Pharmaceutical composition containing immunoconjugate and use thereof

Abstract

The present invention relates to a pharmaceutical composition containing an immunoconjugate, and the use thereof. Specifically, the present invention relates to a pharmaceutical composition, which contains an immunoconjugate and a buffer.

Description

A pharmaceutical composition comprising an immunoconjugate and its use

[0001] The present disclosure claims priority to the Chinese patent application “A pharmaceutical composition comprising an immunoconjugate and its use” (Application No.: 2023110361039) filed on August 16, 2023, which is incorporated herein by reference. Technical Field

[0002] The present disclosure belongs to the field of pharmaceutical preparations, and particularly relates to a pharmaceutical composition comprising an immunoconjugate, and its use as a medicine. Background Art

[0003] Human interleukin-2 (IL-2), also known as T cell growth factor (TCGF), has a gene located on chromosome 4 (4q27) and encompasses a 7kb sequence consisting of 133 amino acids with a molecular weight of approximately 15kD. Initial in vitro cell experiments demonstrated that T cells, after activation through the TCR and CD28, secrete IL-2 and express the IL-2 receptor (IL-2R) on their cell surface. Binding of IL-2 to its receptor induces T cell proliferation and effector activity. This model established IL-2 as a central molecule in T cell immune responses. However, subsequent in vivo experiments revealed that knockout of IL-2 or its receptor resulted in autoimmunity in animals. Subsequent experiments demonstrated that IL-2 not only activates effector cells such as T cells and NK cells, but also regulatory T cells (Tregs), thereby suppressing excessive self-immunity.

[0004] IL-2 acts through IL-2R on the cell surface. IL-2R consists of three subunits, IL-2Rα (i.e. CD25), IL-2Rβ (i.e. CD122) and IL-2Rγ (i.e. CD132). The three subunits can form three receptor forms: the high binding receptor contains all three subunits IL-2Rα / β / γ, the medium binding receptor contains IL-2Rβ / γ, and the low binding receptor is IL-2Rα. Among them, IL-2Rβ and IL-2Rγ are necessary for IL-2 to activate downstream signaling pathways. When IL-2 binds to IL-2Rβ and IL-2Rγ at the same time, the two receptor subunits form heterodimers, phosphorylate STAT5 in the cell, enter the cell nucleus and lead to the transcription and expression of corresponding genes; IL-2Rα is not necessary for signal transduction, but can enhance the binding of IL-2 to IL-2Rβ and IL-2Rγ. Treg continuously expresses the high binding receptors IL-2Rα / β / γ, while unactivated CD8 +T cells and NK cells only express the medium-binding receptor IL-2Rβ / γ. Therefore, low-dose IL-2 preferentially binds to and activates Treg, while high-dose IL-2 can also activate CD8 + Immune effector cells such as T cells and NK cells. Due to its role in activating immune effector cells, high-dose IL-2 can be used for tumor immunotherapy. However, high-dose IL-2 treatment is also accompanied by serious toxic side effects, including fever, diarrhea, capillary leak syndrome, etc. These side effects are related to the excessive activation of peripheral T cells and NK cells. Reducing or eliminating the activity of IL-2 on peripheral T cells and NK cells, and specifically targeting the tumor microenvironment or tumor-associated immune cells, can exert its anti-tumor effect while avoiding the systemic toxicity caused by IL-2.

[0005] PD-1 (Programmed Cell death-1) belongs to the CD28 receptor family and is an immunosuppressive receptor (Riley et al. 2009, Immunol. Rev. 29: 114-25). The family also includes CD28, CTLA-4, ICOS, PD-1 and BTLA. PD-1 is mainly expressed in activated B cells, T cells and bone marrow cells (Chen et al. 2013, Nat. Rev. Immunol. 13: 227-42), and has two cell surface glycoprotein ligands, PD-L1 and PD-L2. PD-L1 is widely expressed in lymphocytes (such as CD4 + T cells and CD8 + T cells, macrophages, etc.) as well as peripheral tissues, various tumor cells, and virus-infected cells. PD-L2 is primarily expressed on activated dendritic cells and macrophages (Dong et al. 1999, Nat. Med. 5:1365-9). Upon binding to its ligands PD-L1 or PD-L2, PD-1 downregulates T cell function, including reducing T cell activation, differentiation and proliferation, and cytokine secretion.

[0006] The present disclosure provides an immunoconjugate comprising a PD-1 antibody and IL-2 or a variant thereof, wherein the IL-2 variant has reduced affinity for high-affinity receptors (IL-2Rα / β / γ) and intermediate-affinity receptors (IL-2Rβ / γ) compared to wild-type IL-2, has eliminated affinity for low-affinity receptors (IL-2Rα) compared to wild-type IL-2, and has a greater reduction in affinity for high-affinity receptors than for intermediate-affinity receptors. Due to the reduced affinity for IL-2Rα / β / γ and IL-2Rβ / γ relative to wild-type IL-2, the immunoconjugate is inactive against immune cells that low or do not express PD-1, but can activate immune cells that high express PD-1 from two aspects. On the one hand, the PD-1 antibody blocks the interactions between PD-1 / PD-L1 and PD-1 / PD-L2, thereby relieving tumor immune suppression on T cells. On the other hand, the immunoconjugate binds to PD-1 on the surface of T cells. Only when bound to PD-1 can its IL-2 variant bind to the IL-2 receptor on the cell surface through an in-cis interaction, thereby exerting the IL-2 variant's activation effect on PD-1-positive T cells. Through this design, the IL-2 variant can be used to enhance the PD-1 antibody's effects on T cell activation and proliferation, as well as its anti-tumor effects. It also increases the specificity of IL-2 for PD-1-positive immune cells and avoids the systemic toxicity common in wild-type IL-2, providing a more effective solution for clinical tumor treatment.

[0007] However, due to their large molecular weight and complex structure, immunoconjugates are prone to degradation and aggregation, becoming unstable, leading to reduced activity or even ineffectiveness. To ensure that fusion proteins are suitable for administration and maintain stability during storage and subsequent use, thereby achieving optimal efficacy, the development of stable drug formulations is particularly important.

[0008] Summary of the Invention

[0009] The present disclosure provides a pharmaceutical composition comprising an immunoconjugate (e.g., a fusion protein of a PD-1 binding domain and a cytokine), a method for preparing the pharmaceutical composition, and methods for treating or preventing diseases or related pharmaceutical uses thereof.

[0010] The present disclosure provides a pharmaceutical composition comprising an immunoconjugate (e.g., a fusion protein of a PD-1 binding domain and a cytokine) and a buffer. In some embodiments, the buffer is a histidine salt buffer. In some embodiments, the buffer is selected from one or more of acetate-histidine, succinate-histidine, and histidine-histidine hydrochloride. "One or more" in the present disclosure includes one, two, or more.

[0011] 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 1 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 In some embodiments, the concentration of the buffer agent is about 5mM to about 35mM. In some embodiments, the concentration of the buffer agent is about 5mM to about 20mM. In some embodiments, the concentration of the buffer agent is about 5mM to about 20mM. In some embodiments, the concentration of the buffer agent is about 10mM to about 20mM. In some embodiments, the concentration of the buffer agent is about 10mM to about 20mM.

[0012] 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, about 36 mM, about 37 mM, about 38 mM, about 39 mM, about 40 mM, about 41 mM, about 42 mM, about 43 mM, about 44 mM, about 45 mM, about 46 mM, about 47 mM, about 48 mM, about 49 mM, about 50 mM, about 51 mM, about 52 mM, about 53 mM, about 54 mM, about 55 mM, about 56 mM, about 57 mM, about 58 mM, about 59 mM, about 60 mM, about 61 mM, about 62 mM, about 63 mM, about 64 mM, about 65 mM, about 66 mM, about 67 mM, about 68 mM, about 69 mM, about 70 mM, about 71 mM, about 72 mM mM, about 40mM, about 43mM, about 45mM, about 48mM, about 50mM, about 55mM, about 57mM, about 58mM, about 60mM, about 62mM, about 65mM, about 68mM, about 70mM, about 72mM, about 75mM, about 78mM, about 80mM, about 82mM, about 85mM, about 88mM, about 90mM, about 92mM, about 95mM, about 100mM.

[0013] In some embodiments, the buffer is a histidine salt and the concentration of the histidine salt is about 1 mM to about 100 mM, for example, about 1 mM to about 50 mM, about 1 mM to about 55 mM, about 1 mM to about 60 mM, about 1 mM to about 70 mM, about 1 mM to about 80 mM, about 1 mM to about 85 mM, about 1 mM to about 90 mM, about 5 mM to about 100 mM, about 1 mM to about 80 mM, about 5 mM to about 70 mM, about 5 mM to about 60 mM, about 5 mM to about 50 mM, about 5 mM to about 35 mM, about 5 mM to about 20 mM, about 5 mM to about 10 mM, about 10 mM to about 20 mM. In some embodiments, the concentration of the histidine salt 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, about 36 mM, about 37 mM, about 38 mM, about 39 mM, about 40 mM, about 41 mM, about 42 mM, about 43 mM, about 44 mM, about 45 mM, about 46 mM, about 47 mM, about 48 mM, about 49 mM, about 50 mM, about 51 mM, about 52 mM, about 53 mM, about 54 mM, about 55 mM, about 56 mM, about 57 mM, about 58 mM, about 59 mM, about 60 mM, about 61 mM, about 62 mM, about 63 mM, about 64 mM, about 65 mM, about 66 mM, about 67 mM, about 68 mM, about 69 mM, about 70 mM, about 71 mM, about 72 mM mM, about 40mM, about 43mM, about 45mM, about 48mM, about 50mM, about 55mM, about 57mM, about 58mM, about 60mM, about 62mM, about 65mM, about 68mM, about 70mM, about 72mM, about 75mM, about 78mM, about 80mM, about 82mM, about 85mM, about 88mM, about 90mM, about 92mM, about 95mM, about 100mM.

[0014] In some specific embodiments, the buffer is acetate-histidine, and the concentration of acetate-histidine is about 1 mM to about 100 mM, for example, about 1 mM to about 50 mM, about 1 mM to about 55 mM, about 1 mM to about 60 mM, about 1 mM to about 70 mM, about 1 mM to about 80 mM, about 1 mM to about 85 mM, about 1 mM to about 90 mM, about 5 mM to about 100 mM, about 1 mM to about 80 mM, about 5 mM to about 70 mM, about 5 mM to about 60 mM, about 5 mM to about 50 mM, about 5 mM to about 35 mM, about 5 mM to about 20 mM, about 5 mM to about 10 mM, about 10 mM to about 20 mM. In some embodiments, the concentration of the acetate-histidine is 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 25 mM, about 30 mM, about 35 mM. M, about 40mM, about 50mM, about 55mM, about 57mM, about 58mM, about 60mM, about 62mM, about 65mM, about 68mM, about 70mM, about 72mM, about 75mM, about 78mM, about 80mM, about 82mM, about 85mM, about 88mM, about 90mM, about 92mM, about 95mM, about 100mM.

[0015] In some specific embodiments, the buffer is histidine-histidine hydrochloride, and the concentration of the histidine-histidine hydrochloride is about 1 mM to about 100 mM, for example, about 1 mM to about 50 mM, about 1 mM to about 55 mM, about 1 mM to about 60 mM, about 1 mM to about 70 mM, about 1 mM to about 80 mM, about 1 mM to about 85 mM, about 1 mM to about 90 mM, about 5 mM to about 100 mM, about 1 mM to about 80 mM, about 5 mM to about 70 mM, about 5 mM to about 60 mM, about 5 mM to about 50 mM, about 5 mM to about 35 mM, about 5 mM to about 20 mM, about 5 mM to about 10 mM, about 10 mM to about 20 mM. About 5mM, about 6mM, about 7mM, about 8mM, about 9mM, about 10mM, about 11mM, about 12mM, about 13mM, about 14mM, about 15mM, about 16mM, about 17mM, about 18mM, about 19mM, about 20mM, about 25mM, about 30mM, about 35mM, about 40mM, about 50mM, about 55mM, about 57mM, about 58mM, about 60mM, about 62mM, about 65mM, about 68mM, about 70mM, about 72mM, about 75mM, about 78mM, about 80mM, about 82mM, about 85mM, about 88mM, about 90mM, about 92mM, about 95mM, about 100mM.

[0016] In some specific embodiments, the buffer is succinate-histidine, and the concentration of succinate-histidine is about 1 mM to about 100 mM, for example, about 1 mM to about 50 mM, about 1 mM to about 55 mM, about 1 mM to about 60 mM, about 1 mM to about 70 mM, about 1 mM to about 80 mM, about 1 mM to about 85 mM, about 1 mM to about 90 mM, about 5 mM to about 100 mM, about 1 mM to about 80 mM, about 5 mM to about 70 mM, about 5 mM to about 60 mM, about 5 mM to about 50 mM, about 5 mM to about 35 mM, about 5 mM to about 20 mM, about 5 mM to about 10 mM, about 10 mM to about 20 mM. About 5mM, about 6mM, about 7mM, about 8mM, about 9mM, about 10mM, about 11mM, about 12mM, about 13mM, about 14mM, about 15mM, about 16mM, about 17mM, about 18mM, about 19mM, about 20mM, about 25mM, about 30mM, about 35mM, about 40mM, about 50mM, about 55mM, about 57mM, about 58mM, about 60mM, about 62mM, about 65mM, about 68mM, about 70mM, about 72mM, about 75mM, about 78mM, about 80mM, about 82mM, about 85mM, about 88mM, about 90mM, about 92mM, about 95mM, about 100mM.

