Pharmaceutical composition comprising BTN3a binding protein, and use thereof

Abstract

Provided are a pharmaceutical composition comprising a BTN3A binding protein, and a use thereof. Specifically, the present invention relates to a pharmaceutical composition, comprising a BTN3A binding protein and a buffer, and further comprising one or more of a surfactant, a saccharide, a complexing agent, or an amino acid.

Description

Pharmaceutical compositions containing BTN3A binding protein and their uses

[0001] This disclosure claims priority to Chinese patent application filed on February 5, 2025 (application number: 2025101299023), which is incorporated herein by reference. Technical Field

[0002] This disclosure pertains to the field of pharmaceutical formulations, and specifically relates to a pharmaceutical composition comprising BTN3A binding protein and its use therein, as well as its use as a medicine. Background Technology

[0003] The advent of immunotherapy has transformed the traditional landscape of cancer treatment. Currently, most immunotherapies focus on enhancing the anti-tumor immune response of αβT cells; however, other lymphocyte subsets with anti-tumor potential deserve attention. γδT cells are a special subset of T cells with innate immune characteristics, accounting for approximately 0.5%-5% of total T cells in the human body. They can be activated by directly recognizing antigen signals transmitted by specific ligands without relying on the major histocompatibility complex (MHC) and antigen-presenting cells (APCs). All T cells express TCRs, and based on the different TCR genes, they are divided into αβT and γδT cells. αβT cells express Vα and VβTCR chains, while γδT cells express Vγ and VδTCR chains. Human γδT cells comprise seven VγTCR chains (Vγ2, 3, 4, 5, 8, 9, and 11) and four Vδ chains (Vδ1, 2, 3, and 5). Based on the different δ chains, human γδT cells are divided into three main subsets: Vδ1T, Vδ2T, and Vδ3T. Vδ1T cells are mainly found in mucosal epithelial tissues, Vδ2T cells are mainly found in peripheral blood, and Vδ3T cells are mainly distributed in the liver and intestine. The Vγ9Vδ2T cell subset, which accounts for approximately 1-5% of total peripheral blood T cells, is the most studied subset. Studies have shown that activated Vγ9Vδ2T cells can kill cancer cells by secreting cytokines such as IFN-γ and TNF-α or by releasing perforin and granzymes. Activated Vγ9Vδ2T cells also proliferate, further contributing to their anti-tumor effect.

[0004] In recent years, BTN3A (B3A), a member of the lactolipoprotein subfamily 3, has become an important molecule regulating the function of Vγ9Vδ2T cells. The BTN3A family includes three isoforms: BTN3A1 (also called CD277), BTN3A2, and BTN3A3, each expressed by a different gene on human chromosome 6. They are expressed in humans and non-human primates (NHPs). The BTN3A family consists of type I transmembrane proteins. The extracellular segment of each BTN3A isoform comprises an IgV domain distant from the cell membrane and an IgC domain close to the cell membrane. The extracellular segment sequences of the three isoforms share up to 95% homology. The cytoplasmic regions of BTN3A1 and BTN3A3 contain a B30.2 domain, while BTN3A2 lacks this domain. Recent studies have found that under bacterial or viral stimulation and stress conditions, phosphorylated antigens (pAg) gradually accumulate in cells and are recognized and bound by the B30.2 domain of the intracellular segment of the BTN3A protein. After binding to pAg, the extracellular segment of the BTN3A protein undergoes a conformational change, further promoting the formation of a complex with the cell membrane surface BTN2A1 protein. This complex then binds to the Vγ9 chain of Vγ9Vδ2T cells through the IgV domain of BTN2A1, thereby activating Vγ9Vδ2T cells to perform their functions. Because BTN3A3 has a poor affinity for pAg, only BTN3A1 can sense the increase in intracellular pAg and activate Vγ9Vδ2T cells.

[0005] Recent studies have found that by designing antibodies that target and bind to the extracellular domain of BTN3A1, intracellular pAg can be replaced, thus more efficiently activating Vγ9Vδ2 T cells. Furthermore, BTN3A1 antibodies can enhance the anti-tumor activity of αβT cells.

[0006] The novel anti-BTN3A antibody with the disclosed sequence structure exhibits excellent activity in activating γδT cells and promoting the release of cytokines from γδT cells, thereby enhancing the killing of tumor cells by γδT cells and demonstrating good druggability and potential for clinical application. However, due to the large molecular weight and complex structure of the antibody, it is prone to degradation and polymerization, leading to instability and reduced or even ineffective activity. Therefore, the development of stable formulations of the drug is particularly important to ensure that the binding protein is suitable for drug delivery and maintains stability during storage and subsequent use, thus achieving better efficacy. Summary of the Invention

[0007] This disclosure provides a pharmaceutical composition comprising BTN3A binding protein. It also provides a method for preparing said pharmaceutical composition, and methods for treating or preventing diseases, or related pharmaceutical uses.

[0008] This disclosure provides a pharmaceutical composition comprising a BTN3A binding protein and a buffer. In some embodiments, the buffer is selected from one or more of acetate buffers, citrate buffers, histidine buffers, and phosphate buffers. In some specific embodiments, the buffer is selected from one or more of acetate-sodium acetate, citrate-sodium citrate, histidine-histidine hydrochloride, and disodium hydrogen phosphate-sodium dihydrogen phosphate. "One or more" in this disclosure includes one, two, or more.

[0009] In some embodiments, the concentration of the buffer is from about 1 mM to about 100 mM, for example, from about 1 mM to about 50 mM, from about 1 mM to about 55 mM, from about 1 mM to about 60 mM, from about 1 mM to about 70 mM, from about 1 mM to about 80 mM, from about 5 mM to about 100 mM, from about 5 mM to about 80 mM, from about 5 mM to about 70 mM, from about 5 mM to about 50 mM, from about 5 mM to about 45 mM, from about 5 mM to about 40 mM, from about 5 mM to about 35 mM, from about 5 mM to about 30 mM, from about 5 mM to about 25 mM, about 5 mM to about 20 mM, about 5 mM to about 10 mM, about 10 mM to about 20 mM, about 10 mM to about 30 mM, about 10 mM to about 35 mM, about 10 mM to about 40 mM, about 10 mM to about 50 mM, about 15 mM to about 20 mM, about 15 mM to about 25 mM, about 15 mM to about 35 mM, about 15 mM to about 50 mM, about 20 mM to about 35 mM, about 1 mM to about 10 mM, about 1 mM to about 20 mM, or any range between these values. In some embodiments, the concentration of the buffer is about 2 mM to about 50 mM. In some embodiments, the concentration of the buffer is about 5 mM to about 30 mM.

[0010] In some embodiments, the concentration of the buffer is about 1 mM, about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, about 10 mM, about 11 mM, about 12 mM, about 13 mM, about 14 mM, about 15 mM, about 16 mM, about 17 mM, about 18 mM, about 19 mM, about 20 mM, about 22 mM, about 25 mM, about 26 mM, about 28 mM, about 30 mM, about 35 mM. mM, approximately 40mM, approximately 43mM, approximately 45mM, approximately 48mM, approximately 50mM, approximately 55mM, approximately 57mM, approximately 58mM, approximately 60mM, approximately 62mM, approximately 65mM, approximately 68mM, approximately 70mM, approximately 72mM, approximately 75mM, approximately 78mM, approximately 80mM, approximately 82mM, approximately 85mM, approximately 88mM, approximately 90mM, approximately 92mM, approximately 95mM, approximately 100mM.

[0011] In some embodiments, the buffer or the pharmaceutical composition has a pH value of about 3.0 to about 7.0, for example, about 3.5 to about 7.0, about 4.0 to about 7.0, about 4.5 to about 7.0, about 4.5 to about 6.8, about 4.5 to about 6.5, about 4.5 to about 6.2, about 4.5 to about 6.0, about 4.0 to about 6.5, about 5.5 to about 7.0, about 5.5 to about 6.8, about 5.5 to about 6.5, about 5 pH values ​​ranging from about 0.5 to about 6.2, about 5.5 to about 6.0, about 6.0 to about 7.0, about 6.0 to about 6.8, about 6.0 to about 6.5, about 6.0 to about 6.2, about 4.0 to about 6.0, about 4.0 to about 5.5, about 4.0 to about 5.0, about 4.0 to about 4.8, 4.0 to about 4.5, about 4.8 to about 5.0, about 5.0 to about 5.5, about 4.5 to about 4.8, or any range between these values. In some embodiments, the buffer or the pharmaceutical composition has a pH value of about 3.5 to about 7.5. In some embodiments, the buffer or the pharmaceutical composition has a pH value of about 4.0 to about 7.0. In some embodiments, the buffer or the pharmaceutical composition has a pH value of about 4.5 to about 7.0.

[0012] In some embodiments, the buffer or the pharmaceutical composition has a pH value of about 3.0, about 3.1, about 3.2, about 3.3, about 3.4, about 3.5, about 3.6, about 3.7, about 3.8, about 3.9, about 4.0, about 4.1, about 4.2, about 4.3, about 4.4, about 4.5, about 4.6, about 4.7, about 4.8, about 4.9, about 5.0, about 5.1, about 5.2, about 5.3, about 5.4, about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, about 6.0, about 6.1, about 6.2, about 6.3, about 6.4, about 6.5, about 6.7, about 6.8, about 6.9, or about 7.0.

[0013] Typically, the pH of a pharmaceutical composition obtained by replacing a buffer is almost identical to the pH of the buffer. However, it is known to those skilled in the art that pH drift may sometimes occur during pharmaceutical formulation, but the pH drift of the pharmaceutical formulation is generally small (e.g., within ±0.8). In some embodiments, the pH drift of the pharmaceutical formulation is within ±0.5.

[0014] In some embodiments, the concentration of the BTN3A binding protein is from about 0.01 mg / mL to about 200 mg / mL, for example, from about 0.1 mg / mL to about 200 mg / mL, from about 0.5 mg / mL to about 200 mg / mL, from about 1 mg / mL to about 200 mg / mL, from about 5 mg / mL to about 200 mg / mL, from about 10 mg / mL to about 200 mg / mL, from about 10 mg / mL to about 180 mg / mL, from about 10 mg / mL to about 160 mg / mL, from about 10 mg / mL to about 150 mg / mL. mL, about 10 mg / mL to about 120 mg / mL, about 10 mg / mL to about 110 mg / mL, about 10 mg / mL to about 100 mg / mL, about 10 mg / mL to about 80 mg / mL, about 10 mg / mL to about 60 mg / mL, about 10 mg / mL to about 40 mg / mL, about 20 mg / mL to about 200 mg / mL, about 20 mg / mL to about 180 mg / mL, about 20 mg / mL to about 150 mg / mL, about 20 mg / mL to about 120 mg / mL, about 20 mg / mL to Approximately 110 mg / mL, approximately 20 mg / mL to approximately 100 mg / mL, approximately 20 mg / mL to approximately 80 mg / mL, approximately 20 mg / mL to approximately 60 mg / mL, approximately 20 mg / mL to approximately 40 mg / mL, approximately 40 mg / mL to approximately 120 mg / mL, approximately 40 mg / mL to approximately 110 mg / mL, approximately 40 mg / mL to approximately 100 mg / mL, approximately 40 mg / mL to approximately 80 mg / mL, 40 mg / mL to approximately 60 mg / mL, approximately 60 mg / mL to approximately 80 mg / mL, approximately 60 mg / The concentrations are approximately 120 mg / mL, 60 mg / mL, 150 mg / mL, 80 mg / mL, 200 mg / mL, 50 mg / mL, 110 mg / mL, 100 mg / mL, 150 mg / mL, 80 mg / mL, 110 mg / mL, or any range between these values. In some embodiments, the concentration of the BTN3A binding protein is approximately 0.01 mg / mL, approximately 0.05 mg / mL, approximately 0.1 mg / mL, or approximately 0.5 mg / mL (approx. 1 mg / mL), approximately 5 mg / mL, approximately 10 mg / mL, approximately 15 mg / mL, approximately 20 mg / mL, approximately 25 mg / mL, approximately 30 mg / mL, approximately 35 mg / mL, approximately 40 mg / mL, approximately 45 mg / mL, approximately 50 mg / mL, approximately 55 mg / mL, approximately 60 mg / mL, approximately 61 mg / mL, approximately 62 mg / mL, approximately 63 mg / mL, approximately 64 mg / mL, approximately 65 mg / mL, approximately 66 mg / mL, approximately 67 mg / mL, approximately 68 mg / mL, approximately 69 mg / mL, approximately 70 mg / mL, approximately 71 mg / mL, approximately 72 mg / mL, approximately 73 mg / mL, approximately 7 4 mg / mL, approximately 75 mg / mL, approximately 76 mg / mL, approximately 77 mg / mL, approximately 78 mg / mL, approximately 79 mg / mL, approximately 80 mg / mL, approximately 81 mg / mL, approximately 82 mg / mL, approximately 83 mg / mL, approximately 84 mg / mL, approximately 85 mg / mL, approximately 86 mg / mL, approximately 87 mg / mL, approximately 88 mg / mL, approximately 89 mg / mL, approximately 90 mg / mL, approximately 91 mg / mL, approximately 92 mg / mL, approximately 93 mg / mL, approximately 94 mg / mL, approximately 95 mg / mL, approximately 96 mg / mL, approximately 97 mg / mL, approximately 98 mg / mL, approximately 99 mg / mL, approximately 100 mg / mL Approximately 101 mg / mL, approximately 102 mg / mL, approximately 103 mg / mL, approximately 104 mg / mL, approximately 105 mg / mL, approximately 106 mg / mL, approximately 107 mg / mL, approximately 108 mg / mL, approximately 109 mg / mL, approximately 110 mg / mL, approximately 111 mg / mL, approximately 112 mg / mL, approximately 113 mg / mL, approximately 114 mg / mL, approximately 115 mg / mL, approximately 116 mg / mL, approximately 117 mg / mL, approximately 118 mg / mL, approximately 119 mg / mL, approximately 120 mg / mL, approximately 121 mg / mL, approximately 122 mg / mL, approximately 123 mg / mL, approximately 124 mg / mL Approximately 125 mg / mL, approximately 126 mg / mL, approximately 127 mg / mL, approximately 128 mg / mL, approximately 129 mg / mL, approximately 130 mg / mL, approximately 131 mg / mL, approximately 132 mg / mL, approximately 133 mg / mL, approximately 134 mg / mL, approximately 135 mg / mL, approximately 140 mg / mL, approximately 145 mg / mL, approximately 150 mg / mL, approximately 155 mg / mL, approximately 160 mg / mL, approximately 165 mg / mL, approximately 170 mg / mL, approximately 175 mg / mL, approximately 180 mg / mL, approximately 185 mg / mL, approximately 190 mg / mL, approximately 195 mg / mL, approximately 200 mg / mL.

[0015] In some embodiments, the pharmaceutical composition as described in any of the preceding embodiments comprises one or more of a surfactant, sugar, complexing agent, or amino acid.

[0016] In some embodiments, the surfactant is a nonionic surfactant. In some embodiments, the surfactant is selected from poloxamer (e.g., poloxamer 188), polysorbates (e.g., polysorbate 20 (i.e., PS20), polysorbate 80 (i.e., PS80)), polyhydroxyalkanoates, Triton, sodium lauryl sulfonate, sodium lauryl sulfonate, sodium octyl glycoside, lauryl-sulfobetaine, myristyl-sulfobetaine, linoleyl-sulfobetaine, stearyl-sulfobetaine, lauryl-sarcosine, myristyl-sarcosine, linoleyl-sarcosine, stearyl-sarcosine, linoleyl-saccharide, succinate ... Betaine, myristyl-betaine, cetyl-betaine, lauramidopropyl-betaine, cocarbamate-propyl-betaine, linoleamide-propyl-betaine, myristamidopropyl-betaine, palmitoamide-propyl-betaine, isostearamidopropyl-betaine, myristamidopropyl-dimethylamine, palmitoamide-propyl-dimethylamine, isostearamidopropyl-dimethylamine, sodium methyl cocoyl, sodium methyl oleate, polyethylene glycol, polypropylene glycol, copolymers of ethylene and propylene glycol, etc., or any combination thereof. In some embodiments, the surfactant is sodium dodecyl sulfate (SDS) or polysorbate. In some embodiments, the surfactant is polysorbate 80.

[0017] In some embodiments, the concentration of the surfactant is from about 0.01% (w / v) to about 20% (w / v), for example from about 0.01% (w / v) to about 18% (w / v), from about 0.01% (w / v) to about 15% (w / v), from about 0.01% (w / v) to about 12% (w / v), from about 0.01% (w / v) to about 8% (w / v), from about 0.01% (w / v) to about 5% (w / v), or from about 0.01% (w / v) to about 4% (w / v). From about 0.01% (w / v) to about 3% (w / v), from about 0.01% (w / v) to about 2% (w / v), from about 0.01% (w / v) to about 1.8% (w / v), from about 0.01% (w / v) to about 1.5% (w / v), from about 0.01% (w / v) to about 1.2% (w / v), from about 0.01% (w / v) to about 1% (w / v), from about 0.01% (w / v) to about 0.08% (w / v), and any range between these point values. In some embodiments, the concentration of the surfactant is about 0.001% (w / v), 0.002% (w / v), 0.005% (w / v), 0.01% (w / v), 0.02% (w / v), 0.03% (w / v), about 0.04% (w / v), or about 0.05% (w / v).

[0018] In some embodiments, the sugar is selected from glucose, sucrose, trehalose, lactose, fructose, and maltose. In some embodiments, the sugar is sucrose or trehalose; in some specific embodiments, the sugar is sucrose.

