Anti-BTLA antibody pharmaceutical composition and use thereof

Abstract

Provided are a stable anti-BTLA (B and T lymphocyte attenuator) antibody pharmaceutical composition and its application in medicine. The pharmaceutical composition contains an anti-BTLA antibody, a buffer, and may also contain at least one stabilizer, and optionally may also contain a surfactant.

Description

Technical Field

[0001] The present invention relates to the field of therapeutic pharmaceutical compositions, and in particular to the field of pharmaceutical preparations, wherein the pharmaceutical composition comprises a humanized antibody that specifically binds to B and T lymphocyte attenuators (BTLA). Background Art

[0002] Positive and negative co-stimulatory signals play a decisive role in the regulation of B cell and T cell activity, and the molecules that mediate these signals have been shown to be effective targets for immunomodulators. In addition to T cell receptor (TCR) engagement, optimal activation of naive T cells also requires positive co-stimulation, while negative co-stimulation is believed to be required for the acquisition of autoimmune tolerance and the termination of effector T cell function. When interacting with B7.1 or B7.2 on the surface of antigen presenting cells (APCs), the prototypical T cell co-stimulatory molecule CD28 sends signals that promote T cell proliferation and differentiation in response to TCR engagement, but the CD28 homolog cytotoxic T lymphocyte antigen-4 (CTLA-4) mediates the inhibition of T cell proliferation and effector function (Chambers et al., Ann. Rev. Immunol., 19: 565-594, 2001; Egen et al., Nature Immunol, 3: 611-618, 2002). Several new molecules homologous to the B7 family have been discovered (Abbas et al., Nat. Med., 5:1345-6, 1999; Coyle et al., Nat. Immunol., 2:203-9, 2001; Carreno et al., Annu. Rev. Immunol., 20:29-53, 2002; Liang et al., Curr. Opin. Immunol., 14:384-90, 2002), and their roles in T cell activation are beginning to be elucidated.

[0003] B and T lymphocyte attenuators (BTLA) are members of the CD28 family, which also includes CD28, ICOS, CTLA-4 and PD-1. Based on the functional effects of adding monoclonal antibodies to increase T cell proliferation, it was found that the original members of the family, CD28 and ICOS, have immune activation effects (Hutloff et al., 1999). BTLA, CTLA-4 and PD-1 are described as negative regulatory proteins. Several in vivo studies have confirmed the inhibitory effect of BTLA in lymphocyte responses. BTLA-deficient mice prepared by Murphy and colleagues (Washington University St. Louis) showed a 3-fold increase in IgG produced in response to T-dependent antigens. In addition, T cells and B cells isolated from BTLA-mice showed a greater proliferation response to antigen-receptor stimulation using CD3- and anti-IgM, respectively (Watanabe, 2003). In overexpression studies, BTLA was found to associate with B cell receptor complexes and with T cell receptors. Consistent with the results of this study, antigen-receptor-independent stimulation with ConA (T cells) or LPS (B cells) was not affected in BTLA-deficient lymphocytes and could not be modulated with anti-BTLA antibodies. BTLA knockout mice have been shown to develop spontaneous autoimmune disease over time and have a shortened lifespan (Oya, 2008). BTLA knockout mice show increased disease severity in models of autoimmune encephalomyelitis (EAE) and allergic airway inflammation, both of which rely on T cell activity (Watanabe, 2005; Deppong, 2006).

[0004] It has been shown that herpes virus entry mediator (HVEM) is a ligand for BTLA (Scully et al., 2005). HVEM is a type I transmembrane glycoprotein and a member of the TNF receptor superfamily with four extracellular cysteine-rich regions (CDRs) containing six pseudo-repeated cysteines. BTLA and HVEM regulate the functions of T cells and APCs mainly through dynamic expression on the cell surface. BTLA binding to the ligand not only inhibits T cell proliferation and downregulates the T cell activation marker CD25, but also inhibits the production of IFN-γ, IL-2, IL-4, and IL-10, but cannot induce cell apoptosis. HVEM binding to BTLA leads to downregulation of T cell activation and proliferation (Sedy, 2005). These research results indicate that the expression of BTLA or the binding of BTLA-HVEM is closely related to the activation and proliferation of T cells.

[0005] Antibodies can be used as therapeutic drugs. Certain antibodies can cause unwanted antibody immunogenicity when used as therapeutic drugs in vivo. Because most monoclonal antibodies are derived from rodents, repeated use in humans leads to the production of immune responses against therapeutic antibodies (e.g., human anti-mouse antibodies or HAMA). Such immune responses at least lead to loss of therapeutic efficacy, and at best lead to potentially lethal allergic reactions. One method of reducing the immunogenicity of rodent antibodies includes the production of chimeric antibodies in which mouse variable regions (Fv) are fused to human constant regions (Liu et al. (1987) Proc. Natl. Acad. Sci. USA 84: 3439-43). However, mice injected with hybrids of human variable regions and mouse constant regions developed strong antibody responses against human variable regions, indicating that the retention of intact rodent Fv regions in such chimeric antibodies can still cause harmful immunogenicity in patients.

[0006] In addition, grafting the complementarity determining region (CDR) loops of rodent variable domains onto a human framework (i.e., humanization) has been used to further minimize rodent sequences. Jones et al. (1986) Nature 321:522; Verhoeyen et al. (1988) Science 239:1534. However, CDR loop swapping still does not uniformly produce antibodies with the same binding properties as the starting antibody. In humanized antibodies, framework residues (FR) (residues involved in CDR loop support) are often also required to maintain antigen binding affinity. Kabat et al. (1991) J. Immunol. 147:1709. Although the use of CDR grafting and framework residue retention in many humanized antibody constructs has been reported, it is difficult to predict whether a particular sequence will produce an antibody with the desired binding properties and, occasionally, biological properties. See, e.g., Queen et al., (1989) Proc. Natl. Acad. Sci. USA 86:10029; Gorman et al., (1991) Proc. Natl. Acad. Sci. USA 88:4181; and Hodgson, (1991) Biotechnology (NY), 9:421-5. In addition, most existing studies have used different human sequences for the animal light and heavy chain variable sequences, rendering the predictability of such studies questionable. Sequences of known antibodies have been used, or more commonly, sequences of antibodies with known X-ray crystal structures, such as antibodies NEW and KOL. See, e.g., 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 disorders, autoimmune disorders, and proliferative disorders. Such antibodies may preferably have low immunogenicity in human subjects, allowing repeated administration without adverse immune responses.

[0008] Therefore, there is a need in the art for protein formulations with high stability. Summary of the invention

[0009] The pharmaceutical composition of the present invention is a highly stable pharmaceutical composition containing human antibodies that specifically bind to BTLA. In particular, the present invention finds 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 an antigen-binding fragment thereof.

[0011] In some embodiments, the above-mentioned anti-BTLA antibody or its antigen-binding fragment has HCDR1, HCDR2 and HCDR3 with amino acid sequences as shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, respectively, and LCDR1, LCDR2 and LCDR3 with amino acid sequences as shown 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 its antigen-binding fragment in the pharmaceutical composition is about 1-200 mg / mL, preferably about 5-100 mg / mL, preferably about 10-50 mg / mL; more preferably, the concentration of the above-mentioned anti-BTLA antibody or its antigen-binding fragment 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, preferably about 10 mg / mL, 20 mg / mL, 40 mg / mL.

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

[0014] In some embodiments, the above buffer is a histidine buffer, preferably, the histidine buffer is selected from histidine-hydrochloride buffer or histidine-acetate buffer, preferably histidine-hydrochloride buffer.

[0015] In some embodiments, the above-mentioned histidine-hydrochloride buffer is composed of histidine and histidine hydrochloride, preferably L-histidine and L-histidine monohydrochloride. In some embodiments, the histidine buffer is made of 1-20mM L-histidine and 1-20mM L-histidine monohydrochloride. In some embodiments, the histidine buffer is composed of histidine and histidine hydrochloride in a molar ratio of 1:1 to 1:4. In some embodiments, the histidine buffer is composed of histidine and histidine hydrochloride in a molar ratio of 1:1. In some embodiments, the histidine buffer is composed of histidine and histidine hydrochloride in a molar ratio of 1:3. In some embodiments, the histidine preparation is: a histidine buffer with a pH of 5.5 made of 4.5mM L-histidine and 15.5mM L-histidine monohydrochloride. In some embodiments, the histidine preparation is: a histidine buffer with a pH of 6.0 made of 15mM histidine and 15mM histidine hydrochloride.

[0016] In some embodiments, the above-mentioned histidine buffer is a histidine-acetate buffer, preferably, the molar ratio of the two is 1:1 to 1.5:1, preferably, the pH of such buffer is 5.5±0.3, preferably about 5.5, preferably, such buffer contains 15-20mM histidine and 12-15mM acetic acid.

[0017] In some embodiments, the above buffer is an acetate buffer, preferably, the acetate buffer is an acetate-sodium acetate buffer or an acetate-potassium acetate buffer, preferably an acetate-sodium acetate buffer.

[0018] In some embodiments, the buffer is a citric acid buffer, and preferably, the citric acid buffer is a citric acid-sodium citrate buffer.

[0019] In some embodiments, the concentration of the above buffer is about 1-100mM, preferably about 5-50mM, preferably about 10-30mM, preferably about 20-30mM; preferably about 10-20mM, and a non-limiting example of the concentration of the above buffer is about 5mM, 10mM, 15mM, 20mM, 25mM, 30mM, 40mM, 45mM, 50mM, 55mM, 60mM, 65mM, 70mM, 75mM, 80mM, 85mM, 90mM, 95mM or 100mM or a range formed by any two values ​​within these ranges as endpoints, preferably 10mM, 20mM or 30mM.

[0020] In some embodiments, the pH of the above buffer is about 5.0-6.5, preferably about 5.0-6.0, preferably about 5.5-6.5, preferably about 5.0-5.5, preferably about 5.5-6.0, preferably about 6.0-6.5, and non-limiting examples of the pH of the above buffer 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.

[0021] In some embodiments, the pharmaceutical composition further comprises a stabilizer, wherein the stabilizer is 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.

[0022] 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.

[0023] 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; the stabilizer is a combination of about 30-100 mM sodium chloride and about 50-200 mM sorbitol; the stabilizer is a combination of about 30-100 mM sodium chloride and about 50-200 mM sucrose; the stabilizer is a combination of about 30-100 mM sodium chloride and about 50-200 mM trehalose.