[0017] The present disclosure provides a pharmaceutical composition comprising an immunoconjugate, wherein the pH of the pharmaceutical composition is about 4.0 to about 6.5. In some embodiments, the pharmaceutical composition comprises an immunoconjugate and a buffer.

[0018] In some embodiments, the pH of the buffer or the pharmaceutical composition is from about 4.0 to about 6.5, for example, from about 4.0 to about 6.0, from about 4.0 to about 5.5, from about 4.0 to about 5.0, from about 4.0 to about 4.8, from 4.0 to about 4.5, from about 4.8 to about 5.0, from about 5.0 to about 5.5, from about 4.5 to about 4.8, or any range therebetween. In some embodiments, the pH of the buffer or the pharmaceutical composition is from about 4.0 to about 5.0. In some embodiments, the pH of the buffer or the pharmaceutical composition is from about 4.0 to about 4.5.

[0019] In some embodiments, the pH of the buffer or the pharmaceutical composition is 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.

[0020] Typically, the pH of the pharmaceutical composition obtained by replacing the buffer is substantially consistent with the pH of the buffer. It is also well known to those skilled in the art that pH drift may sometimes occur during the pharmaceutical preparation process, but the pH drift of the pharmaceutical preparation is generally small (e.g., within a range of ±0.8). In some embodiments, the pH drift of the pharmaceutical preparation is within a range of ±0.5.

[0021] In some embodiments, the concentration of the immunoconjugate is from about 0.01 mg / mL to about 200 mg / mL, for example, 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 150 mg / mL, from about 10 mg / mL to about 120 mg / mL, from about 10 mg / mL to about 110 mg / mL, from about 10 mg / mL to about 100 mg / mL, from about 10 mg / mL to about 80 mg / mL, from about 10 mg / mL to about 60 mg / mL, from about 10 mg / mL to about 40 mg / mL, from about 20 mg / mL to about 200 mg / mL, from about In some embodiments, the present invention provides an aqueous / modified aqueous solution of at least one embodiment of the present invention. The aqueous / modified aqueous solution of at least one embodiment of the present invention can be a mixture of at least one embodiment of the present invention. In some embodiments, the concentration of the immunoconjugate is about 0.01 mg / mL, about 0.05 mg / mL, about 0.1 mg / mL, about 0.5 mg / mL about 1 mg / mL, about 5 mg / mL, about 10 mg / mL, about 15 mg / mL, about 20 mg / mL, about 25 mg / mL, about 30 mg / mL, about 35 mg / mL, about 40 mg / mL, about 45 mg / mL, about 50 mg / mL, about 55 mg / mL, about 60 mg / mL, about 61 mg / mL, about 62 mg / mL, about 63 mg / mL, about 64 mg / mL, about 65 mg / mL, about 66 mg / mL, about 67 mg / mL, about 68 mg / mL, about 69 mg / mL, about 70 mg / mL, about 71 mg / mL, about 72 mg / mL, about 73 mg / mL, about 74 mg / mL 4 mg / mL, about 75 mg / mL, about 76 mg / mL, about 77 mg / mL, about 78 mg / mL, about 79 mg / mL, about 80 mg / mL, about 81 mg / mL, about 82 mg / mL, about 83 mg / mL, about 84 mg / mL, about 85 mg / mL, about 86 mg / mL, about 87 mg / mL, about 88 mg / mL, about 89 mg / mL, about 90 mg / mL, about 91 mg / mL, about 92 mg / mL, about 93 mg / mL, about 94 mg / mL, about 95 mg / mL, about 96 mg / mL, about 97 mg / mL, about 98 mg / mL, about 99 mg / mL, about 100 mg / mL , about 101 mg / mL, about 102 mg / mL, about 103 mg / mL, about 104 mg / mL, about 105 mg / mL, about 106 mg / mL, about 107 mg / mL, about 108 mg / mL, about 109 mg / mL, about 110 mg / mL, about 111 mg / mL, about 112 mg / mL, about 113 mg / mL, about 114 mg / mL, about 115 mg / mL, about 116 mg / mL, about 117 mg / mL, about 118 mg / mL, about 119 mg / mL, about 120 mg / mL, about 121 mg / mL, about 122 mg / mL, about 123 mg / mL, about 124 mg / mL , about 125 mg / mL, about 126 mg / mL, about 127 mg / mL, about 128 mg / mL, about 129 mg / mL, about 130 mg / mL, about 131 mg / mL, about 132 mg / mL, about 133 mg / mL, about 134 mg / mL, about 135 mg / mL, about 140 mg / mL, about 145 mg / mL, about 150 mg / mL, about 155 mg / mL, about 160 mg / mL, about 165 mg / mL, about 170 mg / mL, about 175 mg / mL, about 180 mg / mL, about 185 mg / mL, about 190 mg / mL, about 195 mg / mL, about 200 mg / mL.

[0022] In some embodiments, the pharmaceutical composition as described in any one of the above, it comprises a surfactant. In some embodiments, the surfactant is a nonionic surfactant. In some embodiments, the surfactant is selected from poloxamer (e.g., poloxamer 188), polysorbate (e.g., polysorbate 20 (i.e., PS20), polysorbate 80 (i.e., PS80)), polyhydroxyalkyl, Triton, sodium dodecyl sulfate, sodium lauryl sulfate, sodium octyl glucoside, lauryl-sulfobetaine, myristyl-sulfobetaine, linoleyl-sulfobetaine, stearyl-sulfobetaine, lauryl-sarcosine, myristyl-sarcosine, linoleyl-sarcosine, stearyl-sarcosine, linoleyl-sweet In some embodiments, the surfactant is a copolymer of sodium lauryl sulfate (SDS) or polysorbate. In some embodiments, the surfactant is a polysorbate 80.

[0023] In some embodiments, the concentration of the surfactant is from about 0.001% (w / v) to about 1.0% (w / v), for example, from about 0.001% (w / v) to about 0.8% (w / v), from about 0.001% (w / v) to about 0.5% (w / v), from about 0.001% (w / v) to about 0.2% (w / v), from about 0.001% (w / v) to about 0.1% (w / v), from about 0.01% (w / v) to about 1.0% (w / v), about 0.01% (w / v) to about 0.8% (w / v), about 0.01% (w / v) to about 0.5% (w / v), about 0.01% (w / v) to about 0.2% (w / v), about 0.01% (w / v) to about 0.1% (w / v), about 0.01% (w / v) to about 0.18% (w / v), about 0.01% ( w / v) to about 0.15% (w / v), about 0.01% (w / v) to about 0.1% (w / v), about 0.01% (w / v) to about 0.08% (w / v), about 0.01% (w / v) to about 0.06% (w / v), about 0.01% (w / v) to about 0.04% (w / v), about 0.02% (w / v) to about 0.2% (w / v), about 0.02% (w / v) to about 0.15% (w / v) ), about 0.02% (w / v) to about 0.1% (w / v), about 0.02% (w / v) to about 0.08% (w / v), about 0.02% (w / v) to about 0.04% (w / v), about 0.04% (w / v) to about 0.2% (w / v), about 0.04% (w / v) to about 0.1% (w / v), about 0.04% (w / v) to about 0.08% (w / v), and any ranges therebetween. In some embodiments, the concentration of the surfactant is about 0.001% (w / v), about 0.05% (w / v), about 0.01% (w / v), about 0.02% (w / v), about 0.03% (w / v), about 0.04% (w / v), about 0.05% (w / v), about 0.06% (w / v), about 0.07% (w / v), about 0.08% (w / v), about 0.09% (w / v), about 0.1% (w / v), about 0.11% (w / v), about 0.12% (w / v), about 0.13% (w / v), about 0.14% (w / v), about 0.15% (w / v), about 0.16% (w / v), about 0.17% (w / v), about 0.18% (w / v), about 0.19% (w / v), about 0.2% (w / v)), about 0.3% (w / v)), about 0.4% (w / v)), about 0.5% (w / v)), about 0.6% (w / v)), about 0.7% (w / v)), about 0.8% (w / v).

[0024] In some embodiments, the pharmaceutical composition as described in any one of the above items comprises an osmotic pressure regulator. In some embodiments, the osmotic pressure regulator is a sugar (including monosaccharides, disaccharides, trisaccharides, polysaccharides, sugar alcohols, reducing sugars, non-reducing sugars, etc.), an amino acid (including arginine, glycine, cysteine, histidine, etc.) or a salt (sodium chloride, potassium chloride, calcium chloride, etc.). In some embodiments, the osmotic pressure regulator is a sugar selected from the group consisting of glucose, sucrose, trehalose, lactose, fructose, maltose, dextran, glycerol, erythritol, glycerol, arabitol, xylitol, sorbitol (also known as sorbitol), mannitol, milibiose, melezitose, raffinose, mannotriose, stachyose, maltose, lactulose, maltulose, maltitol, lactitol and iso-maltulose. In some embodiments, the osmotic pressure regulator is selected from one or more of the group consisting of sucrose, trehalose, sorbitol, arginine, proline, glycine and sodium chloride. In some embodiments, the osmotic pressure regulator is a non-reducing disaccharide; in some embodiments, the osmotic pressure regulator is trehalose and / or sucrose; in some embodiments, the osmotic pressure regulator is trehalose; in some embodiments, the trehalose is trehalose.

[0025] In some embodiments, the concentration of the osmotic pressure regulating agent is about 1% (w / v) to about 20% (w / v), for example, about 1% (w / v) to about 18% (w / v), about 1% (w / v) to about 15% (w / v), about 1% (w / v) to about 12 (w / v), about 1% (w / v) to about 8% (w / v), about 1% (w / v) to about 8.8% (w / v), about 2% (w / v) to about 20% (w / v), about 2% (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) / 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) / v), about 5% (w / v) to about 8% (w / v), about 5% (w / v) to about 8.8% (w / v), about 4.4% (w / v) to about 20% (w / v), about 4.4% (w / v) to about 15% (w / v), about 4.4% (w / v) to about 10% (w / v), about 4.4% (w / v) to about 8% (w / v), about 4.4% (w / v) to about 8.8% (w / v), and any ranges therebetween. In some embodiments, the concentration of the osmotic pressure regulating agent 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), about 7% (w / v), about 7.8% (w / v), about 8% (w / v), about 8.2% (w / v), about 8.4% (w / v), about 8.8% (w / v), about 9% (w / v), about 10% (w / v), about 12% (w / v), about 14% (w / v), about 16% (w / v), about 18% (w / v), about 20% (w / v).

[0026] In some embodiments, any of the above pharmaceutical compositions further comprises a stabilizer. In some embodiments, the stabilizer is arginine or a salt thereof. In some embodiments, the pharmaceutical composition comprises arginine hydrochloride. In some embodiments, the pharmaceutical composition does not comprise arginine.

[0027] In some embodiments, the concentration of the stabilizer is from about 5 mM to about 200 mM, for example, from about 10 mM to about 150 mM, from about 10 mM to about 180 mM, from about 10 mM to about 120 mM, from about 10 mM to about 100 mM, from about 10 mM to about 80 mM, from about 10 mM to about 70 mM, from about 10 mM to about 50 mM, from about 10 mM to about 30 mM, from about 30 mM to about 50 mM, from about 30 mM to about 80 mM, from about 30 mM to about 100 mM, from about 50 mM to about 70 mM, from about 50 mM to about 100 mM, and any ranges therebetween. In some embodiments, the concentration of the stabilizer is about 10 mM, about 20 mM, about 30 mM, about 40 mM, about 50 mM, about 60 mM, about 70 mM, about 80 mM, about 90 mM, about 100 mM, about 110 mM, about 120 mM, about 150 mM.