[0019] In some embodiments, the concentration of the sugar is from about 0.1% (w / v) to about 20% (w / v), for example from about 1% (w / v) to about 18% (w / v), from about 1% (w / v) to about 15% (w / v), from about 1% (w / v) to about 12% (w / v), from about 1% (w / v) to about 8% (w / v), from about 1% (w / v) to about 8.8% (w / v), from about 2% (w / v) to about 20% (w / v), from about 2% (w / v) to about 20% (w / v). (w / v) to about 15% (w / v), about 2% (w / v) to about 10% (w / v), about 2% (w / v) to about 8% (w / v), about 2% (w / v) to about 8.8% (w / v), about 3% (w / v) to about 20% (w / v), about 3% (w / v) to about 15% (w / v), about 3% (w / v) to about 10% (w / v), about 3% (w / v) to about 8% (w / v), about 3% (w / v) to about 8%. 8% (w / v), about 4% (w / v) to about 20% (w / v), about 4% (w / v) to about 15% (w / v), about 4% (w / v) to about 10% (w / v), about 4% (w / v) to about 8% (w / v), about 4% (w / v) to about 8.8% (w / v), about 5% (w / v) to about 20% (w / v), about 5% (w / v) to about 15% (w / v), about 5% (w / v) to about 10% (w / v) Approximately 5% (w / v) to approximately 8% (w / v), approximately 5% (w / v) to approximately 8.8% (w / v), approximately 4.4% (w / v) to approximately 20% (w / v), approximately 4.4% (w / v) to approximately 15% (w / v), approximately 4.4% (w / v) to approximately 10% (w / v), approximately 4.4% (w / v) to approximately 8% (w / v), approximately 4.4% (w / v) to approximately 8.8% (w / v), and any range between these point values. In some embodiments, the concentration of the sugar is about 1% (w / v), 1.2% (w / v), 1.4% (w / v), about 2% (w / v), about 3% (w / v), about 4% (w / v), about 4.2% (w / v), about 4.4% (w / v), about 4.8% (w / v), about 5% (w / v), about 6% (w / v), or about 7%. (w / v), approximately 7.8% (w / v), approximately 8% (w / v), approximately 8.2% (w / v), approximately 8.4% (w / v), approximately 8.8% (w / v), approximately 9% (w / v), approximately 10% (w / v), approximately 12% (w / v), approximately 14% (w / v), approximately 16% (w / v), approximately 18% (w / v), approximately 20% (w / v).

[0020] In some embodiments, the amino acid is selected from methionine.

[0021] In some embodiments, the concentration of the amino acid is from about 0.01% (w / v) to about 20% (w / v), for example, from about 0.01% (w / v) to about 18% (w / v), from about 0.01% (w / v) to about 15% (w / v), from about 0.01% (w / v) to about 12% (w / v), from about 0.01% (w / v) to about 8% (w / v), from about 0.01% (w / v) to about 5% (w / v), from about 0.01% (w / v) to about 4% (w / v), from about 0.01% (w / v) to about 3% (w / v), from about 0.01% (w / v) to about 2% (w / v), from about 0.01% (w / v) to about 1% (w / v). (w / v), about 0.02% (w / v) to about 2% (w / v), about 0.03% (w / v) to about 2% (w / v), about 0.04% (w / v) to about 2% (w / v), about 0.05% (w / v) to about 2% (w / v), about 0.06% (w / v) to about 2% (w / v), about 0.07% (w / v) to about 2% (w / v), about 0.08% (w / v) to about 2% (w / v), about 0.09% (w / v) to about 2% (w / v), about 0.10% (w / v) to about 2% (w / v), about 0.12% (w / v) to about 2% (w / v), and any range between these point values. In some embodiments, the concentration of the amino acid is about 0.01% (w / v), 0.02% (w / v), 0.05% (w / v), 0.1% (w / v), 0.12% (w / v), 0.13% (w / v), about 0.14% (w / v), about 0.148% (w / v), about 0.149% (w / v), or about 0.15% (w / v). (v), approximately 0.16% (w / v), approximately 0.18% (w / v), approximately 0.2% (w / v), approximately 0.22% (w / v), approximately 0.25% (w / v), approximately 0.3% (w / v), approximately 0.32% (w / v), approximately 0.35% (w / v), approximately 0.4% (w / v), approximately 0.45% (w / v), approximately 0.5% (w / v).

[0022] In one embodiment, the complexing agent is selected from disodium edetate.

[0023] In some embodiments, the concentration of the complexing agent is from about 0.001% (w / v) to about 0.1% (w / v), for example, from about 0.001% (w / v) to about 0.01% (w / v), from about 0.002% (w / v) to about 0.01% (w / v), from about 0.003% (w / v) to about 0.01% (w / v), from about 0.004% (w / v) to about 0.01% (w / v), from about 0.004% (w / v) to about 0.008% (w / v), and any range between these values. In some embodiments, the concentration of the complexing agent is from about 0.001% (w / v), 0.002% (w / v), 0.004% (w / v), 0.005% (w / v), 0.006% (w / v), and 0.008% (w / v).

[0024] In some embodiments, the pharmaceutical composition comprises:

[0025] (1) BTN3A binding protein at concentrations of approximately 0.01 g / L to approximately 200 g / L.

[0026] Buffers ranging from approximately 1 mM to approximately 100 mM

[0027] Surfactants ranging from approximately 0.01% (w / v) to approximately 10% (w / v),

[0028] Sugars of approximately 0.1% (w / v) to approximately 20% (w / v), and,

[0029] The amino acids present may be selected from approximately 0.01% (w / v) to approximately 10% (w / v);

[0030] The complexing agent may be present in an amount of about 0.001% (w / v) to about 5% (w / v);

[0031] The pH of the pharmaceutical composition is from about 3.0 to about 7.0; or,

[0032] (2) BTN3A binding protein at concentrations of approximately 0.01 g / L to approximately 200 g / L.

[0033] Acetate buffer, citrate buffer, histidine buffer or phosphate buffer at a concentration of about 1 mM to about 100 mM;

[0034] Polysorbate 80, from about 0.01% (w / v) to about 10% (w / v),

[0035] Sucrose from about 0.1% (w / v) to about 20% (w / v), and,

[0036] The methionine present optionally is about 0.01% (w / v) to about 10% (w / v);

[0037] Sodium edetate may be present in an optional amount of about 0.001% (w / v) to about 5% (w / v);

[0038] The pH of the pharmaceutical composition is from about 3.0 to about 7.0; or,

[0039] (3) BTN3A binding protein at approximately 20 g / L to approximately 180 g / L,

[0040] Acetic acid-sodium acetate, citric acid-sodium citrate, histidine-histidine hydrochloride or disodium hydrogen phosphate-sodium dihydrogen phosphate buffers of about 1 mM to about 100 mM.

[0041] Polysorbate 80 at approximately 0.01% (w / v) to approximately 2% (w / v)

[0042] Sucrose of about 0.5% (w / v) to about 12% (w / v), and,

[0043] The methionine present optionally is about 0.01% (w / v) to about 2% (w / v);

[0044] Sodium edetate may be present in an optional amount of about 0.001% (w / v) to about 0.1% (w / v);

[0045] The pH of the pharmaceutical composition is from about 3.0 to about 7.0; or,

[0046] (4) BTN3A binding protein at approximately 50 g / L to approximately 150 g / L,

[0047] Acetic acid-sodium acetate, citric acid-sodium citrate, histidine-histidine hydrochloride or disodium hydrogen phosphate-sodium dihydrogen phosphate buffers of about 1 mM to about 50 mM.

[0048] Polysorbate 80 at approximately 0.01% (w / v) to approximately 1% (w / v),

[0049] Sucrose of about 1% (w / v) to about 12% (w / v), and,

[0050] The presence of methionine is optionally about 0.01% (w / v) to about 1% (w / v);

[0051] The pH of the pharmaceutical composition is from about 4.5 to about 7.0.

[0052] In some embodiments, the pharmaceutical composition comprises:

[0053] (1-1) BTN3A binding protein at approximately 20 g / L to approximately 180 g / L,

[0054] Approximately 1 mM to approximately 100 mM histidine-histidine hydrochloride,

[0055] Approximately 0.5% (w / v) to approximately 12% (w / v) sucrose,

[0056] From about 0.01% (w / v) to about 2% (w / v) polysorbate 80,

[0057] The pH of the pharmaceutical composition is from about 5.0 to about 7.0; or

[0058] (1-2) BTN3A binding protein at approximately 20 g / L to approximately 180 g / L,

[0059] Approximately 1 mM to approximately 100 mM histidine-histidine hydrochloride,

[0060] Approximately 0.5% (w / v) to approximately 12% (w / v) sucrose,

[0061] From about 0.01% (w / v) to about 2% (w / v) polysorbate 80,

[0062] Approximately 0.001% (w / v) to approximately 5% (w / v) disodium edetate;

[0063] The pH of the pharmaceutical composition is from about 5.0 to about 7.0; or

[0064] (1-3) BTN3A binding protein of approximately 20 g / L to approximately 180 g / L,

[0065] Approximately 1 mM to approximately 100 mM histidine-histidine hydrochloride,

[0066] Approximately 0.5% (w / v) to approximately 12% (w / v) sucrose,

[0067] From about 0.01% (w / v) to about 2% (w / v) polysorbate 80,

[0068] Approximately 0.01% (w / v) to approximately 10% (w / v) methionine;

[0069] The pH of the pharmaceutical composition is from about 5.0 to about 7.0; or

[0070] (1-4) Approximately 50 g / L to approximately 150 g / L of BTN3A binding protein,

[0071] Approximately 1 mM to approximately 50 mM histidine-histidine hydrochloride,

[0072] From approximately 1% (w / v) to approximately 12% (w / v) sucrose,

[0073] From about 0.01% (w / v) to about 1% (w / v) polysorbate 80,

[0074] The pH of the pharmaceutical composition is about 6.0 to about 7.0; or

[0075] (1-5) Approximately 50 g / L to approximately 150 g / L of BTN3A binding protein,

[0076] Approximately 1 mM to approximately 50 mM histidine-histidine hydrochloride,

[0077] From approximately 1% (w / v) to approximately 12% (w / v) sucrose,

[0078] From about 0.01% (w / v) to about 1% (w / v) polysorbate 80,

[0079] Approximately 0.001% (w / v) to approximately 5% (w / v) disodium edetate;

[0080] The pH of the pharmaceutical composition is about 6.0 to about 7.0; or

[0081] (1-6) Approximately 50 g / L to approximately 150 g / L of BTN3A-binding protein,

[0082] Approximately 1 mM to approximately 50 mM histidine-histidine hydrochloride,

[0083] From approximately 1% (w / v) to approximately 12% (w / v) sucrose,

[0084] From about 0.01% (w / v) to about 1% (w / v) polysorbate 80,

[0085] Approximately 0.01% (w / v) to approximately 1% (w / v) methionine;

[0086] The pH of the pharmaceutical composition is about 6.0 to about 7.0; or

[0087] (2-1) BTN3A binding protein at approximately 20 g / L to approximately 180 g / L,

[0088] Approximately 1 mM to approximately 100 mM of acetate-sodium acetate,

[0089] Approximately 0.5% (w / v) to approximately 12% (w / v) sucrose,

[0090] From about 0.01% (w / v) to about 2% (w / v) polysorbate 80,

[0091] The pH of the pharmaceutical composition is from about 4.5 to about 7.0; or

[0092] (2-2) Approximately 50 g / L to approximately 150 g / L of BTN3A-binding protein,

[0093] Approximately 1 mM to approximately 50 mM of acetate-sodium acetate

[0094] From approximately 1% (w / v) to approximately 12% (w / v) sucrose,

[0095] From about 0.01% (w / v) to about 1% (w / v) polysorbate 80,

[0096] The pH of the pharmaceutical composition is from about 4.5 to about 7.0; or

[0097] (3-1) BTN3A binding protein at approximately 20 g / L to approximately 180 g / L,

[0098] Approximately 1 mM to approximately 100 mM citric acid-sodium citrate

[0099] Approximately 0.5% (w / v) to approximately 12% (w / v) sucrose,

[0100] From about 0.01% (w / v) to about 2% (w / v) polysorbate 80,

[0101] The pH of the pharmaceutical composition is from about 5.0 to about 7.0; or

[0102] (3-2) Approximately 50 g / L to approximately 180 g / L of BTN3A-binding protein,

[0103] Approximately 1 mM to approximately 50 mM citric acid-sodium citrate

[0104] From approximately 1% (w / v) to approximately 12% (w / v) sucrose,

[0105] From about 0.01% (w / v) to about 1% (w / v) polysorbate 80,

[0106] The pH of the pharmaceutical composition is from about 5.0 to about 7.0; or

[0107] (4-1) BTN3A binding protein at approximately 20 g / L to approximately 180 g / L,

[0108] Approximately 1 mM to approximately 100 mM disodium hydrogen phosphate - sodium dihydrogen phosphate

[0109] Approximately 0.5% (w / v) to approximately 12% (w / v) sucrose,

[0110] From about 0.01% (w / v) to about 2% (w / v) polysorbate 80,

[0111] The pH of the pharmaceutical composition is from about 5.0 to about 7.0; or,

[0112] (4-2) Approximately 50 g / L to approximately 150 g / L of BTN3A-binding protein,

[0113] Approximately 1 mM to approximately 50 mM disodium hydrogen phosphate - sodium dihydrogen phosphate

[0114] From approximately 1% (w / v) to approximately 12% (w / v) sucrose,

[0115] From about 0.01% (w / v) to about 1% (w / v) polysorbate 80,

[0116] The pH of the pharmaceutical composition is from about 5.0 to about 7.0.

[0117] BTN3A binding protein

[0118] This disclosure provides a BTN3A-binding protein comprising a BTN3A-specific binding domain, said domain including a heavy chain variable region (VH) and / or a light chain variable region (VL). In some embodiments, the BTN3A-specific binding domain in the BTN3A-binding protein is one or more.

[0119] This disclosure provides a BTN3A binding protein comprising a heavy chain variable region (VH) and / or a light chain variable region (VL).

[0120] In some embodiments, the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 with amino acid sequences as shown in SEQ ID NO: 17-19, respectively, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3 with amino acid sequences as shown in SEQ ID NO: 20-22, respectively.

[0121] HCDR1 (SEQ ID NO: 17): X1YYX2X3, where X1 is selected from D or N, X2 is selected from I or M, and X3 is selected from T or N;

[0122] HCDR2 (SEQ ID NO: 18): X4IYGSX5NTX6YASWAKG, wherein X4 is selected from V or I, X5 is selected from S or D, and X6 is selected from V or Y;

[0123] HCDR3(SEQ ID NO:19):X7X8X9X 10 SSAX 11 X 12 X 13 X 14 Where X7 is selected from G or N, X8 is selected from Y or L, X9 is selected from L or D, and X 10 Selected from A or Y, X 11 Selected from D or Y, X 12 Selected from I or F, X 13 It does not exist or is H, X 14 It does not exist or is I;

[0124] LCDR1 (SEQ ID NO: 20): QX 15SQSVYNNNRLA, where X 15 Selected from S or A;

[0125] LCDR2 (SEQ ID NO: 21): X 16 ASX 17 LAS, where X 16 Selected from D or E, X 17 Selected from T or K;

[0126] LCDR3 (SEQ ID NO: 22): QX 18 YYSGX 19 IX 20 X 21 , where X 18 Selected from T or G, X 19 Selected from Y or F, X 20 Selected from W or Y, X 21 Choose from A or P.

[0127] In some embodiments, the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 of any of the amino acid sequences shown in SEQ ID NO: 1, 3, 31-35, and 39-44, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3 of any of the amino acid sequences shown in SEQ ID NO: 2, 4, 36-38, and 45-47.

[0128] In some embodiments, the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 of any of the amino acid sequences shown in SEQ ID NO: 1, 31-35, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3 of any of the amino acid sequences shown in SEQ ID NO: 2, 36-38.

[0129] In some embodiments, the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 with amino acid sequences as shown in SEQ ID NO: 5-7, respectively, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3 with amino acid sequences as shown in SEQ ID NO: 8-10, respectively.

[0130] In some embodiments, the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 of any of the amino acid sequences shown in SEQ ID NO: 3, 39-44, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3 of any of the amino acid sequences shown in SEQ ID NO: 4, 45-47.

[0131] In some embodiments, the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 with amino acid sequences as shown in SEQ ID NO: 11-13, respectively, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3 with amino acid sequences as shown in SEQ ID NO: 14-16, respectively.

[0132] The CDRs mentioned above are defined according to the Kabat, IMGT, Chothia, AbM, or Contact numbering systems, for example, the Kabat numbering system.

[0133] In some embodiments, the BTN3A binding protein comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises an amino acid sequence as shown in or having at least 80% or at least 90% identity with any of SEQ ID NO: 1, 3, 31-35, 39-44, and / or the light chain variable region comprises an amino acid sequence as shown in or having at least 80% or at least 90% identity with any of SEQ ID NO: 2, 4, 36-38, 45-47, and / or the light chain variable region comprises an amino acid sequence as shown in or having at least 80% or at least 90% identity with any of SEQ ID NO: 2, 4, 36-38, 45-47, and / or the light chain variable region.

[0134] In some embodiments, the BTN3A binding protein comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises an amino acid sequence as shown in or having at least 80% or at least 90% identity with any of SEQ ID NO: 1, 31-35, and the light chain variable region comprises an amino acid sequence as shown in or having at least 80% or at least 90% identity with any of SEQ ID NO: 2, 36-38.

[0135] In some specific implementations, the BTN3A binding protein includes a heavy chain variable region and a light chain variable region, wherein:

[0136] The heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 1 or having at least 80% or at least 90% identity with it, and the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 2 or having at least 80% or at least 90% identity with it;

[0137] The heavy chain variable region comprises an amino acid sequence as shown in any of SEQ ID NO: 31-35 or having at least 80% or at least 90% identity with it, and the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 36 or having at least 80% or at least 90% identity with it;

[0138] The heavy chain variable region comprises an amino acid sequence as shown in any of SEQ ID NO: 31-35 or having at least 80% or at least 90% identity with it, and the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 37 or having at least 80% or at least 90% identity with it; or

[0139] The heavy chain variable region comprises an amino acid sequence as shown in any of SEQ ID NO: 31-35 or having at least 80% or at least 90% identity with it, and the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 38 or having at least 80% or at least 90% identity with it.