[0024] In some embodiments, the stabilizer is sodium chloride. In some embodiments, the stabilizer is sodium chloride at a concentration of about 50-200 mM, preferably about 100-190 mM, preferably about 120-180 mM, preferably about 130-170 mM, preferably about 130-150 mM, and 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, preferably 135 mM or 140 mM.

[0025] In some embodiments, the stabilizer is mannitol. In some embodiments, the stabilizer is mannitol at a concentration of about 100-300 mM, preferably about 150-300 mM, preferably about 200-280 mM, and a non-limiting example of the concentration of mannitol is about 200 mM, 210 mM, 220 mM, 230 mM, 240 mM, 250 mM, 260 mM, 270 mM, 280 mM, preferably 240 mM.

[0026] 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, preferably about 200-280 mM, and a non-limiting example of the sorbitol concentration is about 200 mM, 210 mM, 220 mM, 230 mM, 240 mM, 250 mM, 260 mM, 270 mM, 280 mM, preferably 240 mM.

[0027] In some embodiments, the stabilizer is sucrose. In some embodiments, the stabilizer is sucrose at a concentration of about 100-300 mM, preferably about 150-300 mM, preferably about 200-280 mM, and a non-limiting example of the sucrose concentration is about 200 mM, 210 mM, 220 mM, 230 mM, 240 mM, 250 mM, 260 mM, 270 mM, 280 mM, preferably 220 mM.

[0028] 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, preferably about 200-280 mM, and a non-limiting example of the concentration of trehalose is about 180 mM, 200 mM, 210 mM, 220 mM, 230 mM, 240 mM, 250 mM, 260 mM, 270 mM, 280 mM, preferably 220 mM.

[0029] In some embodiments, the stabilizer is a combination of sodium chloride and mannitol. In some embodiments, the stabilizer is a combination of about 30-100mM sodium chloride and about 50-200mM mannitol, preferably a combination of about 40-80mM sodium chloride and about 100-180mM mannitol, preferably a combination of about 40-60mM sodium chloride and about 120-160mM mannitol, and a non-limiting example of the stabilizer is a combination of about 54mM sodium chloride and about 144mM mannitol, and a combination of about 50mM sodium chloride and about 140mM mannitol.

[0030] 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 a combination of about 40-80 mM sodium chloride and about 100-180 mM sorbitol, preferably a combination of about 40-60 mM sodium chloride and about 120-160 mM sorbitol, and non-limiting examples of the stabilizer are 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.

[0031] In some embodiments, the stabilizer is a combination of sodium chloride and sucrose. In some embodiments, the stabilizer is a combination of about 30-100mM sodium chloride and about 50-200mM sucrose, preferably a combination of about 40-80mM sodium chloride and about 100-180mM sucrose, preferably a combination of about 40-60mM sodium chloride and about 120-160mM sucrose, and a non-limiting example of the stabilizer is a combination of about 54mM sodium chloride and about 132mM sucrose, and a combination of about 50mM sodium chloride and about 150mM sucrose.

[0032] 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 a combination of about 40-80 mM sodium chloride and about 100-180 mM trehalose, preferably a combination of about 40-60 mM sodium chloride and about 120-160 mM trehalose, and non-limiting examples of the stabilizer are 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, a combination of about 60 mM sodium chloride and about 120 mM trehalose, preferably a combination of about 54 mM sodium chloride and about 132 mM trehalose, or a combination of about 50 mM sodium chloride and about 140 mM trehalose.

[0033] In some embodiments, the pharmaceutical composition further comprises a surfactant selected from polysorbate 80, polysorbate 20 or poloxamer 188.

[0034] In some embodiments, the surfactant is selected from polysorbate 80.

[0035] In some embodiments, the surfactant is selected from polysorbate 20.

[0036] In some embodiments, the concentration of the above-mentioned surfactant is about 0.001%-0.1%, preferably about 0.01%-0.05%, preferably about 0.02%-0.04%, calculated on a w / v basis; as a non-limiting example, the concentration of the above-mentioned surfactant is about 0.02%, 0.03% or 0.04%, preferably 0.02%.

[0037] In some embodiments, the anti-BTLA antibody or antigen-binding fragment thereof is selected from a murine antibody, a chimeric antibody, a humanized antibody, preferably a humanized antibody.

[0038] In some embodiments, the anti-BTLA antibody or antigen-binding fragment thereof has a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO:7, and a light chain variable region with an amino acid sequence as shown in SEQ ID NO:8.

[0039] In some embodiments, the anti-BTLA antibody or antigen-binding fragment thereof has a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO:7, and a light chain variable region with an amino acid sequence as shown in SEQ ID NO:9.

[0040] In some embodiments, the anti-BTLA antibody or antigen-binding fragment thereof has a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO:7, and a light chain variable region with an amino acid sequence as shown in SEQ ID NO:10.

[0041] In some embodiments, the anti-BTLA antibody or antigen-binding fragment thereof has a heavy chain amino acid sequence as shown in SEQ ID NO:11, and a light chain amino acid sequence as shown in SEQ ID NO:12.

[0042] In some embodiments, the above-mentioned pharmaceutical composition comprises the components shown in any one of the following (1)-(8), or is composed of the components shown in any one of the following (1)-(8); wherein the anti-BTLA antibody or antigen-binding fragment thereof is as described in any embodiment of the present invention:

[0043] (1) (a) about 10 mg / mL-50 mg / mL of an anti-BTLA antibody or antigen-binding fragment thereof; (b) about 10-30 mM histidine buffer, pH about 5.0-6.0; (c) a combination of about 30 mM to about 100 mM sodium chloride and about 50-200 mM trehalose; (d) and about 0.01%-0.05% polysorbate 80; or

[0044] (2) (a) about 10 mg / mL to 50 mg / mL of an anti-BTLA antibody or antigen-binding fragment thereof; (b) about 10-30 mM histidine buffer, pH about 5.0-6.0; (c) a combination of about 30 mM to about 100 mM sodium chloride and about 50-200 mM mannitol; (d) and about 0.01% to 0.05% polysorbate 80; or

[0045] (3) (a) about 10 mg / mL-50 mg / mL of an anti-BTLA antibody or antigen-binding fragment thereof; (b) about 10-30 mM histidine buffer, pH about 5.0-6.0; (c) a combination of about 30 mM to about 100 mM sodium chloride and about 50-200 mM sucrose; (d) and about 0.01%-0.05% polysorbate 80; or

[0046] (4) (a) about 10 mg / mL-50 mg / mL of an anti-BTLA antibody or antigen-binding fragment thereof; (b) about 10-30 mM histidine buffer, pH about 5.0-6.0; (c) a combination of about 30 mM to about 100 mM sodium chloride and about 50-200 mM sorbitol; (d) and about 0.01%-0.05% polysorbate 80; or

[0047] (5) (a) about 10 mg / mL-50 mg / mL of an anti-BTLA antibody or an antigen-binding fragment thereof; (b) about 10-30 mM histidine buffer, pH about 5.0-6.0; (c) about 100 mM to about 300 mM trehalose; (d) and about 0.01%-0.05% polysorbate 80;

[0048] (6) (a) about 10 mg / mL-50 mg / mL of an anti-BTLA antibody or an antigen-binding fragment thereof; (b) about 10-30 mM citric acid buffer, pH about 5.5-6.5; (c) about 100 mM to about 300 mM trehalose; (d) and about 0.01%-0.05% polysorbate 80.

[0049] (7) (a) about 10 mg / mL-50 mg / mL of an anti-BTLA antibody or an antigen-binding fragment thereof; (b) about 10-30 mM citric acid buffer, pH about 5.5-6.5; (c) a combination of about 30 mM to about 100 mM sodium chloride and about 50-200 mM mannitol; (d) and about 0.01%-0.05% polysorbate 80.

[0050] (8) (a) about 10 mg / mL-50 mg / mL of an anti-BTLA antibody or an antigen-binding fragment thereof; (b) about 10-30 mM acetate buffer, pH about 5.0-6.0; (c) a combination of about 30 mM to about 100 mM sodium chloride and about 50-200 mM mannitol; (d) and about 0.01%-0.05% polysorbate 80.

[0051] In some embodiments, the pharmaceutical composition comprises or consists of the components shown in any one of the following (9)-(13);

[0052] (9) (a) about 20 mg / mL of an anti-BTLA antibody or antigen-binding fragment thereof; (b) about 20 mM histidine buffer, pH about 6.0; (c) a combination of about 54 mM sodium chloride and about 132 mM trehalose; (d) and about 0.02% polysorbate 80; or

[0053] (10) (a) about 20 mg / mL of an anti-BTLA antibody or antigen-binding fragment thereof; (b) about 20 mM histidine buffer, pH about 5.5; (c) about 220 mM trehalose; (d) and about 0.02% polysorbate 80; or

[0054] (11) (a) about 20 mg / mL of an anti-BTLA antibody or antigen-binding fragment thereof; (b) about 20 mM histidine buffer, pH about 5.5; (c) a combination of about 50 mM sodium chloride and about 140 mM mannitol; (d) and about 0.02% polysorbate 80;

[0055] (12) (a) about 20 mg / mL of an anti-BTLA antibody or antigen-binding fragment thereof; (b) about 20 mM histidine buffer, pH about 5.5; (c) a combination of about 50 mM sodium chloride and about 140 mM trehalose; (d) and about 0.02% polysorbate 80; and

[0056] (13) (a) about 20 mg / mL of an anti-BTLA antibody or an antigen-binding fragment thereof; (b) about 20 mM histidine buffer, pH about 5.0-6.0; (c) a combination of about 50 mM sodium chloride and about 140 mM trehalose; and (d) about 0.02% polysorbate 80.

[0057] In some embodiments, the pharmaceutical composition is a liquid preparation or a lyophilized preparation.

[0058] In some embodiments, the pharmaceutical composition is a liquid preparation, such as an aqueous solution preparation.

[0059] In some embodiments, the liquid formulation 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.

[0060] 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.

[0061] The present invention also provides use of the above pharmaceutical composition in preparing drugs for treating or preventing BTLA-mediated diseases.