[0028] In some embodiments, the pharmaceutical composition comprises the following:

[0029] (a) about 10 mg / mL to about 200 mg / mL of the immunoconjugate,

[0030] about 1 mM to about 50 mM buffer,

[0031] about 0.01% (w / v) to about 0.2% (w / v) of a surfactant,

[0032] about 1% (w / v) to about 20% (w / v) of an osmotic pressure regulating agent, and,

[0033] optionally, about 10 mM to about 100 mM of a stabilizer;

[0034] The pH of the pharmaceutical composition is from about 4.0 to about 5.5; or,

[0035] (b) about 10 mg / mL to about 120 mg / mL of the immunoconjugate,

[0036] about 5 mM to about 35 mM acetate or histidine buffer;

[0037] about 0.04% (w / v) to about 0.2% (w / v) polysorbate 80,

[0038] about 4% (w / v) to about 10% (w / v) sucrose or trehalose, and,

[0039] optionally, about 30 mM to about 100 mM arginine or arginine hydrochloride;

[0040] The pH of the pharmaceutical composition is from about 4.0 to about 5.5; or,

[0041] (c) about 10 mg / mL to about 120 mg / mL of the immunoconjugate,

[0042] about 5 mM to about 35 mM acetic acid, histidine-histidine hydrochloride, acetate-histidine, or succinate-histidine;

[0043] about 0.04% (w / v) to about 0.2% (w / v) polysorbate 80,

[0044] about 4% (w / v) to about 10% (w / v) sucrose or trehalose,

[0045] The pH of the pharmaceutical composition is from about 4.0 to about 5.0; or,

[0046] (d) about 10 mg / mL to about 120 mg / mL of the immunoconjugate,

[0047] about 5 mM to about 20 mM of histidine acetate or histidine succinate,

[0048] about 0.04% (w / v) to about 0.2% (w / v) polysorbate 80,

[0049] about 4% (w / v) to about 8.8% (w / v) sucrose or trehalose,

[0050] The pH of the pharmaceutical composition is from about 4.0 to about 5.0; or,

[0051] (e) about 10 mg / mL to about 120 mg / mL of the immunoconjugate,

[0052] about 5 mM to about 20 mM succinate-histidine;

[0053] about 0.04% (w / v) to about 0.08% (w / v) polysorbate 80,

[0054] about 1% (w / v) to about 10% (w / v) trehalose, and,

[0055] optionally about 10 mM to about 100 mM arginine hydrochloride;

[0056] The pH of the pharmaceutical composition is from about 4.0 to about 4.5; or,

[0057] (e-1) about 10 mg / mL to about 80 mg / mL of an immunoconjugate,

[0058] about 5 mM to about 10 mM succinate-histidine;

[0059] about 0.04% (w / v) to about 0.08% (w / v) polysorbate 80,

[0060] about 4% (w / v) to about 10% (w / v) trehalose, and,

[0061] optionally about 10 mM to about 100 mM arginine hydrochloride;

[0062] The pH of the pharmaceutical composition is from about 4.0 to about 4.5; or,

[0063] (e-2) about 10 mg / mL to about 40 mg / mL of an immunoconjugate,

[0064] about 10 mM succinate-histidine;

[0065] about 0.04% (w / v) to about 0.08% (w / v) polysorbate 80,

[0066] about 4% (w / v) to about 8.8% (w / v) trehalose,

[0067] The pH of the pharmaceutical composition is from about 4.0 to about 4.5; or,

[0068] (e-3) about 40 mg / mL to about 120 mg / mL of an immunoconjugate,

[0069] about 10 mM to about 20 mM succinate-histidine;

[0070] about 0.04% (w / v) to about 0.08% (w / v) polysorbate 80,

[0071] about 4% (w / v) to about 10% (w / v) trehalose,

[0072] The pH of the pharmaceutical composition is from about 4.0 to about 4.5; or

[0073] (e-4) about 40 mg / mL to about 80 mg / mL of an immunoconjugate,

[0074] about 20 mM succinate-histidine;

[0075] about 0.04% (w / v) to about 0.08% (w / v) polysorbate 80,

[0076] about 4.4% (w / v) to about 8.8% (w / v) trehalose,

[0077] The pH of the pharmaceutical composition is from about 4.0 to about 4.5; or

[0078] (f) about 10 mg / mL to about 120 mg / mL of the immunoconjugate,

[0079] about 5 mM to about 20 mM succinate-histidine;

[0080] about 0.1% (w / v) to about 0.2% (w / v) polysorbate 80,

[0081] about 4% (w / v) to about 8% (w / v) sucrose, and,

[0082] optionally about 10 mM to about 100 mM arginine hydrochloride;

[0083] The pH of the pharmaceutical composition is from about 4.5 to about 5.0; or,

[0084] (f-1) about 10 mg / mL to about 80 mg / mL of an immunoconjugate,

[0085] about 5 mM to about 10 mM succinate-histidine;

[0086] about 0.1% (w / v) to about 0.2% (w / v) polysorbate 80,

[0087] about 4% (w / v) to about 8% (w / v) sucrose, and,

[0088] optionally about 10 mM to about 100 mM arginine hydrochloride;

[0089] The pH of the pharmaceutical composition is from about 4.5 to about 5.0; or,

[0090] (f-2) about 40 mg / mL to about 80 mg / mL of an immunoconjugate,

[0091] approximately 5 mM succinate-histidine;

[0092] about 0.1% (w / v) to about 0.2% (w / v) polysorbate 80,

[0093] about 4% (w / v) to about 8% (w / v) sucrose,

[0094] The pH of the pharmaceutical composition is from about 4.5 to about 5.0; or,

[0095] (f-3) about 40 mg / mL to about 120 mg / mL of an immunoconjugate,

[0096] about 10 mM to about 20 mM succinate-histidine;

[0097] about 0.1% (w / v) to about 0.2% (w / v) polysorbate 80,

[0098] about 4% (w / v) to about 8% (w / v) sucrose,

[0099] The pH of the pharmaceutical composition is from about 4.5 to about 5.0; or,

[0100] (f-4) about 80 mg / mL to about 120 mg / mL of an immunoconjugate,

[0101] about 10 mM succinate-histidine;

[0102] about 0.1% (w / v) to about 0.2% (w / v) polysorbate 80,

[0103] about 4% (w / v) to about 8% (w / v) sucrose,

[0104] The pH of the pharmaceutical composition is from about 4.5 to about 5.0; or,

[0105] (f-5) about 80 mg / mL to about 120 mg / mL of an immunoconjugate,

[0106] About 10 mM succinate-histidine,

[0107] About 0.2% (w / v) polysorbate 80,

[0108] About 8% (w / v) sucrose,

[0109] The pH of the pharmaceutical composition is about 4.8; or,

[0110] (f-6) about 80 mg / mL to about 120 mg / mL of an immunoconjugate,

[0111] About 10 mM succinate-histidine,

[0112] About 0.2% (w / v) polysorbate 80,

[0113] About 8% (w / v) sucrose,

[0114] The pH of the pharmaceutical composition is about 5.0; or,

[0115] (g) about 10 mg / mL to about 120 mg / mL of the immunoconjugate,

[0116] about 5 mM to about 20 mM of histidine acetate or histidine-histidine hydrochloride;

[0117] about 0.04% (w / v) to about 0.08% (w / v) polysorbate 80,

[0118] about 4% (w / v) to about 8% (w / v) sucrose;

[0119] The pH of the pharmaceutical composition is about 4.5 to about 5.0.

[0120] In some embodiments, the pharmaceutical composition of any one of (a), (b), (e), (e-1), (f), and (f-1) comprises about 10 mM to about 100 mM arginine hydrochloride. In some embodiments, the pharmaceutical composition of any one of (a), (b), (e), (e-1), (f), and (f-1) does not comprise about 10 mM to about 100 mM arginine hydrochloride.

[0121] In some embodiments, the pharmaceutical composition comprises any of the following:

[0122] (1) about 10 mg / mL to about 120 mg / mL of the immunoconjugate,

[0123] About 10 mM succinate-histidine,

[0124] About 0.04% (w / v) polysorbate 80,

[0125] About 4.4% (w / v) trehalose,

[0126] The pH of the pharmaceutical composition is from about 4.0 to about 4.5;

[0127] (2) about 10 mg / mL to about 80 mg / mL of the immunoconjugate,

[0128] About 10 mM succinate-histidine,

[0129] About 0.04% (w / v) polysorbate 80,

[0130] About 4.4% (w / v) trehalose,

[0131] The pH of the pharmaceutical composition is from about 4.0 to about 4.5;

[0132] (3) about 10 mg / mL to about 40 mg / mL of the immunoconjugate,

[0133] About 10 mM succinate-histidine,

[0134] About 0.04% (w / v) polysorbate 80,

[0135] About 4.4% (w / v) trehalose,

[0136] The pH of the pharmaceutical composition is about 4.5;

[0137] (7) about 40 mg / mL of immunoconjugate,

[0138] About 10 mM succinate-histidine,

[0139] About 0.04% (w / v) polysorbate 80,

[0140] About 4.4% (w / v) trehalose,

[0141] The pH of the pharmaceutical composition is about 4.5.

[0142] (8) about 40 mg / mL of immunoconjugate,

[0143] About 10 mM succinate-histidine,

[0144] About 0.08% (w / v) polysorbate 80,

[0145] About 8% (w / v) trehalose,

[0146] The pH of the pharmaceutical composition is about 4.5.

[0147] (9) about 40 mg / mL of immunoconjugate,

[0148] About 10 mM succinate-histidine,

[0149] About 0.04% (w / v) polysorbate 80,

[0150] About 8% (w / v) trehalose,

[0151] The pH of the pharmaceutical composition is about 4.0.

[0152] (10) about 40 mg / mL of the immunoconjugate,

[0153] About 10 mM succinate-histidine,

[0154] About 0.08% (w / v) polysorbate 80,

[0155] About 5% (w / v) trehalose,

[0156] The pH of the pharmaceutical composition is about 4.0.

[0157] (11) about 40 mg / mL of immunoconjugate,

[0158] About 10 mM succinate-histidine,

[0159] About 0.08% (w / v) polysorbate 80,

[0160] About 8% (w / v) trehalose,

[0161] The pH of the pharmaceutical composition is about 4.0.

[0162] (12) about 40 mg / mL of immunoconjugate,

[0163] About 10 mM succinate-histidine,

[0164] About 0.08% (w / v) polysorbate 80,

[0165] About 8% (w / v) trehalose,

[0166] About 50 mM arginine hydrochloride

[0167] The pH of the pharmaceutical composition is about 4.0.

[0168] or,

[0169] (4) about 10 mg / mL to about 120 mg / mL of the immunoconjugate,

[0170] About 20 mM succinate-histidine,

[0171] About 0.08% (w / v) polysorbate 80,

[0172] About 8.8% (w / v) trehalose,

[0173] The pH of the pharmaceutical composition is from about 4.0 to about 4.5;

[0174] (5) about 40 mg / mL to about 120 mg / mL of the immunoconjugate,

[0175] About 20 mM succinate-histidine,

[0176] About 0.08% (w / v) polysorbate 80,

[0177] About 8.8% (w / v) trehalose,

[0178] The pH of the pharmaceutical composition is from about 4.0 to about 4.5;

[0179] (6) about 40 mg / mL to about 80 mg / mL of the immunoconjugate,

[0180] About 20 mM succinate-histidine,

[0181] About 0.08% (w / v) polysorbate 80,

[0182] About 8.8% (w / v) trehalose,

[0183] The pH of the pharmaceutical composition is about 4.5.

[0184] (13) about 80 mg / mL of immunoconjugate,

[0185] About 20 mM succinate-histidine,

[0186] About 0.08% (w / v) polysorbate 80,

[0187] About 8.8% (w / v) trehalose,

[0188] The pH of the pharmaceutical composition is about 4.5.

[0189] In some embodiments, the immunoconjugate in the pharmaceutical composition of the present disclosure is a fusion protein of an antigen binding domain and a cytokine. In some embodiments, the immunoconjugate is a fusion protein of a PD-1 binding domain and a cytokine.

[0190] In some embodiments, the PD-1 binding domain comprises at least one immunoglobulin single variable domain, wherein the immunoglobulin single variable domain comprises: CDR1, CDR2, and CDR3 in the amino acid sequence shown in any one of SEQ ID NOs: 2, 8-15.

[0191] The CDR1, CDR2 and CDR3 are defined according to the Kabat, IMGT, Chothia, AbM or Contact numbering systems. The immunoglobulin single variable structure specifically binds to the PD-1 antigen or a fragment thereof.

[0192] PD-1 binding domain

[0193] In some embodiments, the aforementioned PD-1 binding domain, according to the Kabat numbering system, the amino acid sequences of CDR1, CDR2 and CDR3 of the immunoglobulin single variable domain are shown as SEQ ID NO: 3, 16 and 17, respectively.

[0194] In some embodiments, the amino acid sequence of CDR1 of the immunoglobulin single variable domain in the aforementioned PD-1 binding domain is shown in SEQ ID NO: 3, the amino acid sequence of CDR2 is shown in SEQ ID NO: 4 or 18, and the amino acid sequence of CDR3 is shown in any one of SEQ ID NOs: 5, 19-20.

[0195] In some embodiments, the amino acid sequences of CDR1, CDR2, and CDR3 of the immunoglobulin single variable domain in the aforementioned PD-1 binding domain are:

[0196] As shown in SEQ ID NO: 3, 18, 19,

[0197] As shown in SEQ ID NO: 3, 4, 5, or

[0198] As shown in SEQ ID NO: 3, 4, 20.

