Anti-BTLA antibody pharmaceutical composition and use thereof

Abstract

The present invention provides a stable anti-BTLA (B and T lymphocyte attenuator) antibody pharmaceutical composition and its pharmaceutical use. The pharmaceutical composition comprises an anti-BTLA antibody, a buffer, and may further comprise at least one stabilizer and, optionally, a surfactant.

Description

[Technical Field]

[0001] The present invention relates to the field of therapeutic pharmaceutical compositions. In particular, the present invention relates to the field of pharmaceutical formulations, which pharmaceutical compositions comprise a humanized antibody that specifically binds to B and T lymphocyte attenuator (BTLA). [Background technology]

[0002] Positive and negative costimulatory signals play crucial roles in regulating B cell and T cell activity, and molecules mediating these signals have proven to be effective targets for immunomodulatory agents. In addition to T cell receptor (TCR) engagement, positive costimulation is required for optimal activation of naive T cells, while negative costimulation is considered necessary for the acquisition of autoimmune tolerance and the termination of effector T cell function. Upon interacting with B7.1 or B7.2 on the surface of antigen-presenting cells (APCs), the prototypic T cell costimulatory molecule CD28 responds to TCR engagement by signaling to promote T cell proliferation and differentiation, whereas the CD28 homolog cytotoxic T lymphocyte antigen-4 (CTLA-4) mediates the suppression of T cell proliferation and effector function (Non-Patent Document 1, Non-Patent Document 2). Several new molecules with homology to the B7 family have already been discovered (Non-Patent Document 3, Non-Patent Document 4, Non-Patent Document 5, Non-Patent Document 6), and their role in T cell activation has been described.

[0003] B and T lymphocyte attenuator (BTLA) is a member of the CD28 family, which also includes CD28, ICOS, CTLA-4, and PD-1. The first family members, CD28 and ICOS, were discovered to have immunostimulatory functions due to their ability to enhance T cell proliferation upon the addition of monoclonal antibodies (Hutloff et al., 1999). BTLA, CTLA-4, and PD-1 have been described as negative regulatory proteins. Several in vivo studies have demonstrated the inhibitory effect of BTLA on lymphocyte responses. BTLA-deficient mice, generated by Murphy and colleagues (Washington University, St. Louis), produced a three-fold increase in IgG in response to T-dependent antigens. Furthermore, T cells and B cells isolated from BTLA-deficient mice exhibited enhanced proliferative responses following antigen-receptor stimulation with CD3 and anti-IgM, respectively (Watanabe, 2003). Overexpression studies revealed that BTLA associates with the B cell receptor complex and T cell receptor. Consistent with these findings, BTLA-deficient lymphocytes are unaffected by antigen-receptor-independent stimulation with ConA (T cells) or LPS (B cells), and are not modulated by anti-BTLA antibodies. BTLA knockout mice develop spontaneous autoimmune disease over time and exhibit shortened lifespans (Oya, 2008). BTLA knockout mice exhibit progressive disease severity in models of autoimmune encephalomyelitis (EAE) and allergic airway inflammation, both of which are dependent on T cell activation (Watanabe, 2005; Deppong, 2006).

[0004] Herpesvirus entry mediator (HVEM) has been identified as a ligand for BTLA (Scully et al., 2005). HVEM is a type I transmembrane glycoprotein and a member of the TNF receptor superfamily. It has four extracellular cysteine-rich domains (CDRs) and six pseudo-repeated cysteines. BTLA and HVEM regulate T cell and APC functions primarily through dynamic cell surface expression. Upon binding to its ligand, BTLA suppresses T cell proliferation and downregulates the T cell activation marker CD25. It also suppresses the production of IFN-γ, IL-2, IL-4, and IL-10, but fails to induce cell apoptosis. Upon binding to BTLA, HVEM downregulates T cell activation and proliferation (Sedy, 2005). These findings demonstrate that BTLA expression and BTLA-HVEM binding are closely related to T cell activation and proliferation.

[0005] Antibodies can be used as therapeutic agents. Some antibodies can cause unwanted antibody immunogenicity when used as therapeutic agents in vivo. Because most monoclonal antibodies are derived from rodents, repeated use in humans can lead to immune responses against the therapeutic antibody (e.g., human anti-mouse antibodies, or HAMA). Such immune responses can, at the very least, cause therapeutic failure and, at worst, potentially fatal allergic reactions. One method for reducing the immunogenicity of rodent antibodies involves the production of chimeric antibodies, in which the mouse variable region (Fv) is fused to a human constant region (Non-Patent Document 7). However, mice injected with heterozygotes of human variable and mouse constant regions generated strong antibody responses against the human variable region, demonstrating that retention of the complete rodent Fv region in such chimeric antibodies can still cause adverse immunogenicity in patients.

[0006] In addition, grafting complementarity-determining region (CDR) loops from rodent variable domains onto human frameworks (i.e., humanization) has been used to minimize rodent sequences (Non-Patent Document 8). However, CDR loop exchange does not consistently produce antibodies with binding properties homologous to those of the starting antibody. In humanized antibodies, framework residues (FRs) (residues involved in CDR loop support) often need to be altered to maintain antigen-binding affinity (Non-Patent Document 9). While numerous humanized antibody constructions have been reported using CDR grafting and framework residue maintenance, it is difficult to predict whether a particular sequence will generate an antibody with the desired binding and / or biological properties. See, for example, Non-Patent Document 10, Non-Patent Document 11, and Non-Patent Document 12. However, the predictability of such studies is compromised by the use of different human sequences for the animal's light and heavy chain variable sequences. The sequences of known antibodies have been used, or more commonly, the sequences of antibodies with known X-ray crystal structures, such as antibodies NEW and KOL, have been used. See, eg, Jones et al., supra; Verhoeyen et al., supra; and Gorman et al., supra. Exact sequence information for a few humanized constructs has been reported.

[0007] There is a need for anti-BTLA antibodies, particularly anti-BTLA monoclonal antibodies, for treating human disorders, such as inflammatory, autoimmune, and proliferative disorders. Such antibodies preferably have low immunogenicity in human subjects, allowing for repeated administration without eliciting an adverse immune response. Therefore, there is a need in the art for highly stable protein formulations. [Prior art documents] [Non-patent literature]

[0008] [Non-Patent Document 1] Chambers et al., Ann. Rev. Immunol., 19:565-594, 2001 [Non-patent document 2] Egen et al., Nature Immunol, 3:611-618, 2002 [Non-patent document 3] Abbas et al., Nat. Med., 5:1345-6, 1999 [Non-patent document 4] Coyle et al., Nat. Immunol., 2:203-9, 2001 [Non-Patent Document 5] Carreno et al., Annu. Rev. Immunol., 20:29-53, 2002 [Non-patent document 6] Liang et al., Curr. Opin. Immunol., 14:384-90, 2002 [Non-Patent Document 7] Liu et al. (1987) Proc. Natl. Acad. Sci. USA 84:3439-43 [Non-patent document 8] Jones et al. (1986) Nature 321:522; Verhoeyen et al. (1988) Science 239:1534 [Non-Patent Document 9] Kabat et al. (1991) J. Immunol. 147:1709 [Non-Patent Document 10] Queen et al. (1989) Proc. Natl. Acad. Sci. USA 86:10029 [Non-Patent Document 11] Gorman et al. (1991) Proc. Natl. Acad. Sci. USA 88:4181 [Non-Patent Document 12] Hodgson, (1991) Biotechnology (NY), 9:421-5 Summary of the Invention

[0009] The pharmaceutical composition of the present invention is a highly stable pharmaceutical composition comprising a humanized antibody that specifically binds to BTLA. In particular, it has been discovered in the present invention that the combination of trehalose and sodium chloride can significantly improve the stability of the pharmaceutical composition.

[0010] The present invention provides a pharmaceutical composition comprising: (1) a buffer; and (2) an anti-BTLA antibody or antigen-binding fragment thereof.

[0011] In some embodiments, the anti-BTLA antibody or antigen-binding fragment thereof has HCDR1, HCDR2, and HCDR3 of the amino acid sequences set forth in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, respectively, and LCDR1, LCDR2, and LCDR3 of the amino acid sequences set forth in SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, respectively.

[0012] In some embodiments, the concentration of the anti-BTLA antibody or antigen-binding fragment thereof in the pharmaceutical composition is about 1 to 200 mg / mL, preferably about 5 to 100 mg / mL, and preferably about 10 to 50 mg / mL; more preferably, the concentration of the anti-BTLA antibody or antigen-binding fragment thereof is about 10 mg / mL, 15 mg / mL, 20 mg / mL, 25 mg / mL, 30 mg / mL, 35 mg / mL, 40 mg / mL, 45 mg / mL, or 50 mg / mL, and preferably about 10 mg / mL, 20 mg / mL, or 40 mg / mL.

[0013] In some embodiments, the buffer is selected from one or more of an acetate buffer, a citrate buffer, and a histidine buffer. In some embodiments, the buffer is a histidine buffer, preferably the histidine buffer is selected from a histidine-hydrochloride buffer or a histidine-acetate buffer, preferably a histidine-hydrochloride buffer.

[0014] In some embodiments, the histidine-hydrochloride buffer solution is prepared from histidine and histidine hydrochloride, preferably L-histidine and L-histidine monohydrochloride. In some embodiments, the histidine buffer solution is prepared from 1 to 20 mM L-histidine and 1 to 20 mM L-histidine monohydrochloride. In some embodiments, the histidine buffer solution is prepared from histidine and histidine hydrochloride in a molar ratio of 1:1 to 1:4. In some embodiments, the histidine buffer solution is prepared from histidine and histidine hydrochloride in a molar ratio of 1:1. In some embodiments, the histidine buffer solution is prepared from histidine and histidine hydrochloride in a molar ratio of 1:3. In some embodiments, the histidine formulation is a histidine buffer prepared with 4.5 mM L-histidine and 15.5 mM L-histidine monohydrochloride at a pH of 5.5. In some embodiments, the histidine formulation is a histidine buffer prepared with 15 mM histidine and 15 mM histidine hydrochloride at a pH of 6.0.

[0015] In some embodiments, the histidine buffer is a histidine-acetate buffer, preferably in a molar ratio of 1:1 to 1.5:1. The pH of these buffers is preferably 5.5±0.3, preferably about 5.5. The buffers preferably contain 15 to 20 mM histidine and 12 to 15 mM acetic acid.

[0016] In some embodiments, the buffer is an acetate buffer, preferably an acetate-sodium acetate buffer or an acetate-potassium acetate buffer, preferably an acetate-sodium acetate buffer. In some embodiments, the buffer is a citrate buffer, and preferably, the citrate buffer is a citric acid-sodium citrate buffer.