[0062] In some embodiments, the above diseases include tumors, infectious diseases, inflammatory or autoimmune diseases. 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, esophageal cancer, endocrine system cancer, thyroid cancer, adrenal love, soft tissue cancer, urethral cancer, chronic or acute leukemia, chronic myeloid leukemia, acute adult Lymphocytic leukemia, chronic lymphocytic leukemia, solid tumors of childhood, lymphocytic lymphoma, bladder cancer, cancer of the kidney or ureter, cancer of the renal pelvis, 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 said 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, Goodpasture's syndrome, pemphigus vulgaris, pemphigoid, primary biliary cirrhosis, multiple sclerosis, acute idiopathic polyneuritis, etc.; the systemic autoimmune diseases include systemic lupus erythematosus, rheumatoid arthritis, systemic vasculitis, scleroderma, pyroptosis, dermatomyositis, mixed connective tissue disease, autoimmune hemolytic anemia, ulcerative colitis, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0063] Figure 1 : Comparative experiment on the binding of humanized antibody hu 17 to BTLA of different species.

[0064] Figure 2 : Effect of hu18 on tumor volume in B-hBTLA mice transplanted with MC38-hHVEM cells.

[0065] Figure 3: Effect of the test article on body weight of B-hBTLA mice transplanted with MC38-hHVEM cells.

[0066] Definition and Description

[0067] In order to more easily understand the present invention, some technical and scientific terms are specifically defined below. Unless otherwise clearly defined in this article, all other technical and scientific terms used herein have the meanings commonly understood by those of ordinary skill in the art to which the present invention belongs. It should be understood that the present invention is not limited to specific methods, reagents, compounds, compositions or biosystems, and certainly the above can be changed. It should also be understood that the terms used in this application are only for the purpose of describing specific embodiments and are not intended to be limited.

[0068] As used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to "a polypeptide" includes combinations of two or more polypeptides, etc.

[0069] The term "pharmaceutical composition" or "preparation" refers to a mixture containing one or more compounds described herein or their pharmaceutically acceptable salts or prodrugs and other components, such as physiologically pharmaceutically acceptable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration to an organism, facilitate the absorption of the active ingredient, and thus exert biological activity.

[0070] The term "liquid formulation" refers to a formulation in a liquid state, and is not intended to refer to a resuspended lyophilized formulation. The liquid formulation of the present invention is stable upon storage, and its stability is not dependent on lyophilization (or other state-changing methods, such as spray drying).

[0071] The term "aqueous liquid formulation" refers to a liquid formulation using water as a solvent. In some embodiments, the aqueous liquid formulation is a formulation that does not require lyophilization, spray drying and / or freezing to maintain stability (eg, chemical and / or physical stability and / or biological activity).

[0072] The term "excipient" refers to an agent that can be added to a formulation to provide desired properties (e.g., consistency, increased 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.

[0073] As used herein, "about" when referring to a measurable value (eg, an amount, a duration, etc.) is intended to encompass variations of ±20% or ±10% relative to the particular value, including ±5%, ±1%, and ±0.1%, as such variations are suitable for performing the disclosed methods.

[0074] The term "buffer pH of about 5.0-6.5" refers to an agent that, through the action of its acid / base conjugate components, renders the solution containing the agent resistant to changes in pH. The buffer used in the formulations of the present invention may have a pH in the range of about 5.0 to about 6.5, or a pH in the range of about 5.5 to about 6.5, or a pH in the range of about 5.0 to about 6.0.

[0075] Herein, examples of "buffers" that control the pH within this range include acetates (e.g., sodium acetate), succinates (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.

[0076] "Histidine buffer" is a buffer containing histidine ions. Examples of histidine buffers include histidine and histidine salts, such as histidine hydrochloride, histidine acetate, histidine phosphate and histidine sulfate, such as a histidine buffer containing histidine and histidine hydrochloride; the histidine buffer of the present invention also includes a histidine buffer containing histidine and acetate (such as sodium salt or potassium salt).

[0077] "Citrate buffer" is a buffer comprising citrate ions. Examples of citrate buffers include citric acid-sodium citrate, citric acid-potassium citrate, citric acid-calcium citrate, citric acid-magnesium citrate, and the like. A preferred citrate buffer is citric acid-sodium citrate buffer.

[0078] "Acetate buffer" is a buffer comprising 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.

[0079] The term "stabilizer" refers to a pharmaceutically acceptable excipient that protects the active pharmaceutical ingredient and / or formulation from chemical and / or physical degradation during manufacture, storage and use. 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, alanine, octopine, strombine) and trimethylamine N-oxide (TMAO), human serum albumin (hsa), bovine serum albumin (bsa), α-casein, globulin, α-lactalbumin, LDH, lysozyme, myoglobin, ovalbumin and RNAaseA. Some stabilizers, such as sodium chloride, calcium chloride, magnesium chloride, mannitol, sorbitol, sucrose, etc., can also play the role of controlling osmotic pressure. The stabilizer specifically used in the present invention is selected from one or more of polyols, salts, and sugars. Preferred salt is sodium chloride, preferred sugar is sucrose and trehalose, and preferred polyols are sorbitol and 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, more preferably sodium chloride-trehalose.

[0080] The term "surfactant" generally includes agents that protect proteins such as antibodies from air / solution interface-induced stress, solution / surface-induced stress to reduce the aggregation of antibodies or minimize the formation of particulate matter in the formulation. Exemplary surfactants include, but are not limited to, nonionic surfactants such as polyoxyethylene sorbitan fatty acid esters (such as polysorbate 20 and polysorbate 80), polyethylene-polypropylene copolymers, polyethylene-polypropylene glycol, polyoxyethylene-stearate, polyoxyethylene alkyl ethers, such as polyoxyethylene monolauryl ether, alkylphenyl polyoxyethylene ether (Triton-X), polyoxyethylene-polyoxypropylene copolymers (poloxamers, Pluronic), sodium lauryl sulfate (SDS).

[0081] The term "isotonic" means that the formulation has substantially the same osmotic pressure as human blood. Isotonic formulations generally have an osmotic pressure of about 250 to 350 mOsm. Isotonicity can be measured using a vapor pressure or freezing point depression osmometer.

[0082] The term "stable" formulation is one in which the antibody substantially maintains its physical stability and / or chemical stability and / or biological activity during the manufacturing process and / or upon storage. A pharmaceutical formulation may be stable even if the antibody contained therein does not retain 100% of its chemical structure or biological function after a period of storage. In some cases, an antibody formulation that maintains about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% of its structure or function after a period of storage may also be considered "stable." Various analytical techniques for measuring protein stability are available in the art and are reviewed in Peptide and Protein Drug Delivery, 247-301, Vincent Lee, ed., Marcel Dekker, Inc., New York, NY, Pubs. (1991), and Jones, A. (1993) Adv. Drug Delivery Rev. 10: 29-90 (both incorporated by reference).

[0083] The stability of the formulation can be measured by determining the percentage of natural antibodies remaining therein (and other methods) after the formulation has been stored at a certain temperature for a certain period of time. Among other methods, the percentage of natural antibodies can be measured by size exclusion chromatography (e.g., size exclusion high performance liquid chromatography [SEC-HPLC]), and "natural" means not aggregated and not degraded. In some embodiments, the stability of the protein is determined by the percentage of monomeric protein in a solution with a low percentage of degradation (e.g., fragmentation) 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 longer, with no more than about 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.1% aggregated form of antibodies.

[0084] Stability can be measured by determining the percentage of antibody that migrates in the more acidic fraction of the main fraction of the antibody (the "major charged form") during ion exchange (among other methods), where stability is inversely proportional to the percentage of the antibody in the acidic form. The percentage of "acidified" antibody can be measured by ion exchange chromatography (e.g., cation exchange high performance liquid chromatography [CEX-HPLC]), among other methods. In some embodiments, an acceptable level of stability means that after a formulation is stored at a certain temperature for a certain period of time, no more than about 49%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1%, 0.5% or 0.1% of the antibody is detectable in the acidic form. The storage period before measuring stability can 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 longer. When evaluating stability, the temperature at which the pharmaceutical preparation is allowed to be stored can be any temperature in the range of about -80°C to about 45°C, such as storage at 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.

[0085] An antibody "retains its physical stability" in the pharmaceutical composition if it shows substantially no signs of, for example, aggregation, precipitation, and / or denaturation when visually inspected for color and / or clarity or when measured by UV light scattering or by size exclusion chromatography. Aggregation is the process by which individual molecules or complexes associate covalently or non-covalently to form aggregates. Aggregation may proceed to the extent that a visible precipitate is formed.

[0086] The stability of the formulation, e.g., physical stability, can be assessed by methods known in the art, including measuring the apparent extinction (absorbance or optical density) of a sample. Such extinction measurements are related to the turbidity of the formulation. The turbidity of a formulation is, in part, an inherent property of the protein dissolved in solution, and is typically measured by turbidimetry and measured in nephelometric turbidity units (NTU).

[0087] The turbidity level that varies with, for example, the concentration of one or more components in the solution (e.g., protein and / or salt concentration) is also referred to as the "emulsification" or "emulsified appearance" of the formulation. The turbidity level can be calculated with reference to a standard curve generated using a suspension of known turbidity. The reference standard for determining the turbidity level of a pharmaceutical composition can be based on 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 less than or equal to the turbidity of a reference suspension having about 3 according to the European Pharmacopoeia standard. Turbidimetric turbidity measurements can detect Rayleigh scattering in the absence of association or non-ideal effects, which generally varies linearly with concentration. Other methods for evaluating physical stability are well known in the art.

[0088] An antibody "retains its chemical stability" in a pharmaceutical composition if its chemical stability at a given time point is such that the antibody is considered to still retain its biological activity as defined below. Chemical stability can be assessed, for example, by detecting or quantifying chemically altered forms of the antibody. Chemical alterations can include size changes (e.g., shortening), which can be assessed using, for example, size exclusion chromatography, SDS-PAGE, and / or matrix-assisted laser desorption ionization / time of flight mass spectrometry (MALDI / TOF MS). Other types of chemical alterations include charge changes (e.g., occurring as a result of deamidation or oxidation), which can be assessed by, for example, ion exchange chromatography.

[0089] An antibody "retains its biological activity" in a pharmaceutical composition if the antibody in the pharmaceutical composition is biologically active for its intended purpose. For example, a formulation of the invention is considered stable if, after storage at a temperature such as 5°C, 25°C, 45°C, etc. for a certain period of time (e.g., 1 to 12 months), the anti-BTLA antibody contained in the formulation binds to BTLA with an affinity that is at least 90%, 95% or more of the binding affinity of the antibody before the storage. Binding affinity can also be determined, for example, by ELISA or plasma resonance techniques.