[0140] In some embodiments, the BTN3A binding protein comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises an amino acid sequence as shown in or having at least 80% or at least 90% identity with any of SEQ ID NO: 3, 39-44, and the light chain variable region comprises an amino acid sequence as shown in or having at least 80% or at least 90% identity with any of SEQ ID NO: 4, 45-47.

[0141] In some specific implementations, the BTN3A binding protein includes both heavy chain variable regions and light chain variable regions:

[0142] The heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 3 or having at least 80% or at least 90% identity with it, and the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 4 or having at least 80% or at least 90% identity with it;

[0143] The heavy chain variable region comprises an amino acid sequence as shown in any of SEQ ID NO: 39-44 or having at least 80% or at least 90% identity with it, and the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 45 or having at least 80% or at least 90% identity with it;

[0144] The heavy chain variable region comprises an amino acid sequence as shown in any of SEQ ID NO: 39-44 or having at least 80% or at least 90% identity with it, and the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 46 or having at least 80% or at least 90% identity with it; or,

[0145] The heavy chain variable region comprises an amino acid sequence as shown in any of SEQ ID NO: 39-44 or having at least 80% or at least 90% identity with it, and the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 47 or having at least 80% or at least 90% identity with it.

[0146] In some implementations, the aforementioned BTN3A binding protein is an anti-BTN3A antibody or its antigen-binding fragment.

[0147] In some implementation schemes, the aforementioned antibodies are rabbit-derived antibodies, chimeric antibodies, humanized antibodies, or fully human antibodies.

[0148] In some implementation schemes, the aforementioned antibody is a recombinant antibody.

[0149] In some implementations, the aforementioned antibody is a monospecific antibody or a multispecific antibody (e.g., a bispecific antibody, a trispecific antibody, or a tetraspecific antibody).

[0150] In some embodiments, the aforementioned antibody or its antigen-binding fragment is a single-chain antibody (i.e., full-length heavy chain and light chain); Fab, modified Fab, Fab', modified Fab', F(ab')2, Fv, Fab-Fv, Fab-dsFv, single-domain antibody (e.g., VH or VL or VHH), scFv, bivalent or trivalent or tetravalent antibody, Bis-scFv, diabody, tribody, triabody, tetrabody, and epitope-binding fragments of any of the above.

[0151] In some implementations, the aforementioned antibody or its antigen-binding fragment is humanized, reversed mutation, affinity matured, T-cell epitope removed / reduced, antibody deamidated, and / or antibody isomerized.

[0152] In some implementations, after TCE removal / reduction modification, there are one or more changes in one or more CDRs, which result in a reduction in the immunogenicity of the BTN3A binding protein.

[0153] In some implementation schemes, the heavy-chain framework region of the genus template used in the humanization process is derived from IGHV3-66, and the light-chain framework region is derived from IGkV1-27.

[0154] In some embodiments, the BTN3A binding protein further includes an Fc region of an immunoglobulin. In some specific embodiments, the Fc region is the Fc region of human IgG1, human IgG2, human IgG3, or human IgG4. In some specific embodiments, the Fc region may be an Fc region with reduced effector function; for example, the Fc region may have a mutation, resulting in reduced effector function in an exemplary IgG. The Fc region includes substitutions with the following: N297A or N297Q (IgG1); L234A / L235A (IgG1); V234A / G237A (IgG2); L235A / G237A / E318A (IgG4); H268Q / V309L / A330S / A331S (IgG2); C220S / C226S / C229S / P238S (IgG1); C226S / C229S / E233P / L234V / L235A (IgG1); L234F / L235E / P331S (IgG1); or S267E / L328F (IgG1), where “ / ” indicates “and”. In some specific embodiments, the Fc region is the Fc region of human IgG1 with L234F, L235E and / or P331S mutations, and optionally, K446 is removed. In some specific embodiments, the Fc region has an amino acid sequence having at least 80% or 90% identity with SEQ ID NO: 23, 24.

[0155] In some embodiments, the Fc region is stability-enhancing, or has a mutation that increases the stability of the Fc region compared to the wild-type Fc region. In some embodiments, the Fc region can cause the binding protein to form a dimer molecule. In some embodiments, the Fc region can prolong the in vivo half-life of the binding protein.

[0156] In some embodiments, the BTN3A binding protein comprises a heavy chain and a light chain, wherein the heavy chain comprises an amino acid sequence as shown in or having at least 80% or at least 90% identity with any of SEQ ID NO: 25, 27, 48-52, 56-61, and / or the light chain comprises an amino acid sequence as shown in or having at least 80% or at least 90% identity with any of SEQ ID NO: 26, 28, 53-55, 62-64.

[0157] In some specific implementations, the BTN3A binding protein contains both a heavy chain and a light chain:

[0158] The heavy chain comprises an amino acid sequence as shown in SEQ ID NO: 25 or having at least 80% or at least 90% identity with it, and the light chain comprises an amino acid sequence as shown in SEQ ID NO: 26 or having at least 80% or at least 90% identity with it;

[0159] The heavy chain comprises an amino acid sequence as shown in any of SEQ ID NO: 48-52 or having at least 80% or at least 90% identity with it, and the light chain comprises an amino acid sequence as shown in SEQ ID NO: 53 or having at least 80% or at least 90% identity with it;

[0160] The heavy chain comprises an amino acid sequence as shown in any of SEQ ID NO: 48-52 or having at least 80% or at least 90% identity with it, and the light chain comprises an amino acid sequence as shown in SEQ ID NO: 54 or having at least 80% or at least 90% identity with it; or,

[0161] The heavy chain comprises an amino acid sequence as shown in any of SEQ ID NO: 48-52 or having at least 80% or at least 90% identity with it, and the light chain comprises an amino acid sequence as shown in SEQ ID NO: 55 or having at least 80% or at least 90% identity with it.

[0162] In some specific implementations, the BTN3A binding protein includes:

[0163] The heavy chain comprises an amino acid sequence as shown in SEQ ID NO: 27 or having at least 80% or at least 90% identity with it, and the light chain comprises an amino acid sequence as shown in SEQ ID NO: 28 or having at least 80% or at least 90% identity with it;

[0164] The heavy chain comprises an amino acid sequence as shown in any of SEQ ID NO: 56-61 or having at least 80% or at least 90% identity with it, and the light chain comprises an amino acid sequence as shown in SEQ ID NO: 62 or having at least 80% or at least 90% identity with it;

[0165] The heavy chain comprises an amino acid sequence as shown in any of SEQ ID NO: 56-61 or having at least 80% or at least 90% identity with it, and the light chain comprises an amino acid sequence as shown in SEQ ID NO: 63 or having at least 80% or at least 90% identity with it; or,

[0166] The heavy chain comprises an amino acid sequence as shown in any of SEQ ID NO: 56-61 or having at least 80% or at least 90% identity with it, and the light chain comprises an amino acid sequence as shown in SEQ ID NO: 64 or having at least 80% or at least 90% identity with it.

[0167] In this disclosure, "at least 80% (sequence) identity" encompasses at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% (sequence) identity; "at least 90% (sequence) identity" encompasses at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% (sequence) identity.

[0168] In some implementations, the BTN3A binding protein specifically binds to one or more of BTN3A1, BTN3A2, and BTN3A3, for example:

[0169] It specifically binds to BTN3A1, but does not specifically bind to BTN3A2 or BTN3A3.

[0170] It specifically binds to BTN3A2, but does not specifically bind to BTN3A1 or BTN3A3.

[0171] It specifically binds to BTN3A3, but does not specifically bind to BTN3A1 or BTN3A2.

[0172] It specifically binds to BTN3A1 and BTN3A2, but does not specifically bind to BTN3A3.

[0173] It specifically binds to BTN3A2 and BTN3A3, but does not specifically bind to BTN3A1.

[0174] It specifically binds to BTN3A1 and BTN3A3, but does not specifically bind to BTN3A2.

[0175] It also specifically binds to BTN3A1, BTN3A2, and BTN3A3.

[0176] In some specific implementation schemes, the aforementioned BTN3A1, BTN3A2, and BTN3A3 are primate or human BTN3A1, BTN3A2, and BTN3A3.

[0177] In some embodiments, the BTN3A binding protein of this disclosure has one or more of the following characteristics:

[0178] (i) EC50 measured by ELISA, with concentrations of 0.1 nM, 0.05 nM, 0.02 nM, 0.01 nM or lower. 50In combination with human BTN3A1, the ELSIA detection method is well known in the art, for example, as described in Part 1 of Embodiment 3 of this disclosure;

[0179] (ii) EC measured by FACS, with values ​​of 50 nM, 20 nM, 15 nM, 10 nM, 8 nM, 5 nM or lower. 50 The FACS detection method is known in the art, for example, as described in Part 2 of Example 3 of this disclosure, for binding to human BTN3A1 on cells;

[0180] (iii) As measured by Biacore, its K values ​​are 5E-10M, 2E-10M, 1E-10M or lower. D In conjunction with human BTN3A1, the Biacore detection method is well known in the art, for example, as described in Part 3 of Embodiment 3 of this disclosure;

[0181] (iv) EC at 10 nM, 5 nM, 2 nM, 1 nM or lower 50 Inducing activation and proliferation of γδT cells (e.g., Vγ9Vδ2 T cells), the EC 50 The detection methods are well known in the art, such as those described in Part 4 of Embodiment 3 of this disclosure; in some embodiments, the induction is generated when γδT cells are co-cultured with cancer cells (e.g., SKOV-3 cells);

[0182] (v) Inducing γδT cells (e.g., Vγ9Vδ2T cells) to secrete cytokines (e.g., IFNγ); in some embodiments, the induction is generated when γδT cells are co-cultured with cancer cells (e.g., SKOV-3 cells).

[0183] This disclosure also provides a BTN3A binding protein comprising any one or any combination of the following CDRs: HCDR1, HCDR2, and HCDR3 with amino acid sequences as shown in SEQ ID NO: 17-19, respectively, and LCDR1, LCDR2, and LCDR3 with amino acid sequences as shown in SEQ ID NO: 20-22, respectively.

[0184] In some embodiments, this disclosure provides the aforementioned BTN3A binding protein variants having one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) amino acid mutations compared to the heavy chain variable region of any of the amino acid sequences shown in SEQ ID NO: 1, 3, 31-35, 39-44 and / or the light chain variable region of any of the amino acid sequences shown in SEQ ID NO: 2, 4, 36-38, 45-47; said amino acid mutations may be conserved substitutions, substitutions, or modifications, and / or deletions or additions that do not affect function; said amino acid mutations may occur in the CDR region and / or FR region.

[0185] In some embodiments, a protein or molecule is provided that binds to or competitively binds to the same epitope as the aforementioned BTN3A-binding protein of this disclosure.

[0186] In some embodiments, a protein or molecule is provided that blocks the binding of the aforementioned BTN3A-binding protein of this disclosure to BTN3A (including BTN3A1, BTN3A2 and / or BTN3A3).

[0187] In some embodiments, a protein or molecule is provided whose binding to BTN3A (including BTN3A1, BTN3A2 and / or BTN3A3) is blocked by the aforementioned BTN3A binding protein of this disclosure.

[0188] In some embodiments, a protein or molecule is provided comprising any combination of one or more heavy chain variable regions and light chain variable regions as described in this disclosure, wherein the heavy chain variable region comprises a sequence selected from any of SEQ ID NO: 1, 3, 31-35, 39-44, and the light chain variable region comprises a sequence selected from any of SEQ ID NO: 2, 4, 36-38, 45-47. For example, the protein or molecule is a conjugate, which may, for example, contain any detectable tag.

[0189] In some embodiments, the BTN3A binding protein in this disclosure is a protein or molecule disclosed in WO2025031287A, which is incorporated herein by reference.

[0190] This disclosure also provides a lyophilized formulation, which is obtained by freeze-drying the pharmaceutical composition, or the lyophilized formulation can be reconstituted to form the pharmaceutical composition.

[0191] This disclosure also provides a reconstituted solution, which is prepared by reconstituted the lyophilized formulation.

[0192] In some embodiments, the reconstituted solution is obtained by reconstituted the lyophilized preparation with a solvent; preferably, the solvent is water, physiological saline or glucose.

[0193] In some embodiments, the pharmaceutical composition or reconstituted solution is an intravenous injection, subcutaneous injection, intraperitoneal injection, or intramuscular injection; preferably an intravenous or subcutaneous injection.

[0194] This disclosure also provides an article comprising a container containing the pharmaceutical composition, the lyophilized formulation, and the reconstituted solution.

[0195] Treatment

[0196] This disclosure also provides a method of treating or preventing a disease, comprising administering to a subject in need a therapeutic or preventative amount of the pharmaceutical composition, the lyophilized formulation, the reconstituted solution, or the article thereof.

[0197] In some implementations, the aforementioned disease is a disease or condition associated with BTN3A overexpression.

[0198] In some implementations, the aforementioned disease is a disease or condition mediated by BTN3A.

[0199] In some implementation schemes, the aforementioned disease is cancer.

[0200] Terminology Definition

[0201] To facilitate understanding of this disclosure, certain technical and scientific terms are specifically defined below. Unless otherwise expressly defined in this disclosure, all other technical and scientific terms used in this disclosure shall have the meaning commonly understood by one of ordinary skill in the art to which this disclosure pertains.

[0202] "Buffer" refers to a buffer that is resistant to pH changes through the action of its acid-base conjugate components. Examples of buffers that maintain pH within an appropriate range include acetate, succinate, gluconate, histidine, oxalate, lactate, phosphate, citrate, tartrate, fumarate, glycylglycine, and other organic acid buffers.

[0203] "Histidine buffer" is a buffer containing histidine ions. Examples of histidine buffers include acetate-histidine, succinate-histidine, histidine-histidine hydrochloride, hydrochloride-histidine, sulfate-histidine, and other buffers. For example, a histidine-hydrochloride buffer is prepared by reacting histidine with hydrochloric acid or histidine with histidine hydrochloride.

[0204] "Pharmaceutical composition" means a mixture containing one or more of the compounds described herein or their physiologically / pharmacologically acceptable salts or prodrugs, along with other chemical components, such as physiologically / pharmacologically acceptable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration to a living organism, thereby promoting the absorption of the active ingredient and its biological activity. In this document, "pharmaceutical composition" and "formulation" are not mutually exclusive.

[0205] Unless otherwise specified, the solvent in the solution form of the pharmaceutical compositions described in this disclosure is water.

[0206] As used herein, the term "about" means a numerical value within the acceptable error range of a specific value determined by a person skilled in the art, the numerical value depending in part on how it is measured or determined (i.e., the limits of the measurement system). For example, in every practice in the art, "about" may mean within or above 1 standard deviation. Alternatively, "about" or "substantially includes" may mean a range of up to 20%. Furthermore, particularly for biological systems or processes, the term may mean up to an order of magnitude or up to five times the numerical value. Unless otherwise stated, when a specific value appears in this disclosure and claims, the meaning of "about" or "substantially includes" should be assumed to be within the acceptable error range of that specific value.

[0207] The pharmaceutical compositions described in this disclosure achieve a stable effect: the antibodies therein substantially retain their physical and / or chemical stability and / or biological activity after storage; preferably, the pharmaceutical compositions substantially retain their physical and chemical stability and their biological activity after storage. The storage period is generally selected based on the intended shelf life of the pharmaceutical composition. Currently, various analytical techniques are available for measuring protein stability, which can measure stability after storage at a selected temperature for a selected period of time.

[0208] Stable drug antibody formulations are those in which no significant changes are observed when stored at refrigerated temperatures (2-8°C) for at least 3 months, preferably 6 months, more preferably 1 year, and even more preferably up to 2 years. Additionally, stable liquid formulations include those that exhibit the desired characteristics after storage at 25°C for 1 month, 3 months, 6 months, or at 40°C for 1 month. Typical acceptable criteria for stability are as follows: degradation of antibody monomers typically not exceeding about 10%, preferably not exceeding about 5%, as determined by SEC-HPLC. Visually, the drug antibody formulation is colorless to yellow, clear to slightly opalescent. The concentration, pH, and osmotic pressure of the formulation exhibit variations not exceeding ±10%. Truncation typically not exceeding about 10%, preferably not exceeding about 5%, and aggregation typically not exceeding about 10%, preferably not exceeding about 5%, are observed.

[0209] If, after visual inspection of color and / or clarity, or by means of UV light scattering, size exclusion chromatography (SEC), and dynamic light scattering (DLS), the antibody does not show significant increase in aggregation, precipitation, and / or denaturation, then the antibody “retains its physical stability” in the pharmaceutical formulation. Changes in protein conformation can be evaluated by fluorescence spectroscopy (which determines the tertiary structure of the protein) and by FTIR spectroscopy (which determines the secondary structure of the protein).

[0210] If an antibody does not show significant chemical changes, then the antibody "retains its chemical stability" in the pharmaceutical formulation. Chemical stability can be assessed by detecting and quantifying the chemically altered form of the protein. Degradation processes that frequently alter the chemical structure of a protein include hydrolysis or truncation (evaluated by methods such as size exclusion chromatography and SDS-PAGE), oxidation (evaluated by methods such as peptide mapping combined with mass spectrometry or MALDI / TOF / MS), deamidation (evaluated by methods such as ion exchange chromatography, capillary isoelectric focusing, peptide mapping, and isofpartate measurement), and isomerization (evaluated by measuring isofpartate content, peptide mapping, etc.).

[0211] If the antibody's biological activity at a given time is within a predetermined range of the biological activity exhibited when the pharmaceutical formulation is prepared, then the antibody "retains its biological activity" in the pharmaceutical formulation. The biological activity of an antibody can be determined, for example, by an antigen-binding assay.