[0199] In some embodiments, the immunoglobulin single variable domain of the aforementioned PD-1 binding domain is humanized, affinity matured, T cell epitope removed, antibody deamidation reduced, and / or antibody isomerization reduced. In some embodiments, the immunoglobulin single variable domain is obtained through affinity maturation and has one or more changes in one or more CDRs, which result in increased affinity for PD-1 compared to the parent.

[0200] In some embodiments, the humanization process uses the heavy chain framework region of the human germline template IGHV3-23*01 or IGHV3-23*04.

[0201] In some embodiments, the amino acid sequence of the immunoglobulin single variable domain in the aforementioned PD-1 binding domain is as shown in any one of SEQ ID NOs: 2, 8-15, or has at least 80%, at least 85%, or at least 90% sequence identity with any of the aforementioned sequences.

[0202] In some embodiments, the amino acid sequence of the immunoglobulin single variable domain in the aforementioned PD-1 binding domain is as shown in SEQ ID NO: 13, or has at least 80%, at least 85%, or at least 90% sequence identity with any of the aforementioned sequences.

[0203] In some embodiments, at least one immunoglobulin single variable domain in the aforementioned PD-1 binding domain is a VHH.

[0204] In some embodiments, the aforementioned PD-1 binding domain comprises one or more (e.g., 2, 3, 4, 5, 6) aforementioned immunoglobulin single variable domains, which may be the same or different and may form a dimer or multimer molecule.

[0205] In some embodiments, the aforementioned PD-1 binding domain comprises or is an anti-PD-1 antibody or antigen-binding fragment thereof that specifically binds to PD-1 or a fragment thereof; such antibodies or antigen-binding fragments are, for example, camelid antibodies, chimeric antibodies, humanized antibodies, fully human antibodies, or antigen-binding fragments thereof; such antibodies or antigen-binding fragments are, for example, recombinant antibodies or fragments thereof. In some specific embodiments, the antigen-binding fragment is a linear antibody, a single-chain antibody, a nanobody, a peptibody, a domain antibody, and a multispecific antibody (bispecific antibody, diabody, triabody, tetrabody, tandem di-scFv, tandem tri-scFv).

[0206] In some embodiments, the aforementioned anti-PD-1 antibody or antigen-binding fragment thereof further comprises a human immunoglobulin Fc region; for example, the Fc region is an Fc region of human IgG1, IgG2, or IgG4. The Fc region may have mutations, exemplary mutations being L234A / L235A on IgG1, V234A / G237A / P238S / H268A / V309L / A330S / P331S on IgG2, F234A / L235A on IgG4, S228P / F234A / L235A on IgG4, N297A on IgG1, IgG2, IgG3, or IgG4, V234A / G237A on IgG2, K214T / E233P / L234V / L235A / G236 deletion on IgG1, A327G / P331A / D365E / L358M, H268Q / V309L / A330S / P331S on IgG2, L234F / L235E / D265A on IgG1, L234A / L235A / G237A / P238S / H268A / A330S / P331S on IgG1, S228P / F234A / L235A / G237A / P238S on IgG4, and S228P / F234A / L235A / G236 deletion / G237A / P238S on IgG4. Hybrid IgG2 / 4 Fc domains, such as an Fc having residues 117-260 from IgG2 and residues 261-447 from IgG4, can also be used. In some specific embodiments, the Fc region of the human IgG4 has S228P, F234A, L235A and / or K447A mutations. In some specific embodiments, the Fc region of the human IgG1 has L234A / L235A or L234A / L235A / P329G mutations.

[0207] In some embodiments, the Fc region contained in the aforementioned PD-1 antibody or antigen-binding fragment thereof can enable the binding protein to form a dimeric molecule and prolong the in vivo half-life of the binding protein.

[0208] In some embodiments, the immunoglobulin single variable domain in the aforementioned PD-1 antibody or antigen-binding fragment thereof is connected to the Fc region via a linker. The linker can be a non-functional amino acid sequence of 1-20 or more amino acids in length and without secondary or higher structure. For example, the linker is a flexible linker, such as G4S, GS, GAP, (G4S)2, (G4S)3, (G4S)4, (G4S)5, ASGS, etc.

[0209] In some embodiments, the aforementioned PD-1 antibody or antigen-binding fragment thereof has at least one activity selected from the following:

[0210] (a)≤10 -7 K D The value binds to human PD-1 or its epitope;

[0211] (b) inhibiting the binding of PD-1 to PD-L1;

[0212] (c) inhibiting the binding of PD-1 to PD-L2;

[0213] (d) Induction of CD4 + T cells secrete IFN-γ;

[0214] (e) enhance PBMC activation;

[0215] (f) enhancing T cell activation;

[0216] (g) Inhibit tumor growth.

[0217] In some embodiments, the aforementioned PD-1 binding domain of the present disclosure binds to the K D The value can be ≤1×10 -7 M, for example, ≤1×10 -8 M, or ≤1×10 -9 M, or ≤1×10 -10 M.

[0218] In some embodiments, the PD-1 binding domain of the present disclosure can specifically bind to human PD-1 and block the interaction between PD-1 and PD-L1, and / or PD-1 and PD-L2.

[0219] In some embodiments, the aforementioned PD-1 binding domains of the present disclosure are capable of inhibiting tumor growth by at least about 10%, such as at least about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, or about 80%.

[0220] In some embodiments, the aforementioned PD-1 binding domains disclosed herein encompass variants, for example, comprising one or more amino acid substitutions, preferably conservative amino acid substitutions, compared to any one of SEQ ID NOs: 2, 8-15, for example, comprising 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 conservative amino acid substitutions, which may occur in the CDR region and / or the FR region.

[0221] In some embodiments, the aforementioned PD-1 binding domain of the present disclosure is resistant to heat treatment or has high stability. For example, it is treated at 40°C for up to 30 days without significant aggregation or degradation, and is stable at at least 60°C.

[0222] In some embodiments, a PD-1 antibody or an antigen-binding fragment thereof is provided, which binds to or competes for binding to the same epitope as the immunoglobulin single variable domain of the aforementioned PD-1 binding domain of the present disclosure.

[0223] In some embodiments, a PD-1 antibody or an antigen-binding fragment thereof is provided, which blocks the binding of the immunoglobulin single variable domain of the aforementioned PD-1 binding domain disclosed herein to PD-1 (e.g., human PD-1).

[0224] In some embodiments, a PD-1 antibody or an antigen-binding fragment thereof is provided, wherein the binding of the antibody to PD-1 (e.g., human PD-1) is blocked by the immunoglobulin single variable domain in the aforementioned PD-1 antibody or antigen-binding fragment thereof of the present disclosure.

[0225] In some embodiments, the aforementioned PD-1 binding domain of the present disclosure reduces the binding of PD-1 to PD-L1 and / or PD-L2 by at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 98%.

[0226] In some embodiments, the present disclosure provides a PD-1 binding protein comprising at least one of the aforementioned immunoglobulin single variable domains (such as VHH) that binds to PD-1.

[0227] As previously stated, and in this disclosure, "at least 90% identity" includes 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% identity.

[0228] IL-2 or its variants

[0229] In some embodiments, the cytokine is IL-2 or a variant thereof. The IL-2 is preferably human IL-2, and the amino acid sequence of wild-type human IL-2 is exemplified in SEQ ID NO: 50. The positions of the IL-2 variants disclosed herein are numbered according to the wild-type IL-2 amino acid sequence set forth in SEQ ID NO: 50.

[0230] In some embodiments, the present disclosure provides IL-2 variants having reduced affinity for high-affinity receptors (IL-2Rα / β / γ) and medium-affinity receptors (IL-2Rβ / γ) compared to wild-type IL-2, and / or having an affinity for low-affinity receptors (IL-2Rα) eliminated compared to wild-type IL-2. In some specific embodiments, the affinity of the IL-2 variant for IL-2Rα / β / γ is more reduced than the affinity for IL-2Rβ / γ. The affinity can be detected by conventional methods, such as ELISA, ForteBio, SPR, such as the SPR detection method provided in Example 13 of the present disclosure.

[0231] In some embodiments, the present disclosure provides an IL-2 variant comprising or consisting of mutation (i), mutation (ii), mutation (iii), mutation (iv) or any combination thereof.

[0232] Regarding mutation (i):

[0233] The present disclosure provides mutations that confer upon IL-2 variants an altered (eg, increased or decreased) affinity for IL-2Rβ / γ compared to wild-type IL-2.

[0234] In some embodiments, the IL-2 variant has a mutation (i) that reduces affinity for IL-2Rβ / γ compared to wild-type IL-2. In some embodiments, the IL-2 variant has a 1-1000-fold, 2-800-fold, 5-500-fold, 5-200-fold, 10-200-fold, 10-100-fold, 10-50-fold, 20-200-fold, 20-100-fold, 5-20-fold, 2-20-fold, 50-100-fold, 50-70-fold, e.g., about 5, about 10, about 13, about 15, about 20, about 30, about 40, about 50, about 60, about 63, about 70, about 80, about 90, about 100, about 110, about 120, about 130, about 140, about 150, about 180, about 200, about 250, about 300, about 400, about 500-fold, compared to wild-type IL-2. In some specific embodiments, the affinity of the IL-2 variant to IL-2Rβ / γ is reduced by 10-100 times compared to wild-type IL-2. The affinity can be detected by conventional methods, such as the SPR detection method provided in Example 13 of the present disclosure.

[0235] In some embodiments, the aforementioned mutation (i) is located at positions 12-20, positions 84-95, and positions 126-130 of IL-2.

[0236] In some embodiments, the aforementioned mutation (i) is selected from positions 12, 15, 16, 18, 19, 20, 23, 43, 69, 84, 87, 88, 91, 92, 95, 123, 126, 127, 129, 130 or any combination thereof; In some embodiments, the mutation is selected from positions L12, E15, H16, L18, L19, D20, M23, Q22, K43, V69, D84, S87, N88, V91, I92, E95, T123, Q126, S127, I129 and S130 or any combination thereof, such as N88 / D20; In some specific embodiments, the Mutations selected from the group consisting of L12R, L12K, L12E, L12Q, E15Q, E15R, E15A, E15S, H16N, H16A, H16E, H16D, H16G, H16S, H16T, H16V, H16P, D20A, D20H, D20Y, D20N, D20Q, D20E, D20R, D20S, M23A, M23R, M23K, M23G, M23S, M23T, M23V, M23P, S87K, S87A, D84S, D84L, D84N, D84V, D84H, D84Y, D84R, D84K, D84G , D84A, D84T, D84P, N88D, N88A, N88S, N88E, N88T, N88R, N88I, V91A, V91T, V91E, V91I, V91L, V91D, V91N, V91Q, V91S, V91H, I92E, I92T, I92K, I92R, I92L, E95S, E95A, E95R, E95Q, E95G, E95T, E95V, E95P, E95H, E95N, T123A, T123E, T123K, T123Q, Q126D, Q126L, Q126A, Q126S , Q126T, Q126E, S127A, S127E, S127K, S127Q, D84N / E95Q, D84E / E95Q, D84T / E95Q, D84Q / E95Q, D84T / H16T, D84N / V91I, D84T / Q126E, D84N / Q126E, H16T / D84Q, H16T / V91I; in some embodiments, the mutation (i) is selected from N88D, N88R, D20A, D20N, V91T, Q126D or H16A / D84S; in some embodiments, the mutation (i) is N88D or D20A.

[0237] Regarding mutation (ii):

[0238] The present disclosure provides mutations that result in IL-2 variants having altered (eg, decreased) affinity for IL-2Rα compared to wild-type IL-2, or the inability to bind to IL-2Rα.

[0239] In some embodiments, the IL-2 variant has a reduced affinity for IL-2Rα compared to wild-type IL-2, or a mutation (ii) that does not bind to IL-2Rα. The affinity can be detected by conventional methods, such as the SPR detection method provided in Example 13 of the present disclosure.

[0240] In some specific embodiments, the aforementioned mutation (ii) is selected from positions 35, 37, 38, 41, 42, 43, 45, 61, 62, 65, 68, 72 or any combination thereof; in some embodiments, the mutation (ii) is selected from positions K35, T37, R38, E62, P65, E68, F42, Y45, L72 or any combination thereof; in some embodiments, the mutation is selected from R38A, R38D, R38E, E62Q, F42A, F42G, F42S, F42T, F42Q, F42E, F42N, F42D, F42R, F42K, Y45A, Y45G, Y45S , Y45T, Y45Q, Y45E, Y45N, Y45D, Y45R, Y45K, P65R, P65E, P65K, P65H, P65Y, P65Q, P65D, P65N, E68A, E68Q, E68K, E68R, L72G, L72A, L72S, L72T, L72Q, L72E, L72N, L72D, L72R, L72K; In some embodiments, the mutation (ii) is selected from F42A, Y45A, L72G, F42A / L72G, F42A / Y45A, Y45A / L72G or F42A / Y45A / L72G.

[0241] Regarding mutation (iii):

[0242] The present disclosure provides mutations (iii) that improve the stability, expression level, purity, and drugability of IL-2 variants.