[0090] In the context of the present invention, in a pharmacological sense, a "therapeutically effective amount" or "effective amount" of an antibody refers to an amount that is effective in preventing or treating or alleviating the 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 the drug that protects a subject from the onset of the disease or promotes disease regression when used alone or in combination with another therapeutic agent, as evidenced by a reduction in the severity of disease symptoms, an increase in the frequency and duration of disease symptom-free periods, or the prevention of damage or disability caused by disease pain. The ability of a drug to promote disease regression can be evaluated using a variety of methods known to those skilled in the art, such as in human subjects during clinical trials, in animal model systems that predict human efficacy, or by measuring the activity of the agent in an in vitro assay. A therapeutically effective amount of a drug includes a "prophylactically effective amount," i.e., any amount of the drug that inhibits the development or recurrence of a disease when given alone or in combination with other therapeutic drugs to a subject at risk of the disease or a subject with a recurring disease.

[0091] The term "subject" or "patient" is intended to include mammalian organisms. 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 transgenic non-human animals. In a specific embodiment of the invention, the subject is a human.

[0092] The terms "administering", "administering" and "treating" refer to introducing a composition comprising a therapeutic agent into a subject using any of a variety of methods or delivery systems known to those skilled 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 administration other than enteral or topical administration, usually by injection, including but not limited to intravenous, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraframe, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcutaneous, intraarticular, subcapsular, subarachnoid, intraspinal, intradural and intrasternal injection and infusion and in vivo electroporation.

[0093] Anti-BTLA antibodies

[0094] The term "antibody" as used herein should be understood to include complete antibody molecules and antigen-binding fragments thereof. The term "antigen-binding portion" or "antigen-binding fragment" of an antibody as used herein (or simply "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 its epitope.

[0095] The term "full-length antibody" or "complete antibody molecule" as used herein refers to an immunoglobulin molecule comprising four peptide chains, two heavy (H) chains (about 50-70 kDa at full length) and two light (L) chains (about 25 kDa at full length) interconnected 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 complementary determining regions (CDRs) and regions separated by more conservative framework regions (FRs). 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 antibodies mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system.

[0096] As used herein, the term "CDR" refers to the complementary determining region within the variable sequence of an antibody. There are three CDRs in each variable region of the heavy and light chains, which are named HCDR1, HCDR2 and HCDR3 or LCDR1, LCDR2 and LCDR3 for each heavy and light chain variable region. The exact boundaries of these CDRs are defined differently in different systems.

[0097] The precise amino acid sequence boundaries of the variable region CDRs of the antibodies of the invention can be determined using any of a number of well-known schemes, including Chothia (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)), based on the three-dimensional structure of the antibody and the topology of the CDR loops, Kabat (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), the international ImMunoGeneTics database (IMGT) (1999 Nucleic Acids Research, 27, 209-212), and the North CDR definition based on affinity propagation clustering using a large number of crystal structures. The CDRs of the antibodies of the present invention can be defined by those skilled in the art according to any scheme in the art (e.g., different assignment systems or combinations).

[0098] As used herein, "antigen-binding fragments" include fragments of antibodies or derivatives thereof, generally including at least one fragment of the antigen-binding region or variable region (e.g., one or more CDRs) of a parent antibody, which retains 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 and multispecific antibodies formed from antibody fragments. When the binding activity of an antibody is expressed on a molar concentration basis, the binding fragment or its derivative generally retains at least 10% of the antigen-binding activity of the parent antibody. Preferably, the binding fragment or its derivative retains at least 20%, 50%, 70%, 80%, 90%, 95%, or 100% or more of the antigen-binding affinity of the parent antibody. It is also contemplated that the antigen-binding fragment of an antibody may include conservative or non-conservative amino acid substitutions that do not significantly change its biological activity (referred to as "conservative variants" or "functional conservative variants" of antibodies).

[0099] The anti-BTLA antibodies or antigen-binding fragments thereof described in the present invention include any one of the anti-BTLA antibodies described in application number CN201810870514.0, and all the contents disclosed herein are incorporated herein by introduction. In some embodiments, the CDR sequences of the antibodies used in the methods and compositions of the present invention include the CDR sequences of the antibody hu18 described in CN201810870514.0. In some embodiments, the CDR sequences of the antibodies used in the methods and compositions of the present invention include the CDR sequences of the antibody hu17 described in CN201810870514.0. In some embodiments, the CDR sequences of the antibodies used in the methods and compositions of the present invention include the CDR sequences of the antibody hu19 described in CN201810870514.0.

[0100] The non-limiting, exemplary antibodies used in the examples herein are selected from hu17, hu18 and hu19 described in CN201810870514.0, all of which are fully humanized antibodies that specifically bind to human BTLA, wherein the antibodies hu17, hu18 and hu19 all have HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, respectively, and LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, respectively; preferably, the antibody hu17 has a heavy chain variable region as shown in SEQ ID NO: 7, respectively, and a light chain variable region as shown in SEQ ID NO: 8, respectively; the antibody hu18 has a heavy chain variable region as shown in SEQ ID NO: 7, respectively, and a light chain variable region as shown in SEQ ID NO: 9, respectively; the antibody hu19 has a heavy chain variable region as shown in SEQ ID NO: 7, respectively, and an amino acid sequence as shown in SEQ ID NO: 9, respectively. Preferably, the antibody hu18 has a heavy chain amino acid sequence as shown in SEQ ID NO: 11 and a light chain amino acid sequence as shown in SEQ ID NO: 12. The CDR amino acid sequence of the above humanized antibody is defined by the IMGT system.

[0101] Pharmaceutical preparations

[0102] The pharmaceutical composition of the present invention is a highly stable pharmaceutical composition containing human antibodies that specifically bind to BTLA. In particular, the present invention finds that the combination of trehalose and sodium chloride can significantly improve the stability of the pharmaceutical composition.

[0103] The present invention provides a pharmaceutical composition comprising: (1) a buffer; and (2) an anti-BTLA antibody or an antigen-binding fragment thereof.

[0104] 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 as shown in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively, and LCDR1, LCDR2, and LCDR3 as shown 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 as shown in SEQ ID NO: 7 and a light chain variable region as shown in SEQ ID NO: 8; or has a heavy chain variable region as shown in SEQ ID NO: 7 and a light chain variable region as shown in SEQ ID NO: 9; or has a heavy chain variable region as shown in SEQ ID NO: 7 and a light chain variable region as shown in SEQ ID NO: 10; more preferably, the antibody in the pharmaceutical composition of the present invention has a heavy chain amino acid sequence as shown in SEQ ID NO: 11 and a light chain amino acid sequence as shown in SEQ ID NO: 12, respectively.

[0105] In the pharmaceutical composition of the present invention, the concentration of the anti-BTLA antibody or its antigen-binding fragment is about 1-200 mg / mL, preferably about 5-100 mg / mL, preferably about 10-50 mg / mL, and more preferably 15-25 mg / mL. As a non-limiting example, the concentration of the anti-BTLA antibody or its antigen-binding fragment 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; preferably about 10 mg / mL, 20 mg / mL or 40 mg / mL.

[0106] The buffer in the pharmaceutical composition of the present invention can be selected from acetate buffer, citrate buffer and histidine buffer to provide the pharmaceutical composition of the present invention with a pH of 5.0 to 6.5, preferably 5.0 to 6.0. On the other hand, the pH of the buffer used in the pharmaceutical composition of the present invention can be 5.0-6.5, preferably 5.0-6.0.

[0107] A particularly preferred buffer in the pharmaceutical composition of the present invention is a histidine buffer. Preferably, the pH of the histidine buffer used in the present invention is 5.0-6.0, more preferably 5.5±0.3, 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 composed of histidine and histidine hydrochloride, preferably L-histidine and L-histidine monohydrochloride. In some embodiments, the histidine buffer is made of 1-20mM L-histidine and 1-20mM L-histidine monohydrochloride. In some embodiments, the histidine buffer is composed of histidine and histidine hydrochloride in a molar ratio of 1:1 to 1:4. In some embodiments, the histidine buffer is composed of histidine and histidine hydrochloride in a molar ratio of 1:1. In some embodiments, the histidine buffer is composed of histidine and histidine hydrochloride in a molar ratio of 1:3. In some embodiments, the histidine preparation is a histidine buffer with a pH of 5.5 made from 4.5 mM L-histidine and 15.5 mM L-histidine monohydrochloride. In some embodiments, the histidine preparation is a histidine buffer with a pH of 6.0 made from 15 mM histidine and 15 mM histidine hydrochloride.

[0108] The histidine buffer in the pharmaceutical composition of the present invention can be a histidine-acetate buffer, preferably, the molar ratio of the two is 1:1 to 1.5:1, preferably, the pH of such buffer is 5.5±0.3, preferably about 5.5, preferably, such buffer contains 15-20mM histidine and 12-15mM acetic acid.

[0109] Therefore, the pharmaceutical composition of the present invention may contain: a histidine-histidine hydrochloride buffer having a pH of 5.0-6.0, the concentration of which in the pharmaceutical composition is 10-30 mM; and 15-25 mg / mL of the anti-BTLA antibody or antigen-binding fragment thereof described in any of the foregoing embodiments, in particular hu17, hu18 or hu19 or antigen-binding fragments thereof described herein.

[0110] In some embodiments, the pharmaceutical composition of the present invention further contains 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 contains at least sodium chloride, and optionally contains one or more of mannitol, sorbitol, sucrose and trehalose. For example, the pharmaceutical composition may contain sodium chloride and mannitol, sodium chloride and sorbitol, sodium chloride and sucrose, or sodium chloride and trehalose. When contained, the concentration of the stabilizer in the pharmaceutical composition is about 20mM-300mM, preferably 50mM-300mM, more preferably 120mM-250mM. In some embodiments, the stabilizer is sodium chloride at a concentration of about 50-200mM; or the stabilizer is mannitol at a concentration of about 100-300mM; or the stabilizer is sorbitol at a concentration of about 100-300mM; or the stabilizer is sucrose at a concentration of about 100-300mM; or the stabilizer is trehalose at a concentration of about 100-300mM; or the stabilizer is a combination of about 30-100mM sodium chloride and about 50-200mM mannitol; the stabilizer is a combination of about 30-100mM sodium chloride and about 50-200mM sorbitol; the stabilizer is a combination of about 30-100mM sodium chloride and about 50-200mM sucrose; the stabilizer is a combination of about 30-100mM sodium chloride and about 50-200mM trehalose.