[0017] In some embodiments, the concentration of the buffer solution is about 1 to 100 mM, preferably about 5 to 50 mM, preferably about 10 to 30 mM, preferably about 20 to 30 mM, preferably about 10 to 20 mM; non-limiting examples of the concentration of the buffer solution are about 5 mM, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 40 mM, 45 mM, 50 mM, 55 mM, 60 mM, 65 mM, 70 mM, 75 mM, 80 mM, 85 mM, 90 mM, 95 mM, 100 mM, or a range defined by any two endpoints within these ranges, preferably 10 mM, 20 mM, or 30 mM.

[0018] In some embodiments, the pH value of the buffer solution is about 5.0 to 6.5, preferably about 5.0 to 6.0, preferably about 5.5 to 6.5, preferably about 5.0 to 5.5, preferably about 5.5 to 6.0, preferably about 6.0 to 6.5; non-limiting examples of pH values ​​of the buffer solution are about 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, preferably about 5.0, 5.5, or 6.0.

[0019] In some embodiments, the pharmaceutical composition further comprises a stabilizer selected from one or more of sodium chloride, mannitol, sorbitol, sucrose, maltose, xylitol, and trehalose. Preferably, the stabilizer is a combination of trehalose and sodium chloride.

[0020] In some embodiments, the concentration of the stabilizer is about 20 mM to 300 mM, preferably 50 mM to 300 mM, and more preferably 120 mM to 250 mM.

[0021] In some embodiments, the stabilizer is sodium chloride at a concentration of about 50 to 200 mM, or the stabilizer is mannitol at a concentration of about 100 to 300 mM, or the stabilizer is sorbitol at a concentration of about 100 to 300 mM, or the stabilizer is sucrose at a concentration of about 100 to 300 mM, or the stabilizer is trehalose at a concentration of about 100 to 300 mM, or the stabilizer is a combination of about 30 to 100 mM sodium chloride and about 50 to 200 mM mannitol, or a combination of about 30 to 100 mM sodium chloride and about 50 to 200 mM sorbitol, or a combination of about 30 to 100 mM sodium chloride and about 50 to 200 mM sucrose, or a combination of about 30 to 100 mM sodium chloride and about 50 to 200 mM trehalose.

[0022] In some embodiments, the stabilizer is sodium chloride. In some embodiments, the stabilizer is sodium chloride having a concentration of about 50 to 200 mM, and the concentration of the sodium chloride is preferably about 100 to 190 mM, preferably about 120 to 180 mM, preferably about 130 to 170 mM, preferably about 130 to 150 mM. Non-limiting examples of the sodium chloride concentration are about 100 mM, 110 mM, 120 mM, 125 mM, 130 mM, 135 mM, 140 mM, 145 mM, 150 mM, 155 mM, 160 mM, 170 mM, 180 mM, 190 mM, 200 mM, and preferably 135 mM or 140 mM.

[0023] In some embodiments, the stabilizer is mannitol. In some embodiments, the stabilizer is mannitol at a concentration of about 100 to 300 mM, preferably about 150 to 300 mM, and preferably about 200 to 280 mM, with non-limiting examples of mannitol concentrations being about 200 mM, 210 mM, 220 mM, 230 mM, 240 mM, 250 mM, 260 mM, 270 mM, and 280 mM, and preferably 240 mM.

[0024] In some embodiments, the stabilizer is sorbitol. In some embodiments, the stabilizer is sorbitol at a concentration of about 100-300 mM, preferably about 150-300 mM, and preferably about 200-280 mM, with non-limiting examples of sorbitol concentrations being about 200 mM, 210 mM, 220 mM, 230 mM, 240 mM, 250 mM, 260 mM, 270 mM, 280 mM, and preferably 240 mM.

[0025] In some embodiments, the stabilizer is sucrose. In some embodiments, the stabilizer is sucrose at a concentration of about 100 to 300 mM, preferably about 150 to 300 mM, and preferably about 200 to 280 mM. Non-limiting examples of sucrose concentrations are about 200 mM, 210 mM, 220 mM, 230 mM, 240 mM, 250 mM, 260 mM, 270 mM, 280 mM, and preferably 220 mM.

[0026] In some embodiments, the stabilizer is trehalose. In some embodiments, the stabilizer is trehalose at a concentration of about 100-300 mM, preferably about 150-300 mM, and preferably about 200-280 mM, with non-limiting examples of trehalose concentrations being about 180 mM, 200 mM, 210 mM, 220 mM, 230 mM, 240 mM, 250 mM, 260 mM, 270 mM, 280 mM, and preferably 220 mM.

[0027] In some embodiments, the stabilizer is a combination of sodium chloride and mannitol. In some embodiments, the stabilizer is a combination of about 30-100 mM sodium chloride and about 50-200 mM mannitol, preferably about 40-80 mM sodium chloride and about 100-180 mM mannitol, preferably about 40-60 mM sodium chloride and about 120-160 mM mannitol; non-limiting examples of the stabilizer include a combination of about 54 mM sodium chloride and about 144 mM mannitol, and a combination of about 50 mM sodium chloride and about 140 mM mannitol.

[0028] In some embodiments, the stabilizer is a combination of sodium chloride and sorbitol. In some embodiments, the stabilizer is a combination of about 30-100 mM sodium chloride and about 50-200 mM sorbitol, preferably about 40-80 mM sodium chloride and about 100-180 mM sorbitol, preferably about 40-60 mM sodium chloride and about 120-160 mM sorbitol; non-limiting examples of the stabilizer include a combination of about 54 mM sodium chloride and about 144 mM sorbitol, and a combination of about 40 mM sodium chloride and about 160 mM sorbitol.

[0029] In some embodiments, the stabilizer is a combination of sodium chloride and sucrose. In some embodiments, the stabilizer is a combination of about 30-100 mM sodium chloride and about 50-200 mM sucrose, preferably about 40-80 mM sodium chloride and about 100-180 mM sucrose, preferably about 40-60 mM sodium chloride and about 120-160 mM sucrose; non-limiting examples of the stabilizer include a combination of about 54 mM sodium chloride and about 132 mM sucrose, and a combination of about 50 mM sodium chloride and about 150 mM sucrose.

[0030] In some embodiments, the stabilizer is a combination of sodium chloride and trehalose. In some embodiments, the stabilizer is a combination of about 30-100 mM sodium chloride and about 50-200 mM trehalose, preferably about 40-80 mM sodium chloride and about 100-180 mM trehalose, and preferably about 40-60 mM sodium chloride and about 120-160 mM trehalose. Non-limiting examples of the stabilizer include a combination of about 54 mM sodium chloride and about 132 mM trehalose, a combination of about 50 mM sodium chloride and about 140 mM trehalose, or a combination of about 60 mM sodium chloride and about 120 mM trehalose, preferably about 54 mM sodium chloride and about 132 mM trehalose, or a combination of about 50 mM sodium chloride and about 140 mM trehalose.

[0031] In some embodiments, the pharmaceutical composition further comprises a surfactant, wherein the surfactant is selected from polysorbate 80, polysorbate 20, or poloxamer 188. In some embodiments, the surfactant is selected from polysorbate 80. In some embodiments, the surfactant is selected from polysorbate 20.

[0032] In some embodiments, the concentration of the surfactant, calculated on a w / v basis, is about 0.001% to 0.1%, preferably about 0.01% to 0.05%, preferably about 0.02% to 0.04%, and by way of non-limiting example, the concentration of the surfactant is about 0.02%, 0.03%, or 0.04%, preferably 0.02%.

[0033] In some embodiments, the anti-BTLA antibody or antigen-binding fragment thereof is selected from a murine antibody, a chimeric antibody, or a humanized antibody, preferably a humanized antibody.

[0034] In some embodiments, the anti-BTLA antibody or antigen-binding fragment thereof has a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO:7 and a light chain variable region having the amino acid sequence set forth in SEQ ID NO:8. In some embodiments, the anti-BTLA antibody or antigen-binding fragment thereof has a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO:7 and a light chain variable region having the amino acid sequence set forth in SEQ ID NO:9. In some embodiments, the anti-BTLA antibody or antigen-binding fragment thereof has a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO:7 and a light chain variable region having the amino acid sequence set forth in SEQ ID NO:10. In some embodiments, the anti-BTLA antibody or antigen-binding fragment thereof has a heavy chain amino acid sequence set forth in SEQ ID NO:11 and a light chain amino acid sequence set forth in SEQ ID NO:12.

[0035] In some embodiments, the pharmaceutical composition comprises any one of the components set forth in any one of (1) to (8) below, or is prepared from any one of the components set forth in any one of (1) to (8) below, and the anti-BTLA antibody or antigen-binding fragment thereof is as described in any one of the embodiments of the present invention.

[0036] (1) (a) about 10 mg / mL to 50 mg / mL of an anti-BTLA antibody or antigen-binding fragment thereof; (b) about 10 to 30 mM histidine buffer having a pH value of about 5.0 to 6.0; (c) a combination of about 30 mM to about 100 mM sodium chloride and about 50 to 200 mM trehalose; (d) about 0.01% to 0.05% polysorbate 80; or

[0037] (2) (a) about 10 mg / mL to 50 mg / mL of an anti-BTLA antibody or antigen-binding fragment thereof; (b) about 10 to 30 mM histidine buffer having a pH value of about 5.0 to 6.0; (c) a combination of about 30 mM to about 100 mM sodium chloride and about 50 to 200 mM mannitol; (d) about 0.01% to 0.05% polysorbate 80; or

[0038] (3) (a) about 10 mg / mL to 50 mg / mL of an anti-BTLA antibody or antigen-binding fragment thereof; (b) about 10 to 30 mM histidine buffer having a pH value of about 5.0 to 6.0; (c) a combination of about 30 mM to about 100 mM sodium chloride and about 50 to 200 mM sucrose; (d) about 0.01% to 0.05% polysorbate 80; or

[0039] (4) (a) about 10 mg / mL to 50 mg / mL of an anti-BTLA antibody or antigen-binding fragment thereof; (b) about 10 to 30 mM histidine buffer having a pH value of about 5.0 to 6.0; (c) a combination of about 30 mM to about 100 mM sodium chloride and about 50 to 200 mM sorbitol; (d) about 0.01% to 0.05% polysorbate 80; or

[0040] (5) (a) about 10 mg / mL to 50 mg / mL of an anti-BTLA antibody or antigen-binding fragment thereof; (b) about 10 to 30 mM histidine buffer having a pH value of about 5.0 to 6.0; (c) about 100 mM to about 300 mM trehalose; (d) about 0.01% to 0.05% polysorbate 80; or

[0041] (6) (a) about 10 mg / mL to 50 mg / mL of an anti-BTLA antibody or antigen-binding fragment thereof; (b) about 10 to 30 mM citrate buffer having a pH value of about 5.5 to 6.5; (c) about 100 mM to about 300 mM trehalose; (d) about 0.01% to 0.05% polysorbate 80; or

[0042] (7) (a) about 10 mg / mL to 50 mg / mL of an anti-BTLA antibody or antigen-binding fragment thereof; (b) about 10 to 30 mM citrate buffer having a pH value of about 5.5 to 6.5; (c) a combination of about 30 mM to about 100 mM sodium chloride and about 50 to 200 mM mannitol; (d) about 0.01% to 0.05% polysorbate 80; or

[0043] (8) (a) about 10 mg / mL to 50 mg / mL of an anti-BTLA antibody or antigen-binding fragment thereof; (b) about 10 to 30 mM acetate buffer having a pH value of about 5.0 to 6.0; (c) a combination of about 30 mM to about 100 mM sodium chloride and about 50 to 200 mM mannitol; and (d) about 0.01% to 0.05% polysorbate 80.