[0243] The present disclosure incorporates the IL-2 variants in WO2020125743A in its entirety.

[0244] In some embodiments, the IL-2 variant has a mutation (iii) selected from positions 11, 26, 27, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 70, 71, 72, 78, 82, 88, 132, or any combination thereof; in some embodiments, the mutation (iii) is selected from positions N26, N29, N30, N71, Q11, L132, L70, P82, G27, F78, or any combination thereof; in some embodiments, The mutation (iii) is selected from N26Q, N29S, N30S, N71Q, Q11C / L132C, L70C / P82C, G27C / F78C or any combination thereof; in some embodiments, the mutation (iii) is selected from N26Q, N29S, N30S, N26Q / N29S / N71Q, N26Q / N29S, N26Q / N30S, N26Q / N30S / Q11C / L132C, N26Q / N29S / L70C / P82C, N26Q / N30S / G27C / F78C.

[0245] Regarding mutation (iv):

[0246] In some embodiments, the IL-2 variant contains a mutation (iv), wherein the mutation (iv) is selected from position 3 and / or 125; in some embodiments, the mutation (iv) is selected from position T3 and / or C125; in some embodiments, the mutation (iv) is selected from T3A, T3G, T3Q, T3E, T3N, T3D, T3R, T3K, T3P and / or C125A, C125S, C125T, C125V mutations.

[0247] I. Exemplary IL-2 variants

[0248] The present disclosure provides IL-2 comprising or only comprising the following mutations:

[0249] (a) F42A and N88D, N88A, N88S, N88E, N88T, N88R, or N88I;

[0250] (b) F42A and D20A, D20H, D20Y, D20N, D20Q, D20E, D20R or D20S;

[0251] (c) F42A and Q126D, Q126L, Q126A, Q126S, Q126T, or Q126E;

[0252] (d) F42A and V91A, V91T, V91E, V91I, V91L, V91D, V91N, V91Q, V91S, or V91H;

[0253] (e) F42A / L72G and N88D, N88A, N88S, N88E, N88T, N88R, or N88I;

[0254] (f) F42A / L72G and D20A, D20H, D20Y, D20N, D20Q, D20E, D20R or D20S;

[0255] (g) F42A / L72G and Q126D, Q126L, Q126A, Q126S, Q126T, or Q126E;

[0256] (h) F42A / L72G and V91A, V91T, V91E, V91I, V91L, V91D, V91N, V91Q, V91S or V91H;

[0257] (i) any one of the above (a) to (h) and N26Q;

[0258] (j) any one of the above (a) to (h) and N29S;

[0259] (k) any one of the above (a) to (h) and N71Q;

[0260] (l) any one of the above (a) to (h) and N26Q / N29S;

[0261] (m) any one of the above (a) to (h) and N26Q / N29S / N71Q;

[0262] (n) any one of the above (a)-(h) and Q11C / L132C;

[0263] (o) any one of the above (a) to (h) and L70C / P82C;

[0264] (p) is any one of the above (a)-(h) and G27C / F78C;

[0265] (q) any one of the above (a)-(h) and T3A;

[0266] (r) is any one of the above (a) to (h) and T3A / C125A.

[0267] II. Exemplary IL-2 variants

[0268] The present disclosure provides IL-2 comprising or only comprising the following mutations:

[0269] 42A / 72G / 88D,

[0270] 42A / 72G / 88R,

[0271] 42A / 72G / 20A,

[0272] 42A / 72G / 20N,

[0273] 42A / 72G / 91T,

[0274] 42A / 72G / 126D,

[0275] 42A / 72G / 16A / 84S,

[0276] 42A / 72G / 26Q / 29S / 71Q,

[0277] 38E / 42A / 91T,

[0278] 42A / 72G / 88D / 26Q / 29S / 71Q,

[0279] 42A / 72G / 88R / 26Q / 29S / 71Q,

[0280] 42A / 72G / 20A / 26Q / 29S / 71Q,

[0281] 42A / 72G / 20N / 26Q / 29S / 71Q,

[0282] 42A / 72G / 91T / 26Q / 29S / 71Q,

[0283] 42A / 72G / 126D / 26Q / 29S / 71Q, or

[0284] 42A / 72G / 16A / 84S / 26Q / 29S / 71Q.

[0285] III. Exemplary IL-2 variants

[0286] The present disclosure provides IL-2 comprising or only comprising the following mutations:

[0287] T3A / F42A / L72G / N88D / C125A,

[0288] T3A / F42A / L72G / N88R / C125A,

[0289] T3A / F42A / L72G / D20A / C125A,

[0290] T3A / F42A / L72G / D20N / C125A,

[0291] T3A / F42A / L72G / V91T / C125A,

[0292] T3A / F42A / L72G / Q126D / C125A,

[0293] T3A / F42A / L72G / H16A / D84S / C125A,

[0294] T3A / F42A / L72G / N26Q / N29S / N71Q / C125A,

[0295] T3A / R38E / F42A / V91T / C125A,

[0296] T3A / F42A / L72G / N88D / N26Q / N29S / N71Q / C125A,

[0297] T3A / F42A / L72G / N88R / N26Q / N29S / N71Q / C125A,

[0298] T3A / F42A / L72G / D20A / N26Q / N29S / N71Q / C125A,

[0299] T3A / F42A / L72G / D20N / N26Q / N29S / N71Q / C125A,

[0300] T3A / F42A / L72G / V91T / N26Q / N29S / N71Q / C125A,

[0301] T3A / F42A / L72G / Q126D / N26Q / N29S / N71Q / C125A, or

[0302] T3A / F42A / L72G / H16A / D84S / N26Q / N29S / N71Q / C125A.

[0303] In some embodiments, the present disclosure provides an IL-2 variant comprising or having an amino acid sequence as set forth in any one of SEQ ID NOs: 23-31, or having at least 80%, at least 85%, or at least 90% sequence identity thereto.

[0304] In some embodiments, any of the aforementioned IL-2 variants containing mutation (i) has any one or more of 1) to 5) relative to wild-type IL-2:

[0305] 1) have reduced affinity for the high-affinity IL-2R receptor (IL-2Rαβγ);

[0306] 2) have reduced affinity for the intermediate-affinity IL-2R receptor (IL-2Rβγ);

[0307] 3) have reduced T cells (such as CD4 + and CD8 + T cell) activation;

[0308] 4) has reduced activation of NK cells;

[0309] 5) having reduced release of inflammatory factors from T cells and / or NK cells stimulated by IL-2.

[0310] In some embodiments, any of the aforementioned IL-2 variants containing mutation (i) and mutation (ii) has any one or more of the aforementioned 1)-5) and the following 6)-8) relative to wild-type IL-2:

[0311] 6) have reduced affinity for IL-2Rα, or do not bind to IL-2Rα;

[0312] 7) The affinity for IL-2Rαβγ is reduced more than that for IL-2Rβγ;

[0313] 8) Has reduced CD25 + cells (e.g. CD25 + CD8 + Activation of T cells, Treg cells).

[0314] In some embodiments, any of the aforementioned IL-2 variants containing mutation (i), mutation (ii), mutation (iii), and / or mutation (iv) has any one or more of the aforementioned 1)-8) and the following 9) relative to wild-type IL-2:

[0315] 9) Improved stability, drugability, expression level, purity and / or prolonged in vivo half-life.

[0316] Immunoconjugates

[0317] The present disclosure provides immunoconjugates comprising an antigen binding domain and IL-2 or a variant thereof.

[0318] In some embodiments, the antigen binding domain in the immunoconjugate targets immune effector cells or tumor cells; in some specific embodiments, the immune effector cells are capable of responding to stimulation or activation by IL-2; in some specific embodiments, the immune effector cells have IL-2Rβγ; in some specific embodiments, the immune effector cells are, for example, NK cells, CD4+CD25-T cells, or CD8+CD25-T cells.

[0319] In some embodiments, the antigen binding domain in the immunoconjugate targets PD-1, CTLA-4, LAG3, TIM3, 4-1BB, OX40, GITR, CD8a, CD8b, CD4, NKp30, NKG2A, TIGIT, TGFβR1, TGFβR2, Fas, NKG2D, NKp46, PD-L1, CD107a, ICOS, TNFR2, FAP, TNC A1, TNC A2, CEA, EDB, MCSP or CD16a; in some specific embodiments, the antigen binding domain targets PD-1 (i.e., a PD-1 binding domain).

[0320] In some embodiments, the PD-1 binding domain is any PD-1 binding domain provided herein.

[0321] Exemplarily, in some specific embodiments, the PD-1 binding domain comprises at least one immunoglobulin single variable domain, wherein the immunoglobulin single variable domain comprises: CDR1, CDR2, and CDR3 in the amino acid sequence shown in any one of SEQ ID NOs: 2, 8-15, wherein the CDR1, CDR2, and CDR3 are defined according to the Kabat, IMGT, Chothia, AbM, or Contact numbering systems.

[0322] Exemplarily, in some specific embodiments, the PD-1 binding domain, according to the Kabat numbering system, the amino acid sequences of CDR1, CDR2 and CDR3 of the immunoglobulin single variable domain are shown in SEQ ID NOs: 3, 16 and 17, respectively.

[0323] Exemplarily, in some specific embodiments, the amino acid sequence of CDR1 of the immunoglobulin single variable domain in the PD-1 binding domain is shown in SEQ ID NO: 3, the amino acid sequence of CDR2 is shown in SEQ ID NO: 4 or 18, and the amino acid sequence of CDR3 is shown in any one of SEQ ID NOs: 5, 19-20.

[0324] Exemplarily, in some embodiments, the immunoglobulin single variable domain comprises CDR1, CDR2 and CDR3 as shown in SEQ ID NOs: 3, 4 and 5, or as shown in SEQ ID NOs: 3, 18 and 19, or as shown in SEQ ID NOs: 3, 4 and 20.

[0325] Illustratively, in some specific embodiments, the amino acid sequence of the immunoglobulin single variable domain is shown in any one of SEQ ID NOs: 2, 8-15, respectively; or has at least 80% sequence identity with any of the sequences.

[0326] In some embodiments, the PD-1 binding domain is selected from the PD-1 binding domains of the following PD-1 antibodies: Pembrolizumab, Nivolumab, Sintilimab, Tislelizumab, Cemiplimab, Toripalimab, Camrelizumab, Penpulizumab, Sepalimab, Sasanlimab, limab), spartalizumab, retifanlimab, balstilimab, cetrelimab, dostarlimab (TSR-042), CS-1003, SCT-I10A, SSI-361, MEDI-0680 (AMP-514), BAT-1306, JTX-4014, JTX-4014, BI-754091, AK-103. In other embodiments, the antigen-binding domain in the immunoconjugate is selected from the antigen-binding domain of the following antibodies: PD-L1 antibody (e.g., Durvalumab, Atezolizumab, Durvalumab, Avelumab, Adebrelimab, Envafolimab, STI-A1110, CS-1001, TQB-2450, KL-A167, IMC-001, CX-072); CTLA-4 antibody (e.g., Tremelimumab, Ipilimumab); 4-1BB antibody (e.g., Urelumab, Utomilumab). The antigen-binding domain (e.g., PD-1 binding domain) is the portion of the antibody (e.g., PD-1 antibody) that binds to the antigen (e.g., PD-1), for example, its Fab', F(ab')2, or its heavy chain variable region and light chain variable region, or a domain containing HCDR1, HCDR2, HCDR3 of its heavy chain variable region and LCDR1, LCDR2, LCDR3 of its light chain variable region.

[0327] In some embodiments, the PD-1 binding domain comprises pembrolizumab or an antigen-binding fragment thereof (e.g., Fab), for example, comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 of the amino acid sequences set forth in SEQ ID NOs: 54, 55, and 56, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3 of the amino acid sequences set forth in SEQ ID NOs: 57, 58, and 59. In some specific embodiments, the heavy chain variable region is the heavy chain variable region of the Fab heavy chain of the amino acid sequence set forth in SEQ ID NO: 52, and the light chain variable region comprises the light chain variable region of the Fab light chain of the amino acid sequence set forth in SEQ ID NO: 53.

[0328] In some embodiments, the IL-2 or variant thereof in the immunoconjugate is any IL-2 or variant thereof provided herein.

[0329] Exemplarily, in some specific embodiments, the IL-2 is wild-type human IL-2.

[0330] Exemplarily, in some specific embodiments, the IL-2 variant comprises mutation (i), mutation (ii), mutation (iii), mutation (iv) or any combination thereof provided herein, or consists of mutation (i), mutation (ii), mutation (iii), mutation (iv) or any combination thereof.

[0331] Exemplarily, in some specific embodiments, the IL-2 variant comprises or only comprises the mutation of any one of (a)-(r) provided above in the present disclosure.

[0332] Exemplarily, in some embodiments, the IL-2 variant contains an amino acid sequence as shown in any one of SEQ ID NOs: 23-31, or a sequence having at least 80%, at least 85%, or at least 90% identity thereto, or consists of the above sequence.