[0111] Therefore, in some embodiments, the pharmaceutical composition of the present invention contains: a histidine-histidine hydrochloride buffer having a pH of 5.0-6.0, the concentration of which in the pharmaceutical composition is 10-30 mM; 15-25 mg / mL of the anti-BTLA antibody or antigen-binding fragment thereof described in any of the foregoing embodiments, in particular hu17, hu18 or hu19 or antigen-binding fragment thereof described herein; and 20 mM-300 mM of a stabilizer, preferably, the stabilizer comprises at least sodium chloride, optionally comprising one of mannitol, sorbitol, sucrose and trehalose, 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.

[0112] In some embodiments, the pharmaceutical composition of the present invention also includes a surfactant. Preferred surfactants are selected from polysorbate 80, polysorbate 20 and poloxamer 188. The most preferred surfactant is polysorbate 80. In w / v, the concentration of the surfactant in the pharmaceutical composition of the present invention is about 0.001%-0.1%, preferably about 0.01%-0.05%, preferably about 0.02%-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%, preferably 0.02%.

[0113] Therefore, in some embodiments, the pharmaceutical composition of the present invention contains: a histidine-histidine hydrochloride buffer having a pH of 5.0-6.0, the concentration of which in the pharmaceutical composition is 10-30 mM; 15-25 mg / mL of the anti-BTLA antibody or antigen-binding fragment thereof described in any of the above embodiments, in particular hu17, hu18 or hu19 or antigen-binding fragment thereof described herein; 20 mM-300 mM of a stabilizer, preferably, the stabilizer is trehalose, or at least includes sodium chloride, and optionally includes mannitol. One of alcohol, sorbitol, sucrose and trehalose, preferably 200-300mM trehalose, or 50-150mM sodium chloride, or 40-80mM sodium chloride and 120-150mM mannitol, or 40-80mM sodium chloride and 120-150mM sorbitol, or 40-80mM sodium chloride and 120-150mM sucrose, or 40-80mM sodium chloride and 120-150mM trehalose; and 0.02%-0.04% polysorbate 80 by w / v. In a particularly preferred embodiment, the pharmaceutical composition of the present invention contains the following components, or consists of the following components: histidine-histidine hydrochloride buffer with 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 on a w / v basis; 15-25 mg / mL of antibody hu18 or an antigen-binding fragment thereof.

[0114] The pharmaceutical composition of the present invention can be a liquid preparation or a lyophilized preparation. It should be understood that for a liquid preparation, in addition to the buffer, stabilizer, antibody or antigen-binding fragment thereof and surfactant described herein, it also contains water used to prepare the pharmaceutical composition.

[0115] Medical uses and methods

[0116] The present invention also provides a pharmaceutical composition according to any embodiment of the present invention for treating or preventing a BTLA-mediated disease, use of the pharmaceutical composition according to any embodiment 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 the pharmaceutical composition according to any embodiment of the present invention to an individual or patient in need thereof to treat or prevent a BTLA-mediated disease.

[0117] In the present invention, BTLA-mediated diseases refer to diseases in which BTLA participates in the occurrence and development of the disease, including but not limited to tumors, infectious diseases, and inflammatory or autoimmune diseases. In the present invention, tumors suitable for treatment and prevention by 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, gastric cancer, testicular cancer, esophageal cancer, small intestine cancer, cervical cancer, vaginal cancer, Hodgkin's disease, non-Hodgkin's lymphoma, esophageal cancer, endocrine system cancer, thyroid cancer, adrenal love, soft tissue cancer, urethral cancer, chronic or acute leukemia, chronic Myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, solid tumors of childhood, lymphocytic lymphoma, bladder cancer, cancer of the kidney or ureter, cancer of the renal pelvis, 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 said cancers. Autoimmune diseases suitable for treatment and prevention by the pharmaceutical composition of the present invention 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, Goodpasture's syndrome, pemphigus vulgaris, pemphigoid, primary biliary cirrhosis, multiple cerebrospinal sclerosis, acute idiopathic polyneuritis, etc.; the systemic autoimmune diseases include systemic lupus erythematosus, rheumatoid arthritis, systemic vasculitis, scleroderma, pyroptosis, dermatomyositis, mixed connective tissue disease, autoimmune hemolytic anemia, ulcerative colitis, etc. DETAILED DESCRIPTION

[0118] The present invention will be described below in the form of specific examples. It should be understood that these examples are merely illustrative and are not intended to limit the scope of the present invention. The methods and materials used in the examples are, unless otherwise stated, conventional methods and materials in the art.

[0119] Example 1: Buffer system and pH screening experiment

[0120] In liquid pharmaceutical compositions, the buffer system and pH closely affect the stability of the antibody, and each antibody with unique physicochemical properties has the most suitable type of buffer and pH. This example aims to screen an optimal buffer system and pH so that the anti-BTLA antibody disclosed in the present invention has the best stability for clinical application.

[0121] This embodiment is carried out with antibody hu18 at concentrations of about 10 mg / mL, 20 mg / mL and 40 mg / mL. The sample was ultrafiltrated and concentrated using VIVAFlow200 for liquid exchange. After the liquid exchange, the sample was in the corresponding prescription, and the sample was placed in a closed centrifuge tube for buffer screening. Acetate buffer, citrate buffer and histidine buffer were screened, with pH ranging from 5.0 to 6.5 (as shown in Table 1). The samples were placed in a 40°C environment and taken out for analysis and detection at weeks 0, 2 and 4, respectively. The main pathway of protein degradation is the formation of aggregates, cleavage products and charged variants. Size exclusion chromatography (SEC-HPLC) was used to determine the percentage of native form (protein monomer) and aggregated form, and cation exchange chromatography (CEX-HPLC) was used to determine the percentage of acidic and basic forms of antibodies. The SEC-HPLC monomer content and CEX-HPLC main peak content at the start of the experiment (0W), two weeks (2W) and four weeks (4W) were used to fit a straight line and calculate the downward slope (% / week) to investigate the effect of different buffer systems and pH on the stability of antibody hu18. The results are summarized in Tables 2 and 3.

[0122] Table 1: Prescription information for buffer systems and pH screening experiments

[0123]

[0124]

[0125] Note: “-” means not added.

[0126] Table 2: SEC-HPLC monomer content degradation rate in buffer system and pH screening experiment

[0127]

[0128]

[0129] Table 3: Degradation rate of CEX-HPLC charge isomer content in buffer system and pH screening experiment

[0130]

[0131] As shown in Tables 2 and 3, in the SEC-HPLC experimental detection, after being placed at high temperature of 40°C for 4 weeks, the rate of decrease of the monomer content increased with the increase of protein concentration, but the rate of decrease of the monomer content at different protein concentrations was low in the histidine buffer system of pH 5.5-6.0, and the average rate of decrease was ≤0.27% / week; in the CEX-HPLC experimental detection, it can be seen that the degradation rate of the main peak content of CEX-HPLC has a certain correlation with pH. The higher the pH, the faster the degradation rate of the main peak content. In the buffer system of pH 5.0-6.0, the degradation rate of the main peak content is relatively low, and the correlation between the degradation rate of the main peak content and the protein concentration is not obvious.

[0132] Taking into account the product specifications and the target quality attributes of the product (aggregate content level), a histidine buffer with a protein concentration of 20 mg / ml and a pH of 5.5 or 6.0 was selected for excipient (stabilizer) prescription screening.

[0133] Example 2: Stabilizer screening experiment

[0134] In order to further explore the effect of different excipients on antibody stability, we selected sodium chloride, mannitol, sorbitol, sucrose or trehalose or one or a combination of preparations for comparative testing. That is, the above-mentioned different excipients or their combinations were added to 20mM histidine buffer or citric acid buffer containing about 20mg / mL antibody hu18, and the specific prescription information is shown in Table 4. After each prescription preparation was packaged, it was placed at 40°C and taken out for analysis and detection at week 0, week 2, and week 4. The changes in the monomer content of antibody hu18 were detected by molecular exclusion high performance liquid chromatography (SEC-HPLC), and the main peak content of antibody hu18 charge was detected by weak cation high performance liquid chromatography (CEX-HPLC). The results are shown in Table 5.

[0135] According to the results of the stability study, the antibodies of the formulation samples with different excipients showed strong thermal stability after being placed at 40°C for 2 weeks.

[0136] Comprehensive data analysis, comparison of F13-F17 and F18-F21 found that the excipient combination of mannitol, sorbitol, sucrose and trehalose with sodium chloride is superior to the single excipients of mannitol, sorbitol, sucrose and trehalose, which is manifested in the lower degradation rate of the monomer content of SEC-HPLC and the main peak content of CEX-HPLC. Among the excipients containing trehalose, the histidine buffer system at pH 5.5 is the most stable, the decrease rate of antibody monomer purity is as low as 0.11% / week, and the decrease rate of the main charge of the antibody is only 3.72% / week, which is significantly lower than pH 6.0.

[0137] Therefore, the selection of the excipient combination of pH 5.5, trehalose and sodium chloride is more conducive to the stability of the product. At the same time, it can be seen that for the degradation of monomer content in SEC-HPLC, the histidine buffer system at pH 6.0 (F17) is significantly better than the citric acid buffer system at pH 6.0 (F23).

[0138] Table 4: Prescription information in excipient screening experiments

[0139]

[0140]

[0141] Note: “-” means not added.

[0142] Table 5: Summary of excipient screening test results

[0143]

[0144]

[0145] Example 3: Surfactant screening experiment

[0146] Surfactants are often added to liquid preparations to protect proteins such as antibodies from stress induced by the air / solution interface and stress induced by the solution / surface during storage to reduce the aggregation of antibodies or minimize the formation of particles in the preparation, which is conducive to the stability of the physical and chemical properties of antibodies. Different concentrations of polysorbate 20 or polysorbate 80 were added to the preparation containing 20mM histidine buffer (the molar ratio of histidine to histidine hydrochloride was 1:1, pH 6.0) and 20mg / ml of antibody hu18, and the preparation was placed at 40°C for 4 weeks and then analyzed and tested. The results are shown in Table 6.

[0147] Comprehensive analysis showed that the results of surfactant screening tests (F17, F24, F25 and F26) showed that the addition of different concentrations of polysorbate 80 or polysorbate 20 had no significant effect on the monomer content of SEC-HPLC and the main peak content of CEX-HPLC.