[0044] In some embodiments, the pharmaceutical composition contains any one of the ingredients listed in (9) to (13) below, or is prepared using any one of the ingredients listed in (9) to (13) below.

[0045] (9) (a) about 20 mg / mL of an anti-BTLA antibody or antigen-binding fragment thereof; (b) about 20 mM histidine buffer having a pH value of about 6.0; (c) a combination of about 54 mM sodium chloride and about 132 mM trehalose; (d) about 0.02% polysorbate 80; or

[0046] (10) (a) about 20 mg / mL of an anti-BTLA antibody or antigen-binding fragment thereof; (b) about 20 mM histidine buffer having a pH value of about 5.5; (c) about 220 mM trehalose; and (d) about 0.02% polysorbate 80; or

[0047] (11) (a) about 20 mg / mL of an anti-BTLA antibody or antigen-binding fragment thereof; (b) about 20 mM histidine buffer having a pH value of about 5.5; (c) a combination of about 50 mM sodium chloride and about 140 mM mannitol; (d) about 0.02% polysorbate 80; or

[0048] (12) (a) about 20 mg / mL of an anti-BTLA antibody or antigen-binding fragment thereof; (b) about 20 mM histidine buffer having a pH value of about 5.5; (c) a combination of about 50 mM sodium chloride and about 140 mM trehalose; (d) about 0.02% polysorbate 80; or

[0049] (13) (a) about 20 mg / mL of an anti-BTLA antibody or antigen-binding fragment thereof; (b) about 20 mM histidine buffer having a pH value of about 5.0 to 6.0; (c) a combination of about 50 mM sodium chloride and about 140 mM trehalose; and (d) about 0.02% polysorbate 80.

[0050] In some embodiments, the pharmaceutical composition is a liquid formulation or a lyophilized formulation. In some embodiments, the pharmaceutical composition is a liquid formulation, for example, an aqueous formulation. In some embodiments, the liquid or lyophilized formulation is stable at 2-8°C for at least 3 months, at least 6 months, at least 12 months, at least 18 months, or at least 24 months. In some embodiments, the aqueous solution or lyophilized formulation is stable at 40° C. for at least 7 days, at least 14 days, or at least 28 days.

[0051] The present invention further provides the use of the above pharmaceutical composition in the preparation of a medicament for treating or preventing a BTLA-mediated disease.

[0052] In some embodiments, the disease comprises a tumor, an infectious disease, inflammation, or an autoimmune disease, including melanoma, breast cancer, kidney cancer, prostate cancer, colon cancer, lung cancer, pancreatic cancer, bone cancer, skin cancer, head or neck cancer, uterine cancer, ovarian cancer, rectal cancer, anal cancer, stomach cancer, testicular cancer, esophageal cancer, small intestine cancer, cervical cancer, vaginal cancer, Hodgkin's disease, non-Hodgkin's lymphoma, endocrine system cancer, thyroid cancer, adrenal cancer, soft tissue cancer, urethral cancer, chronic or acute leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, or leukemia. Cancers of the central nervous system include: myeloid leukemia, chronic lymphocytic leukemia, childhood solid tumors, lymphocytic lymphoma, bladder cancer, kidney or ureter cancer, renal pelvis cancer, neoplasms of the central nervous system, primary central nervous system lymphoma, tumor angiogenesis, spinal axis tumors, brain stem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, T-cell lymphoma, environmentally induced cancers including those induced by asbestos, and combinations of the foregoing cancers. The autoimmune diseases include organ-specific autoimmune diseases and systemic autoimmune diseases. The organ-specific autoimmune diseases include chronic lymphocytic thyroiditis, hyperthyroidism, insulin-dependent diabetes mellitus, myasthenia gravis, ulcerative colitis, pernicious anemia with chronic atrophic gastritis, pulmonary hemorrhagic nephritic syndrome, pemphigus vulgaris, pemphigoid, primary biliary cirrhosis, multiple encephalomyelitis, acute idiopathic polyneuropathy, etc. The systemic autoimmune diseases include systemic lupus erythematosus, rheumatoid arthritis, systemic vasculitis, scleroderma, pemphigoid, dermatomyositis, mixed connective tissue disease, autoimmune hemolytic anemia, ulcerative colitis, etc. [Brief explanation of the drawings]

[0053] [Figure 1] This is a comparative study of the binding of the humanized antibody hu17 to BTLA from various species. [Figure 2] Effect of hu18 on tumor volume in B-hBTLA mice transplanted with MC38-hHVEM cells. [Figure 3] The effect of the test product on the body weight of B-hBTLA mice transplanted with MC38-hHVEM cells. DETAILED DESCRIPTION OF THE INVENTION

[0054] <Definitions and Explanations> In order that the present invention may be more readily understood, the following definitions of technical and scientific terms are provided. Unless otherwise clearly defined herein, all other technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art of the present invention. The present invention is not limited to specific methods, reagents, compounds, compositions, or biological systems, and, of course, variations thereon can be made. The terms used herein are for the purpose of describing specific embodiments only and are not intended to be limiting.

[0055] Unless expressly stated otherwise, the singular terms "a," "one," "an," and "the," as used in this specification and the appended claims, include plural references. Thus, for example, reference to "a polypeptide" includes a combination of two or more polypeptides, and the like.

[0056] The term "pharmaceutical composition" or "formulation" refers to a mixture containing one or more compounds described herein or pharmaceutically acceptable salts or precursor drugs thereof and other components, such as physiologically pharmaceutically acceptable vectors and excipients. The pharmaceutical composition is intended to facilitate administration to the body, enhance absorption of the active ingredient, and exert physiological activity.

[0057] The term "liquid formulation" refers to a formulation that is in a liquid state, and not a lyophilized formulation that is resuspended. Liquid formulations of the present invention are stable to storage and do not owe their stability to lyophilization (or other transformation methods, such as spray drying).

[0058] The term "aqueous liquid formulation" refers to a liquid formulation in which water is the solvent. In some embodiments, an aqueous liquid formulation is a formulation that maintains stability (e.g., chemical and / or physical stability and / or biological activity) without the need for lyophilization, spray drying, and / or freezing.

[0059] The term "excipient" refers to an agent that can be added to a formulation to provide desired properties (e.g., consistency, high stability) and / or to adjust osmotic pressure. Examples of commonly used excipients include, but are not limited to, sugars, polyols, amino acids, surfactants, and polymers.

[0060] As used herein, "about" when referring to a measurable value (e.g., quantity, duration, etc.) is meant to include variations applicable to the disclosed method, such as ±20% or ±10% variation from the particular value, e.g., ±5%, ±1%, ±0.1%, etc.

[0061] The term "a buffer solution having a pH value of about 5.0 to 6.5" refers to a reagent whose acid / base conjugate component makes the solution resistant to changes in pH. The buffer solution used in the formulation of the present invention may have a pH value in the range of about 5.0 to about 6.5, or about 5.5 to about 6.5, or about 5.0 to about 6.0.

[0062] As used herein, examples of "buffers" that control the pH value within the range include acetate (e.g., sodium acetate), succinate (e.g., sodium succinate), gluconic acid, histidine, histidine hydrochloride, methionine, citrate, phosphate, citrate / phosphate, imidazole, acetic acid, acetate, citrate, combinations thereof, and other organic acid buffers.

[0063] A "histidine buffer" is a buffer containing histidine ions. An example of a histidine buffer contains histidine and a salt of histidine, such as histidine hydrochloride, histidine acetate, histidine phosphate, or histidine sulfate, e.g., a histidine buffer containing histidine and histidine hydrochloride. The histidine buffer of the present invention may also include a histidine buffer containing histidine and an acetate salt (e.g., a sodium salt or a potassium salt).

[0064] A "citrate buffer" is a buffer containing citrate ions. Examples of citrate buffers include citric acid-sodium citrate, citric acid-potassium citrate, citric acid-calcium citrate, citric acid-magnesium citrate, etc. A preferred citrate buffer is citric acid-sodium citrate buffer.

[0065] An "acetate buffer" is a buffer containing acetate ions. Examples of acetate buffers include acetic acid-sodium acetate, acetic acid-potassium acetate, acetic acid-calcium acetate, acetic acid-magnesium acetate, etc. A preferred acetate buffer is acetic acid-sodium acetate buffer.

[0066] The term "stabilizer" means a pharmaceutically acceptable excipient that protects the active drug ingredient and / or formulation from chemical and / or physical degradation during preparation, storage and application. Stabilizers include, but are not limited to, sugars, amino acids, salts, polyols, and their metabolites, as defined below, such as sodium chloride, calcium chloride, magnesium chloride, mannitol, sorbitol, sucrose, trehalose, arginine hydrochloride, arginine, glycine, alanine (α-alanine, β-alanine), betaine, leucine, lysine, glutamic acid, aspartic acid, proline, 4-hydroxyproline, sarcosine, γ-aminobutyric acid (GABA), opines (alanopine, octopine, strombine), trimethylamine N-oxide (TMAO), human serum albumin (hsa), bovine serum albumin (bsa), α-casein, globulin, α-lactalbumin, LDH, lysozyme, myoglobin, ovalbumin, and RNAase A. Some stabilizers, such as sodium chloride, calcium chloride, magnesium chloride, mannitol, sorbitol, and sucrose, can control osmotic pressure. The stabilizer specifically used in the present invention is selected from one or more of polyols, salts, and sugars. A preferred salt is sodium chloride, a preferred sugar is sucrose or trehalose, and a preferred polyol is sorbitol or mannitol. Preferred stabilizers are sodium chloride, mannitol, sorbitol, sucrose, trehalose, sodium chloride-sorbitol, sodium chloride-mannitol, sodium chloride-sucrose, and sodium chloride-trehalose, more preferably sodium chloride-sorbitol, sodium chloride-mannitol, sodium chloride-sucrose, and sodium chloride-trehalose, and even more preferably sodium chloride-trehalose.