[0333] In some embodiments, the immunoconjugate further comprises a human immunoglobulin Fc region, such as the Fc region of human IgG1, IgG2, IgG3, or IgG4.

[0334] In some embodiments, the antigen binding domain (e.g., PD-1 binding domain) in the immunoconjugate is located at the N-terminus of the Fc region, and the IL-2 or variant thereof is located at the N-terminus or C-terminus of the Fc region. In some specific embodiments, the antigen binding domain is located at the N-terminus of the Fc region, and the IL-2 or variant thereof is located at the C-terminus of the Fc region.

[0335] In some embodiments, the valency ratio of the antigen binding domain (e.g., PD-1 binding domain) to IL-2 or its variant in the immunoconjugate is between 6:1 and 1:3 (e.g., 4:1 to 1:2), specifically 4:1, 2:1 or 1:1.

[0336] In some embodiments, the immunoconjugate contains 1 or 2 IL-2 or variants thereof and 1, 2, 3, 4, 5 or 6 antigen-binding domains (e.g., PD-1 binding domains).

[0337] In some embodiments, the immunoconjugate further comprises an Fc region comprising a first subunit Fc1 and a second subunit Fc2 capable of associating with each other.

[0338] In some embodiments, Fc1 and Fc2 contain amino acid mutations that result in Fc1 preferentially pairing with Fc2 or forming heterodimers with Fc2 compared to Fc1. In some embodiments, the mutations are located in the CH3 of Fc1 and Fc2. In some embodiments, the amino acid mutations in Fc1 and Fc2 result in greater electrostatic complementarity than a wild-type mutant lacking the mutations. In some embodiments, the amino acid mutations in Fc1 and Fc2 result in greater steric complementarity than a wild-type mutant lacking the mutations.

[0339] In some embodiments, in Fc1 and Fc2, for example, within the CH3 / CH3 interface, one or more amino acid residues in the CH3 domain of Fc1 are mutated with one or more amino acid residues having a larger side chain volume, thereby generating a protrusion (or knob) on the surface of the CH3 domain of Fc1, and one or more, preferably two or three, amino acid residues in the CH3 domain of Fc2 that interact with the CH3 domain of Fc1 are mutated with amino acid residues having a smaller side chain volume, thereby generating a depression (or hole) on the surface of the CH3 domain of Fc2 that interacts with the CH3 domain of Fc1. In some embodiments, the import residue with a larger side chain volume is phenylalanine (F), tyrosine (Y), arginine (R), or tryptophan (W). In some embodiments, the import residue with a smaller side chain volume is serine (S), alanine (A), valine (V), or threonine (T).

[0340] In some specific embodiments, the Fc1 comprises at least one or at least two amino acid mutations selected from T366S, L368A and Y407V (hole mutation modification), and the Fc2 comprises T366W (knob mutation modification); or the Fc1 comprises T366W (knob mutation modification), and the Fc2 comprises at least one or at least two amino acid mutations selected from T366S, L368A and Y407V (hole mutation modification).

[0341] In some specific embodiments, the IL-2 or variant thereof is located in the polypeptide chain where Fc1 is located.

[0342] In some embodiments, Fc1 and Fc2, for example, CH3, may contain a natural non-cysteine ​​to cysteine ​​mutation, such as S354C in Fc1 and Y349C in Fc2; or Y349C in Fc1 and S354C in Fc2.

[0343] In some embodiments, Fc1 and Fc2, for example, within the Fc1 CH3 / Fc2 CH3 interface, comprise the following amino acid mutations or a combination thereof:

[0344] T366Y / Y407T;

[0345] T366W / Y407A;

[0346] T366Y / Y407T;

[0347] T394W / F405A;

[0348] T366Y / F405A / T394W / Y407T;

[0349] T366W / F405W / T394S / Y407A;

[0350] F405W / T394S;

[0351] D399C / K392C;

[0352] T366W / T366S / L368A / Y407V;

[0353] T366W / D399C / T366S / L368A / K392C / Y407V;

[0354] T366W / K392C / T366S / D399C / L368A / Y407V;

[0355] S354C / T366W / Y349C / T366S / L368A / Y407V;

[0356] Y349C / T366W / S354C / T366S / L368A / Y407V;

[0357] E356C / T366W / Y349C / T366S / L368A / Y407V;

[0358] Y349C / T366W / E356C / T366S / L368A / Y407V;

[0359] E357C / T366W / Y349C / T366S / L368A / Y407V; and

[0360] Y349C / T366W / E357C / T366S / L368A / Y407V.

[0361] In some specific embodiments, Fc1 and Fc2 further comprise amino acid mutations that form an electrostatic interaction interface between Fc1 and Fc2 (e.g., CH3 and CH3). The amino acid mutations that form the electrostatic interaction interface are, for example, selected from the following:

[0362] K370E / D399K / K439D / D356K / E357K / K409D;

[0363] K409D / D399K;

[0364] K409E / D399K;

[0365] K409E / D399R;

[0366] K409D / D399R;

[0367] D339K / E356K;

[0368] D399K / E356K / K409D / K392D;

[0369] D399K / E356K / K409D / K439D;

[0370] D399K / E357K / K409D / K370D;

[0371] D399K / E356K / E357K / K409D / K392D / K370D;

[0372] D399K / E357K / K409D / K392D;

[0373] K392D / K409D / D399K; and

[0374] K409D / K360D / D399K.

[0375] In some embodiments, Fc1 and / or Fc2 comprise domains from different antibody subtypes, such as CH3 from different antibody subtypes.

[0376] In addition, the present disclosure cites WO96 / 27011, WO98 / 050431, EP1870459, WO2007 / 110205, WO2007 / 147901, WO2009 / 089004, WO2010 / 129304, WO2011 / 90754, WO2011 / 143545, WO2012058768, WO2013157954, and WO2013096291 for modifying the CH3 region of the Fc region to enhance heterodimerization.

[0377] In some embodiments, Fc1 and / or Fc2, such as CH3, contain amino acid mutations for altering effector functions. The antibody effector functions include, but are not limited to, C1q binding and complement-dependent cytotoxicity, Fc receptor (e.g., FcγR) binding, antibody-dependent cellular cytotoxicity (ADCC), phagocytosis, downregulation of cell surface receptors (e.g., B cell receptors), and B cell activation. Amino acid mutations that alter effector functions are, for example, selected from the following:

[0378] S298A / E333A / K334A;

[0379] S239D / I332E / A330L;

[0380] S239D / I332E / G236A;

[0381] G236A / S239D / A330L / I332E;

[0382] F243L / R292P / Y300L / V305I / P396L;

[0383] K326A / E333A;

[0384] K326W / E333S;

[0385] K326M / E333S;

[0386] C221D / D222C;

[0387] S267E / H268F / S324T;

[0388] E345R;

[0389] S298A / E333A / K334A / N434A;

[0390] E294 deletion / T307P / N434Y;

[0391] T256N / A378V / S383N / N434Y;

[0392] T252L / T253S / T254F;

[0393] M252Y / S254T / T256E;

[0394] M428L / N434S;

[0395] L234A / L235A;

[0396] S228P / L235E;

[0397] L234A / L235A / P331S;

[0398] L234A / L235A / P329G;

[0399] D265A / E233P;

[0400] H268Q / V309L / A330S / P331S;

[0401] V234A / G237A / P238S / H268A / V309L / A300S / P331S;

[0402] L234A / L235A / G237A / P238S / H268A / V309L / A300S / P331S;

[0403] S228P / F234A / L235A;

[0404] D270A / P329A;

[0405] L234F / L235E;

[0406] L234F / L235E / P331S;

[0407] F241A / V264A / D265A;

[0408] N297G / D265A; and

[0409] L234Y / G236W / S298A.

[0410] In some embodiments, the Fc1 and / or the Fc2 comprise mutations that reduce effector function (e.g., ADCC), such as L234A / L235A, L234A / L235A / P329G.

[0411] In some embodiments, Fc1 and Fc2, for example, in their CH3, contain amino acid mutations for changing half-life (e.g., extending half-life). In some embodiments, the half-life depends on FcRn binding affinity. Prolonging half-life can allow for reducing the amount of drug administered to the patient and / or reducing the frequency of administration. In some specific embodiments, the Fc region has M252Y, S254T, and / or T256E mutations. In some embodiments, the immunoconjugate has PEGylation modification or is conjugated / fused to (human serum) albumin or albumin-binding protein.

[0412] In some embodiments, Fc1 and / or Fc2, such as CH3, comprise one or more allotypic mutations. In some embodiments, the allotypic mutations are D356E and L358M.

[0413] In some embodiments, the immunoconjugate comprises a combination of polypeptide chains having the following amino acid sequences: SEQ ID NOs: 21 and 22; SEQ ID NOs: 36 and 37; SEQ ID NOs: 38 and 39; SEQ ID NOs: 40 and 41; SEQ ID NOs: 42 and 43; or an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any of the above polypeptide chain combinations.

[0414] In some specific embodiments, the immunoconjugate comprises a polypeptide chain combination having the following amino acid sequences: SEQ ID NO: 42 and 43, or an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any of the above polypeptide chain combinations.

[0415] Exemplary Immunoconjugates

[0416] Regarding immunoconjugates, the present disclosure provides the following embodiments:

[0417] Embodiment 1. An immunoconjugate comprising:

[0418] 1) a PD-1 binding domain, and an IL-2 variant comprising a mutation selected from N88D or D20A; or

[0419] 2) a PD-1 binding domain, and an IL-2 variant having reduced or eliminated affinity for high-affinity receptors (IL-2Rα / β / γ) compared to wild-type IL-2, and reduced affinity for medium-affinity receptors (IL-2Rβ / γ) compared to wild-type IL-2, with affinity for high-affinity receptors being more reduced than affinity for medium-affinity receptors; for example, the IL-2 variant contains a mutation selected from N88D or D20A.

[0420] Embodiment 2. The immunoconjugate of embodiment 1, comprising a mutation selected from F42, Y45, L72, or any combination thereof;

[0421] For example, it contains a mutation selected from F42A, Y45A, L72G or any combination thereof;

[0422] More specifically, it contains mutations of F42A / L72G, F42A / Y45A, Y45A / L72G, and F42A / Y45A / L72G.

[0423] Embodiment 3. The immunoconjugate of embodiment 1 or 2, comprising a mutation selected from the group consisting of N26Q, N29S, N30S, N71Q, Q11C and L132C, L70C and P82C, G27C and F78C, or any combination thereof;

[0424] For example, it contains a mutation or combination selected from the group consisting of:

[0425] N26Q, N29S, N30S, N26Q / N29S / N71Q, N26Q / N29S, N26Q / N30S, N26Q / N30S / Q11C / L132C, N26Q / N29S / L70C / P82C, N26Q / N30S / G27C / F78C.

[0426] Embodiment 4. An immunoconjugate comprising:

[0427] An antigen binding domain, and IL-2 or a variant thereof, wherein the IL-2 variant contains a first mutation, wherein the first mutation causes the IL-2 variant to have reduced affinity for the intermediate affinity receptor (IL-2Rβ / γ) compared to wild-type IL-2;

[0428] For example, the first type of mutation is selected from the group consisting of: L12, E15, H16, L18, L19, D20, M23, Q22, K43, V69, D84, S87, N88, V91, I92, E95, T123, Q126, S127, I129 and S130;

[0429] For another example, the first type of mutation is selected from the group consisting of: L12R, L12K, L12E, L12Q, E15Q, E15R, E15A, E15S, H16A, H16E, H16D, H16G, H16S, H16T, H16V, H16P, D20A, D20H, D20Y, D20N, M23A, M23R, M23K, M23G, M23S, M23T, M23V, M23P, S87K, S87A, D84S, D84L, D84N, D84V, D84H, D84Y, D84R, D84K, D84G, D84A, D84T, D84P, N88D, N88A, N88S, N88T, N88R, N88I, V91A, V91T, V91E, I92A, E95S, E95A, E95R, E95Q, E95G, E95T, E9 5V, E95P, E95H, E95N, T123A, T123E, T123K, T123Q, Q126D, Q126L, Q126A, Q126S, Q126T, Q126E, S127A, S127E, S127K, S127Q;

[0430] For another example, the first type of mutation is selected from: N88D, N88R, D20A, D20N, V91T, Q126D, H16A / D84S;

[0431] More for example, the first type of mutation is selected from N88D or D20A.

[0432] Embodiment 5. The immunoconjugate of embodiment 4, wherein the IL-2 variant contains a second type of mutation, wherein the second type of mutation causes the IL-2 variant to have reduced or eliminated affinity for high-affinity receptors (IL-2Rα / β / γ) compared to wild-type IL-2;

[0433] For example, the second type of mutation is selected from R38, E62, P65, E68, F42, Y45, L72 or any combination thereof;

[0434] For another example, the second type of mutation is selected from R38A, R38D, R38E, E62Q, P65R, P65E, P65K, P65H, P65Y, P65Q, P65D, P65N, E68A, E68Q, E68K, E68R, F42A, F42G, F42S, F42T, F42Q, F42E, F42N, F42D, F42R, F42K, Y45A, Y45G, Y45S, Y45T, Y45Q, Y45E, Y45N, Y45D, Y45R, Y45K, L72G, L72A, L72S, L72T, L72Q, L72E, L72N, L72D, L72R, L72K;

[0435] More for example, the second type of mutation is selected from F42A, Y45A, L72G, F42A / L72G, F42A / Y45A, Y45A / L72G or F42A / Y45A / L72G.