[0148] Table 6: Surfactant screening results

[0149]

[0150] In summary, we explored the stability of the recombinant humanized anti-BTLA monoclonal antibody hu18 by investigating different buffer systems, different pH conditions, different antibody concentrations, and different excipient and surfactant compositions, and determined the optimal liquid formulation. Antibody hu18 selected histidine buffer to adjust pH, trehalose and sodium chloride to adjust the osmotic pressure of the preparation, and polysorbate 80 to increase the solubility of the preparation.

[0151] Example 4: Long-term stability study of formulations

[0152] Liquid pharmaceutical products containing therapeutic antibodies usually need to be stored at 2-8°C, so it is very important that the preparation can maintain high stability during long-term storage. Based on the above screening results, we designed prescription No. 27 based on the previous 26 prescriptions to conduct long-term stability research on the preparation.

[0153] Four batches of stock solutions were selected to prepare the No. 27 prescription preparations shown in Table 7, which were stored in transparent vials and placed at 2-8°C for 6 months. After that, each sample was analyzed and tested. The stability was evaluated by the following parameters: (a) visual appearance; (b) light obstruction method for detecting insoluble particles (OD405nm); (c) pH; (d) CE-SDS (sodium dodecyl sulfate capillary electrophoresis) method for detecting the molecular weight of the antibody; (d) SEC-HPLC for measuring the content of antibody monomers (quality standard: ≥97.0%), aggregates (quality standard: ≤3.0%) or fragments (quality standard: ≤1.0%); (e) CEX-HPLC for measuring the main charge (quality standard: ≥70.0%), acidic charge (quality standard: ≤30.0%) or basic charge content (quality standard: ≤15.0%) of the antibody; (f) ELISA for detecting antibody binding activity (quality standard: 70%-130% of the reference product); (g) protein content (quality standard: 18-22 mg / ml).

[0154] The results showed that the appearance, pH value, insoluble particles, protein content, purity (SEC-HPLC (size exclusion high performance liquid chromatography), CEX-HPLC (weak cation exchange high performance liquid chromatography), R-CE-SDS (reduction electrophoresis), NR-CE-SDS (non-reduction electrophoresis) and biological activity of the four batches of stock solutions in the No. 27 formulation had no significant changes, as shown in Table 8. The results showed that the above four batches of stock solutions had very good stability during the storage process of 0-24 months when stored at 2-8°C in the No. 27 formulation.

[0155] Table 7: Formulation of the long-term stability study of the drug product

[0156]

[0157] Note: Histidine buffer is prepared by the molar ratio of histidine to histidine hydrochloride of 1:3.

[0158] Table 8: Long-term stability data of the formulation

[0159]

[0160]

[0161]

[0162]

[0163]

[0164] Note: NA in the table means that this time point is a non-key inspection item and no inspection is performed at this sampling point; NR means that this time point has not yet arrived, the same below.

[0165] Example 5: Accelerated stability study of formulations

[0166] Four batches of stock solution were selected to prepare the No. 27 prescription preparations shown in Table 7, which were stored in transparent vials and placed at 25±2°C and 60±5% relative humidity (RH). After 0-12 months, each sample was analyzed and tested.

[0167] As shown in Table 9, formulation No. 27 has higher stability against protein degradation, and the degradation kinetic parameters measured at 25±2°C meet the requirements for storage at room temperature for up to 12 months.

[0168] Table 9: Accelerated stability data of drug formulations

[0169]

[0170]

[0171]

[0172] Note: NA in the table means that this time point is a non-key inspection item and no inspection is performed at this sampling point; NR means that the time point has not yet arrived.

[0173] Example 6: ELISA detection of binding of humanized antibodies to hBTLA

[0174] The binding specificity of humanized antibodies hu17, hu18 and hu19 to hBTLA was detected by conventional ELISA detection method. 96-well ELISA plates were coated with 0.5 μg / ml hBTLA and incubated at 37 degrees Celsius for 60 minutes. Then the solution in the wells was discarded, washed 3 times with washing buffer, and blocked with PBS solution containing 2% BSA for 60 minutes. After washing 3 times with washing buffer, a gradient diluted antibody solution was added (the other components in the solution except the antibody were the same as those in prescription number 27), incubated at 37 degrees Celsius for 60 minutes, rinsed 3 times with washing buffer, and then added 1:10000 times diluted HRP-labeled mouse anti-human IgG4 secondary antibody, incubated at 37 degrees Celsius for 1 hour, rinsed three times with washing buffer, and then 100 μl TMB substrate solution was added for color development. After reacting at room temperature for 30 minutes, the reaction was terminated with 100 μl 2M hydrochloric acid solution and the absorbance was read at 450 nm.

[0175] Its EC 50 The values ​​are shown in the following Table 10. The results showed that humanized antibodies hu17, hu18 and hu19 can specifically bind to hBTLA.

[0176] Table 10: Specific binding data of humanized antibody combinations hu17, hu18 and hu19 to hBTLA

[0177] Humanized Antibodies hu17 hu18 hu19 <![CDATA[EC 50 (ng / mL)]]> 6.3 5.4 6.6

[0178] Example 7: FACS detection of binding of humanized antibodies to hBTLA on 293F cells

[0179] The binding ability of humanized anti-BTLA antibodies hu17, hu18 and hu19 to hBTLA expressed on cells was determined by cell-based flow cytometry (FACS). 293F cells expressing hBTLA were digested, centrifuged and resuspended in FACS buffer. The cell weight was 2.5×10 4 , add 50ul of volume to the wells of a 96-well round-bottom plate, add 50ul of antibody diluent of different concentrations (starting concentration 10ug / ml, 3-fold titration; other components in the diluent except the antibody are the same as prescription number 27), mix well, and incubate at room temperature for 30min; wash the cells twice with FACS buffer and add 100ul goat anti-human IgG-PE antibody, incubate in the dark for 30min; wash twice with FACS buffer and perform 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 analyzed by flow cytometry. Based on the exclusion of PI-positive cells from the FSC / SSC gate, live cells were gated and their geometric mean fluorescence was measured. Use Prism TM The data were analyzed using a sigmoidal dose-response model within the software.

[0180] EC of each antibody 50 The values ​​are shown in the following Table 11. The results showed that humanized antibodies hu17, hu18, and hu19 could effectively bind to hBTLA on 293F cells.

[0181] Table 11

[0182] hu17 hu18 hu19 <![CDATA[EC 50 (ng / mL)]]> 96.74 89.34 95.06

[0183] Example 8: FACS detection of the blocking effect of humanized antibodies on the binding of BTLA to HVEM

[0184] The ability of humanized antibodies hu17, hu18, and hu19 to block hBTLA binding to hHVEM expressed on 293F cells was tested by cell-based flow cytometry (FACS) assay. hHVEM-293F stably expressing cells were digested, centrifuged, and resuspended in FACS buffer. The cells were divided into 2.5 × 10 4 , add 50ul of volume to the wells of a 96-well round-bottom plate, add pre-biotin-labeled hBTLA (1ug / ml) protein and mix, incubate at 4°C for 15 minutes; then add 50ul of different concentrations of humanized antibody diluent (starting at 5ug / ml, 3-fold titration; other components in the diluent except the antibody are the same as prescription number 27) and mix well, incubate at room temperature for 30min; wash the cells twice with FACS buffer and add 100ul goat anti-human IgG-PE antibody, incubate in the dark for 30min; wash twice with FACS buffer and perform 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 analyzed by flow cytometry. Based on the exclusion of PI-positive cells from the FSC / SSC gate, live cells were gated and their geometric mean fluorescence was measured. Use Prism TM The data were analyzed using a sigmoidal dose-response model within the software.

[0185] IC of each antibody 50 The values ​​are shown in Table 12. The results showed that humanized antibodies hu17, hu18, and hu19 can effectively block the binding of BTLA to HVEM on the cell surface.

[0186] Table 12

[0187] hu17 hu18 hu19 <![CDATA[IC 50 (ng / mL)]]> 142.7 172.2 161.5

[0188] Example 9: Humanized anti-BTLA antibodies promote T cell activation

[0189] CHO cells stably expressing hPD-L1 / hHVEM were plated into 96-well plates with 5×10 cells per well.4 , cultured overnight at 37°C and 7% CO2, the cell supernatant was removed, 40ul of humanized anti-BTLA antibody diluent was added to each well (the starting concentration was 60ug / ml, 3-fold concentration gradient dilution; the other components of the diluent except the antibody were the same as those in prescription No. 27), and 40ul of Jurkat reporter cells that can continuously express hPD-1 / hBTLA / NFAT-luciferase were added. The total number of cells was 1×10 5 The cells were cultured at 37°C and 7% CO2 for 6 hours, luciferase reagent was added, and the luminescence value was detected by a microplate reader.

[0190] EC of each antibody 50 The values ​​are shown in the following Table 13. The results showed that humanized anti-BTLA antibodies hu17, hu18 and hu19 can effectively promote T cell activation.

[0191] Table 13

[0192] hu17 hu18 hu19 <![CDATA[EC 50 (ng / mL)]]> 256.7 277.3 290.4

[0193] Example 10: Affinity of humanized anti-BTLA antibodies to hBTLA

[0194] The detection experiment was carried out using the Biacore T200 instrument of GE Healthcare Life Sciences, and the Series S CM5 chip was loaded onto the instrument. The system buffer used was HBS-EP+ (10mM HEPES, pH 7.4, 150mM NaCl, 3mM EDTA, 0.05% surfactant P20). The BTLA-Fc antigen was coupled to the chip detection channel, and a mixture of 400mM EDC and 100mM NHS was injected at 10μL / min for 420s to activate the chip surface. The BTLA-Fc antigen was diluted in 10mM sodium acetate / acetic acid (pH 5.5) buffer to a final concentration of 20μg / mL and injected at 10μL / min for coupling, and 1M ethanolamine-hydrochloric acid solution (pH 8.5) was injected at 10μL / min for 420s for blocking.

[0195] The antibody was diluted 2-fold with Biacore system buffer (the other ingredients in the diluent except the antibody were the same as those in prescription number 27), with a total of 6 concentration points. The concentration gradient was 24nM, 12nM, 6nM, 3nM, 1.5nM and 0.75nM, of which 24nM was a repeated test. Data analysis used GE Healthcare Life Sciences' data analysis software Biacore T200Evaluation Software version 3.0. The data fitting model used was 1:1 Binding. The kinetic constants of binding between the antibody and the antigen, binding rate ka (1 / Ms), dissociation rate kd (1 / s), and affinity constant KD (M) were fitted, and the results are shown in Table 14.