[0067] The term "surfactant" generally includes agents that protect proteins, such as antibodies, from the effects of air / solution interface and solution / surface induced stresses, for example, to reduce antibody aggregation or minimize particulate formation in the formulation. Exemplary surfactants include, but are not limited to, non-ionic surfactants, such as polyoxyethylene sorbitan fatty acid esters (e.g., polysorbate 20 and polysorbate 80), polyethylene-polypropylene copolymers, polyethylene-polypropylene glycols, polyoxyethylene-stearic acid esters, polyoxyethylene alkyl ethers such as polyoxyethylene monolauryl ether, polyoxyethylene alkylphenyl ethers (Triton-X), polyoxyethylene-polypropylene oxide copolymers (poloxamers, Pluronics), and sodium lauryl sulfate (SDS).

[0068] The term "isotonic" means that the formulation has an osmotic pressure approximately equal to that of human blood. Isotonic formulations generally have an osmotic pressure of approximately 250-350 mOsm. Isotonicity can be measured using a vapor pressure or freezing point depression osmometer.

[0069] The term "stable" formulation refers to a formulation in which the physical and / or chemical stability and / or physiological activity of the antibody contained therein are essentially maintained during manufacturing and / or storage. A pharmaceutical formulation may be stable even if the chemical structure or physiological function of the antibody contained therein is not 100% maintained after storage for a certain period of time. A formulation may be considered "stable" if it maintains about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% of the antibody structure or function after storage for a certain period of time. Analytical techniques for measuring protein stability are available in the art and are described in Peptide and Protein Drug Delivery, 247-301, Vincent Lee, Editor, Marcel Dekker, Inc., New York, NY, Pubs. (1991), and Jones, A. (1993) Adv. Drug Delivery Rev. 10:29-90 (both incorporated by reference).

[0070] The stability of a formulation can be measured by determining the percentage of native antibody remaining in the formulation (and other methods) after storage at a certain temperature and period of time. Among other methods, the percentage of native antibody can be measured by size exclusion chromatography (e.g., size exclusion high performance liquid chromatography [SEC-HPLC]), where "native" means non-aggregated and non-degraded. In some embodiments, protein stability is determined by the percent of intact protein in a solution having a low percentage of degraded (e.g., fragmented) and / or aggregated protein. In some embodiments, the formulation can be stably stored at room temperature, about 25-30°C, or 40°C for at least 2 weeks, at least 28 days, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months, at least 18 months, at least 24 months, or even longer, with at most about 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.1% aggregated antibody.

[0071] Stability can be measured by measuring the percentage of antibody ("acid form") that transitions during ion exchange in a fraction that is slightly more acidic than the main fraction of the antibody ("mainly charged form") (and other methods), with stability being inversely proportional to the percentage of antibody in the acid form. Among other methods, the percentage of "oxidized" antibody can be measured by ion exchange chromatography (e.g., cation exchange high-performance liquid chromatography [CEX-HPLC]). In some embodiments, acceptable stability means that the amount of detectable antibody in the acid form does not exceed about 49%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.1% after storage of the formulation at a given temperature and period. The storage period before measuring stability may be at least 2 weeks, at least 28 days, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months, at least 18 months, at least 24 months, or even longer. The temperature at which the pharmaceutical formulation can be stored during stability assessment may be any temperature within the range of about −80° C. to about 45° C., for example, about −80° C., about −30° C., about −20° C., about 0° C., about 2-8° C., about 5° C., about 25° C., or about 40° C.

[0072] An antibody "retains its physical stability" in the pharmaceutical composition if it shows essentially no signs of aggregation, precipitation, and / or denaturation, as measured, for example, by visual inspection of color and / or clarity, or by UV light scattering or size exclusion chromatography. Aggregation is the process by which single molecules or complexes associate with each other through covalent or non-covalent bonds to form aggregates. Aggregation may be allowed to proceed until a precipitate is observed.

[0073] The stability, e.g., physical stability, of a formulation can be assessed by methods well known in the art, such as measuring the surface extinction coefficient (absorbance or optical density) of a sample. This extinction coefficient measurement correlates with the turbidity of the formulation. The turbidity of a formulation is due in part to the inherent properties of the dissolved protein in solution and is typically measured nephelometrically and in nephelometric turbidity units (NTU).

[0074] For example, the turbidity level, which varies depending on the concentration of one or more components in a solution (e.g., protein and / or salt concentration), is also referred to as the "turbidity" or "cloudy appearance" of the formulation. The turbidity level can be calculated by reference to a calibration curve constructed using suspensions of known turbidity. Reference standards for measuring the turbidity level of pharmaceutical compositions may follow the European Pharmacopoeia standard (European Pharmacopoeia, 4th Edition, Directorate for the Quality of Medicine of the Council of Europe (EDQM), Strasbourg, France). According to the European Pharmacopoeia standard, a clear solution is defined as a solution having a turbidity lower than or equal to that of a reference suspension of about 3 based on the European Pharmacopoeia standard. Turbidity measurements by nephelometry can measure Rayleigh scattering in cases where no binding or non-ideal effects occur, but Rayleigh scattering generally varies linearly with concentration. Other methods for assessing physical stability are well known in the art.

[0075] An antibody "retains its chemical stability" in a pharmaceutical composition if the antibody's chemical stability at a given time indicates that the antibody still retains its biological activity, as defined below. For example, chemical stability can be assessed by detecting or quantifying chemical changes in the antibody. Chemical changes, which can include size changes (e.g., truncations), can be assessed, for example, by size exclusion chromatography, SDS-PAGE, and / or matrix-assisted laser desorption / ionization / time-of-flight mass spectrometry (MALDI / TOF MS). Other chemical changes, including charge changes (e.g., occurring as a result of deamidation or oxidation), can be assessed, for example, by ion exchange chromatography.

[0076] An antibody in a pharmaceutical composition "retains its biological activity" if the antibody is biologically active for its intended purpose. For example, a formulation of the present invention is stable if, after storage at temperatures such as 5°C, 25°C, or 45°C for a period of time (e.g., 1-12 months), the binding affinity of the anti-BTLA antibody contained in the formulation to BTLA is at least 90%, 95%, or greater than the binding affinity of the antibody prior to storage. Binding affinity can be measured, for example, by ELISA or plasma resonance techniques.

[0077] In an embodiment of the present invention, a "therapeutically effective amount" or "effective amount" of an antibody, from a pharmacological perspective, is an amount that is effective for preventing, treating, or alleviating symptoms of a disorder that the antibody can effectively treat. In the present invention, a "therapeutically effective amount" or "therapeutically effective dose" of a drug is any amount of a drug, used alone or in combination with other therapeutic agents, that protects a subject from disease attacks or promotes disease regression, as evidenced by a decrease in the severity of disease symptoms, an increase in the frequency and duration of disease-asymptomatic periods, or prevention of damage or functional impairment due to disease affliction. The disease regression-promoting effect of a drug can be assessed by several methods known to those skilled in the art, for example, by measuring the activity of the agent in human subjects during clinical trials, in animal model systems predictive of human efficacy, or by in vitro assays. A therapeutically effective amount of a drug includes a "prophylactically effective amount," i.e., any amount of a drug that, when administered alone or in combination with other therapeutic agents, to a subject at risk of developing a disease or experiencing disease recurrence, prevents disease progression or recurrence.

[0078] The terms "subject" or "patient" are meant to include living mammals. Examples of subjects / patients include humans and non-human mammals, such as non-human primates, dogs, cows, horses, pigs, sheep, goats, cats, mice, rabbits, rats, and non-human transgenic animals. In certain embodiments of the invention, the subject is a human.

[0079] The terms "application," "administration," and "treatment" refer to the introduction of a composition containing a therapeutic agent into a subject by any one of a variety of methods or delivery systems known to those of skill in the art. Routes of administration of anti-PD-1 antibodies include intravenous, intramuscular, subcutaneous, peritoneal, spinal, or other parenteral routes of administration, such as injection or infusion. "Parenteral administration" refers to a mode of administration, generally by injection, excluding enteral or topical administration, and includes, but is not limited to, intravenous, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intramyocardial, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, intradural, and intrasternal injection and infusion, and in vivo electroporation.

[0080] <Anti-BTLA antibody> As used herein, the term "antibody" should be understood to include intact antibody molecules and antigen-binding fragments thereof. As used herein, the term "antigen-binding portion" or "antigen-binding fragment" of an antibody (or abbreviated as "antibody portion" or "antibody fragment") refers to one or more fragments of an antibody that retain the ability to specifically bind to human BTLA (B and T lymphocyte attenuator) or an epitope thereof.

[0081] As used herein, the term "full-length antibody" or "complete antibody molecule" refers to an immunoglobulin molecule comprising four peptide chains: two heavy (H) chains (full-length, approximately 50-70 kDa) and two light (L) chains (full-length, approximately 25 kDa) linked by disulfide bonds. Each heavy chain consists of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region (abbreviated herein as CH). The heavy chain constant region consists of three domains: CH1, CH2, and CH3. Each light chain consists of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region consists of one domain, CL. The VH and VL regions can be further subdivided into highly variable complementarity-determining regions (CDRs) and more conserved regions, called framework regions (FRs), between them. Each VH or VL region consists of three CDRs and four FRs arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant regions of the antibody mediate the binding of the immunoglobulin to host tissues or factors, including cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system.

[0082] As used herein, the term "CDR" refers to a complementarity-determining region in an antibody variable sequence. Three CDRs are present in each heavy and light chain variable region, designated HCDR1, HCDR2, and HCDR3, or LCDR1, LCDR2, and LCDR3, respectively. The precise boundaries of these CDRs are defined by the system.

[0083] The precise amino acid sequence boundaries of the variable region CDRs of the antibodies described herein can be determined by any of many well-known methods, including the Chothia conformation, based on the three-dimensional structure of the antibody and the topology of the CDR ring (Chothia et al. (1989) Nature 342:877-883; Al-Lazikani et al., "Standard conformations for the canonical structures of immunoglobulins", Journal of Molecular Biology, 273, 927-948 (1997)), the Kabat conformation, based on antibody sequence variability (Kabat et al., Sequences of Proteins of Immunological Interest, 4th edition, US Department of Health and Human Services, National Institutes of Health (1987)), AbM (University of Bath), Contact (University College London), and the international ImMunoGeneTics database (IMGT) (1999 Nucleic Acids Research, 27, 209-212), and North CDR definitions based on affinity propagation clustering using a large number of crystal structures. The CDRs of the antibodies of the present invention can be determined by those skilled in the art using any method known in the art (e.g., different assignment systems or combinations thereof).

[0084] As used herein, "antigen-binding fragment" includes antibody fragments or derivatives thereof, typically comprising at least a fragment of the antigen-binding or variable region (e.g., one or more CDRs) of the parent antibody and retaining at least some of the binding specificity of the parent antibody. Examples of antigen-binding fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments, diabodies, linear antibodies, single-chain antibody molecules such as sc-Fv, nanobodies formed from antibody fragments, and multispecific antibodies. When antibody binding activity is expressed in molar concentrations, binding fragments or derivatives thereof typically retain at least 10% of the antigen-binding activity of the parent antibody. It is preferred that binding fragments or derivatives thereof retain at least 20%, 50%, 70%, 80%, 90%, 95%, 100%, or more of the antigen-binding affinity of the parent antibody. Antigen-binding fragments of antibodies are expected to contain conservative or non-conservative amino acid substitutions (referred to as "conservative variants" or "function-conservative variants" of the antibody) that do not appreciably alter its biological activity.