[0436] Embodiment 6. The immunoconjugate of embodiment 4 or 5, wherein the IL-2 variant contains a third class of mutations, wherein the third class of mutations imparts increased stability to the IL-2 variant compared to wild-type IL-2;

[0437] For example, the third type of mutation is selected from N26, N29, N30, N71, Q11, L132, L70, P82, G27, F78 or any combination thereof;

[0438] For another example, the third type of mutation is selected from N26Q, N29S, N30S, N71Q, Q11C / L132C, L70C / P82C, G27C / F78C or any combination thereof;

[0439] For example, the third type of mutation is selected from: N26Q, N29S, N30S, N26Q / N29S / N71Q, N26Q / N29S, N26Q / N30S, N26Q / N30S / Q11C / L132C, N26Q / N29S / L70C / P82C, N26Q / N30S / G27C / F78C.

[0440] Embodiment 7. An immunoconjugate comprising: a PD-1 binding domain, and IL-2 or a variant thereof;

[0441] The PD-1 binding domain comprises at least one immunoglobulin single variable domain, wherein the immunoglobulin single variable domain comprises:

[0442] CDR1, CDR2 and CDR3 in the amino acid sequence of any one of SEQ ID NOs: 2, 8-15; wherein the CDR1, CDR2 and CDR3 are defined according to the Kabat, IMGT, Chothia, AbM or Contact numbering systems;

[0443] For example, according to the Kabat numbering system, the amino acid sequences of CDR1, CDR2 and CDR3 of the immunoglobulin single variable domain are shown in SEQ ID NOs: 3, 16 and 17, respectively.

[0444] For another example, the amino acid sequence of CDR1 of the immunoglobulin single variable domain is shown in SEQ ID NO: 3, the amino acid sequence of CDR2 is shown in SEQ ID NO: 4 or 18, and the amino acid sequence of CDR3 is shown in any one of SEQ ID NOs: 5, 19-20;

[0445] For example, the amino acid sequences of CDR1, CDR2, and CDR3 of the immunoglobulin single variable domain are shown in SEQ ID NOs: 7, 18, and 19, respectively.

[0446] Embodiment 8. The immunoconjugate of any one of embodiments 1 to 7, wherein the IL-2 variant comprises a combination of mutations selected from Group 1) and Group 2):

[0447] Group 1) mutations are selected from the group consisting of: F42A / L72G, F42A / Y45A, Y45A / L72G and F42A / Y45A / L72G;

[0448] Group 2) mutations selected from the group consisting of: N88R, N88G, N88I, N88D, D20H, D20Y, V91T, Q126L, Q126D, H16A / D84S;

[0449] For example, the IL-2 variant contains the following mutation combinations: F42A / L72G / N88D, F42A / L72G / N88R, F42A / L72G / D20A, F42A / L72G / D20N, F42A / L72G / V91T, F42A / L72G / Q126D, F42A / L72G / H16A / D84S.

[0450] Embodiment 9. The immunoconjugate of embodiment 8, wherein the IL-2 variant further comprises a Group 3) mutation selected from the group consisting of: N26Q, N29S, N30S, N26Q / N29S / N71Q, N26Q / N29S, N26Q / N30S, N26Q / N30S / Q11C / L132C, N26Q / N29S / L70C / P82C, and N26Q / N30S / G27C / F78C;

[0451] For example, the IL-2 variant contains the following mutation combination:

[0452] F42A / L72G / N88D / N26Q / N29S / N71Q,

[0453] F42A / L72G / N88R / N26Q / N29S / N71Q,

[0454] F42A / L72G / D20A / N26Q / N29S / N71Q,

[0455] F42A / L72G / D20N / N26Q / N29S / N71Q,

[0456] F42A / L72G / V91T / N26Q / N29S / N71Q,

[0457] F42A / L72G / Q126D / N26Q / N29S / N71Q,

[0458] F42A / L72G / H16A / D84S / N26Q / N29S / N71Q.

[0459] Embodiment 10. The immunoconjugate of any one of embodiments 1 to 9, comprising a mutation selected from T3 and / or C125, or a combination thereof;

[0460] For example, T3A and / or C125A, C125S, C125T, C125V mutations.

[0461] Embodiment 11. The immunoconjugate of any one of embodiments 1 to 10, wherein the IL-2 variant has an amino acid sequence as shown in any one of SEQ ID NOs: 23-31.

[0462] Embodiment 12. The immunoconjugate of any one of embodiments 4 to 6, 8 to 10,

[0463] 1) The antigen is selected from:

[0464] 1-1) Antigens on tumor cells or immune cells in the tumor microenvironment;

[0465] 1-2) PD-1, CTLA-4, LAG3, TIM3, 4-1BB, OX40, GITR, CD8a, CD8b, CD4, NKp30, NKG2A, TIGIT, TGFβR1, TGFβR2, Fas, NKG2D, NKp46, PD-L1, CD107a, ICOS, TNFR2, FAP, TNC A1, TNC A2, CEA, EDB, MCSP or CD16a;

[0466] 1-3) Immune checkpoint antigens, such as PD-1, PD-L1, and CTLA-4;

[0467] 2) The antigen-binding domain comprises a VH domain and a VL domain of an antibody selected from the following:

[0468] PD-1 antibodies (e.g., Pembrolizumab, Nivolumab, Sintilimab, Tislelizumab, Cemiplimab, Toripalimab, Camrelizumab, Penpulizumab, Sepalimumab, Sasanlimab, Spardizumab) artalizumab, retifanlimab, balstilimab, cetrelimab, dostarlimab (TSR-042), CS-1003, SCT-I10A, SSI-361, MEDI-0680 (AMP-514), BAT-1306, JTX-4014, JTX-4014, BI-754091, AK-103);

[0469] CTLA-4 antibodies (e.g., Tremelimumab, Ipilimumab);

[0470] 4-1BB antibodies (e.g., Urelumab, Utomilumab);

[0471] PD-L1 antibodies (e.g., Durvalumab, Atezolizumab, Durvalumab, Avelumab, Adebrelimab, Envafolimab, STI-A1110, CS-1001, TQB-2450, KL-A167, IMC-001, CX-072).

[0472] Embodiment 13. The immunoconjugate of any one of Embodiments 1 to 3 and 12, wherein the PD-1 binding domain comprises at least one immunoglobulin single variable domain, wherein the immunoglobulin single variable domain comprises:

[0473] CDR1, CDR2 and CDR3 in the amino acid sequence of any one of SEQ ID NOs: 2 and 8-15, wherein the CDR1, CDR2 and CDR3 are defined according to the Kabat, IMGT, Chothia, AbM or Contact numbering systems;

[0474] For example, according to the Kabat numbering system, the amino acid sequences of CDR1, CDR2 and CDR3 of the immunoglobulin single variable domain are shown in SEQ ID NOs: 3, 16 and 17, respectively;

[0475] For another example, the amino acid sequence of CDR1 of the immunoglobulin single variable domain is shown in SEQ ID NO: 3, the amino acid sequence of CDR2 is shown in SEQ ID NO: 4 or 18, and the amino acid sequence of CDR3 is shown in any one of SEQ ID NOs: 5, 19-20;

[0476] For example, the amino acid sequences of CDR1, CDR2, and CDR3 of the immunoglobulin single variable domain are shown in SEQ ID NOs: 3, 18, and 19, 3, 4, and 5, or 3, 4, and 20, respectively.

[0477] Embodiment 14. The immunoconjugate of embodiment 7 or 13, wherein the immunoglobulin single variable domain is:

[0478] 1) Humanization, affinity maturation, removal of T cell epitopes, reduction of antibody deamidation and / or reduction of antibody isomerization; for example, the heavy chain framework region of the human germline template used in the humanization process is IGHV3-23*01 or IGHV3-23*04;

[0479] 2) Camelid antibodies, chimeric antibodies, humanized antibodies, fully human antibodies or antigen-binding fragments thereof.

[0480] Embodiment 15. The immunoconjugate of embodiment 13 or 14, wherein the amino acid sequence of the immunoglobulin single variable domain is as follows:

[0481] SEQ ID NO: 2, any one of 8-15; or

[0482] Having at least 90% sequence identity with any of the aforementioned sequences.

[0483] Embodiment 17. The immunoconjugate of any of the preceding embodiments, further comprising a human immunoglobulin Fc region; for example, the Fc region is the Fc region of human IgG1, IgG2, IgG3, or IgG4.

[0484] Embodiment 18. The immunoconjugate of embodiment 17, wherein the Fc region of human IgG1 contains one or more amino acid mutations that reduce binding to Fc receptors (FcR, such as FcγR) and / or reduce effector function;

[0485] For example, the Fc region of human IgG1 contains one or more amino acid mutations that reduce binding to Fcγ receptors (FcγR) and / or reduce ADCC effects;

[0486] For another example, the Fc region of the human IgG1 contains mutations in L234, L235, P329 or any combination thereof;

[0487] For example, the Fc region of human IgG1 contains L234A / L235A or L234A / L235A / P329G mutations.

[0488] Embodiment 19. The immunoconjugate of embodiment 17 or 18, wherein the Fc region comprises a mutation that promotes association (or heterodimerization) of the first and second subunits of the Fc region;

[0489] For example, amino acid residues in the CH3 region of the first subunit of the Fc region are replaced with amino acid residues having a larger side chain volume to generate a protrusion in the CH3 region of the first subunit, and the protrusion can be placed in the cavity in the CH3 region of the second subunit, and amino acid residues in the CH3 region of the second subunit of the Fc region are replaced with amino acid residues having a smaller side chain volume to generate a cavity in the CH3 region of the second subunit, in which the protrusion in the CH3 region of the first subunit can be placed; for another example, the amino acid residues with a larger side chain volume are selected from R, F, Y, and W, and the amino acid residues with a smaller side chain volume are selected from A, S, T, and V.

[0490] Embodiment 20. The immunoconjugate of embodiment 19, wherein

[0491] The first subunit of the Fc region contains a T366 mutation, and the second subunit contains a mutation selected from T366, L368, Y407 or any combination thereof;

[0492] The first subunit of the Fc region contains an S354 or E356 mutation, and the second subunit contains a mutation selected from Y349; or

[0493] The first subunit of the Fc region contains S354 and T366 mutations, and the second subunit contains Y349, T366, L368 and Y407 mutations;

[0494] For example,

[0495] The first subunit of the Fc region contains a T366W mutation, and the second subunit contains a mutation selected from T366S, L368A, Y407V or any combination thereof;

[0496] The first subunit of the Fc region contains an S354C or E356C mutation, and the second subunit contains a mutation selected from Y349C; or

[0497] The first subunit of the Fc region contains S354C / T366W mutations, and the second subunit contains Y349C / T366S / L368A / Y407V mutations.

[0498] Embodiment 21. The immunoconjugate of embodiment 19 or 20, wherein the second subunit of the Fc region further contains H435 and / or R436 mutations, preferably H435R / Y436F, H435R, H435K mutations.

[0499] Embodiment 22. The immunoconjugate of any one of embodiments 17 to 21, wherein the Fc region of human IgG1 contains mutations that enhance FcRn-mediated recycling and / or extend half-life;

[0500] For example, the Fc region of the human IgG1 contains M252 and / or M428 mutations;

[0501] For example, the Fc region of human IgG1 contains M252Y / M428V and M252Y / M428L mutations.

[0502] Embodiment 23. The immunoconjugate of any one of the preceding embodiments, wherein the PD-1 binding domain or antigen binding domain is located at the N-terminus of the Fc region, and the IL-2 variant is located at the N-terminus or C-terminus of the Fc region;

[0503] For example, the PD-1 binding domain or antigen binding domain is located at the N-terminus of Fc, and the IL-2 variant is located at the C-terminus of the Fc region.

[0504] Embodiment 24. The immunoconjugate according to any of the preceding embodiments, wherein the valence ratio of the PD-1 binding domain or antigen binding domain to IL-2 or its variant is between 6:1 and 1:3 (e.g., 4:1 to 1:2), preferably 4:1, 2:1 or 1:1; or, one immunoconjugate contains 1 or 2 IL-2 or its variants and 1, 2, 3, 4, 5 or 6 PD-1 binding domains or antigen binding domains.