[0212] The three-letter and single-letter codes for amino acids used in this disclosure are as described in J. biol. chem, 243, p3558 (1968).

[0213] "BTN3A" has its general meaning in the art and refers to human BTN3A polypeptide, exemplarily BTN3A1 as shown in NP_008979.3 (Genbank Accession NO), BTN3A2 as shown in NP_008978.2 (Genbank Accession NO), or BTN3A3 as shown in NP_008925.1 (Genbank Accession NO).

[0214] "BTN3A binding protein" encompasses any protein capable of specifically binding to BTN3A or any molecule containing said protein, including but not limited to anti-BTN3A antibodies against BTN3A as defined in this disclosure, their antigen-binding fragments, or conjugates thereof. In some embodiments, the BTN3A binding protein may include a linker and / or a portion having effector function, such as a half-life extended portion (e.g., an immunoglobulin single variable domain binding serum albumin) and / or a fusion partner (e.g., serum albumin) and / or a conjugated polymer (e.g., PEG) and / or an Fc region.

[0215] The term "antibody" encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, monospecific antibodies, multispecific antibodies (e.g., bispecific antibodies), full-length antibodies, and antibody fragments (or antigen-binding fragments, or antigen-binding portions), as long as they exhibit the desired antigen-binding activity. Antibodies can refer to immunoglobulins, which are tetrapeptide chains composed of two heavy chains and two light chains linked by interchain disulfide bonds. The amino acid composition and sequence of the constant region of the heavy chain of immunoglobulins differ, thus their antigenicity also differs. Based on this, immunoglobulins can be divided into five classes, or isotypes of immunoglobulins: IgM, IgD, IgG, IgA, and IgE, with their corresponding heavy chains being μ, δ, γ, α, and ε chains, respectively. Within the same class of Ig, based on differences in the amino acid composition of the hinge region and the number and position of disulfide bonds in the heavy chain, different subclasses can be distinguished; for example, IgG can be divided into IgG1, IgG2, IgG3, and IgG4. Light chains are classified into κ chains or λ chains based on differences in their constant regions. Each of the five classes of Ig can have either a κ chain or a λ chain. The sequence of approximately 110 amino acids near the N-terminus of the antibody heavy and light chains varies considerably, forming the variable region (V region); the remaining amino acid sequences near the C-terminus are relatively stable, forming the constant region (C region). The variable region includes three hypervariable regions (HVR) and four relatively conserved framework regions (FR). The three hypervariable regions determine the antibody's specificity and are also known as complementarity-determining regions (CDR). Each light chain variable region (VL) and heavy chain variable region (VH) consists of three CDR regions and four FR regions, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The three CDR regions of the light chain refer to LCDR1, LCDR2, and LCDR3; the three CDR regions of the heavy chain refer to HCDR1, HCDR2, and HCDR3.

[0216] The antibodies disclosed herein can be polyclonal, monoclonal, xenogeneic, allogeneic, syngeneic, or modified forms thereof, with monoclonal antibodies being particularly suitable in many embodiments. Generally, the antibodies disclosed herein are recombinant antibodies. As used herein, “recombinant” refers to products such as cells or nucleic acids, proteins, or vectors, indicating that said cells, nucleic acids, proteins, or vectors have been modified by introducing heterologous nucleic acids or proteins or by altering native nucleic acids or proteins, or that said cells are derived from such modified cells. For example, recombinant cells express genes not present in native (non-recombinant) cell forms or express native genes that are abnormally expressed, poorly expressed, or not expressed at all.

[0217] "Antigen-binding fragments" encompass single-chain antibodies (i.e., full-length heavy and light chains); Fab, modified Fab, Fab', modified Fab', F(ab')2, Fv, Fab-Fv, Fab-dsFv, single-domain antibodies (e.g., VH, VL, or VHH), scFv, bivalent, trivalent, or tetravalent antibodies, Bis-scFv, diabody, tribody, triabody, tetrabody, and epitope-binding fragments of any of the above (see, for example, Holliger and Hudson, 2005, Nature Biotech. 23(9): 1126-1136; Adair and Lawson, 2005, Drug Design Reviews-Online 2(3), 209-217). Methods for generating and preparing these antigen-binding fragments are well known in the art (see, for example, Verma et al., 1998, Journal of Immunological Methods, 216, 165-181).

[0218] The determination or definition of a CDR can be accomplished by resolving the structure of the antibody and / or the structure of the antibody-ligand complex, thereby enabling the definitive depiction of the CDR and the identification of residues containing the antibody binding site. This can be achieved using any of the various techniques known to those skilled in the art, such as X-ray crystallography. A variety of analytical methods can be used to identify CDRs, including but not limited to the Kabat numbering system, the Chothia numbering system, the AbM numbering system, the IMGT numbering system, contact definition, and conformation definition. The Kabat numbering system is the standard for numbering residues in antibodies and is commonly used to identify CDR regions (see, for example, Johnson & Wu, 2000, Nucleic Acids Res., 28: 214-8). The Chothia numbering system is similar to the Kabat numbering system, but it takes into account the location of certain structural loop regions (see, for example, Chothia et al., 1986, J. Mol. Biol., 196: 901-17; Chothia et al., 1989, Nature, 342: 877-83). The AbM numbering system uses a computer program integration suite produced by the Oxford Molecular Group to model antibody structures (see, for example, Martin et al., 1989, ProcNatl Acad Sci (USA), 86: 9268-9272; "AbMTM, A Computer Program for Modeling Variable Regions of Antibodies," Oxford, UK; Oxford Molecular, Ltd). The AbM numbering system uses a combination of knowledge databases and a de novo approach to model the tertiary structure of antibodies from basic sequences (see those described in Samudrala et al., 1999, "Ab Initio Protein Structure Prediction Using a Combined Hierarchical Approach" in PROTEINS, Structural, Function and Genetics Suppl., 3: 194-198). Contact definitions are based on the analysis of available complex crystal structures (see, for example, MacCallum et al., 1996, J. Mol. Biol., 5: 732-45). In the conformational definition, the position of CDR can be identified as a residue that contributes enthalpy to antigen binding (see, for example, Makabe et al., 2008, Journal of Biological Chemistry, 283: 1156-1166).Other CDR boundary definitions may not strictly follow one of the methods described above, but still overlap with at least a portion of the Kabat CDR, although they may be shortened or lengthened depending on the predicted or experimental results that a particular residue or group of residues does not significantly affect antigen binding. As used in this disclosure, a CDR may refer to a CDR defined by any method (including combinations of methods) known in the art. The correspondence between various numbering systems is well known to those skilled in the art.

[0219] A "domain" of a polypeptide or protein refers to a folded protein structure that can maintain its tertiary structure independently of the rest of the protein. Generally, a domain is responsible for a single functional property of a protein and, in many cases, can be added to, removed from, or transferred to other proteins without losing the function of the rest of the protein and / or the domain itself.

[0220] "Antibody framework (FR)" refers to a portion of a variable domain that serves as a scaffold for the antigen-binding loop (CDR) of that variable domain.

[0221] "Humanized antibody," also known as CDR-grafted antibody, refers to an antibody generated by grafting a non-human CDR sequence into the variable region framework of a human antibody. This overcomes the strong immune response induced by chimeric antibodies due to their carrying of large amounts of non-human protein components. To avoid a decrease in activity along with a decrease in immunogenicity, minimal reverse mutations can be performed on the variable region of the fully human antibody to maintain activity. Examples of "humanization" include the "humanization" of a camelid-derived VHH domain by replacing one or more amino acid residues in the original VHH sequence with one or more amino acid residues present at the corresponding position in the VH domain of a conventional human tetrapeptide chain antibody. The humanized VHH domain may contain one or more fully human framework region sequences, and in some specific embodiments, may contain the human framework region sequence of IGHV3. Humanization methods include protein surface amino acid resurfacing and antibody humanization using a universal framework grafting method (CDR grafting to a universal framework), which involves "grafting" the CDR onto other "scaffolds" (including but not limited to human scaffolds or non-immunoglobulin scaffolds). Suitable scaffolds and techniques for CDR grafting are known in the art. Germline DNA sequences of human heavy and light chain variable region genes, for example, can be found in the VBase human germline sequence database and in Kabat, EA et al., 1991, Sequences of Proteins of Immunological Interest, 5th edition. The humanized antibodies disclosed herein also include humanized antibodies further matured by phage display with affinity for the CDR. Furthermore, to avoid a decrease in activity along with a decrease in immunogenicity, minimal reverse or reversion mutations can be performed on the human antibody variable region framework sequence to maintain activity.

[0222] "Affinity-matured" antibodies are those that have one or more alterations in one or more hypervariable regions (HVRs) compared to parental antibodies that do not possess such alterations, resulting in improved affinity of the antibody for the antigen. For example, an "affinity-matured" BTN3A binding protein or anti-BTN3A antibody has one or more alterations in one or more CDRs, resulting in increased affinity for the antigen compared to its parent antibody. Affinity-matured antibodies can be prepared, for example, by methods known in the art as described below: Marks et al., 1992, Biotechnology 10: 779-783 or Barbas et al., 1994, Proc. Nat. Acad. Sci, USA 91: 3809-3813; Shier et al., 1995, Gene 169: 147-155; Yelton et al., 1995, Immunol. 155: 1994-2004; Jackson et al., 1995, J. Immunol. 154(7): 3310-9; and Hawkins et al., 1992, J. MoI. Biol. 226(3): 889896; KS Johnson and RE Hawkins, “Affinity maturation of antibodies using phage display”, Oxford University Press 1996.

[0223] Typically, the BTN3A binding protein of this disclosure will be measured in a preferred 10 as in a Biacore, KinExA, or Fortibio assay. -7 Up to 10 -10 mol / L (M), more preferably 10 -8 Up to 10 -10 moles per liter, or even more preferably 10 -9 Up to 10 -10 or a lower dissociation constant (K) D ), and / or at least 10 -7 M, preferably at least 10 -8 M, more preferably at least 10 -9 M, more preferably at least 10 -10 The association constant (KA) of M binds to the antigen or target protein it is intended to bind to (i.e., BTN3A1, BTN3A2, BTN3A3). Any protein greater than 10... -4 M of K DValues ​​are generally considered to indicate nonspecific binding. The specific binding of antigen-binding proteins to antigens or epitopes can be determined in any suitable manner known, including, for example, surface plasmon resonance (SPR) assays, Scatchard assays, and / or competitive binding assays (e.g., radioimmunoassay (RIA), enzyme immunoassay (EIA), and sandwich competitive assays) as described in this disclosure.

[0224] "Binding affinity" or "affinity" is used in this disclosure as a measure of the strength of a non-covalent interaction between two molecules (e.g., an antibody or a portion thereof with an antigen). The binding affinity between two molecules can be determined by determining the dissociation constant (K). D Quantification can be achieved by using methods such as surface plasmon resonance (SPR) (Biacore) to measure the kinetics of complex formation and dissociation. D The rate constants corresponding to the binding and dissociation of monovalent complexes are called the binding rate constant ka (or kon) and the dissociation rate constant kd (or koff), respectively. D Through equation K D =kd / ka is related to ka and kd. The value of the dissociation constant can be determined directly by well-known methods, and even for complex mixtures, it can be calculated using methods such as those described by Caceci et al. (1984, Byte 9: 340-362). For example, K can be determined using a double-filtered nitrocellulose filter combined with determinations such as those disclosed in Wong & Lohman (1993, Proc. Natl. Acad. Sci. USA 90: 5428-5432). D Other standard assays for assessing the binding ability of antibodies to target antigens are known in the art, including, for example, ELISA, Western blotting, RIA, and flow cytometry, as well as other assays exemplified elsewhere in this disclosure. Antibody binding kinetics and binding affinity can also be determined using standard assays known in the art, such as surface plasmon resonance (SPR), for example, by using Biacore. TM Evaluation can be performed using a system or KinExA. The Kelvin values ​​of individual antibody / antigen complexes can be compared. D The binding affinity is used to compare the binding affinity of different molecules associated with their interactions, for example, comparing the binding affinity of different antibodies for a given antigen. Similarly, the specificity of an interaction can be determined and compared by identifying and comparing the K-value of the target interaction (e.g., the specific interaction between an antibody and an antigen). D The value of K for non-target interactions (e.g., control antibodies known not to bind BTN3A) D The value is evaluated.

[0225] "Conservative substitution" refers to the substitution with another amino acid residue that has properties similar to the original amino acid residue. For example, lysine, arginine, and histidine have similar properties in that they have basic side chains, and aspartic acid and glutamic acid have similar properties in that they have acidic side chains. Furthermore, glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, and tryptophan have similar properties in that they have uncharged polar side chains, and alanine, valine, leucine, threonine, isoleucine, proline, phenylalanine, and methionine have similar properties in that they have nonpolar side chains. Additionally, tyrosine, phenylalanine, tryptophan, and histidine have similar properties in that they have aromatic side chains. Therefore, it will be apparent to those skilled in the art that even when amino acid residues in the group exhibiting similar properties as described above are substituted, it will not show a specific change in properties.

[0226] "Homology," "identity," or "sequence identity" refers to the sequence similarity between two polynucleotide sequences or two polypeptides. When positions in two compared sequences are occupied by the same nucleotide or amino acid monomer—for example, if every position in two DNA molecules is occupied by the same nucleotide—then the molecules are homologous at that position. The percentage of homology between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of positions compared multiplied by 100%. For example, at optimal sequence alignment, if six out of ten positions in two sequences match or are homologous, then the two sequences are 60% homologous. Generally, comparisons are made when the highest percentage of homology is obtained by aligning the two sequences.

[0227] The terms "nucleic acid" and "polynucleotide" are used interchangeably in this disclosure and refer to any single-stranded or double-stranded DNA or RNA molecule, and in the case of a single-stranded molecule, its complementary sequence, preferably double-stranded DNA. When a nucleic acid is placed in a functional relationship with another nucleic acid sequence, the nucleic acid is "effectively linked." For example, if a promoter or enhancer affects the transcription of a coding sequence, then the promoter or enhancer is effectively linked to said coding sequence.

[0228] "Host cell" includes individual cells or cell cultures that may be, or have been, recipients of vectors for incorporating polynucleotide inserts. Host cells include progeny of a single host cell, and progeny may not necessarily be identical to the original parent cell (in morphology or genomic DNA complementation) due to natural, accidental, or intentional mutations. Host cells include cells transfected and / or transformed in vivo with the polynucleotides of this disclosure. "Cell," "cell line," and "cell culture" are used interchangeably, and all such names include their progeny. It should also be understood that, due to intentional or unintentional mutations, all progeny may not be exactly identical in DNA content. This includes mutant progeny with the same function or biological activity as those screened from the originally transformed cells.

[0229] "Inhibit" or "block" are used interchangeably and cover both partial and complete inhibition / blockage. "Inhibit growth" (e.g., involving cells) is intended to include any measurable reduction in cell growth.

[0230] "Giving," "applying," and "treatment," when applied to animals, humans, experimental subjects, cells, tissues, organs, or biological fluids, refer to the contact of an exogenous drug, therapeutic agent, diagnostic agent, or composition with the animal, human, subject, cell, tissue, organ, or biological fluid, such as in therapeutic, pharmacokinetic, diagnostic, research, and experimental methods. Cellular treatment includes contact between a reagent and a cell, as well as contact between a reagent and a fluid, wherein the fluid is in contact with the cell. "Giving," "applying," and "treatment" also mean treatment, such as of cells, by means of a reagent, diagnostic agent, conjugate composition, or by means of another cell in vitro and ex vivo. When applied to humans, veterinary, or research subjects, it refers to therapeutic treatment, preventative or prophylactic measures, research, and diagnostic applications.

[0231] "Treatment" means administering, either internally or externally, a therapeutic agent, such as a pharmaceutical composition comprising any of the binding proteins of this disclosure or thereof, to a subject who has, is suspected of having, or is predisposed to having one or more proliferative diseases or their symptoms, and the therapeutic agent is known to have a therapeutic effect on these symptoms. Typically, the therapeutic agent is administered in a treated subject or population in an amount that effectively relieves symptoms of one or more diseases, whether by inducing the regression of such symptoms or inhibiting their development to any clinically measurable degree. The amount of therapeutic agent that effectively relieves symptoms of any specific disease (also referred to as the "therapeuticly effective amount") can vary depending on a variety of factors, such as the subject's disease state, age, and weight, and the drug's ability to produce the desired therapeutic effect in the subject. Whether the disease symptoms have been relieved can be evaluated using any clinical test method commonly used by a physician or other healthcare professional to assess the severity or progression of the symptoms. Although the embodiments of this disclosure (e.g., treatment methods or products) may be ineffective in alleviating the symptoms of the target disease in a particular subject, they should reduce the symptoms of the target disease in a statistically significant number of subjects, as determined by any statistical test known in the art, such as the Student t-test, chi-square test, U-test according to Mann and Whitney, Kruskal-Wallis test (H-test), Jonckheere-Terpstra test, and Wilcoxon test.

[0232] An "effective amount" includes an amount sufficient to improve or prevent the symptoms or condition of a medical condition. An effective amount also means an amount sufficient to allow or facilitate a diagnosis. The effective amount used on a subject can vary depending on factors such as the condition to be treated, the subject's overall health, the route and dosage of administration, and the severity of side effects. An effective amount can be the maximum dose or administration regimen that avoids significant side effects or toxicity.

[0233] "Optional" or "optionally" means that the event or circumstance described subsequently may, but does not necessarily, occur, and the description includes the possibility that the event or circumstance may or may not occur. "And / or" should be considered as specifically disclosing that each of the two specified features or components has or does not have the other. Therefore, the term "and / or" as used in phrases such as "A and / or B" in this disclosure includes "A and B," "A or B," "A" (alone), and "B" (alone). Unless the context clearly requires otherwise, throughout the specification and claims, the words "comprising," "having," "including," etc., should be understood to have an inclusive meaning rather than an exclusive or exhaustive meaning; that is, the meaning of "including but not limited to." In the context of mutations contained in the Fc region in this disclosure, " / " means "and," for example, "L234A / L235A" means "L234A and L235A," that is, the Fc region contains the L234A and L235A mutations; the amino acid positions of the mutations in the Fc region of this disclosure are defined according to the EU numbering system.