[0085] Anti-BTLA antibodies or antigen-binding fragments thereof described herein include any one of the anti-BTLA antibodies described in Application No. CN201810870514.0, the entire disclosure of which is incorporated herein by reference. In some embodiments, the CDR sequences of antibodies used in methods and compositions of the invention comprise CDR sequences derived from antibody hu18 described in CN201810870514.0. In some embodiments, the CDR sequences of antibodies used in methods and compositions of the invention comprise CDR sequences derived from antibody hu17 described in CN201810870514.0. In some embodiments, the CDR sequences of antibodies used in methods and compositions of the invention comprise CDR sequences derived from antibody hu19 described in CN201810870514.0.

[0086] Non-limiting exemplary antibodies used in the examples herein are selected from hu17, hu18, and hu19, all of which are fully humanized antibodies that specifically bind to human BTLA, as set forth in CN201810870514.0. Antibodies hu17, hu18, and hu19 have HCDR1, HCDR2, and HCDR3 sequences set forth in SEQ ID NOs:1, 2, and 3, respectively, and LCDR1, LCDR2, and LCDR3 sequences set forth in SEQ ID NOs:4, 5, and 6, respectively. Preferably, antibody hu17 has a heavy chain variable region set forth in SEQ ID NO:7 and a light chain variable region set forth in SEQ ID NO:8, respectively. Antibody hu18 has a heavy chain variable region set forth in SEQ ID NO:7 and a light chain variable region set forth in SEQ ID NO:9, respectively. Antibody hu19 has a heavy chain variable region and a light chain variable region whose amino acid sequence is shown in SEQ ID NO:7 and SEQ ID NO:10, respectively. Preferably, antibody hu18 has a heavy chain amino acid sequence shown in SEQ ID NO:11 and SEQ ID NO:12, respectively. The CDR amino acid sequences of the above humanized antibodies are defined according to the IMGT system.

[0087] <Pharmaceutical preparations> The pharmaceutical composition of the present invention is a highly stable pharmaceutical composition comprising a humanized antibody that specifically binds to BTLA. In particular, it has been discovered in the present invention that the combination of trehalose and sodium chloride can significantly improve the stability of the pharmaceutical composition.

[0088] The present invention provides a pharmaceutical composition comprising: (1) a buffer; and (2) an anti-BTLA antibody or antigen-binding fragment thereof.

[0089] The antibody in the pharmaceutical composition of the present invention may be a murine antibody, a chimeric antibody, or a humanized antibody, preferably a humanized antibody, and may have HCDR1, HCDR2, and HCDR3 set forth in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, respectively, and LCDR1, LCDR2, and LCDR3 set forth in SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, respectively. Preferably, the antibody in the pharmaceutical composition of the present invention has a heavy chain variable region set forth in SEQ ID NO:7 and a light chain variable region set forth in SEQ ID NO:8, or a heavy chain variable region set forth in SEQ ID NO:7 and a light chain variable region set forth in SEQ ID NO:9, respectively, or a heavy chain variable region set forth in SEQ ID NO:7 and a light chain variable region set forth in SEQ ID NO:10, respectively. More preferably, the antibody in the pharmaceutical composition of the present invention has a heavy chain amino acid sequence set forth in SEQ ID NO:11 and a light chain amino acid sequence set forth in SEQ ID NO:12, respectively.

[0090] In pharmaceutical compositions of the present invention, the concentration of the anti-BTLA antibody or antigen-binding fragment thereof is about 1 to 200 mg / mL, preferably about 5 to 100 mg / mL, preferably about 10 to 50 mg / mL, and more preferably 15 to 25 mg / mL. Non-limiting examples of the concentration of the anti-BTLA antibody or antigen-binding fragment thereof are about 10 mg / mL, 15 mg / mL, 20 mg / mL, 25 mg / mL, 30 mg / mL, 35 mg / mL, 40 mg / mL, 45 mg / mL, or 50 mg / mL, preferably about 10 mg / mL, 20 mg / mL, or 40 mg / mL.

[0091] The buffer in the pharmaceutical composition of the present invention may be selected from acetate buffer, citrate buffer, and histidine buffer to provide the pharmaceutical composition of the present invention with a pH value of 5.0 to 6.5, preferably 5.0 to 6.0. On the other hand, the pH value of the buffer used in the pharmaceutical composition of the present invention may be 5.0 to 6.5, preferably 5.0 to 6.0.

[0092] In the pharmaceutical composition of the present invention, a particularly preferred buffer is a histidine buffer. Preferably, the pH value of the histidine buffer used in the present invention is 5.0 to 6.0, more preferably 5.5±0.3, and preferably about 5.5. Preferably, the histidine buffer comprises a histidine-hydrochloride buffer or a histidine-acetate buffer, preferably a histidine-hydrochloride buffer. More preferably, the histidine-hydrochloride buffer is prepared from histidine and histidine hydrochloride, preferably L-histidine and L-histidine monohydrochloride. In some embodiments, the histidine buffer is prepared from 1 to 20 mM L-histidine and 1 to 20 mM L-histidine monohydrochloride. In some embodiments, the histidine buffer is prepared from histidine and histidine hydrochloride in a molar ratio of 1:1 to 1:4. In some embodiments, the histidine buffer is prepared with histidine and histidine hydrochloride in a molar ratio of 1:1. In some embodiments, the histidine buffer is prepared with histidine and histidine hydrochloride in a molar ratio of 1:3. In some embodiments, the histidine formulation is a histidine buffer prepared with 4.5 mM L-histidine and 15.5 mM L-histidine monohydrochloride to a pH of 5.5. In some embodiments, the histidine formulation is a histidine buffer prepared with 15 mM histidine and 15 mM histidine hydrochloride to a pH of 6.0.

[0093] The histidine buffer in the pharmaceutical composition of the present invention is a histidine-acetate buffer, preferably in a molar ratio of 1:1 to 1.5:1. The pH value of such a buffer is preferably 5.5±0.3, preferably about 5.5. Preferably, these buffers contain 15 to 20 mM histidine and 12 to 15 mM acetic acid.

[0094] Therefore, the pharmaceutical composition of the present invention may comprise a histidine-histidine hydrochloride buffer solution having a pH value of 5.0 to 6.0 and a concentration in the pharmaceutical composition of 10 to 30 mM, and 15 to 25 mg / mL of an anti-BTLA antibody or antigen-binding fragment thereof described in any one of the above embodiments, particularly hu17, hu18, hu19, or an antigen-binding fragment thereof described herein.

[0095] In some embodiments, the pharmaceutical composition of the present invention further comprises a stabilizer. Preferably, the stabilizer is selected from one or more of sodium chloride, mannitol, sorbitol, sucrose, maltose, xylitol, and trehalose. Preferably, the stabilizer in the pharmaceutical composition comprises at least sodium chloride, and optionally comprises one or more of mannitol, sorbitol, sucrose, and trehalose. For example, the pharmaceutical composition may comprise sodium chloride and mannitol, sodium chloride and sorbitol, sodium chloride and sucrose, or sodium chloride and trehalose. When included, the concentration of the stabilizer in the pharmaceutical composition is about 20 to 300 mM, preferably 50 to 300 mM, and more preferably 120 to 250 mM. In some embodiments, the stabilizer is sodium chloride at a concentration of about 50-200 mM, or the stabilizer is mannitol at a concentration of about 100-300 mM, or the stabilizer is sorbitol at a concentration of about 100-300 mM, or the stabilizer is sucrose at a concentration of about 100-300 mM, or the stabilizer is trehalose at a concentration of about 100-300 mM, or the stabilizer is a combination of about 30-100 mM sodium chloride and about 50-200 mM mannitol, or a combination of about 30-100 mM sodium chloride and about 50-200 mM sorbitol, or a combination of about 30-100 mM sodium chloride and about 50-200 mM sucrose, or a combination of about 30-100 mM sodium chloride and about 50-200 mM trehalose.

[0096] Therefore, in some embodiments, the pharmaceutical composition of the present invention comprises a histidine-histidine hydrochloride buffer solution having a pH value of 5.0 to 6.0 and a concentration in the pharmaceutical composition of 10 to 30 mM, 15 to 25 mg / mL of an anti-BTLA antibody or antigen-binding fragment thereof described in any one of the above embodiments, particularly hu17, hu18, hu19, or an antigen-binding fragment thereof described herein, and 20 to 300 mM of a stabilizer. Preferably, the stabilizer contains at least sodium chloride and optionally contains one of mannitol, sorbitol, sucrose, and trehalose, and is preferably 50-150 mM sodium chloride, or 40-80 mM sodium chloride and 120-150 mM mannitol, or 40-80 mM sodium chloride and 120-150 mM sorbitol, or 40-80 mM sodium chloride and 120-150 mM sucrose, or 40-80 mM sodium chloride and 120-150 mM trehalose. In some embodiments, the stabilizer is 200-300 mM trehalose.

[0097] In some embodiments, the pharmaceutical composition of the present invention further comprises a surfactant. Preferred surfactants are selected from polysorbate 80, polysorbate 20, and poloxamer 188. The most preferred surfactant is polysorbate 80. Calculated on a w / v basis, the concentration of the surfactant in the pharmaceutical composition of the present invention is about 0.001% to 0.1%, preferably about 0.01% to 0.05%, and preferably about 0.02% to 0.04%. As a non-limiting example, the concentration of the surfactant in the pharmaceutical composition of the present invention is about 0.02%, 0.03%, or 0.04%, and preferably 0.02%.

[0098] Thus, in some embodiments, the pharmaceutical composition of the present invention comprises a histidine-histidine hydrochloride buffer solution having a pH of 5.0-6.0 and a concentration in the pharmaceutical composition of 10-30 mM; 15-25 mg / mL of an anti-BTLA antibody or antigen-binding fragment thereof described in any one of the above embodiments, particularly hu17, hu18, hu19, or an antigen-binding fragment thereof described herein; 20-300 mM of a stabilizer; and 0.02%-0.04% polysorbate 80, calculated on a w / v basis. Preferably, the stabilizer is trehalose, or contains at least sodium chloride and optionally contains one of mannitol, sorbitol, sucrose, and trehalose, and is preferably 200 to 300 mM trehalose, or 50 to 150 mM sodium chloride, or 40 to 80 mM sodium chloride and 120 to 150 mM mannitol, or 40 to 80 mM sodium chloride and 120 to 150 mM sorbitol, or 40 to 80 mM sodium chloride and 120 to 150 mM sucrose, or 40 to 80 mM sodium chloride and 120 to 150 mM trehalose. In a particularly preferred embodiment, the pharmaceutical composition of the present invention comprises or is prepared from the following components: a histidine-histidine hydrochloride buffer solution having a pH of 5.5±0.3 and a concentration of 20±5 mM, 40-80 mM sodium chloride, 120-150 mM trehalose, 0.02-0.04% polysorbate 80 (calculated on a w / v basis), and 15-25 mg / mL of antibody hu18 or an antigen-binding fragment thereof.