[0196] Table 14: Affinity data of humanized antibodies

[0197] ka(1 / Ms) kd(1 / s) KD(M) hu17 6.24E+05 1.16E-04 1.86E-10 hu18 6.39E+05 8.67E-05 1.36E-10

[0198] Example 11: Characterization of the binding kinetics of humanized antibodies to BTLA of different species

[0199] To detect the cross-reaction between chimeric antibodies and cynomolgus monkey and mouse BTLA, the Fortebio assay was used. Briefly, human BTLA, cynomolgus monkey BTLA or mouse BTLA were coupled to an activated CM5 biosensor chip to achieve approximately 100-200 response units (RU), and then unreacted groups were blocked with 1M ethanolamine. Humanized antibody samples (the antibody in the sample was hu17, and the other components were the same as recipe number 27) were injected at 30 times / min in SPR running buffer at increasing concentrations from 0.12nM to 90nM, and the binding response to BTLA of different origins was calculated by subtracting the RU from the blank flow chamber.

[0200] The results are as follows Figure 1 The comparison of hu17 binding to BTLA from different species showed that it not only had high affinity to human BTLA, but also had similar affinity to cynomolgus monkey BTLA, but it had little binding to mouse BTLA.

[0201] Example 12: Inhibitory effect of humanized antibody (prescription number: 27) on tumor growth in mice

[0202] The Hxp-hHVEM plasmid was electroporated into mouse colon cancer (MC38) cells (ATCC) to construct an MC38-hHVEM cell bank. Then, the cells were subcloned by limiting dilution method, and single clones were screened by flow cytometry to obtain MC38-HVEM cells. Then, MC38-hHVEM cells were cultured at 1×10 6The concentration of 0.1 mL / min was inoculated subcutaneously on the right side of B-hBTLA humanized female mice. 3 The mice were randomly divided into groups according to the tumor volume, with 8 mice in each group, for a total of 5 groups, namely: G1 0.9% sodium chloride injection solvent control group, G2 KLH (anti-keyhole limpet hemocyanin antibody) (10 mg / kg) negative control group, G3 hu18 (1 mg / kg) group, G4 hu18 (3 mg / kg) group and G5 hu18 (10 mg / kg) group. All groups were administered by intraperitoneal injection, twice a week, for 7 consecutive times, and the experiment was terminated 4 days after the last administration. The tumor volume and body weight were measured twice a week, and the mouse body weight and tumor volume were recorded. At the end of the experiment, the animals were euthanized, the tumors were removed, weighed, and photographed, and the relative tumor inhibition rate (TGI%, TGI% = (1-(T i -T0) / (V i -V0))×100%,T i The tumor volume at the end of drug administration in the treatment group, the tumor volume at the beginning of drug administration in the treatment group T0, V i The tumor volume at the end of administration in the blank group (V0), the tumor volume at the beginning of administration in the blank group (V1).

[0203] The results of the effects of the test products in each group on the tumor volume of B-hBTLA mice transplanted with MC38-hHVEM cells are shown in Tables 15 and Figure 2 21 days after the first administration, the average tumor volume of the KLH (10 mg / kg) negative control group was 1560 ± 256 mm 3 The average tumor volumes of the other treatment groups were 1073±224mm 3 、747±268mm 3 and 868±211mm 3 Compared with the KLH negative control group, the TGI% of each drug-treated group was 33.7%, 56.4% and 48.0%, respectively, and the P values ​​were 0.175, 0.046 and 0.056, respectively, indicating that the test drug hu18 had a certain inhibitory effect on tumor growth.

[0204] Table 15: Effect of hu18 on tumor volume in B-hBTLA mice transplanted with MC38-hHVEM cells

[0205]

[0206]

[0207] Note: a: mean ± standard error; b: statistical comparison of tumor volume in the drug-treated group and that in the KLH negative control group 21 days after drug administration, t-test.

[0208] The results of the effects of the test products in each group on the body weight of MC38-hHVEM cell transplanted B-hBTLA mice are shown in Tables 16 and Figure 3 All experimental animals were active and ate well during the administration period, and the weight of animals in each administration group increased to a certain extent. No experimental animals died during the experiment. 21 days after administration, the weight of mice in each administration group did not change significantly compared with the weight of mice in the KLH negative control group (P>0.05), indicating that the experimental animals tolerated the test product well.

[0209] Table 16: Effects of the test article on the body weight of MC38-hHVEM cell transplanted B-hBTLA mice

[0210]

[0211] Note: a: mean ± standard error; b: statistical comparison of body weight of drug-treated group and body weight of KLH negative control group 21 days after drug administration, t-test.

[0212] Example 13: Humanized antibody hu18 (prescription number: 27) has no ADCC effector function

[0213] ADCC is initiated when antibodies bind to cell surface target proteins and then connect to Fcγ receptors (FcγRs) expressed on effector cells. It is clearly documented that human IgG1 has significantly higher binding affinity to FcγRs than IgG4, especially binding to FcγR-I and FcγR-IIIA, and this affinity is associated with the strength of IgG1 activation of ADCC. Reminiscent of ADCC, CDC is activated when antibodies cross-link cell surface targets and C1q proteins, followed by a cascade of complement complex formation and target cell lysis. As a proxy for ADCC and CDC, the detection of antibody binding to FcγRs and C1q can serve as a basic indicator of ADCC and CDC. Therefore, the present invention uses biacore T200 (GE) to evaluate the kinetic affinity of monoclonal antibodies for binding to major FcγRs.

[0214] GE's anti-His antibody was immobilized on the sensor chip. Various Fc receptors, including recombinant human FcγRIIIA (CD16a) V176, recombinant human FcγRIIA (CD32a) R167, recombinant human FcγRI (CD64) and recombinant human FcRn were captured, and then a series of dilutions of recombinant human anti-BTLA antibody (i.e., hu18) were injected to detect and analyze the binding properties of the interaction. hu18 is an IgG4 subtype antibody, and hu18-IgG1 is an IgG1 subtype that shares the same Fab as hu18 and serves as a positive control.

[0215] The results are shown in Table 17. Compared with the control antibody of the IgG1 subtype, the binding of the recombinant human anti-BTLA antibody of the IgG4 subtype to the Fc receptor is relatively weak, and the interaction of the hu18 antibody with FcγRIIIA (CD16a) V176 is 400 times weaker than that of the IgG1 subtype control antibody. This shows that hu18 has a weak or no ADCC effect. As shown in Table 17, the hu18 antibody does not bind to C1q, while the hu18-IgG1 antibody can bind to C1q.

[0216] Table 17: Binding affinity of the interaction between anti-BTLA antibodies and Fc receptors

[0217]

[0218] Table 18: Binding affinity of the interaction between anti-BTLA antibodies and human C1q

[0219] Sequence Listing <110> Shanghai Junshi Biopharmaceuticals Co., Ltd. Suzhou Junmeng Biopharmaceutical Technology Co., Ltd. <120> Anti-BTLA antibody pharmaceutical composition and use thereof <130> 19A872 <150> CN 202010066932.1 <151> 2020-01-20 <160> 12 <170> SIPOSequenceListing 1.0 <210> 1 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> HCDR1 <400> 1 His Thr Tyr Ala His 1 5 <210> 2 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> HCDR2 <400> 2 Ile Asp Pro Ala Asn Gly Asn Thr 1 5 <210> 3 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> HCDR3 <400> 3 Val Ala Asp His Tyr Gly Ser Ser Leu Leu Asp Tyr 1 5 10 <210> 4 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> LCDR1 <400> 4 Gln Ser Leu Leu Asp Ser Asp Gly Lys Thr Tyr 1 5 10 <210> 5 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> LCDR2 <400> 5 Leu Val Ser 1 <210> 6 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> LCDR3 <400> 6 Trp Gln Gly Thr Tyr Phe Pro Tyr Thr 1 5 <210> 7 <211> 119 <212> PRT <213> Artificial Sequence <220> <223> Heavy chain variable region <400> 7 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Asn Phe Lys His Thr 20 25 30 Tyr Ala His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Arg Ile Asp Pro Ala Asn Gly Asn Thr Lys Tyr Asp Pro Lys Phe 50 55 60 Gln Gly Arg Ala Thr Met Thr Ala Asp Thr Ala Ser Asn Thr Ala Tyr 65 70 75 80 Leu Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Val Ala Asp His Tyr Gly Ser Ser Leu Leu Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 8 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> Light chain variable region <400> 8 Asp Ile Val Met Thr Gln Thr Pro Leu Ser Leu Ser Val Thr Pro Gly 1 5 10 15 Gln Pro Ala Ser Ile Ser Cys Lys Ser Ser Gln Ser Leu Leu Asp Ser 20 25 30 Asp Gly Lys Thr Tyr Leu Asn Trp Phe Gln Gln Arg Pro Gly Gln Ser 35 40 45 Pro Arg Arg Leu Ile Tyr Leu Val Ser Lys Leu Asp Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Trp Gln Gly 85 90 95 Thr Tyr Phe Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 9 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> Light chain variable region <400> 9 Asp Val Val Met Thr Gln Thr Pro Leu Ser Leu Ser Val Thr Pro Gly 1 5 10 15 Gln Pro Ala Ser Ile Ser Cys Lys Ser Ser Gln Ser Leu Leu Asp Ser 20 25 30 Asp Gly Lys Thr Tyr Leu Asn Trp Phe Gln Gln Arg Pro Gly Gln Ser 35 40 45 Pro Arg Arg Leu Ile Tyr Leu Val Ser Lys Leu Asp Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Trp Gln Gly 85 90 95 Thr Tyr Phe Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 10 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> Light chain variable region <400> 10 Asp Ile Val Met Thr Gln Thr Pro Leu Ser Leu Ser Val Thr Pro Gly 1 5 10 15 Gln Pro Ala Ser Ile Ser Cys Lys Ser Ser Gln Ser Leu Leu Asp Ser 20 25 30 Asp Gly Lys Thr Tyr Leu Asn Trp Phe Gln Gln Arg Pro Gly Gln Ser 35 40 45 Pro Arg Arg Leu Met His Leu Val Ser Lys Leu Asp Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Trp Gln Gly 85 90 95 Thr Tyr Phe Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 11 <211> 446 <212> PRT <213> Artificial Sequence <220> <223> Heavy Chain <400> 11 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Asn Phe Lys His Thr 20 25 30 Tyr Ala His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Arg Ile Asp Pro Ala Asn Gly Asn Thr Lys Tyr Asp Pro Lys Phe 50 55 60 Gln Gly Arg Ala Thr Met Thr Ala Asp Thr Ala Ser Asn Thr Ala Tyr 65 70 75 80 Leu Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Val Ala Asp His Tyr Gly Ser Ser Leu Leu Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro 210 215 220 Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe 225 230 235 240 Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro 245 250 255 Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val 260 265 270 Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr 275 280 285 Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val 290 295 300 Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys 305 310 315 320 Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser 325 330 335 Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro 340 345 350 Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val 355 360 365 Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly 370 375 380 Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp 385 390 395 400 Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp 405 410 415 Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His 420 425 430 Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys 435 440 445 <210> 12 <211> 219 <212> PRT <213> Artificial Sequence <220> <223> Light chain <400> 12 Asp Val Val Met Thr Gln Thr Pro Leu Ser Leu Ser Val Thr Pro Gly 1 5 10 15 Gln Pro Ala Ser Ile Ser Cys Lys Ser Ser Gln Ser Leu Leu Asp Ser 20 25 30 Asp Gly Lys Thr Tyr Leu Asn Trp Phe Gln Gln Arg Pro Gly Gln Ser 35 40 45 Pro Arg Arg Leu Ile Tyr Leu Val Ser Lys Leu Asp Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Trp Gln Gly 85 90 95 Thr Tyr Phe Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 110 Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 115 120 125 Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 130 135 140 Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln 145 150 155 160 Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 165 170 175 Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 180 185 190 Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 195 200 205 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215