[0234] In this disclosure, "subject" and "patient" refer to mammals, especially primates, and particularly humans. Attached Figure Description

[0235] Figure 1 shows the ELISA results of the binding ability of the anti-BTN3A1 chimeric antibody to recombinant human BTN3A1 protein.

[0236] Figure 2 shows the FACS results of the binding ability of the anti-BTN3A1 chimeric antibody to human BTN3A1 overexpressing cells.

[0237] Figure 3 shows the activation ability of anti-BTN3A1 chimeric antibody on γδT cells. Vγ9Vδ2T cells were co-cultured with A375 target cells, incubated with the antibody to be tested, and the content of human IFNγ secreted in the cell supernatant was measured by ELISA.

[0238] Figure 4 shows the results of detecting the killing ability of BTN3A1 chimeric antibody-mediated γδT cells against A375 cells. Vγ9Vδ2T cells and A375 cells were co-cultured, incubated with the test antibody, and after cell lysis, the luminescent activity was measured using an ELISA reader.

[0239] Figure 5 shows the activation ability of the 20A3 humanized antibody on γδT cells. Vγ9Vδ2T cells were co-cultured with A375 target cells, incubated with the antibody to be tested, and the content of secreted human IFNγ in the cell supernatant was measured by ELISA.

[0240] Figure 6A shows the FACS results of the binding ability of the humanized antibody 17G4 to human BTN3A1 overexpressing cells, and Figure 6B shows the FACS results of the binding ability of the humanized antibody 20A3 to human BTN3A1 overexpressing cells.

[0241] Figure 7A shows the FACS results of the binding ability of 20A3-H2L2 to human BTN3A1 overexpressing cells; Figure 7B shows the FACS results of the binding ability of 20A3-H2L2 to human BTN3A2 overexpressing cells; and Figure 7C shows the FACS results of the binding ability of 20A3-H2L2 to human BTN3A3 overexpressing cells.

[0242] Figure 8 shows the activation ability of 20A3-H2L2 on γδT cells. Vγ9Vδ2T cells were co-cultured with SKOV-3 target cells, incubated with the test antibody, and the content of secreted human IFNγ in the cell supernatant was measured by ELISA.

[0243] Figure 9 shows the results of detecting the killing ability of 20A3-H2L2-mediated γδT cells against SKOV-3 cells. Vγ9Vδ2T cells and SKOV-3 cells were co-cultured, incubated with the test antibody, and after cell lysis, the luminescent activity was measured using an ELISA reader. Detailed Implementation

[0244] The following embodiments are used to further describe this disclosure, but these embodiments are not intended to limit the scope of this disclosure. Experimental methods in the embodiments of this disclosure that do not specify specific conditions are generally performed under conventional conditions, such as those described in Cold Spring Harbor's Antibody Technology Manual or Molecular Cloning Manual; or under conditions recommended by the raw material or commercial manufacturer. Reagents that do not specify a specific source are commercially available, conventional reagents.

[0245] Example 1. Screening and preparation of rabbit-derived monoclonal antibodies against human BTN3A1

[0246] 1. Immunogenic antigens, screening antigens

[0247] The recombinant human BTN3A1 protein (h-BTN3A1-mFc-his) tagged with mouse IgG2a Fc and his, the biotinylated recombinant human BTN3A1 protein (Bio-h-BTN3A1-his-avi) tagged with his and his, and the recombinant human BTN3A1 protein (h-BTN3A1-his) tagged with his are all commercially available protein reagents, and their respective sequence sources are shown in Table 1. These protein reagents can be used in the following experiments.

[0248] Table 1. Sources of recombinant protein amino acid sequences

[0249] 2. Screening of rabbit-derived monoclonal antibodies against human BTN3A1

[0250] Antibodies are produced by immunizing New Zealand white rabbits.

[0251] Experimental Methods: The immunogen was recombinant human BTN3A1 (h-BTN3A1-mFc-his) protein tagged with mouse IgG2a Fc and his. For the initial immunization, an equal volume of Freund's complete adjuvant (CFA) was emulsified with the antigen, at a dose of 300 μg / rabbit. Subsequent booster immunizations used an equal volume of Freund's incomplete adjuvant (IFA) emulsified with the antigen, at a dose of 150 μg / rabbit. Immunization was administered on days 0, 22, 36, and 50. Blood samples were collected on day 42 for serum analysis. New Zealand white rabbit serum was analyzed using ELISA to determine antibody titers. Spleens from rabbits with high ELISA titers were collected, and spleen cells were isolated. B cells binding to biotinylated human BTN3A1 (Bio-h-BTN3A1-his-avi) recombinant protein were sorted using flow cytometry and seeded into 96-well plates at a density of less than one B cell per well. After two weeks of culture, the B cell supernatant was collected.

[0252] 3. ELISA binding assay of rabbit monoclonal antibody against recombinant BTN3A1 protein.

[0253] The binding characteristics of rabbit B cell supernatant to human BTN3A1 were detected by ELISA.

[0254] Experimental Methods: A 96-well ELISA plate was coated with his-tagged human BTN3A1 (h-BTN3A1-his) recombinant protein. After adding rabbit monoclonal antibody supernatant, the antibody-antigen binding activity was detected by adding secondary antibody (HRP-conjugated anti-primary Fc antibody) and HRP substrate TMB. The his-tagged human BTN3A1 (h-BTN3A1-his) recombinant protein was coated at a concentration of 1 μg / mL and incubated overnight at 4°C. The coated plate was then shaken dry and patted dry on absorbent paper. 150 μL / well blocking buffer was added, and the plate was incubated at 37°C for 1 hour. The plate was then shaken dry and patted dry on absorbent paper. Anti-BTN3A1 test antibody was added to each well, and the plate was incubated at 37°C for 1 hour. Discard the supernatant and blot dry. Add 100 μL of HRP-labeled goat anti-rabbit IgG secondary antibody diluted 1:5000 with dilution buffer to each well and incubate at 37°C for 1 h. Wash the ELISA plate with PBS, repeating the wash 5 times with a 30 s immersion interval each time. Add 100 μL of substrate solution (TMB) per well and react for 5 min to terminate the reaction. Finally, add 100 μL of 1 mol / L sulfuric acid to terminate the reaction. Measure the OD450 value at 450 nm using a Tecan-infinite F50 microplate reader.

[0255] We found that two rabbit monoclonal antibodies, r17G4 and r20A3, had high binding activity to recombinant human BTN3A1 protein.

[0256] Table 2. ELISA binding results of rabbit-derived anti-BTN3A1 monoclonal antibody against recombinant human BTN3A1 protein.

[0257] 4. Binding assay of rabbit monoclonal antibody against BTN3A1-expressing cells

[0258] The binding properties of rabbit B cell supernatant to HEK293 cells expressing human BTN3A1 protein were detected by flow cytometry (FACS).

[0259] Experimental Methods: A cell line transiently expressing human BTN3A1 was constructed. After adding antibody, the antibody-antigen binding characteristics were detected by adding secondary antibody. Expression plasmids carrying the human BTN3A1 gene sequence were transiently transfected into HEK293 cells. 2 × 10⁶ cells were seeded per well in a 96-well plate. 5 HEK293 cells were transiently transfected. Centrifuged at 300g for 5 minutes, supernatant removed, and 100 μL of the test antibody added. Incubated at 4°C for 1 hour. Centrifuged again to remove supernatant, washed three times with 200 μL of washing buffer (PBS + 2% FBS), and added 100 μL of 1:500 diluted anti-rabbit IgG secondary antibody. Incubated at 4°C for 1 hour. Centrifuged again to remove supernatant, and washed three times with 200 μL of washing buffer (PBS + 2% FBS). Resuspended the cells in 100 μL of PBS and analyzed by flow cytometry.

[0260] The results showed that r17G4 and r20A3 could significantly bind to HEK293 cells expressing BTN3A1.

[0261] Table 3. Binding results of anti-BTN3A1 rabbit monoclonal antibody to HEK293 cells expressing BTN3A1.

[0262] 5. γδT cell activation assay using anti-BTN3A1 rabbit monoclonal antibody

[0263] The activation capacity of rabbit B cell supernatant was detected by measuring the IFNγ content secreted by Vγ9Vδ2T cells through functional experiments.

[0264] Experimental method: Vγ9Vδ2T cells and A375 target cells were co-cultured, and rabbit B cell supernatant was added and mixed. The mixture was incubated in a 5% CO2 incubator at 37°C for 24 hours. The content of human IFNγ secreted in the cell supernatant was measured using an ELISA kit.

[0265] Table 4 shows that r17G4 and r20A3 can significantly produce IFNγ after incubation with Vγ9Vδ2T cells, indicating that the antibody has the function of activating Vγ9Vδ2T cells.

[0266] Table 4. Results of activation assay of Vγ9Vδ2T cells by anti-BTN3A1 rabbit monoclonal antibody.

[0267] Example 2. Preparation of human-rabbit chimeric antibody against rabbit-derived monoclonal antibody against BTN3A1

[0268] 1. Sequencing of anti-BTN3A1 rabbit monoclonal antibody

[0269] B cells in the logarithmic growth phase corresponding to the above antibody strain were taken, RNA was extracted with Trizol, reverse transcribed into cDNA, amplified by PCR using rabbit Ig-Primer, subcloned into a vector, and finally sequenced to obtain the rabbit monoclonal antibody sequence.

[0270] The heavy chain variable region (HCVR) and light chain variable region (LCVR) sequences of rabbit monoclonal antibodies r17G4 and r20A3 are as follows:

[0271] >r17G4 HCVR

[0272] >r17G4 LCVR

[0273] >r20A3 HCVR

[0274] >r20A3 LCVR

[0275] The underlined characters represent the CDR sequence determined according to the Kabat numbering system, as shown in Table 5.

[0276] Table 5. CDR sequences of r17G4 and r20A3 (according to the Kabat numbering system)

[0277] The two antibodies have the following general formula CDR structure:

[0278] HCDR1 (SEQ ID NO: 17): X1YYX2X3, where X1 is selected from D or N, X2 is selected from I or M, and X3 is selected from T or N;

[0279] HCDR2 (SEQ ID NO: 18): X4IYGSX5NTX6YASWAKG, wherein X4 is selected from V or I, X5 is selected from S or D, and X6 is selected from V or Y;

[0280] HCDR3(SEQ ID NO:19):X7X8X9X 10 SSAX 11 X 12 X 13 X 14Where X7 is selected from G or N, X8 is selected from Y or L, X9 is selected from L or D, and X 10 Selected from A or Y, X 11 Selected from D or Y, X 12 Selected from I or F, X 13 It does not exist or is H, X 14 It does not exist or is I;

[0281] LCDR1 (SEQ ID NO: 20): QX 15 SQSVYNNNRLA, where X 15 Selected from S or A;

[0282] LCDR2 (SEQ ID NO: 21): X 16 ASX 17 LAS, where X 16 Selected from D or E, X 17 Selected from T or K;

[0283] LCDR3 (SEQ ID NO: 22): QX 18 YYSGX 19 IX 20 X 21 , where X 18 Selected from T or G, X 19 Selected from Y or F, X 20 Selected from W or Y, X 21 Choose from A or P.

[0284] 2. Construction and preparation of human-rabbit chimeric antibodies

[0285] The obtained heavy chain variable region and light chain variable region were linked to the human IgG1 heavy chain constant region (Fc region) and human Cκ light chain constant region containing the L234F / L235E / P331S mutation (numbered according to the Eu nomenclature system), respectively, to construct human-rabbit chimeric full-length antibodies 17G4 and 20A3. The sequences are as follows:

[0286] >IgG1 Fc region (L234F / L235E / P331S)

[0287] >IgG1 Fc region (L234F / L235E / P331S with K446 removed)

[0288] >17G4 HC

[0289] >17G4 LC

[0290] >20A3 HC

[0291] >20A3 LC

[0292] The sequence of the positive anti-BTN3A1 antibody mAb1 is also provided here (derived from WO2020025703, SEQ ID NO: 4 and 6 in that patent), whose heavy chain constant region is the IgG1 Fc region (L234F / L235E / P331S with K446 removed).

[0293] >mAb1 HC

[0294] >mAb1 LC

[0295] In the above sequences, the underlined region for the full-length heavy chain is the human IgG1 Fc region, and the underlined region for the full-length light chain is the human Cκ region.

[0296] The encoding gene sequence of the above antibody was synthesized and subcloned into the pcDNA3.1 expression vector. The expression vector and transfection reagent PEI were transfected into CHO cells at a 1:2 ratio and incubated in a CO2 incubator for 4-5 days. The expressed antibody was purified by centrifugation after recovering the supernatant, following standard methods. The target antibody was then obtained through detection.

[0297] Example 3. Functional and efficacy verification of anti-BTN3A1 human-rabbit chimeric antibody

[0298] 1. ELISA binding assay of chimeric antibody to recombinant BTN3A1 protein

[0299] The binding characteristics of the anti-BTN3A1 human-rabbit chimeric antibody to human BTN3A1 were detected by ELISA.

[0300] Experimental Methods: A 96-well ELISA plate was coated with his-tagged human BTN3A1 (h-BTN3A1-his) recombinant protein. Different concentrations of anti-BTN3A1 human-rabbit chimeric antibody were added. The antibody-antigen binding activity was detected by adding secondary antibody (HRP-conjugated anti-primary Fc antibody) and HRP substrate TMB. The his-tagged human BTN3A1 (h-BTN3A1-his) recombinant protein was coated at a concentration of 1 μg / mL and incubated overnight at 4°C. The coated plate was then shaken dry and patted dry on absorbent paper. 150 μL / well blocking buffer was added, and the plate was incubated at 37°C for 1 hour. The plate was then shaken dry and patted dry on absorbent paper. The anti-BTN3A1 test antibody was added to each well and incubated at 37°C for 1 hour. Discard the supernatant and blot dry. Add 100 μL of HRP-labeled anti-human IgG secondary antibody diluted 1:5000 with dilution buffer to each well and incubate at 37°C for 1 h. Wash the ELISA plate with PBS 5 times, soaking for 30 s each time. Add 100 μL of substrate solution (TMB) per well and react for 5 min to terminate the reaction. Finally, add 100 μL of 1 mol / L sulfuric acid to terminate the reaction. Measure the OD value at 450 nm using a Tecan-infinite F50 ELISA reader.

[0301] The results are shown in Table 6 and Figure 1. IgG1 is an isotype control antibody, as is the case in other embodiments of this disclosure. The results show that 17G4 and 20A3 bind significantly to the recombinant human BTN3A1 protein, and their binding activity is comparable to mAb1.

[0302] Table 6. ELISA binding results of anti-BTN3A1 human-rabbit chimeric antibody to recombinant human BTN3A1 protein.

[0303] 2. FACS binding assay of chimeric antibody to BTN3A1 overexpressing cells

[0304] The binding properties of the anti-BTN3A1 human-rabbit chimeric antibody were detected using flow cytometry (FACS).

[0305] Experimental methods: A cell line overexpressing human BTN3A1 with BTN3A protein knocked out was constructed. After adding antibody, the antibody-antigen binding characteristics were detected by adding secondary antibody.

[0306] The cell line construction process involved infecting HEK293 cells with lentiviruses carrying short hairpin RNA sequences that knock out human BTN3A (BTN3A1, BTN3A2, BTN3A3), and obtaining stably overexpressing monoclonal cell lines through antibiotic selection and indefinite dilution. Next, expression plasmids carrying the human BTN3A1 gene sequence were transfected into cells that had already knocked out BTN3A basal expression, and stably overexpressing monoclonal cell lines were obtained through antibiotic selection and indefinite dilution.

[0307] In a 96-well plate, inoculate 2 × 10⁶ cells per well. 5 Overexpressing cells were centrifuged at 300g for 5 minutes, the supernatant was removed, and 100 μL of the test antibody was added. The cells were incubated at 4°C for 1 hour. After centrifugation to remove the supernatant, the cells were washed three times with 200 μL of washing buffer (PBS + 2% FBS). Then, 100 μL of 1:500 diluted anti-human IgG secondary antibody (Invitrogen, A-11013) labeled with Alexa Fluor 488 was added, and the cells were incubated at 4°C for 1 hour. After centrifugation to remove the supernatant, the cells were washed three times with 200 μL of washing buffer (PBS + 2% FBS). The cells were resuspended in 100 μL of PBS and analyzed by flow cytometry.

[0308] The results are shown in Table 7 and Figure 2. The results show that 17G4 and 20A3 bind significantly to human BTN3A1-overexpressing cells, and the binding strength is slightly stronger than that of mAb1.

[0309] Table 7. Results of binding assays of anti-BTN3A1 human-rabbit chimeric antibody to cells overexpressing human BTN3A1.

[0310] 3. Determination of the affinity of chimeric antibody for recombinant BTN3A1 protein

[0311] Experimental Methods: Detection was performed using a Biacore 8K (GE Healthcare) instrument. A Protein A sensor chip was selected, and the mobile phase consisted of HBS-EP+ buffer (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.05% surfactant P2O). Each antibody was prepared as a ligand using HBS-EP+ buffer and captured by the Protein A protein on the chip channels. Human BTN3A1 antigen protein (BTN-HM1A1) was used as the analyte and prepared with HBS-EP+ buffer. The analyte was serially diluted 2-fold from 100 nM to seven concentration points. The analyte was flowed through the experimental and reference channels at a flow rate of 30 μL / min, with a binding time of 90 s and a dissociation time of 500 s. Regeneration buffer (10 mM Glycine, pH 1.5, GE Healthcare, 29238268-AA) was used at a flow rate of 10 μl / min for 30 s. Data processing was performed using Biacore 8K Evaluation software. The corrected signal curve was obtained by subtracting the corresponding reference channel (Fc 1) signal value from the detection channel (Fc 2) signal value. The affinity kinetics curve was fitted according to the 1:1 Langmuir binding model, and the binding rate Ka, dissociation rate Kd, and dissociation constant (i.e., affinity KD value) were calculated.