[0099] The pharmaceutical compositions of the present invention may be liquid or lyophilized formulations. With respect to liquid formulations, it should be understood that in addition to the buffers, stabilizers, antibodies or antigen-binding fragments thereof, and surfactants described herein, the liquid formulations further contain water for preparing the pharmaceutical compositions.

[0100] <Medicinal Use and Method> The present invention further provides a pharmaceutical composition according to any one of the embodiments of the present invention for treating or preventing a BTLA-mediated disease, a use of a pharmaceutical composition according to any one of the embodiments of the present invention in the preparation of a medicament for treating or preventing a BTLA-mediated disease, and a method of administering a therapeutically effective amount of a pharmaceutical composition according to any one of the embodiments of the present invention to an individual or patient in need thereof for treating or preventing a BTLA-mediated disease.

[0101] In the present invention, a BTLA-mediated disease refers to a disease in which BTLA is involved in the onset and progression of the disease, including, but not limited to, tumors, infectious diseases, inflammation, or autoimmune diseases. Tumors that can be treated and prevented using the pharmaceutical composition of the present invention include melanoma, breast cancer, kidney cancer, prostate cancer, colon cancer, lung cancer, pancreatic cancer, bone cancer, skin cancer, head or neck cancer, uterine cancer, ovarian cancer, rectal cancer, anal cancer, stomach cancer, testicular cancer, esophageal cancer, small intestine cancer, cervical cancer, vaginal cancer, Hodgkin's disease, non-Hodgkin's lymphoma, endocrine system cancer, thyroid cancer, adrenal cancer, soft tissue cancer, urethral cancer, chronic or acute leukemia, and other cancers. cancer, chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, childhood solid tumors, lymphocytic lymphoma, bladder cancer, kidney or ureter cancer, renal pelvis cancer, neoplasms of the central nervous system, primary central nervous system lymphoma, tumor angiogenesis, spinal axis tumors, brain stem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, T-cell lymphoma, environmentally induced cancers including those induced by asbestos, and combinations of the foregoing cancers. Autoimmune diseases that can be treated and prevented by the pharmaceutical composition according to the present invention include organ-specific autoimmune diseases and systemic autoimmune diseases. Organ-specific autoimmune diseases include chronic lymphocytic thyroiditis, hyperthyroidism, insulin-dependent diabetes mellitus, myasthenia gravis, ulcerative colitis, pernicious anemia with chronic atrophic gastritis, pulmonary hemorrhagic nephritic syndrome, pemphigus vulgaris, pemphigoid, primary biliary cirrhosis, multiple encephalomyelitis, and acute idiopathic polyneuropathy. Systemic autoimmune diseases include systemic lupus erythematosus, rheumatoid arthritis, systemic vasculitis, scleroderma, pemphigoid, dermatomyositis, mixed connective tissue disease, autoimmune hemolytic anemia, and ulcerative colitis. [Example]

[0102] The present invention will be described below with reference to specific examples. Note that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Unless otherwise specified, the methods and materials used in the examples are conventional methods and materials in the art.

[0103] Example 1: Screening test of buffer systems and pH values In liquid pharmaceutical compositions, the buffer system and pH value have a significant impact on antibody stability, and each antibody with specific physical and chemical properties has its own optimal buffer type and pH value. The purpose of this example is to screen for the optimal buffer system and pH value so that the anti-BTLA antibodies disclosed in the present invention have optimal stability for clinical applications.

[0104] This experiment was performed with antibody hu18 at concentrations of approximately 10 mg / mL, 20 mg / mL, and 40 mg / mL. The samples were subjected to ultrafiltration, concentration, and liquid exchange using a VIVAFlow 200. After liquid exchange, the samples were placed in the corresponding formulations and placed in sealed centrifuge tubes to screen buffers. Acetate buffer, citrate buffer, and histidine buffer were screened, and the pH values ​​were adjusted to 5.0–6.5 (e.g., as shown in Table 1). Samples were stored at 40°C and removed at weeks 0, 2, and 4 for analysis and measurement. The main pathways of protein degradation are the formation of aggregates, degradation products, and charge variants. Size exclusion chromatography (SEC-HPLC) was used to measure the percentage of native and aggregated antibody, and cation exchange chromatography (CEX-HPLC) was used to measure the percentage of acidic and basic antibody forms. Linear fitting was performed using the SEC-HPLC single content and CEX-HPLC main peak content at the start of the test (0W), 2 weeks (2W), and 4 weeks (4W) of incubation to calculate the decline slope (% / week). The effect of each buffer system and each pH value on the antibody stability of antibody hu18 was examined, and the results are shown in Tables 2 and 3.

[0105] [Table 1-1]

[0106] [Table 1-2] Note: "-" indicates no addition.

[0107] [Table 2]

[0108] [Table 3]

[0109] As shown in Tables 2 and 3, SEC-HPLC analysis showed that after 4 weeks of storage at 40°C, the higher the protein concentration, the greater the decline in the concentration of the monomer. However, in histidine buffer systems with pH values ​​of 5.5 to 6.0, the decline in the concentration of the monomer at each protein concentration was small, with an average decline of ≤0.27% / week. CEX-HPLC analysis showed that the decline in the concentration of the monomer was correlated with the pH, with the decline increasing with increasing pH. However, in buffer systems with pH values ​​of 5.0 to 6.0, the decline in the concentration of the monomer was relatively small, indicating that the correlation between the decline in the concentration of the monomer and the protein was unclear.

[0110] Taking into consideration the product specifications and the target quality attributes of the product (polymer content level), a histidine buffer solution with a protein concentration of 20 mg / ml and a pH value of 5.5 or 6.0 was selected to screen the additive (stabilizer) formulation.

[0111] <Example 2: Screening test of stabilizers> To further investigate the effect of each additive on antibody stability, comparative studies were conducted using formulations containing one or a combination of sodium chloride, mannitol, sorbitol, sucrose, or trehalose. Specifically, each of the additives or their combinations was added to 20 mM histidine buffer or citrate buffer containing approximately 20 mg / mL of antibody hu18. The specific formulation information is shown in Table 4. Each formulation was dispensed and incubated at 40°C. The samples were removed at 0, 2, and 4 weeks for analysis and measurement. The change in antibody hu18 content was measured using molecular exclusion high-performance liquid chromatography (SEC-HPLC), and the main charge peak content of antibody hu18 was measured using weak cation exchange high-performance liquid chromatography (CEX-HPLC). The results are shown in Table 5.

[0112] The stability of the antibodies was evaluated by placing the formulation samples containing each additive at a high temperature of 40°C for two weeks, and all of the antibodies exhibited relatively strong thermal stability.

[0113] Comprehensive analysis of the data and comparison of F13-F17 with F18-F21 revealed that the combination of mannitol, sorbitol, sucrose, and trehalose with sodium chloride was superior to the single additives mannitol, sorbitol, sucrose, and trehalose, with smaller declines in the antibody purity by SEC-HPLC and the main peak content by SEC-HPLC. The histidine buffer system at pH 5.5 was the most stable additive, with a small decline in antibody purity of 0.11% / week and a decline in the main antibody charge of only 3.72% / week, significantly smaller than that at pH 6.0.

[0114] Therefore, the combination of trehalose and sodium chloride at pH 5.5 is more advantageous in terms of product stability. At the same time, the histidine buffer system (F17) at pH 6.0 is significantly superior to the citrate buffer system (F23) at pH 6.0 in terms of the reduction in monounsaturated fatty acid content by SEC-HPLC.

[0115] [Table 4-1]

[0116] [Table 4-2] Note: "-" indicates no addition.

[0117] [Table 5]

[0118] <Example 3: Surfactant screening test> Surfactants added to liquid formulations are generally agents that protect proteins, such as antibodies, from the effects of air / solution interface and solution / surface-induced stress during storage, for example, to reduce antibody aggregation or minimize particulate formation in the formulation, and are beneficial for stabilizing the physical and chemical properties of antibodies. Different concentrations of polysorbate 20 or polysorbate 80 were added to a formulation containing 20 mM histidine buffer (1:1 molar ratio of histidine to histidine hydrochloride, pH 6.0) and 20 mg / ml antibody hu18, and the formulation was incubated at 40°C for 4 weeks before analysis and measurement. The results are shown in Table 6.

[0119] Overall, the results of the surfactant screening tests (F17, F24, F25, and F26) showed that the effects of adding different concentrations of polysorbate 80 or polysorbate 20 on the SEC-HPLC content and the main peak content of SEC-HPLC were unclear.

[0120] [Table 6]

[0121] In summary, various buffer systems, pH values, antibody concentrations, additives, and surfactants were considered to study the stability of the recombinant humanized anti-BTLA monoclonal antibody hu18 and determine the optimal liquid formulation. For antibody hu18, the pH was adjusted using a histidine buffer, trehalose and sodium chloride were used to adjust the formulation's osmotic pressure, and polysorbate 80 was added to improve the formulation's solubility.

[0122] Example 4: Study of long-term stability of formulations Liquid drug products containing therapeutic antibodies usually need to be stored at 2-8°C, so it is very important that the formulation can maintain high stability over long periods of storage. Based on the above screening results, formulation number 27 was designed based on the previous 26 formulations to study the formulation's long-term stability.

[0123] Four lots of the stock solution were selected, and formulation No. 27 shown in Table 7 was prepared and stored in transparent vials. After leaving the solution at 2 to 8°C for 6 months, each sample was analyzed and measured. Stability was evaluated based on the following parameters: (a) appearance by macroscopic observation, (b) insoluble particles (OD 405 nm) measured by light obscuration method, (c) pH value, (d) molecular weight of the antibody measured by CE-SDS (sodium lauryl sulfate capillary electrophoresis), (d) content of the antibody alone (quality standard: ≥ 97.0%), polymer (quality standard: ≤ 3.0%), or fragment (quality standard: ≤ 1.0%) measured by SEC-HPLC, (e) content of the antibody's main charge (quality standard: ≥ 70.0%), acidic charge (quality standard: ≤ 30.0%), or basic charge (quality standard: ≤ 15.0%) measured by CEX-HPLC, (f) binding activity of the antibody measured by ELISA (quality standard: 70% to 130% of the control), and (g) protein content (quality standard: 18 to 22 mg / ml).

