated host cell according to claim 19, and a pharmaceutically acceptable carrier.

22. The pharmaceutical composition according to claim 21, wherein the pharmaceutical composition further comprises an antigen-binding protein that specifically recognizes PD-1.

23. A method for treating a disease or disorder in an individual in need thereof, comprising administering to the individual an effective amount of the anti-BTLA antibody according to any one of claims 1-16, the nucleic acid according to claim 17, the vector according to claim 18, the isolated host cell according to claim 19, the antibody prepared by the method according to claim 20, or the pharmaceutical composition according to any one of claims 21-22.

24. The method according to claim 23, wherein the method further comprises administering to the individual an effective amount of an antigen-binding protein that specifically recognizes PD-1.

25. The method according to claim 23, wherein the anti-BTLA antibody and the antigen-binding protein that specifically recognizes PD-1 are administered simultaneously or sequentially.

26. The method according to any one of claims 23-25, wherein the disease or disorder is cancer or an infectious disease, optionally, the disease or disorder is related to the BTLA signaling pathway.

27. The method according to claim 26, wherein the disease or disorder is selected from non-small cell lung cancer, adrenal cancer, bladder cancer, brain cancer, pancreatic cancer, breast cancer, colorectal cancer, melanoma, esophageal cancer, gastric cancer, cervical cancer, head and neck cancer, hepatocellular carcinoma, kidney cancer, liver cancer, ovarian cancer, pancreatic cancer, prostate cancer, small cell lung cancer, testicular cancer, thyroid cancer, uterine cancer, and any type of leukemia, lymphoma, and myeloma, and infectious diseases, including but not limited to Human Papilloma Virus (HPV), Human Immunodeficiency Virus (HIV), Herpes Simplex Virus (HSV), Varicella Zoster Virus (VSV), Cytomegalovirus (CMV), Epstein Barr Virus (EBV), Chlamydia, Rickettsia bacteria, Mycobacterium, Staphylococcus, Streptococcus, Pneumococcus, Neisseria meningitidis, and Gonococcus (conococci), Klebsiella, Proteus, Serratia, Pseudomonas, Legionella, Diphtheria, Salmonella, Bacillus, Cholera, Tetanus, Clostridium botulinum, Anthrax, Plague, Leptospirosis, and Lyme disease bacteria.

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