[0312] The affinity of anti-BTN3A1 antibodies for human BTN3A1 is shown in Table 8. The results showed that 17G4 and 20A3 bound to human BTN3A1 protein with high affinity, and stronger than mAb1 affinity for human BTN3A1.

[0313] Table 8. Affinity of anti-BTN3A1 human-rabbit chimeric antibody to human BTN3A1

[0314] 4. Activation assay of chimeric antibodies on γδT cells

[0315] The in vitro activation function of anti-BTN3A1 human-rabbit chimeric antibody on γδT cells was detected by measuring the IFNγ secretion content of γδT cells.

[0316] Experimental methods: Vγ9Vδ2T cells were co-cultured with A375 target cells, and gradient concentrations of anti-BTN3A1 human-rabbit chimeric antibody were added. The cells were incubated in a 5% CO2 incubator at 37°C. The content of human IFNγ secreted in the cell supernatant was measured using an ELISA kit.

[0317] The results are shown in Table 9 and Figure 3. 17G4 and 20A3 have a significant activating effect on Vγ9Vδ2T cells.

[0318] Table 9. Results of activation assay of γδT cells by anti-BTN3A1 human-rabbit chimeric antibody

[0319] Example 4. Humanization of anti-BTN3A1 rabbit antibody

[0320] Based on the typical VH / VL structures of the obtained rabbit monoclonal antibodies 17G4 and 20A3, this embodiment compares the variable region sequences of the heavy and light chains with antibody germline databases to obtain human germline templates with high homology. The human germline heavy chain framework region is derived from the human heavy chain, and the human germline light chain framework region is derived from the human κ light chain. The preferred human germline heavy chain templates for antibodies 17G4 and 20A3 are IGHV3-66 and IGkV1-27. The CDR region of the rabbit monoclonal antibody is transplanted onto the selected humanized template, replacing the original CDR region of the humanized template, to construct the variable region. Then, based on the three-dimensional structure of the rabbit monoclonal antibody, reverse mutations are performed on embedded residues, residues that directly interact with the CDR region, and residues that significantly affect the conformation of VH and VL. The chemically unstable amino acid residues in the CDR region are optimized to produce a series of humanized antibodies.

[0321] The heavy chain variable regions of various humanized antibodies to 17G4 are as follows:

[0322] >17G4 H1CVR

[0323] >17G4 H2CVR

[0324] >17G4 H3CVR

[0325] >17G4 H4CVR

[0326] >17G4 H5CVR

[0327] The light chain variable regions of various humanized antibodies of 17G4 are as follows:

[0328] >17G4 L1CVR

[0329] >17G4 L2CVR

[0330] >17G4 L3CVR

[0331] In the above sequence, the underlined characters represent CDR, which are the same as SEQ ID NO: 5-10.

[0332] The heavy chain variable regions of each humanized antibody of 20A3 are as follows:

[0333] >20A3 H1CVR

[0334] >20A3 H2CVR

[0335] >20A3 H3CVR

[0336] >20A3 H4CVR

[0337] >20A3 H5CVR

[0338] >20A3 H6CVR

[0339] The light chain variable regions of various humanized antibodies of 20A3 are as follows: >20A3 L1CVR

[0340] >20A3 L2CVR

[0341] >20A3 L3CVR

[0342] In the above sequence, the underlined part is CDR, which is the same as SEQ ID NO: 11-16.

[0343] The aforementioned heavy chain variable regions and light chain variable regions were respectively linked to the human IgG1 heavy chain constant region and the human Cκ light chain constant region with the L234F / L235E / P331S mutation to obtain the full-length sequence.

[0344] The full-length heavy chain sequences of various humanized antibodies of 17G4 are as follows:

[0345] >17G4 H1C

[0346] >17G4 H2C

[0347] >17G4 H3C

[0348] >17G4 H4C

[0349] >17G4 H5C

[0350] The full-length light chain sequences of various humanized antibodies of 17G4 are as follows:

[0351] >17G4 L1C

[0352] >17G4 L2C

[0353] >17G4 L3C

[0354] The full-length heavy chain sequences of the various humanized antibodies of 20A3 are as follows:

[0355] >20A3 H1C

[0356] >20A3 H2C

[0357] >20A3 H3C

[0358] >20A3 H4C

[0359] >20A3 H5C

[0360] >20A3 H6C

[0361] The full-length light chain sequences of the various humanized antibodies of 20A3 are as follows:

[0362] >20A3 L1C

[0363] >20A3 L2C

[0364] >20A3 L3C

[0365] By combining the above-mentioned humanized heavy chain full-length sequence and light chain full-length sequence respectively, the following humanized molecules were obtained:

[0366] Table 10. Anti-BTN3A1 humanized antibodies

[0367] Example 5. Functional and efficacy verification of anti-BTN3A1 humanized antibody

[0368] 1. Antibody activation assay for γδT cells

[0369] The aforementioned humanized molecules 17G4 and 20A3 were expressed and purified, and the activation ability of the humanized antibody on γδT cells was detected according to the method in Part 4 of Example 3.

[0370] The results are shown in Table 11 and Figure 5. The results show that the activity of all humanized molecules of 20A3 was not significantly reduced compared with the parent 20A3.

[0371] Table 11. Results of the activation experiment of γδT cells by the 20A3 humanized antibody.

[0372] 2. Antibody binding assay on BTN3A1 overexpressing cells

[0373] Following the method in Part 2 of Example 3, the binding ability of the anti-BTN3A1 humanized antibody to cells overexpressing human BTN3A1 was tested.

[0374] The results are shown in Table 12, Figures 6A and 6B. The binding ability of the humanized molecules 17G4 and 20A3 to cells was comparable to that of their corresponding chimeric antibodies, and both were superior to the control antibodies.

[0375] Table 12. Results of binding experiments of 17G4 and 20A3 humanized antibodies to cells overexpressing human BTN3A1.

[0376] 3. Affinity determination of antibody to recombinant BTN3A1 protein

[0377] The affinity of 20A3-H2L2 for recombinant human BTN3A1 protein was tested according to the method in Part 3 of Example 3.

[0378] The results are shown in Table 13. The results show that 20A3-H2L2 has a high binding affinity for recombinant human BTN3A1 protein, and the affinity of 20A3-H2L2 for human BTN3A1 is stronger than that for mAb1.

[0379] Table 13. Affinity of 20A3-H2L2 to recombinant human BTN3A1 protein

[0380] 4. Antibody binding assay to BTN3A overexpressing cells

[0381] Flow cytometry (FACS) was used to detect the binding ability of 20A3-H2L2 to other proteins in the BTN3A family. Following the method described in Part 2 of Example 3, HEK293 cell lines overexpressing human BTN3A2 and human BTN3A3 with background BTN3A protein knockout were constructed. The binding characteristics of 20A3-H2L2 to HEK293 cells overexpressing human BTN3A1, human BTN3A2, and human BTN3A3 with background BTN3A protein knockout were then detected.

[0382] The results are shown in Table 14 and Figures 7A, 7B, and 7C. The results show that both 20A3-H2L2 and mAb1 can bind to human BTN3A1, human BTN3A2, and human BTN3A3, and the binding ability of 20A3-H2L2 is stronger than that of mAb1.

[0383] Table 14. Results of binding assays of 20A3-H2L2 to cells overexpressing BTN3A family proteins.

[0384] 5. Antibody-induced activation of γδT cells

[0385] The in vitro activation function of 20A3-H2L2 on γδT cells was detected by measuring the IFNγ secreted by γδT cells. Vγ9Vδ2T cells and SKOV-3 cells expressing high levels of BTN3A protein were co-cultured in vitro, with gradient concentrations of the test antibody added. The cells were incubated at 37°C in a 5% CO2 incubator for 24 hours, and the secreted human IFNγ content in the cell supernatant was measured using an ELISA kit.

[0386] The results are shown in Table 15 and Figure 8. The results indicate that 20A3-H2L2 has a stronger activation ability on Vγ9Vδ2T cells than mAb1 and EC. 50 The expression decreased by 2.6 times. Compared with the experimental results of A375 cells with low BTN3A expression in Part 1 of Example 5, 20A3-H2L2 showed stronger activation activity than mAb1 in this example, indicating that higher BTN3A expression on tumor cells helps to further enhance the activation activity of 20A3-H2L2 on Vγ9Vδ2T cells.

[0387] Table 15. Activation experiment of γδT cells by 20A3-H2L2

[0388] 6. Antibody-activated γδT cell killing experiment against SKOV-3

[0389] The ability of 20A3-H2L2-mediated γδT cell killing of target cells was evaluated by measuring the killing effect of γδT cells on tumor target cells. Vγ9Vδ2T cells and SKOV-3 cells (luc-labeled) were seeded in 96-well plates, and gradient concentrations of 20A3-H2L2, mAb1, or allotype control (IgG1) were added. After co-culturing for 24 hours, cells were lysed with Bright-Glo™ reagent (Promega) for 5 min, and luminescence activity was measured using an Envision multimode microplate reader (PerkinElmer).

[0390] The results are shown in Table 16 and Figure 9. The results indicate that 20A3-H2L2-activated Vγ9Vδ2T cells exhibited stronger SKOV-3 killing ability than mAb1, and 20A3-H2L2 EC... 50 It is a control antibody EC 50 About one-third of it.

[0391] Table 16. Experimental results of 20A3-H2L2 activation of γδT cells to kill SKOV-3 cells.

[0392] The following describes the formulation development of antibody 20A3-H2L2 (heavy chain as shown in SEQ ID NO: 57, light chain as shown in SEQ ID NO: 63).

[0393] Exemplary antibody drug composition (formulation) preparation process

[0394] Step 1: The humanized anti-BTN3A specific monoclonal antibody and stabilizer were formulated into a stock solution, which was then filtered through a 0.22μm filter cartridge for sterilization, and the filtrate was collected.

[0395] Step 2: Adjust the filling volume. Use vials for filling and full capping. Take samples at the beginning, middle and end of filling to test the difference in filling volume.

[0396] Step 3: Turn on the capping machine, add the aluminum cap, and perform capping.

[0397] Step 4: Visual inspection to confirm that the product has no defects such as inaccurate filling or poor appearance. Print paper box labels, fold the paper boxes, pack them, and affix paper box labels.

[0398] Example 6. Screening of different buffer systems

[0399] A formulation with a protein concentration of 150 g / L was prepared by using buffer systems of 20 mM acetate-sodium acetate (pH 5.5), 20 mM citric acid-sodium citrate (pH 5.5), 20 mM histidine-histidine hydrochloride (pH 5.5), and 20 mM phosphate buffer (pH 6.5 and 7.0). 8.0% (w / v) sucrose and 0.02% (w / v) polysorbate 80 were added as formulation stabilizers.

[0400] 1) 20mM acetate-sodium acetate, pH 5.5, 8.0% (w / v) sucrose, 0.02% (w / v) polysorbate 80, antibody protein 150g / L

[0401] 2) 20mM citric acid-sodium citrate, pH 5.5, 8.0% (w / v) sucrose, 0.02% (w / v) polysorbate 80, antibody protein 150g / L

[0402] 3) 20mM histidine-histidine hydrochloride, pH 5.5, 8.0% (w / v) sucrose, 0.02% (w / v) polysorbate 80, antibody protein 150g / L

[0403] 4) 20mM disodium hydrogen phosphate, pH 6.5, 8.0% (w / v) sucrose, 0.02% (w / v) polysorbate 80, antibody protein 150g / L

[0404] 5) 20mM disodium hydrogen phosphate, pH 7.0, 8.0% (w / v) sucrose, 0.02% (w / v) polysorbate 80, antibody protein 150g / L

[0405] The conformational and colloidal stability of the antibody was assessed by determining its melting temperature (Tm), aggregation temperature (Tagg), and particle size. Simultaneously, the stability of the sample under conditions of shaking (200 rpm, 25℃), light exposure (4500±500 lx, 25℃), freeze-thaw cycle (-35℃ / room temperature (RT)), -35℃ storage, and 40℃ was evaluated by detecting the sample's appearance, SEC, IEC, and NRCE. The detection methods are as follows:

[0406] (1) Appearance

[0407] The clarity meter is mainly used for inspecting the appearance of samples. Using a visual method, the sample bottle is wiped clean, and under a certain light intensity, the sample color, clarity, and visible foreign matter are observed against both a white and black background of the clarity meter. Testing instrument: Jingtuo Instruments YB-2A Clarity Meter.

[0408] (2) pH

[0409] The pH value was measured using a potentiometric method, with a 100 μL sample taken and a micro pH meter calibrated with standard solutions. Instrument: Mettler Toledo pH meter, model S210.

[0410] (3) Ultraviolet spectrophotometry

[0411] The NanoDrop spectrophotometer is primarily used for protein concentration detection. When the NanoDrop 2000 was used to determine the protein concentration of a sample, the extinction coefficient was 1.537 (mg / ml). -1 cm -1 Ultrapure water was used as a control. Detection instrument: Thermo Fisher Scientific UV-Vis spectrophotometer, model: Nano Drop 2000.

[0412] (4) Melting temperature (Tm) and aggregation temperature (Tagg)

[0413] Take a UNi tube and add the sample (9 μl) to the corresponding well of the UNi tube according to the pre-set sample position. Measure the sample's dissolution temperature (Tm) and aggregation temperature (Tagg) using a high-throughput multifunctional protein stability analyzer. Instrument: UNCHAINED multifunctional protein stability analysis system, model: Uncle.

[0414] (5) Insoluble particles

[0415] A 96-well plate was used. Baseline testing was performed first with purified water, followed by a preliminary optical adjustment test with 1 mL of sample. Only after passing this test were the samples analyzed. Data analysis was conducted using filters to remove air bubbles and silicone oil. Finally, the concentrations of particles larger than 2 μm were statistically analyzed. Instrument used: Protein Simple flow cytometer, model: MFI 5200.

[0416] (6) Size Exclusion Chromatography (SEC)

[0417] Proteins are separated based on solute molecule size, dispersion coefficient, and surface properties. The stationary phase particles contain micropores that allow only molecules of a corresponding size to pass through. Molecules larger than the micropores cannot enter and are eluted in the initial stage due to limited contact with the stationary phase. Molecules smaller than the micropores can diffuse into the micropores, and their retention time depends on the distance the molecules travel through the pores. After separation, monomers, high-molecular-weight substances, and low-molecular-weight substances are detected at 280 nm using a UV detector, and quantification is performed using peak area normalization. The instrument used for SEC determination was a Waters H-class ultra-high performance liquid chromatograph (UPLC); the column was an ACQUITY UPLC Portein BEH SEC Column 200A, 1.7 μm (4.6 mm * 150 mm).

[0418] (7) Ion exchange chromatography (IEC)

[0419] Ion exchange resin is used as the stationary phase, with both fixed and exchangeable ionic groups on the resin. In a mobile phase with a pH lower than the isoelectric point of the protein, the protein carries a positive charge. Under low ionic strength, when positively charged protein molecules flow through the column, they exchange with the exchangeable ionic groups on the resin, thus adsorbing onto the fixed ionic groups. By gradually increasing the salt ion concentration in the mobile phase, the proteins are eluted sequentially according to the increasing affinity of the charged proteins for the fixed ionic groups. In the SHR-4506 project's IEC-HPLC purity determination, the peak with the highest content is defined as the main peak, with acidic peaks preceding the main peak and alkaline peaks following it. Detection is performed at 280 nm using a UV detector, and quantification is achieved using peak area normalization. IEC testing instrument: Agilent 1260 high-performance liquid chromatograph, model: Agilent 1260 HPLC; chromatographic column: BioPro IEX SF 4.6×250mm, 6μm.

[0420] (8) Whole-column imaging capillary isofocusing electrophoresis (icIEF)

[0421] This method uses a capillary as the separation channel and a high-voltage electric field as the driving force to separate components based on the difference in their isoelectric points. The principle is that a DC voltage is applied across the capillary, creating a pH gradient within the capillary's amphoteric electrolyte solution. Components migrate to their respective isoelectric points based on their charge differences, focusing into a very narrow segment, thus achieving separation. Real-time scanning and imaging of the entire capillary column are performed, and the collected signals are analyzed to calculate the isoelectric point of each component. Peak area percentage: (Peak)% = (Peak Area of ​​Peak) / Total Peak Area (Total Peak Area) × 100%. icIEF detection instrument: Maurice full-column imaging capillary isoelectric focusing electrophoresis system, model: Maurice;

[0422] (9) Non-reducing capillary gel electrophoresis (NR-CE)

[0423] Protein samples of a certain concentration are bound to SDS, denatured by heating, and separated according to molecular weight in a capillary containing an SDS-polymer gel. In the non-reducing state, the sample may contain a series of impurities, including polymers, two-chain-one-light-chain (HHL), two-chain (HH), one-chain-one-light-chain (HL), one-chain (HC), and one-chain (LC), as well as various isomeric impurities. The relative area percentage is calculated using the area normalization method after PDA detection. The instrument used for NR-CE determination is a Beckman PA800 plus capillary electrophoresis system.

[0424] Table 17: Screening Results of Different Buffer Systems - Tm, Tagg, and Particle Size Note: This table shows the sample test results when antibody level is 0.