[0124] The results showed that when the four batches of concentrate were used in formulation No. 27, there were no significant changes in appearance, pH, insoluble particulates, protein content, purity (SEC-HPLC (molecular exclusion high performance liquid chromatography), CEX-HPLC (weak cation exchange high performance liquid chromatography), R-CE-SDS (reduced electrophoresis), NR-CE-SDS (non-reduced electrophoresis)) and biological activity, all of which are shown in Table 8. The results showed that the above four batches of concentrate had very good stability when used in formulation No. 27 at 2-8°C for 0-24 months.

[0125] [Table 7] NOTE: Histidine buffer is prepared from histidine and histidine hydrochloride in a molar ratio of 1:3.

[0126] [Table 8-1]

[0127] [Table 8-2]

[0128] [Table 8-3]

[0129] [Table 8-4] Note: NA in the table indicates that the detection item is not important at this point and is not detected at the sampling point. NR indicates that the point has not been reached. The same applies below.

[0130] Example 5: Accelerated Stability Study of Formulations Four lots of the stock solution were selected, and formulation No. 27 shown in Table 7 was prepared and stored in transparent vials. After leaving the solution at 25±2°C and 60±5% relative humidity (RH) for 0 to 12 months, each sample was analyzed and measured. As shown in Table 9, formulation No. 27 had even higher stability against proteolysis, and the degradation kinetic parameters measured at 25±2° C. met the requirements for 12-month storage at room temperature.

[0131] [Table 9-1]

[0132] [Table 9-2]

[0133] [Table 9-3] Note: NA in the table indicates that the item is not significant at this point and is not detected at that sampling point. NR indicates that the point has not been reached.

[0134] Example 6: Detection of binding between humanized antibodies and hBTLA by ELISA The binding specificity of humanized antibodies hu17, hu18, and hu19 to hBTLA was detected using a conventional ELISA detection method. A 96-well microplate was coated with 0.5 μg / ml hBTLA and incubated at 37°C for 60 minutes. The wells were then discarded, washed three times with wash buffer, and blocked with 2% BSA in PBS for 60 minutes. After washing three times with wash buffer, a gradient-diluted antibody solution (same components as in Formulation No. 27 except for the antibody) was added and incubated at 37°C for 60 minutes. The plate was then rinsed three times with wash buffer. A 1:10,000 diluted HRP-labeled mouse anti-human IgG4 secondary antibody was then added and incubated at 37°C for 1 hour. After rinsing three times with wash buffer, 100 μl of TMB substrate solution was added for color development. The reaction was allowed to proceed at room temperature for 30 minutes, after which 100 μl of 2 M hydrochloric acid was added to terminate the reaction. The absorbance was measured at 450 nm.

[0135] The EC50 values ​​are shown below in Table 10. The results show that the humanized antibodies hu17, hu18, and hu19 can specifically bind to hBTLA.

[0136] [Table 10]

[0137] Example 7: Detection of binding of humanized antibodies to hBTLA on 293F cells by FACS The binding ability of the humanized anti-BTLA antibodies hu17, hu18, and hu19 to hBTLA expressed in cells was measured by cell-based flow cytometry (FACS). 293F cells expressing hBTLA were digested, centrifuged, and resuspended in FACS buffer. 2.5 × 10 cells were cultured. 4The cells were added to wells of a 96-well round-bottom plate to a volume of 50 μl, and 50 μl of antibody dilutions (starting at 10 μg / ml, titrated 3-fold; all other components except the antibody in the dilution solution were the same as in Formulation No. 27) were added and mixed uniformly. After incubation at room temperature for 30 min, the cells were washed twice with FACS buffer, and then 100 μl of goat anti-human IgG-PE antibody was added. The cells were incubated in the dark for 30 min, washed twice with FACS buffer, and then subjected to FACS detection. The washed cells were resuspended in a 4°C buffer containing propidium iodide (PI) and 0.02% sodium azide to prevent receptor internalization, and then analyzed by flow cytometry. PI-positive cells were excluded from the FSC / SSC gate, and live cells were gated, and their geometric mean fluorescence was measured. Data were analyzed using an S-shaped dose-response model in Prism™ software.

[0138] The EC50 value of each antibody is shown in Table 11 below. The results show that the humanized antibodies hu17, hu18, and hu19 can effectively bind to hBTLA on 293F cells.

[0139] [Table 11]

[0140] Example 8: Detection of blocking effect of humanized antibodies on the binding of BTLA to HVEM by FACS The blocking ability of humanized antibodies hu17, hu18, and hu19 on the binding of hBTLA to hHVEM expressed in 293F cells was determined by cell-based flow cytometry (FACS). Cells stably expressing hHVEM-293F were digested, centrifuged, and resuspended in FACS buffer. The cell volume was 2.5 × 10 4Each well was added to a 96-well round-bottom plate to a volume of 50 μl, and pre-biotinylated hBTLA (1 μg / ml) protein was added and mixed. The mixture was incubated at 4°C for 15 minutes. 50 μl of different concentrations of humanized antibody dilutions (starting at 5 μg / ml, titrated 3-fold; all other components except the antibody in the dilution solution were the same as in formulation #27) were added and mixed uniformly. The mixture was then incubated at room temperature for 30 minutes. After washing the cells twice with FACS buffer, 100 μl of goat anti-human IgG-PE antibody was added and incubated for 30 minutes in a dark place. After washing twice with FACS buffer, the cells were subjected to FACS detection. The washed cells were resuspended in a 4°C buffer containing propidium iodide (PI) and 0.02% sodium azide to prevent receptor internalization, and then analyzed by flow cytometry. PI-positive cells were excluded from the FSC / SSC gate, and live cells were gated and their geometric mean fluorescence was measured. Prism TM The data were analyzed using the S-shaped dose-response model of the software.

[0141] The IC50 value of each antibody is shown in Table 12 below. The results show that the humanized antibodies hu17, hu18, and hu19 can effectively block the binding of BTLA to HVEM on the cell surface.

[0142] [Table 12]

[0143] Example 9: Promotion of T cell activation by humanized anti-BTLA antibodies CHO cells stably expressing hPD-L1 / hHVEM were plated in a 96-well plate, with the cell volume per well being 5 × 10 4The cells were cultured overnight at 37°C and 7% CO2, the cell supernatant was removed, and 40 μl of humanized anti-BTLA antibody dilution solution (starting concentration 60 μg / ml, diluted 3-fold, the other components except for the antibody in the dilution solution are the same as in Formulation No. 27) was added to each well, and 40 μl of Jurkat reporter cells capable of persistently expressing hPD-1 / hBTLA / NFAT-luciferase were added to bring the total cell number to 1 × 10 5 The cells were cultured at 37°C and 7% CO2 for 6 hours, luciferase reagent was added, and luminescence was detected using a microplate reader.

[0144] The EC50 value of each antibody is shown in Table 13 below. The results show that the humanized anti-BTLA antibodies hu17, hu18, and hu19 can effectively promote T cell activation.

[0145] [Table 13]

[0146] Example 10: Affinity of humanized anti-BTLA antibodies to hBTLA Detection tests were performed using a GE Healthcare Life Sciences Biacore T200 instrument. A Series S CM5 chip was inserted into the instrument and HBS-EP+ (10 mM HEPES, pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.05% surfactant P20) was used as the system buffer. BTLA-Fc antigen was bound to the chip detection channel by injecting a mixture of 400 mM EDC and 100 mM NHS at 10 μL / min for 420 s over the activated chip surface. BTLA-Fc antigen was then diluted in 10 mM sodium acetate / acetic acid (pH 5.5) buffer to a final concentration of 20 μg / mL and injected at 10 μL / min for binding. It was then blocked by injecting 1 M ethanolamine-HCl solution (pH 8.5) at 10 μL / min for 420 s.

[0147] The antibody was diluted two-fold using the Biacore buffer (other components in the diluent were the same as those in Formulation No. 27, except for the antibody), resulting in a total of six concentration points. The concentration gradient was 24 nM, 12 nM, 6 nM, 3 nM, 1.5 nM, and 0.75 nM, with 24 nM being a replicate. Data analysis was performed using the Biacore T200 Evaluation Software (version 3.0) manufactured by GE Healthcare Life Sciences. A 1:1 Binding model was used for data fitting. The fitting yielded the reaction rate constants for the binding between the antibody and the antigen: the binding rate Ka (1 / Ms), the dissociation rate Kd (1 / s), and the affinity constant KD (M). The results are shown in Table 14.

[0148] [Table 14]

[0149] Example 11: Characterization of the kinetics of binding of humanized antibodies to BTLA from various species To detect cross-reactivity between chimeric antibodies and cynomolgus monkey-derived and mouse-derived BTLA, a ForteBio assay was used. Briefly, human BTLA, cynomolgus monkey-derived BTLA, or mouse-derived BTLA was coupled to an activated CM5 biological sensor chip to achieve approximately 100-200 response units (RU), and then unreacted groups were blocked with 1M ethanolamine. Humanized antibody samples (hu17 antibody, other components identical to formulation #27) were injected at increasing concentrations from 0.12 nM to 90 nM in SPR running buffer at 30 times per minute. Binding responses for different species of BTLA were calculated by subtracting the RU from the blank flow chamber.

[0150] The results are shown in Figure 1. Comparison of the binding of hu17 to BTLA from various species reveals that hu17 not only has high affinity for human BTLA, but also has similar affinity to cynomolgus monkey-derived BTLA, but hardly binds to mouse BTLA.

[0151] Example 12: Inhibitory effect of humanized antibody (formulation number: 27) on tumor growth in mice Mouse colon carcinoma (MC38) cells (ATCC) were electrotransfected with the Hxp-hHVEM plasmid to establish an MC38-hHVEM cell bank. Subsequently, the cells were subcloned by limiting dilution and single clones were screened by flow cytometry to obtain MC38-HVEM cells. The MC38-hHVEM cells were then cultured at 1 × 10 6 The tumor was subcutaneously inoculated into the right flank of a B-hBTLA-humanized female mouse at a concentration of 0.1 ml / 100 cells / 0.1 mL. 3 Once tumors reached the tumor volume, they were randomly assigned to five groups, each consisting of eight mice, based on tumor volume. These groups were: G1, a 0.9% sodium chloride injection vehicle control group; G2, a KLH (anti-keyhole limpet hemocyanin antibody) (10 mg / kg) negative control group; G3, G4, and G5, a hu18 (1 mg / kg) group. All groups were administered intraperitoneally, with two doses administered weekly for a total of seven consecutive doses. The study was terminated four days after the final dose. Tumor volume and body weight were measured twice weekly, and mouse weights and tumor volumes were recorded. At the end of the study, the animals were euthanized, and tumors were excised, weighed, and photographed to determine the relative tumor inhibition rate (TGI%, TGI% = (1-(T i -T0) / (V i -V0)) × 100%, T i Tumor volume at the end of treatment, tumor volume at the start of treatment in the T0 treatment group, V i The tumor volume at the end of administration in the blank group, V0 (tumor volume at the start of administration in the blank group) was calculated.