Claims

1. A pharmaceutical composition comprising: (1) histidine buffer; and (2) an anti-BTLA antibody or an antigen-binding fragment thereof, wherein the anti-BTLA antibody or the antigen-binding fragment thereof has HCDR1, HCDR2 and HCDR3 with amino acid sequences as shown in SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3, respectively, and LCDR1, LCDR2 and LCDR3 with amino acid sequences as shown in SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6, respectively.

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

3. The pharmaceutical composition according to claim 2, characterized in that The histidine buffer is a histidine-histidine hydrochloride buffer.

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

5. The pharmaceutical composition according to claim 4, characterized in that The pH of the buffer is 5.0-6.

5.

6. The pharmaceutical composition according to claim 5, characterized in that The pH of the buffer is 5.0-6.

0.

7. The pharmaceutical composition according to any one of claims 1 to 6, wherein the pharmaceutical composition further comprises a stabilizer, and the stabilizer is selected from one or more of sodium chloride, mannitol, sorbitol, sucrose, maltose, xylitol and trehalose.

8. The pharmaceutical composition according to claim 7, characterized in that The stabilizer is a combination of trehalose and sodium chloride.

9. The pharmaceutical composition of claim 7, wherein the stabilizer is sodium chloride at a concentration of 50-200 mM; or the stabilizer is mannitol at a concentration of 100-300 mM; or the stabilizer is sorbitol at a concentration of 100-300 mM; or the stabilizer is sucrose at a concentration of 100-300 mM; or the stabilizer is trehalose at a concentration of 100-300 mM; or the stabilizer is a combination of 30-100 mM sodium chloride and 50-200 mM mannitol; or the stabilizer is a combination of 30-100 mM sodium chloride and 50-200 mM sorbitol; or the stabilizer is a combination of 30-100 mM sodium chloride and 50-200 mM sucrose; or the stabilizer is a combination of 30-100 mM sodium chloride and 50-200 mM trehalose.

10. The pharmaceutical composition according to claim 9, wherein the stabilizer is a combination of 30-100 mM sodium chloride and 50-200 mM trehalose.

11. The pharmaceutical composition according to claim 9, characterized in that The stabilizer is a combination of 30-100 mM sodium chloride and 50-200 mM trehalose.

12. The pharmaceutical composition of any one of claims 1 to 6, wherein the pharmaceutical composition further comprises a surfactant selected from polysorbate 80, polysorbate 20 or poloxamer 188.

13. The pharmaceutical composition according to claim 12, characterized in that The surfactant is polysorbate 80.

14. The pharmaceutical composition of claim 12, wherein the surfactant concentration is 0.01%-0.1%.

15. The pharmaceutical composition according to claim 14, characterized in that The concentration of the surfactant is 0.01%-0.05%.

16. The pharmaceutical composition of claim 1, wherein the anti-BTLA antibody has a heavy chain variable region as shown in the amino acid sequence of SEQ ID NO: 7, and a light chain variable region as shown in the amino acid sequence of SEQ ID NO: 8; or has a heavy chain variable region as shown in the amino acid sequence of SEQ ID NO: 7, and a light chain variable region as shown in the amino acid sequence of SEQ ID NO: 9; or has a heavy chain variable region as shown in the amino acid sequence of SEQ ID NO: 7, and a light chain variable region as shown in the amino acid sequence of SEQ ID NO:

10.

17. The pharmaceutical composition of claim 1, wherein the anti-BTLA antibody has a heavy chain amino acid sequence as shown in SEQ ID NO:11 and a light chain amino acid sequence as shown in SEQ ID NO:

12.

18. The pharmaceutical composition of any one of claims 1-6, wherein the concentration of the anti-BTLA antibody or antigen-binding fragment thereof is 5-100 mg / mL.

19. The pharmaceutical composition according to claim 18, characterized in that The concentration of the anti-BTLA antibody or antigen-binding fragment thereof is 10-50 mg / mL.

20. The pharmaceutical composition according to claim 19, characterized in that The concentration of the anti-BTLA antibody or antigen-binding fragment thereof is 15-25 mg / mL.

21. The pharmaceutical composition according to claim 1, comprising or consisting of the components shown in any one of the following (1)-(13): (1) (a) 10 mg / mL-50 mg / mL of an anti-BTLA antibody or an antigen-binding fragment thereof; (b) 10-30 mM histidine buffer, pH 5.0-6.0; (c) a combination of 30 mM to 100 mM sodium chloride and 50-200 mM trehalose; (d) and 0.01%-0.05% polysorbate 80; (2) (a) 10 mg / mL-50 mg / mL of an anti-BTLA antibody or an antigen-binding fragment thereof; (b) 10-30 mM histidine buffer, pH 5.0-6.0; (c) a combination of 30 mM to 100 mM sodium chloride and 50-200 mM mannitol; (d) and 0.01%-0.05% polysorbate 80; (3) (a) 10 mg / mL-50 mg / mL of an anti-BTLA antibody or an antigen-binding fragment thereof; (b) 10-30 mM histidine buffer, pH 5.0-6.0; (c) a combination of 30 mM to 100 mM sodium chloride and 50-200 mM sucrose; (d) and 0.01%-0.05% polysorbate 80; (4) (a) 10 mg / mL-50 mg / mL of an anti-BTLA antibody or an antigen-binding fragment thereof; (b) 10-30 mM histidine buffer, pH 5.0-6.0; (c) a combination of 30 mM to 100 mM sodium chloride and 50-200 mM sorbitol; (d) and 0.01%-0.05% polysorbate 80; (5) (a) 10 mg / mL-50 mg / mL of an anti-BTLA antibody or an antigen-binding fragment thereof; (b) 10-30 mM histidine buffer, pH 5.0-6.0; (c) 100 mM to 300 mM trehalose; (d) and 0.01%-0.05% polysorbate 80; (6) (a) 10 mg / mL-50 mg / mL of an anti-BTLA antibody or an antigen-binding fragment thereof; (b) 10-30 mM citric acid buffer, pH 5.5-6.5; (c) 100 mM to 300 mM trehalose; (d) and 0.01%-0.05% polysorbate 80; (7) (a) 10 mg / mL-50 mg / mL of an anti-BTLA antibody or an antigen-binding fragment thereof; (b) 10-30 mM citrate buffer, pH 5.5-6.5; (c) a combination of 30 mM to 100 mM sodium chloride and 50-200 mM mannitol; (d) and 0.01%-0.05% polysorbate 80; (8) (a) 10 mg / mL-50 mg / mL of an anti-BTLA antibody or an antigen-binding fragment thereof; (b) 10-30 mM acetate buffer, pH 5.0-6.0; (c) a combination of 30 mM to 100 mM sodium chloride and 50-200 mM mannitol; (d) and 0.01%-0.05% polysorbate 80; (9) (a) 20 mg / mL of an anti-BTLA antibody or an antigen-binding fragment thereof; (b) 20 mM histidine buffer, pH 6.0; (c) a combination of 54 mM sodium chloride and 132 mM trehalose; (d) and 0.02% polysorbate 80; (10) (a) 20 mg / mL of an anti-BTLA antibody or an antigen-binding fragment thereof; (b) 20 mM histidine buffer, pH 5.5; (c) 220 mM trehalose; (d) and 0.02% polysorbate 80; (11) (a) 20 mg / mL anti-BTLA antibody or antigen-binding fragment thereof; (b) 20 mM histidine buffer, pH 5.5; (c) a combination of 50 mM sodium chloride and 140 mM mannitol; (d) and 0.02% polysorbate 80; (12) (a) 20 mg / mL of an anti-BTLA antibody or an antigen-binding fragment thereof; (b) 20 mM histidine buffer, pH 5.5; (c) a combination of 50 mM sodium chloride and 140 mM trehalose; (d) and 0.02% polysorbate 80; and (13) (a) 20 mg / mL anti-BTLA antibody or antigen-binding fragment thereof; (b) 20 mM histidine buffer, pH 5.0-6.0; (c) a combination of 50 mM sodium chloride and 140 mM trehalose; (d) and 0.02% polysorbate 80.

22. The pharmaceutical composition according to claim 1, characterized in that The pharmaceutical composition contains the following components, or consists of the following components: histidine-histidine hydrochloride buffer with 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 on a w / v basis; and 15-25 mg / mL of an anti-BTLA antibody or an antigen-binding fragment thereof, wherein the anti-BTLA antibody has a heavy chain amino acid sequence as shown in SEQ ID NO:11 and a light chain amino acid sequence as shown in SEQ ID NO:

12.

23. Use of the pharmaceutical composition according to any one of claims 1 to 22 in the preparation of a medicament for treating or preventing BTLA-mediated tumors; the tumor is selected from: melanoma, colon cancer, head or neck cancer, rectal cancer, Hodgkin's disease, non-Hodgkin's lymphoma.

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