[0425] Table 18-1: Screening Results of Different Buffer Systems - Appearance

[0426] Table 18-2: Screening Results of Different Buffer Systems - Appearance

[0427] Table 19-1: Screening Results of Different Buffer Systems - SEC

[0428] Table 19-2: Screening Results of Different Buffer Systems - SEC

[0429] Table 20-1: Screening Results of Different Buffer Systems - IEC

[0430] Table 20-2: Screening Results of Different Buffer Systems - IEC

[0431] Table 21: NRCE Results of Screening Different Buffer Systems

[0432] Example 7 pH Screening of Acetic Acid-Sodium Acetate Buffer System

[0433] Formulas with a protein concentration of 150 g / L were prepared by adding 8.0% (w / v) sucrose and 0.02% (w / v) polysorbate 80 to 20 mM acetate-sodium acetate buffer systems at pH 4.5, 5.0, and 5.5, respectively. The conformational and colloidal stability of the antibodies were investigated by measuring the melting temperature (Tm), aggregation temperature (Tagg), and particle size. The stability of the samples under shaking (300 rpm, 25℃), light exposure (4500±500 lx, 25℃), freeze-thaw cycles (-35℃ / room temperature (RT)), -35℃ storage, and 40℃ was also investigated by measuring the appearance of the samples and their SEC, IEC, and NRCE values ​​under certain conditions.

[0434] 6) 20mM acetate-sodium acetate, pH 4.5, 8.0% (w / v) sucrose, 0.02% (w / v) polysorbate 80, antibody protein 150g / L

[0435] 7) 20mM acetate-sodium acetate, pH 5.0, 8.0% (w / v) sucrose, 0.02% (w / v) polysorbate 80, antibody protein 150g / L

[0436] 8) 20mM acetate-sodium acetate, pH 5.5, 8.0% (w / v) sucrose, 0.02% (w / v) polysorbate 80, antibody protein 150g / L

[0437] Table 22: pH Screening Results of Acetic Acid-Sodium Acetate Buffer System - Tm, Tagg, and Particle Size Detection

[0438] Table 23: pH Screening Results and Appearance of Acetic Acid-Sodium Acetate Buffer System

[0439] Table 24-1 pH Screening-SEC Results for Acetic Acid-Sodium Acetate Buffer System

[0440] Table 24-2: pH Screening-SEC Results for Acetic Acid-Sodium Acetate Buffer System

[0441] Table 25-1: pH Screening Results of Acetic Acid-Sodium Acetate Buffer System - IEC

[0442] Table 25-2: pH screening results of the acetic acid-sodium acetate buffer system - IEC

[0443] Table 26: pH Screening Results of Acetic Acid-Sodium Acetate Buffer System - NRCE

[0444] Example 8. pH screening of histidine-histidine hydrochloride buffer system

[0445] Three buffer systems with different pH values ​​(20 mM histidine-histidine hydrochloride, pH 5.5, 6.0, and 6.5) were selected, and 8.0% (w / v) sucrose and 0.02% (w / v) polysorbate 80 were added as formulation stabilizers to prepare a formulation with a protein concentration of 50 g / L. The stability of the samples at 50 °C and 40 °C was investigated by measuring the appearance of the samples and their SEC, IEC, and NRCE values ​​under certain conditions.

[0446] 9) 20mM histidine-histidine hydrochloride, pH 5.5, 8.0% (w / v) sucrose, 0.02% (w / v) polysorbate 80, antibody protein 50g / L

[0447] 10) 20mM histidine-histidine hydrochloride, pH 6.0, 8.0% (w / v) sucrose, 0.02% (w / v) polysorbate 80, antibody protein 50g / L

[0448] 11) 20mM histidine-histidine hydrochloride, pH 6.5, 8.0% (w / v) sucrose, 0.02% (w / v) polysorbate 80, antibody protein 50g / L

[0449] Table 27: pH Screening Results of Histidine-Histidine Hydrochloride Buffer System - Appearance

[0450] Table 28: Screening Results of Histidine-Histidine Hydrochloride Buffer System - SEC

[0451] Table 29: Screening Results of Histidine-Histidine Hydrochloride Buffer System - NRCE

[0452] Table 30: Screening Results of Histidine-Histidine Hydrochloride Buffer System - IEC

[0453] The results showed that BTN3A monoclonal antibody exhibited good appearance under all tested conditions in a system containing 20 mM histidine-histidine hydrochloride, pH 5.5, 6.0, and 6.5, 8.0% (w / v) sucrose, and 0.02% (w / v) polysorbate 80. At 0°C, there was no significant difference in purity among the groups. At high temperatures and 50°C, both the SEC main peak and the NRCE monomer showed a decreasing trend, but there was no significant difference among the groups. The trend of the IEC alkaline peak decreased with increasing pH, while the trend of the acidic peak increased with increasing pH.

[0454] Example 9. Buffer System Screening

[0455] A formulation with a protein concentration of 50 g / L was prepared by using two buffer systems: 20 mM histidine-histidine hydrochloride (pH 6.5) and 20 mM acetate-sodium acetate (pH 5.5). 8.0% (w / v) sucrose and 0.02% (w / v) polysorbate 80 were added as stabilizers. The stability of the samples at 50 °C and 40 °C was investigated by testing their appearance and SEC, IEC, and NRCE values ​​under certain conditions.

[0456] 12) 20mM acetate-sodium acetate, pH 5.5, 8.0% (w / v) sucrose, 0.02% (w / v) polysorbate 80, antibody protein 50g / L

[0457] 13) 20mM histidine-histidine hydrochloride, pH 6.5, 8.0% (w / v) sucrose, 0.02% (w / v) polysorbate 80, antibody protein 50g / L

[0458] Table 31: Screening and Confirmation of Buffer Systems - Appearance Results

[0459] Table 32: Buffer System Screening and Confirmation - SEC Results

[0460] Table 33: Buffer System Screening and Validation - NRCE Results

[0461] Table 34: Buffer System Screening and Validation - IEC Results

[0462] The results showed that BTN3A monoclonal antibody exhibited good appearance under all testing conditions in formulations 12 and 13. At 0°C, there was no significant difference in purity among the groups; at high temperature and 50°C, both the SEC main peak and NRCE monomer showed a decreasing trend, but there was no significant difference among the groups.

[0463] Example 10. Antibody protein concentration screening

[0464] Formulas with antibody concentrations of 50, 80, and 100 g / L were prepared by adding 20 mM histidine-histidine hydrochloride buffer solution, pH 6.5, 8% (w / v) sucrose, and 0.02% (w / v) polysorbate 80 as stabilizers. The stability of the samples under light (4500±500 lux, 25℃), shaking (200 rpm, 18–26℃), freeze-thaw (-35℃ / RT), cryopreservation (-35℃), 2–8℃, and high temperature (40℃) conditions was investigated by measuring the appearance of the samples and their SEC, IEC, and NRCE values ​​under certain conditions.

[0465] 14) 20mM histidine-histidine hydrochloride, pH 6.5, 8.0% (w / v) sucrose, 0.02% (w / v) polysorbate 80, antibody protein 50g / L

[0466] 15) 20mM histidine-histidine hydrochloride, pH 6.5, 8.0% (w / v) sucrose, 0.02% (w / v) polysorbate 80, antibody protein 80g / L

[0467] 16) 20mM histidine-histidine hydrochloride, pH 6.5, 8.0% (w / v) sucrose, 0.02% (w / v) polysorbate 80, antibody protein 100g / L

[0468] Table 35: Antibody Protein Concentration Screening - Appearance Results

[0469] Table 36-1: Antibody Protein Concentration Screening - SEC Results

[0470] Table 36-2: Antibody Protein Concentration Screening - SEC Results

[0471] Table 37-1: Antibody Protein Concentration Screening - NRCE Results

[0472] Table 37-2: Antibody Protein Concentration Screening - NRCE Results

[0473] Table 38: Antibody Protein Concentration Screening - IEC Results

[0474] The results showed that the sample with a BTN3A monoclonal antibody concentration of 50 g / L exhibited the smallest changes in SEC monomer content and NRCE main peak content under light and high temperature conditions, but these changes were not significantly different from those at protein concentrations of 80 g / L and 100 g / L. Furthermore, under light and high temperature conditions, the percentage of the IEC main peak area decreased in both samples, but there were no significant differences between the groups.

[0475] Example 11. Screening of antibody stabilizers

[0476] The buffer system was selected as 20 mM histidine-histidine hydrochloride buffer solution, pH 6.5. Formulas with an antibody concentration of 80 g / L were prepared by adding 8% (w / v) sucrose, 0.02% (w / v) polysorbate 80, 8% (w / v) sucrose, 0.02% (w / v) polysorbate 80, 0.005% (w / v) disodium edetate, and 8% (w / v) sucrose, 0.02% (w / v) polysorbate 80, and 0.149% (w / v) methionine as stabilizers. The stability of the samples under light (4500±500 lux, 5℃), shaking (200 rpm, 18–26℃), freeze-thaw (-35℃ / RT), cryopreservation (-35℃), 2–8℃, and high temperature (40℃) conditions was investigated by testing the appearance of the samples and their SEC, IEC, and NRCE values ​​under certain conditions.

[0477] 17) 20mM histidine-histidine hydrochloride, pH 6.5, 8.0% (w / v) sucrose, 0.02% (w / v) polysorbate 80, antibody protein 80g / L

[0478] 18) 20mM histidine-histidine hydrochloride, pH 6.5, 8.0% (w / v) sucrose, 0.02% (w / v) polysorbate 80, 0.005% (w / v) disodium edetate, antibody protein 80g / L

[0479] 19) 20mM histidine-histidine hydrochloride, pH 6.5, 8.0% (w / v) sucrose, 0.02% (w / v) polysorbate 80, 0.149% (w / v) methionine, antibody protein 80g / L

[0480] Table 39: Antibody Protein Concentration Screening - Appearance Results

[0481] Table 40-1: Antibody Protein Concentration Screening - SEC Results

[0482] Table 40-2: Antibody Protein Concentration Screening - SEC Results

[0483] Table 41: Antibody Protein Concentration Screening - NRCE Results

[0484] Table 42: Antibody Protein Concentration Screening - IEC Results

[0485] Example 12. Protein concentration and antibody formulation pH investigation

[0486] Based on the selection of buffer systems and stabilizers, and considering the range of variations in formulation specifications and purity, two formulations were selected: 20 mM histidine-histidine hydrochloride, pH 6.2 and 6.8, 7.0% (w / v) sucrose, 0.02% (w / v) polysorbate 80, 0.149% (w / v) methionine, and antibody protein 100 g / L; and 20 mM histidine-histidine hydrochloride, pH 6.5, 7.0% (w / v) sucrose, 0.02% (w / v) polysorbate 80, 0.149% (w / v) methionine, and antibody protein 110 g / L. The stability of the antibody under different pH conditions and protein concentrations was investigated based on these formulations. The stability of the samples under room temperature / light, freeze-thaw (-35℃ / RT), cryopreservation (-35℃), 2–8℃, 25℃, and high temperature (40℃) conditions was examined by detecting the appearance of the samples and the SEC, icIEF, and NRCE values ​​under certain conditions.

[0487] 20) 20mM histidine-histidine hydrochloride, pH 6.2, 7.0% (w / v) sucrose, 0.02% (w / v) polysorbate 80, 0.149% (w / v) methionine, antibody protein 100g / L

[0488] 21) 20mM histidine-histidine hydrochloride, pH 6.5, 7.0% (w / v) sucrose, 0.02% (w / v) polysorbate 80, 0.149% (w / v) methionine, antibody protein 110g / L

[0489] 22) 20mM histidine-histidine hydrochloride, pH 6.8, 7.0% (w / v) sucrose, 0.02% (w / v) polysorbate 80, 0.149% (w / v) methionine, antibody protein 100g / L

[0490] Table 43: Protein Concentration and pH Range Assessment - Appearance Results

[0491] Table 44-1: Protein Concentration and pH Range Assessment - SEC Results

[0492] Table 44-2: Protein Concentration and pH Range Assessment - SEC Results

[0493] Table 45: Protein Concentration and pH Range Investigation - NRCE Results

[0494] Table 46-1: Results of Protein Concentration and pH Range Study - IEC

[0495] Table 46-2: Protein Concentration and pH Range Assessment - IEC Results

[0496] The results showed that, compared with the system of 20 mM histidine-histidine hydrochloride, pH 6.2–6.8, 7.0% (w / v) sucrose, 0.02% (w / v) polysorbate 80, and 0.149% (w / v) methionine, the proportion of SEC monomers in each group of samples did not change significantly under the conditions of 2–8℃, freezing, and freeze-thaw, and the percentage of IEC main peak area and the content of non-reduced CE main peak did not change significantly under the conditions of 2–8℃ and freeze-thaw, indicating that the samples had good stability under the conditions of 2–8℃, freezing, and freeze-thaw. Under light and high temperature conditions, the purity of formulations 18–20 was generally stable, with no significant differences between groups.

[0497] Example 13. Antibody Formulation Investigation

[0498] The prepared sample contained 20 mM histidine-histidine hydrochloride, pH 6.5, 7.0% (w / v) sucrose, 0.02% (w / v) polysorbate 80, 0.149% (w / v) methionine, and 100 g / L antibody protein. The stability of the sample under various conditions was investigated by measuring its appearance and SEC, icIEF, and NRCE values ​​under specific conditions, including room temperature light, light irradiation (4500±500 lux, 5℃), shaking (300 rpm, 18–26℃), freeze-thaw (-35℃ / RT), cryopreservation (-35℃), 2–8℃, 25℃, and high temperature (40℃).

[0499] 23) 20mM histidine-histidine hydrochloride, pH 6.5, 7.0% (w / v) sucrose, 0.02% (w / v) polysorbate 80, 0.149% (w / v) methionine, antibody protein 100g / L

[0500] Table 47: Antibody formulation evaluation - protein concentration, pH value and appearance results Note: NT indicates not detected, the same applies below.

[0501] Table 48: Antibody Prescription Investigation - MFI and DLS Results

[0502] Table 49: Antibody Formulation Analysis - Purity Results

[0503] The results show that the antibody has good stability when the antibody formulation is 20mM histidine-histidine hydrochloride, pH 6.5, 7.0% (w / v) sucrose, 0.02% (w / v) polysorbate 80, 0.149% (w / v) methionine, and antibody protein 100g / L.

[0504] Example 14. Antibody formulation

[0505] This embodiment provides the following prescription:

[0506] (1) 90 g / L BTN3A binding protein, 20 mM histidine-histidine hydrochloride, 7.0% (w / v) sucrose, 0.02% (w / v) polysorbate 80, 0.149% (w / v) methionine, pH 6.5;

[0507] (2) 110 g / L BTN3A binding protein, 20 mM histidine-histidine hydrochloride, 7.0% (w / v) sucrose, 0.02% (w / v) polysorbate 80, 0.149% (w / v) methionine, pH 6.5;

[0508] (3) 100 g / L BTN3A binding protein, 18 mM histidine-histidine hydrochloride, 7.0% (w / v) sucrose, 0.02% (w / v) polysorbate 80, 0.149% (w / v) methionine, pH 6.2;

[0509] (4) 100 g / L BTN3A binding protein, 22 mM histidine-histidine hydrochloride, 7.0% (w / v) sucrose, 0.02% (w / v) polysorbate 80, 0.149% (w / v) methionine, pH 6.8;

[0510] (5) 100 g / L BTN3A binding protein, 20 mM histidine-histidine hydrochloride, 6.0% (w / v) sucrose, 0.02% (w / v) polysorbate 80, 0.149% (w / v) methionine, pH 6.5;

[0511] (6) 100 g / L BTN3A binding protein, 20 mM histidine-histidine hydrochloride, 8.0% (w / v) sucrose, 0.02% (w / v) polysorbate 80, 0.149% (w / v) methionine, pH 6.5;

[0512] (7) 100 g / L BTN3A binding protein, 20 mM histidine-histidine hydrochloride, 7.0% (w / v) sucrose, 0.01% (w / v) polysorbate 80, 0.149% (w / v) methionine, pH 6.5;

[0513] (8) 100 g / L BTN3A binding protein, 20 mM histidine-histidine hydrochloride, 7.0% (w / v) sucrose, 0.03% (w / v) polysorbate 80, 0.149% (w / v) methionine, pH 6.5;

[0514] (9) 100 g / L BTN3A binding protein, 20 mM histidine-histidine hydrochloride, 7.0% (w / v) sucrose, 0.02% (w / v) polysorbate 80, 0.120% (w / v) methionine, pH 6.5;

[0515] (10) 100 g / L BTN3A binding protein, 20 mM histidine-histidine hydrochloride, 7.0% (w / v) sucrose, 0.02% (w / v) polysorbate 80, 0.160% (w / v) methionine, pH 6.5;

[0516] (11) 100 g / L BTN3A binding protein, 20 mM histidine-histidine hydrochloride, 7.0% (w / v) sucrose, 0.02% (w / v) polysorbate 80, pH 6.5;

[0517] (12) 100 g / L BTN3A binding protein, 20 mM histidine-histidine hydrochloride, 7.0% (w / v) sucrose, 0.02% (w / v) polysorbate 80, 0.149% (w / v) methionine, pH 6.2;

[0518] (13) 100 g / L BTN3A binding protein, 20 mM histidine-histidine hydrochloride, 7.0% (w / v) sucrose, 0.02% (w / v) polysorbate 80, 0.149% (w / v) methionine, pH 6.5

[0519] (14) 100 g / L BTN3A binding protein, 20 mM histidine-histidine hydrochloride, 7.0% (w / v) sucrose, 0.02% (w / v) polysorbate 80, 0.149% (w / v) methionine, pH 6.8

[0520] (15) 110 g / L BTN3A binding protein, 20 mM histidine-histidine hydrochloride, 7.0% (w / v) sucrose, 0.02% (w / v) polysorbate 80, 0.149% (w / v) methionine, pH 6.2

[0521] (16) 110 g / L BTN3A binding protein, 20 mM histidine-histidine hydrochloride, 7.0% (w / v) sucrose, 0.02% (w / v) polysorbate 80, 0.149% (w / v) methionine, pH 6.5

[0522] (17) 110 g / L BTN3A binding protein, 20 mM histidine-histidine hydrochloride, 7.0% (w / v) sucrose, 0.02% (w / v) polysorbate 80, 0.149% (w / v) methionine, pH 6.8

Claims

1. A pharmaceutical composition comprising BTN3A binding protein and a buffer; in, The BTN3A-binding protein includes a domain that specifically binds to BTN3A, and the domain includes a heavy chain variable region and a light chain variable region; wherein: The heavy chain variable region includes HCDR1, HCDR2, and HCDR3 as shown in any of SEQ ID NO: 3, 39-44, and the light chain variable region includes LCDR1, LCDR2, and LCDR3 as shown in any of SEQ ID NO: 4, 45-47; or, the heavy chain variable region includes HCDR1, HCDR2, and HCDR3 as shown in any of SEQ ID NO: 1, 31-35, and the light chain variable region includes LCDR1, LCDR2, and LCDR3 as shown in any of SEQ ID NO: 2, 36-38; the CDR is defined according to the Kabat, IMGT, Chothia, AbM, or Contact numbering system; The buffer is selected from one or more of acetate buffer, citrate buffer, histidine buffer and phosphate buffer; more preferably one or more of acetate-sodium acetate, citrate-sodium citrate, histidine-histidine hydrochloride, disodium hydrogen phosphate-sodium dihydrogen phosphate.