[0152] The effects of each test product on tumor volume in MC38-hHVEM cell-implanted B-hBTLA mice are shown in Table 15 and Figure 2. 21 days after the first administration, the mean tumor volume in the KLH (10 mg / kg) negative control group was 1560 ± 256 mm. 3 The mean tumor volumes of the other treatment groups were 1073 ± 224 mm 3 , 747±268mm 3 , and 868±211mm 3When comparing each treatment group with the KLH negative control group, the TGI% was 33.7%, 56.4%, and 48.0%, respectively, with P values ​​of 0.175, 0.046, and 0.056, respectively, indicating that the test drug hu18 had a certain inhibitory effect on tumor growth.

[0153] [Table 15] Note: a: Mean ± standard error; b: Tumor volumes in the treated group and the KLH-negative control group were statistically compared after 21 days of treatment, and t-test was performed.

[0154] The effects of each test article on the body weight of MC38-hHVEM cell-transplanted B-hBTLA mice are shown in Table 16 and Figure 3. All test animals were active and ate well throughout the treatment period, and a certain degree of weight increase was observed in the animals of each treatment group. No test animals died during the test period. After 21 days of treatment, there was no significant change in the body weight of the mice in each treatment group compared with the body weight of the KLH negative control group (P>0.05), indicating that the test animals tolerated the test article well.

[0155] [Table 16] Note: a: Mean ± standard error; b: The weight of the treated group was statistically compared with that of the KLH negative control group 21 days after administration, and t-test was performed.

[0156] Example 13: Humanized antibody hu18 (formulation number: 27) had no ADCC effector function Antibody binding to cell surface target proteins and subsequent ligation with Fcγ receptors (FcγRs) expressed on effector cells triggers ADCC. It has been clearly documented that human IgG1 exhibits significantly higher binding affinity to FcγRs, particularly FcγR-I and FcγR-IIIA, than IgG4. This affinity correlates with the potency of IgG1 to activate ADCC. Regarding ADCC, antibodies crosslink cell surface targets and C1q protein, which then activates CDC during the cascade of complement complex formation and target cell lysis. As a surrogate for ADCC and CDC, detection of antibody binding to FcγRs and C1q can serve as a basic indicator of ADCC and CDCC. Therefore, the present invention used the biacore T200 (GE) to evaluate the kinetic affinity of monoclonal antibody binding to major FcγRs.

[0157] GE anti-His antibodies were immobilized on a sensor wafer. Various Fc receptors, including recombinant human FcγRIIA (CD16a) V176, recombinant human FcγRIIA (CD32a) V167, recombinant human FcγRI (CD64), and recombinant human FcRn, were captured and analyzed by injecting a series of diluted recombinant human anti-BTLA antibodies (i.e., hu18) and measuring the binding properties of the interactions. hu18 is an IgG4 subtype antibody, while hu18-IgG1, an IgG1 subtype antibody that shares the same Fab fragment as hu18, served as a positive control.

[0158] As a result, as shown in Table 17, the IgG4 subtype recombinant human anti-BTLA antibody exhibited weaker binding to Fc receptors than the IgG1 subtype control antibody, and the hu18 antibody exhibited 400-fold weaker interaction with FcγRIIA (CD16a)V176 than the IgG1 subtype control antibody. This indicated that hu18 had low or no ADCC effector activity. As shown in Table 17, the hu18 antibody did not bind to C1q, whereas the hu18-IgG1 antibody could bind to C1q.

[0159] [Table 17]

[0160] Table 18

Claims

1. (1) a buffer solution; (2) an anti-BTLA antibody or an antigen-binding fragment thereof; (3) a stabilizer; (4) A pharmaceutical composition comprising a surfactant, the buffer solution is a histidine buffer solution having a pH value of 5.5 to 6.0; the anti-BTLA antibody has a heavy chain amino acid sequence set forth in SEQ ID NO: 11 and a light chain amino acid sequence set forth in SEQ ID NO: 12; the concentration of the anti-BTLA antibody or antigen-binding fragment thereof is 5 to 100 mg / mL; the stabilizer is a combination of 30 to 100 mM sodium chloride and 50 to 200 mM mannitol, a combination of 30 to 100 mM sodium chloride and 50 to 200 mM sorbitol, a combination of 30 to 100 mM sodium chloride and 50 to 200 mM sucrose, or a combination of 30 to 100 mM sodium chloride and 50 to 200 mM trehalose; The pharmaceutical composition, wherein the surfactant is selected from polysorbate 80 and polysorbate 20, and the concentration of the surfactant is 0.01% to 0.1%.

2. 2. The pharmaceutical composition of claim 1, wherein the buffer is a histidine-hydrochloride buffer.

3. 2. The pharmaceutical composition of claim 1, wherein the buffer is a histidine-histidine hydrochloride buffer.

4. 2. The pharmaceutical composition according to claim 1, wherein the concentration of the buffer solution is 10 to 30 mM.

5. 2. The pharmaceutical composition of claim 1, wherein the stabilizer is a combination of 30-100 mM sodium chloride and 50-200 mM trehalose.

6. 2. The pharmaceutical composition of claim 1, wherein the surfactant is polysorbate 80.

7. 2. The pharmaceutical composition of claim 1, wherein the concentration of the surfactant is 0.01% to 0.05%.

8. 2. The pharmaceutical composition of claim 1, wherein the concentration of the anti-BTLA antibody or antigen-binding fragment thereof is 10 to 50 mg / mL.

9. 2. The pharmaceutical composition of claim 1, wherein the concentration of the anti-BTLA antibody or antigen-binding fragment thereof is 15 to 25 mg / mL.

10. The composition contains a component shown in any one of the following items (1) to (8), or is prepared from a component shown in any one of the following items (1) to (8), namely: (1) (a) 10 mg / mL to 50 mg / mL of an anti-BTLA antibody or antigen-binding fragment thereof; (b) 10 to 30 mM histidine buffer having a pH value of 5.5 to 6.0; (c) a combination of 30 mM to 100 mM sodium chloride and 50 to 200 mM trehalose; (d) 0.01% to 0.05% polysorbate 80; (2) (a) 10 mg / mL to 50 mg / mL of an anti-BTLA antibody or antigen-binding fragment thereof; (b) 10 to 30 mM histidine buffer having a pH value of 5.5 to 6.0; (c) a combination of 30 mM to 100 mM sodium chloride and 50 to 200 mM mannitol; (d) 0.01% to 0.05% polysorbate 80; (3) (a) 10 mg / mL to 50 mg / mL of an anti-BTLA antibody or antigen-binding fragment thereof; (b) 10 to 30 mM histidine buffer having a pH value of 5.5 to 6.0; (c) a combination of 30 mM to 100 mM sodium chloride and 50 to 200 mM sucrose; (d) 0.01% to 0.05% polysorbate 80; (4) (a) 10 mg / mL to 50 mg / mL of an anti-BTLA antibody or antigen-binding fragment thereof; (b) 10 to 30 mM histidine buffer having a pH value of 5.5 to 6.0; (c) a combination of 30 mM to 100 mM sodium chloride and 50 to 200 mM sorbitol; (d) 0.01% to 0.05% polysorbate 80; (5) (a) 20 mg / mL anti-BTLA antibody or antigen-binding fragment thereof; (b) 20 mM histidine buffer having a pH value of 6.0; (c) a combination of 54 mM sodium chloride and 132 mM trehalose; (d) 0.02% polysorbate 80; (6) (a) 20 mg / mL anti-BTLA antibody or antigen-binding fragment thereof; (b) 20 mM histidine buffer having a pH value of 5.5; (c) a combination of 50 mM sodium chloride and 140 mM mannitol; and (d) 0.02% polysorbate 80. (7) (a) 20 mg / mL anti-BTLA antibody or antigen-binding fragment thereof; (b) 20 mM histidine buffer having a pH value of 5.5; (c) a combination of 50 mM sodium chloride and 140 mM trehalose; (d) 0.02% polysorbate 80; (8) The pharmaceutical composition of claim 1, comprising: (a) 20 mg / mL of an anti-BTLA antibody or antigen-binding fragment thereof; (b) 20 mM histidine buffer having a pH value of 5.0 to 6.0; (c) a combination of 50 mM sodium chloride and 140 mM trehalose; and (d) 0.02% polysorbate 80.

11. 10. The pharmaceutical composition of claim 1, comprising or prepared from the following components: a histidine-histidine hydrochloride buffer solution having a pH of 5.5±0.3 and a concentration of 20±5 mM; 40-80 mM sodium chloride; 120-150 mM trehalose; 0.02-0.04% polysorbate 80, calculated on a w / v basis; and 15-25 mg / mL of an anti-BTLA antibody or antigen-binding fragment thereof, wherein the anti-BTLA antibody has a heavy chain amino acid sequence set forth in SEQ ID NO: 11 and a light chain amino acid sequence set forth in SEQ ID NO:

12.

12. Use of the pharmaceutical composition of any one of claims 1 to 11 in the preparation of a medicament for treating or preventing a BTLA-mediated disease.

13. The use according to claim 12, wherein the disease is a tumor, an infectious disease or an autoimmune disease.

14. The tumors include melanoma, breast cancer, kidney cancer, prostate cancer, colon cancer, lung cancer, pancreatic cancer, bone cancer, skin cancer, head or neck cancer, uterine cancer, ovarian cancer, rectal cancer, anal cancer, stomach cancer, testicular cancer, esophageal cancer, small intestine cancer, cervical cancer, vaginal cancer, Hodgkin's disease, non-Hodgkin's lymphoma, endocrine system cancer, thyroid cancer, adrenal gland cancer, soft tissue cancer, urethral cancer, chronic or acute leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, childhood solid tumor, lymphocytic lymphoma, bladder cancer, kidney or ureter cancer, renal pelvis cancer, neoplasms of the central nervous system, primary central nervous system lymphoma, tumor angiogenesis, spinal axis tumor, brain stem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, T-cell lymphoma, and environmentally induced cancers. The autoimmune disease includes organ-specific autoimmune diseases and systemic autoimmune diseases; The organ-specific autoimmune diseases include chronic lymphocytic thyroiditis, hyperthyroidism, insulin-dependent diabetes mellitus, myasthenia gravis, ulcerative colitis, pernicious anemia with chronic atrophic gastritis, pulmonary hemorrhagic nephritic syndrome, pemphigus vulgaris, pemphigoid, primary biliary cirrhosis, multiple cerebrospinal sclerosis, and acute idiopathic polyneuropathy; The use according to claim 13, wherein the systemic autoimmune disease comprises systemic lupus erythematosus, rheumatoid arthritis, systemic vasculitis, scleroderma, pemphigoid, dermatomyositis, mixed connective tissue disease, autoimmune hemolytic anemia, and ulcerative colitis.

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