2. The pharmaceutical composition according to claim 1, wherein: The heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 11-13, respectively, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 14-16, respectively; or The heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, respectively, as shown in SEQ ID NO: 5-7, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3, respectively, as shown in SEQ ID NO: 8-10.

3. The pharmaceutical composition according to claim 1 or 2, wherein, The BTN3A binding protein includes a heavy chain variable region and a light chain variable region. The heavy chain variable region comprises an amino acid sequence as shown in or having at least 90% identity with any of SEQ ID NO: 3, 39-44, and the light chain variable region comprises an amino acid sequence as shown in or having at least 90% identity with any of SEQ ID NO: 4, 45-47; or, The heavy chain variable region comprises an amino acid sequence as shown in any of SEQ ID NO: 1, 31-35 or having at least 90% identity with it, and the light chain variable region comprises an amino acid sequence as shown in any of SEQ ID NO: 2, 36-38 or having at least 90% identity with it. Preferably, The heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 3 or having at least 90% identity with it, and the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 4 or having at least 90% identity with it; The heavy chain variable region comprises an amino acid sequence as shown in any of SEQ ID NO: 39-44 or having at least 90% identity with it, and the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 45 or having at least 90% identity with it. The heavy chain variable region comprises an amino acid sequence as shown in any of SEQ ID NO: 39-44 or having at least 90% identity with it, and the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 46 or having at least 90% identity with it. The heavy chain variable region comprises an amino acid sequence as shown in any of SEQ ID NO: 39-44 or having at least 90% identity with it, and the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 47 or having at least 90% identity with it. The heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 1 or having at least 90% identity with it, and the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 2 or having at least 90% identity with it. The heavy chain variable region comprises an amino acid sequence as shown in any of SEQ ID NO: 31-35 or having at least 90% identity with it, and the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 36 or having at least 90% identity with it. The heavy chain variable region comprises an amino acid sequence as shown in or having at least 90% identity with any of SEQ ID NO: 31-35, and the light chain variable region comprises an amino acid sequence as shown in or having at least 90% identity with any of SEQ ID NO: 37; or, The heavy chain variable region comprises an amino acid sequence as shown in any of SEQ ID NO: 31-35 or having at least 90% identity with it, and the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 38 or having at least 90% identity with it.

4. The pharmaceutical composition according to any one of claims 1-3, wherein, The BTN3A-binding protein includes a domain that specifically binds to BTN3A, the domain comprising a heavy chain variable region and a light chain variable region, wherein: The heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, respectively, as shown in SEQ ID NO: 17-19. The light chain variable region comprises LCDR1, LCDR2, and LCDR3, respectively, as shown in SEQ ID NO: 20-22.

5. The pharmaceutical composition according to any one of claims 1-4, wherein, The BTN3A binding protein is an anti-BTN3A antibody or its antigen-binding fragment, wherein the antibody is preferably a rabbit-derived antibody, a chimeric antibody, a humanized antibody, or a fully human antibody; and the antigen-binding fragment is preferably an scFv, Fab, or F(ab')2 fragment. Preferably, the BTN3A binding protein is a humanized, reversed mutation, affinity-matured, T-cell epitope-removed, antibody deamidation-reduced, and / or antibody isomerization-reduced modified anti-BTN3A antibody or its antigen-binding fragment.

6. The pharmaceutical composition according to any one of claims 1-5, wherein, The BTN3A binding protein further includes the Fc region of an immunoglobulin. Preferably, the Fc region is the Fc region of human IgG1, human IgG2, human IgG3 or human IgG4; More preferably, the Fc region is the Fc region of human IgG1 with L234F, L235E and / or P331S mutations, wherein the position of the mutated amino acid in the Fc region is defined according to the EU numbering system, and optionally, K446 is removed.

7. The pharmaceutical composition according to any one of claims 1-6, wherein, The BTN3A binding protein comprises a heavy chain and a light chain selected from any of the following: The heavy chain comprises an amino acid sequence as shown in SEQ ID NO: 25 or having at least 80% or at least 90% identity with it, and the light chain comprises an amino acid sequence as shown in SEQ ID NO: 26 or having at least 80% or at least 90% identity with it; The heavy chain comprises an amino acid sequence as shown in any of SEQ ID NO: 48-52 or having at least 80% or at least 90% identity with it, and the light chain comprises an amino acid sequence as shown in SEQ ID NO: 53 or having at least 80% or at least 90% identity with it; The heavy chain comprises an amino acid sequence as shown in any of SEQ ID NO: 48-52 or having at least 80% or at least 90% identity with it, and the light chain comprises an amino acid sequence as shown in SEQ ID NO: 54 or having at least 80% or at least 90% identity with it; The heavy chain comprises an amino acid sequence as shown in any of SEQ ID NO: 48-52 or having at least 80% or at least 90% identity with it, and the light chain comprises an amino acid sequence as shown in SEQ ID NO: 55 or having at least 80% or at least 90% identity with it; The heavy chain comprises a heavy chain having at least 80% or at least 90% amino acid sequence identity as shown in SEQ ID NO: 27, and the light chain comprises a light chain having at least 80% or at least 90% amino acid sequence identity as shown in SEQ ID NO:

28. The heavy chain comprises an amino acid sequence as shown in any of SEQ ID NO: 56-61 or having at least 80% or at least 90% identity with it, and the light chain comprises an amino acid sequence as shown in SEQ ID NO: 62 or having at least 80% or at least 90% identity with it; The heavy chain comprises an amino acid sequence as shown in any of SEQ ID NO: 56-61 or having at least 80% or at least 90% identity with it, and the light chain comprises an amino acid sequence as shown in SEQ ID NO: 63 or having at least 80% or at least 90% identity with it; or, The heavy chain comprises an amino acid sequence as shown in any of SEQ ID NO: 56-61 or having at least 80% or at least 90% identity with it, and the light chain comprises an amino acid sequence as shown in SEQ ID NO: 64 or having at least 80% or at least 90% identity with it.

8. The pharmaceutical composition according to any one of claims 1-7, wherein the pH value of the buffer or the pharmaceutical composition is from about 3.0 to about 8.0, preferably from about 3.5 to about 7.5, more preferably from about 4.5 to about 7.

0.

9. The pharmaceutical composition according to any one of claims 1-8, wherein the concentration of the buffer is from about 1 mM to about 100 mM, preferably from about 2 mM to about 50 mM, more preferably from about 5 mM to about 30 mM.

10. The pharmaceutical composition according to any one of claims 1-9, wherein the concentration of the BTN3A binding protein is from about 0.01 g / L to about 200 g / L, preferably from about 10 g / L to about 190 g / L, more preferably from about 50 g / L to about 150 g / L.

11. The pharmaceutical composition according to any one of claims 1-10, further comprising one or more of surfactants, sugars, complexing agents or amino acids.

12. The pharmaceutical composition according to any one of claims 1-11, wherein: (1) The surfactant is polysorbate, preferably polysorbate 20 or polysorbate 80, more preferably polysorbate 80; Preferably, the concentration of the surfactant is from about 0.01% (w / v) to about 10% (w / v), more preferably from about 0.01% (w / v) to about 5% (w / v), and even more preferably from about 0.01% (w / v) to about 2% (w / v); or (2) The sugar is sucrose or trehalose, preferably sucrose; Preferably, the sugar concentration is from about 0.1% (w / v) to about 20% (w / v), more preferably from about 0.2% (w / v) to about 15% (w / v), and even more preferably from about 0.5% (w / v) to about 12% (w / v); or (3) The complexing agent is disodium edetate, preferably, the concentration of disodium edetate is about 0.001% (w / v) to about 5% (w / v), more preferably about 0.001% (w / v) to about 1% (w / v), and more preferably about 0.001% (w / v) to about 0.1% (w / v); or (4) The amino acid is methionine. Preferably, the concentration of methionine is about 0.01% (w / v) to about 10% (w / v), more preferably about 0.01% (w / v) to about 5% (w / v), and more preferably about 0.05% (w / v) to about 2% (w / v).

13. The pharmaceutical composition according to any one of claims 1-12, comprising: (1) BTN3A binding protein at concentrations of approximately 0.01 g / L to approximately 200 g / L. Buffers ranging from approximately 1 mM to approximately 100 mM Surfactants ranging from approximately 0.01% (w / v) to approximately 10% (w / v), Sugars of approximately 0.1% (w / v) to approximately 20% (w / v), and, The amino acids present may be selected from approximately 0.01% (w / v) to approximately 10% (w / v); The complexing agent may be present in an amount of about 0.001% (w / v) to about 5% (w / v); The pH of the pharmaceutical composition is from about 3.0 to about 7.0; or, (2) BTN3A binding protein at concentrations of approximately 0.01 g / L to approximately 200 g / L. Acetate buffer, citrate buffer, histidine buffer or phosphate buffer at a concentration of about 1 mM to about 100 mM; Polysorbate 80, from about 0.01% (w / v) to about 10% (w / v), Sucrose from about 0.1% (w / v) to about 20% (w / v), and, The methionine present optionally is about 0.01% (w / v) to about 10% (w / v); Sodium edetate may be present in an optional amount of about 0.001% (w / v) to about 5% (w / v); The pH of the pharmaceutical composition is from about 3.0 to about 7.0; or, (3) BTN3A binding protein at approximately 20 g / L to approximately 180 g / L, Acetic acid-sodium acetate, citric acid-sodium citrate, histidine-histidine hydrochloride or disodium hydrogen phosphate-sodium dihydrogen phosphate buffers of about 1 mM to about 100 mM. Polysorbate 80 at approximately 0.01% (w / v) to approximately 2% (w / v) Sucrose of about 0.5% (w / v) to about 12% (w / v), and, The methionine present optionally is about 0.01% (w / v) to about 2% (w / v); Sodium edetate may be present in an optional amount of about 0.001% (w / v) to about 0.1% (w / v); The pH of the pharmaceutical composition is from about 3.0 to about 7.0; or, (4) BTN3A binding protein at approximately 50 g / L to approximately 150 g / L, Acetic acid-sodium acetate, citric acid-sodium citrate, histidine-histidine hydrochloride or disodium hydrogen phosphate-sodium dihydrogen phosphate buffers of about 1 mM to about 50 mM. Polysorbate 80 at approximately 0.01% (w / v) to approximately 1% (w / v), Sucrose of about 1% (w / v) to about 12% (w / v), and, The presence of methionine is optionally about 0.01% (w / v) to about 1% (w / v); The pH of the pharmaceutical composition is from about 4.5 to about 7.

0.

14. A pharmaceutical composition comprising any one of the following: (1-1) BTN3A binding protein at approximately 20 g / L to approximately 180 g / L, Approximately 1 mM to approximately 100 mM histidine-histidine hydrochloride, Approximately 0.5% (w / v) to approximately 12% (w / v) sucrose, From about 0.01% (w / v) to about 2% (w / v) polysorbate 80, The pH of the pharmaceutical composition is from about 5.0 to about 7.0; or (1-2) BTN3A binding protein at approximately 20 g / L to approximately 180 g / L, Approximately 1 mM to approximately 100 mM histidine-histidine hydrochloride, Approximately 0.5% (w / v) to approximately 12% (w / v) sucrose, From about 0.01% (w / v) to about 2% (w / v) polysorbate 80, Approximately 0.001% (w / v) to approximately 5% (w / v) disodium edetate; The pH of the pharmaceutical composition is from about 5.0 to about 7.0; or (1-3) BTN3A binding protein of approximately 20 g / L to approximately 180 g / L, Approximately 1 mM to approximately 100 mM histidine-histidine hydrochloride, Approximately 0.5% (w / v) to approximately 12% (w / v) sucrose, From about 0.01% (w / v) to about 2% (w / v) polysorbate 80, Approximately 0.01% (w / v) to approximately 10% (w / v) methionine; The pH of the pharmaceutical composition is from about 5.0 to about 7.0; or (1-4) Approximately 50 g / L to approximately 150 g / L of BTN3A binding protein, Approximately 1 mM to approximately 50 mM histidine-histidine hydrochloride, From approximately 1% (w / v) to approximately 12% (w / v) sucrose, From about 0.01% (w / v) to about 1% (w / v) polysorbate 80, The pH of the pharmaceutical composition is about 6.0 to about 7.0; or (1-5) Approximately 50 g / L to approximately 150 g / L of BTN3A binding protein, Approximately 1 mM to approximately 50 mM histidine-histidine hydrochloride, From approximately 1% (w / v) to approximately 12% (w / v) sucrose, From about 0.01% (w / v) to about 1% (w / v) polysorbate 80, Approximately 0.001% (w / v) to approximately 5% (w / v) disodium edetate; The pH of the pharmaceutical composition is about 6.0 to about 7.0; or (1-6) Approximately 50 g / L to approximately 150 g / L of BTN3A-binding protein, Approximately 1 mM to approximately 50 mM histidine-histidine hydrochloride, From approximately 1% (w / v) to approximately 12% (w / v) sucrose, From about 0.01% (w / v) to about 1% (w / v) polysorbate 80, Approximately 0.01% (w / v) to approximately 1% (w / v) methionine; The pH of the pharmaceutical composition is about 6.0 to about 7.0; or (2-1) BTN3A binding protein at approximately 20 g / L to approximately 180 g / L, Approximately 1 mM to approximately 100 mM of acetate-sodium acetate, Approximately 0.5% (w / v) to approximately 12% (w / v) sucrose, From about 0.01% (w / v) to about 2% (w / v) polysorbate 80, The pH of the pharmaceutical composition is from about 4.5 to about 7.0; or (2-2) Approximately 50 g / L to approximately 150 g / L of BTN3A-binding protein, Approximately 1 mM to approximately 50 mM of acetate-sodium acetate From approximately 1% (w / v) to approximately 12% (w / v) sucrose, From about 0.01% (w / v) to about 1% (w / v) polysorbate 80, The pH of the pharmaceutical composition is from about 4.5 to about 7.0; or (3-1) BTN3A binding protein at approximately 20 g / L to approximately 180 g / L, Approximately 1 mM to approximately 100 mM citric acid-sodium citrate Approximately 0.5% (w / v) to approximately 12% (w / v) sucrose, From about 0.01% (w / v) to about 2% (w / v) polysorbate 80, The pH of the pharmaceutical composition is from about 5.0 to about 7.0; or (3-2) Approximately 50 g / L to approximately 180 g / L of BTN3A-binding protein, Approximately 1 mM to approximately 50 mM citric acid-sodium citrate From approximately 1% (w / v) to approximately 12% (w / v) sucrose, From about 0.01% (w / v) to about 1% (w / v) polysorbate 80, The pH of the pharmaceutical composition is from about 5.0 to about 7.0; or (4-1) BTN3A binding protein at approximately 20 g / L to approximately 180 g / L, Approximately 1 mM to approximately 100 mM disodium hydrogen phosphate - sodium dihydrogen phosphate Approximately 0.5% (w / v) to approximately 12% (w / v) sucrose, From about 0.01% (w / v) to about 2% (w / v) polysorbate 80, The pH of the pharmaceutical composition is from about 5.0 to about 7.0; or, (4-2) Approximately 50 g / L to approximately 150 g / L of BTN3A-binding protein, Approximately 1 mM to approximately 50 mM disodium hydrogen phosphate - sodium dihydrogen phosphate From approximately 1% (w / v) to approximately 12% (w / v) sucrose, From about 0.01% (w / v) to about 1% (w / v) polysorbate 80, The pH of the pharmaceutical composition is from about 5.0 to about 7.0; Preferably, the BTN3A binding protein is as defined in any one of claims 1-8.

15. A lyophilized formulation, wherein the lyophilized formulation is obtained by freeze-drying the pharmaceutical composition according to any one of claims 1 to 14, or the lyophilized formulation can be reconstituted to form the pharmaceutical composition according to any one of claims 1 to 14.

16. A reconstituted solution obtained by reconstituted the lyophilized formulation of claim 15.

17. The reconstituted solution as described in claim 16, wherein the lyophilized formulation is obtained by reconstitution with a solvent; preferably, the solvent is water, physiological saline or glucose.

18. An article comprising a container containing a pharmaceutical composition as claimed in any one of claims 1 to 14, a lyophilized formulation as claimed in claim 15, or a reconstituted solution as claimed in claim 16 or 17.

19. A method of treating or preventing a disease, comprising administering to a subject in need a therapeutically or preventively effective amount of a pharmaceutical composition as described in any one of claims 1 to 14, a lyophilized formulation as described in claim 15, a reconstituted solution as described in claim 16 or 17, or an article as described in claim 18, wherein: Preferably, the disease is cancer.

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