[0505] Embodiment 25. The immunoconjugate of any one of embodiments 1 to 3, 7, 11 to 23, comprising a polypeptide chain combination as shown in any one of the following I) to VI):

[0506] First polypeptide chain: [PD-1 binding domain 1]-[Fc region first subunit]-[linker 1]a-[IL-2 or its variant],

[0507] Second polypeptide chain: [PD-1 binding domain 1]-[Fc region second subunit];

[0508] Wherein, - represents a peptide bond, a linker is a polypeptide capable of achieving a connection function, a is selected from 0 or 1; [PD-1 binding domain 1] is selected from any PD-1 binding domain of Embodiments 11 to 13;

[0509] For example, the linker is (G x S) y Linker, wherein x is selected from an integer of 1-5, and y is selected from an integer of 1-6; for another example, linker 1 is (G4S)3, linker 2 is (G4S)2, and linker 3 is G4S.

[0510] Some other linkers include, but are not limited to, (G m S n ) h or (GGNGT) h or (YGNGT) h or (EPKSS) h The amino acid sequence shown, wherein m and n are each independently selected from an integer of 1-8 (e.g., 1, 2, 3, 4, 5, 6, 7, or 8), and h is independently selected from an integer of 1-20 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20).

[0511] The order of the above polypeptide chains is from N-terminus to C-terminus.

[0512] Embodiment 26. The immunoconjugate of any one of the preceding embodiments, comprising a polypeptide chain combination having the amino acid sequence shown below:

[0513] SEQ ID NO: 42 and 43;

[0514] SEQ ID NO: 21 and 22;

[0515] SEQ ID NO: 36 and 37;

[0516] SEQ ID NO: 38 and 39; or,

[0517] SEQ ID NO: 40 and 41.

[0518] Or an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any of the above polypeptide chains in combination.

[0519] Unless otherwise specified, the mutation sites in the Fc region are coded according to the EU numbering system.

[0520] The present disclosure provides a fusion protein comprising or being any of the aforementioned immunoconjugates.

[0521] In some embodiments, the pharmaceutical composition of any of the foregoing further comprises a solvent. For example, the solvent in the pharmaceutical composition is selected from, but not limited to, a non-toxic physiologically acceptable liquid carrier, such as normal saline, water for injection, glucose solution (such as 5% glucose injection, glucose and sodium chloride injection), and the like.

[0522] In some embodiments, any of the aforementioned pharmaceutical compositions can be administered as an intravenous injection, a subcutaneous injection, an intraperitoneal injection, or an intramuscular injection.

[0523] The present disclosure also provides a liquid preparation comprising any of the aforementioned pharmaceutical compositions.

[0524] The pharmaceutical composition disclosed herein has sufficient drug stability and can be stored stably for a long time.

[0525] The present disclosure also provides a method for preparing a lyophilized formulation of a pharmaceutical composition comprising an immunoconjugate, the method comprising the step of freeze-drying the aforementioned pharmaceutical composition.

[0526] The present disclosure also provides a lyophilized preparation of a pharmaceutical composition comprising an immunoconjugate, which is obtained by freeze-drying a liquid preparation comprising the pharmaceutical composition.

[0527] In some embodiments, the liquid formulation comprises:

[0528] (e-1) about 10 mg / mL to about 80 mg / mL of an immunoconjugate,

[0529] about 5 mM to about 10 mM succinate-histidine;

[0530] about 0.04% (w / v) to about 0.08% (w / v) polysorbate 80,

[0531] about 4% (w / v) to about 10% (w / v) trehalose, and,

[0532] optionally about 10 mM to about 100 mM arginine hydrochloride;

[0533] The pH of the pharmaceutical composition is from about 4.0 to about 4.5; or,

[0534] (e-1-1) about 10 mg / mL to about 80 mg / mL of an immunoconjugate,

[0535] about 5 mM to about 10 mM succinate-histidine;

[0536] about 0.04% (w / v) to about 0.08% (w / v) polysorbate 80,

[0537] about 4% (w / v) to about 10% (w / v) trehalose,

[0538] The pH of the pharmaceutical composition is from about 4.0 to about 4.5; or,

[0539] (e-1-2) about 10 mg / mL to about 80 mg / mL of an immunoconjugate,

[0540] about 5 mM to about 10 mM succinate-histidine;

[0541] about 0.04% (w / v) to about 0.08% (w / v) polysorbate 80,

[0542] about 4% (w / v) to about 10% (w / v) trehalose, and,

[0543] about 10 mM to about 100 mM arginine hydrochloride;

[0544] The pH of the pharmaceutical composition is from about 4.0 to about 4.5; or,

[0545] (e-2) about 10 mg / mL to about 40 mg / mL of an immunoconjugate,

[0546] about 10 mM succinate-histidine;

[0547] about 0.04% (w / v) to about 0.08% (w / v) polysorbate 80,

[0548] about 4% (w / v) to about 8.8% (w / v) trehalose,

[0549] The pH of the pharmaceutical composition is from about 4.0 to about 4.5; or,

[0550] (f-1) about 10 mg / mL to about 80 mg / mL of an immunoconjugate,

[0551] about 5 mM to about 10 mM succinate-histidine;

[0552] about 0.1% (w / v) to about 0.2% (w / v) polysorbate 80,

[0553] about 4% (w / v) to about 8% (w / v) sucrose, and,

[0554] optionally about 10 mM to about 100 mM arginine hydrochloride;

[0555] The pH of the pharmaceutical composition is from about 4.5 to about 5.0; or,

[0556] (f-1-1) about 10 mg / mL to about 80 mg / mL of an immunoconjugate,

[0557] about 5 mM to about 10 mM succinate-histidine;

[0558] about 0.1% (w / v) to about 0.2% (w / v) polysorbate 80,

[0559] about 4% (w / v) to about 8% (w / v) sucrose,

[0560] The pH of the pharmaceutical composition is from about 4.5 to about 5.0; or,

[0561] (f-1-2) about 10 mg / mL to about 80 mg / mL of an immunoconjugate,

[0562] about 5 mM to about 10 mM succinate-histidine;

[0563] about 0.1% (w / v) to about 0.2% (w / v) polysorbate 80,

[0564] about 4% (w / v) to about 8% (w / v) sucrose, and,

[0565] about 10 mM to about 100 mM arginine hydrochloride;

[0566] The pH of the pharmaceutical composition is from about 4.5 to about 5.0; or,

[0567] (f-2) about 40 mg / mL to about 80 mg / mL of an immunoconjugate,

[0568] approximately 5 mM succinate-histidine;

[0569] about 0.1% (w / v) to about 0.2% (w / v) polysorbate 80,

[0570] about 4% (w / v) to about 8% (w / v) sucrose,

[0571] The pH of the pharmaceutical composition is about 4.5 to about 5.0.

[0572] In some embodiments, the liquid formulation comprises any of the following:

[0573] (1) about 10 mg / mL to about 120 mg / mL of the immunoconjugate,

[0574] About 10 mM succinate-histidine,

[0575] About 0.04% (w / v) polysorbate 80,

[0576] About 4.4% (w / v) trehalose,

[0577] The pH of the pharmaceutical composition is from about 4.0 to about 4.5;

[0578] (2) about 10 mg / mL to about 80 mg / mL of the immunoconjugate,

[0579] About 10 mM succinate-histidine,

[0580] About 0.04% (w / v) polysorbate 80,

[0581] About 4.4% (w / v) trehalose,

[0582] The pH of the pharmaceutical composition is from about 4.0 to about 4.5;

[0583] (3) about 10 mg / mL to about 40 mg / mL of the immunoconjugate,

[0584] About 10 mM succinate-histidine,

[0585] About 0.04% (w / v) polysorbate 80,

[0586] About 4.4% (w / v) trehalose,

[0587] The pH of the pharmaceutical composition is about 4.5;

[0588] (7) about 40 mg / mL of immunoconjugate,

[0589] About 10 mM succinate-histidine,

[0590] About 0.04% (w / v) polysorbate 80,

[0591] About 4.4% (w / v) trehalose,

[0592] The pH of the pharmaceutical composition is about 4.5.

[0593] (8) about 40 mg / mL of immunoconjugate,

[0594] About 10 mM succinate-histidine,

[0595] About 0.08% (w / v) polysorbate 80,

[0596] About 8% (w / v) trehalose,

[0597] The pH of the pharmaceutical composition is about 4.5.

[0598] (9) about 40 mg / mL of immunoconjugate,

[0599] About 10 mM succinate-histidine,

[0600] About 0.04% (w / v) polysorbate 80,

[0601] About 8% (w / v) trehalose,

[0602] The pH of the pharmaceutical composition is about 4.0.

[0603] (10) about 40 mg / mL of the immunoconjugate,

[0604] About 10 mM succinate-histidine,

[0605] About 0.08% (w / v) polysorbate 80,

[0606] About 5% (w / v) trehalose,

[0607] The pH of the pharmaceutical composition is about 4.0.

[0608] (11) about 40 mg / mL of the immunoconjugate,

[0609] About 10 mM succinate-histidine,

[0610] About 0.08% (w / v) polysorbate 80,

[0611] About 8% (w / v) trehalose,

[0612] The pH of the pharmaceutical composition is about 4.0.

[0613] (12) about 40 mg / mL of immunoconjugate,

[0614] About 10 mM succinate-histidine,

[0615] About 0.08% (w / v) polysorbate 80,

[0616] About 8% (w / v) trehalose,

[0617] About 50 mM arginine hydrochloride

[0618] The pH of the pharmaceutical composition is about 4.0.

[0619] In some embodiments, the lyophilized formulation of any of the above items, wherein the PD-1 binding domain in the immunoconjugate comprises at least one immunoglobulin single variable domain, wherein the immunoglobulin single variable domain comprises: CDR1, CDR2 and CDR3 of the amino acid sequence shown in any one of SEQ ID NOs: 2, 8-15, wherein the CDR1, CDR2 and CDR3 are defined according to the Kabat, IMGT, Chothia, AbM or Contact numbering systems.

[0620] In some embodiments, the amino acid sequences of CDR1, CDR2, and CDR3 of the immunoglobulin single variable domain are shown in SEQ ID NOs: 3, 16, and 17, respectively.

[0621] In some embodiments, the amino acid sequence of CDR1 of the immunoglobulin single variable domain in the aforementioned PD-1 binding domain is shown in SEQ ID NO: 3, the amino acid sequence of CDR2 is shown in SEQ ID NO: 4 or 18, and the amino acid sequence of CDR3 is shown in any one of SEQ ID NOs: 5, 19-20.

[0622] In some embodiments, the amino acid sequences of CDR1, CDR2, and CDR3 of the immunoglobulin single variable domain in the aforementioned PD-1 binding domain are as shown in SEQ ID NOs: 3, 18, and 19, as shown in SEQ ID NOs: 3, 4, and 5, or as shown in SEQ ID NOs: 3, 4, and 20, respectively.

[0623] In some embodiments, the cytokine is IL-2 or a variant thereof. In some specific embodiments, the IL-2 variant comprises an amino acid sequence as shown in any one of SEQ ID NOs: 23-31 or having at least 90% sequence identity thereto.

[0624] In some embodiments, the immunoconjugate comprises a combination of polypeptide chains having the following amino acid sequences:

[0625] SEQ ID NO: 42 and 43;

[0626] SEQ ID NO: 21 and 22;

[0627] SEQ ID NO: 36 and 37;

[0628] SEQ ID NO: 38 and 39; or,

[0629] SEQ ID NO: 40 and 41.

[0630] In some embodiments, the lyophilized formulation is stable for at least 1 month, at least 3 months, at least 6 months, at least 12 months, at least 18 months, at least 24 months, at least 30 months when stored at 2-8°C protected from light.

[0631] In some embodiments, the lyophilized formulation is stable at 25°C for at least 1 month, at least 3 months, at least 6 months, at least 12 months.

[0632] In some embodiments, the lyophilized formulation is stable at 40°C for at least 7 days, at least 14 days, or at least 30 days.

[0633] The lyophilized preparation provided by the present disclosure can be dissolved quickly, and the solution has a good appearance and good stability after reconstitution.

[0634] The present disclosure also provides a reconstituted solution comprising an immunoconjugate, wherein the reconstituted solution is prepared by reconstituted any one of the aforementioned lyophilized preparations.

[0635] In some embodiments, the reconstituted solution is obtained by reconstituted any of the aforementioned lyophilized formulations with a solvent. In some embodiments, the solvent includes but is not limited to water, physiological saline, glucose, etc., for example, the solvent is water for injection.

[0636] In some embodiments, the concentration of the immunoconjugate in the reconstituted solution is greater than the concentration of the immunoconjugate in the stock solution used to prepare the lyophilized formulation. In some embodiments, the concentration ratio of the immunoconjugate in the reconstituted solution to the immunoconjugate in the stock solution is (1-5): 1, for example, 1: 1, 1.5: 1, 1.6: 1, 1.7: 1, 1.8: 1, 1.9: 1, 2.0: 1, 2.1: 1, 2.2: 1, 2.3: 1, 2.4: 1, 2.5: 1, 2.6: 1, 2.7: 1, 2.8: 1, 2.9: 1, 3.0: 1, 3.5: 1, 4: 1, 4.5: 1, 5: 1, and any ranges between these point values.

[0637] In some embodiments, the reconstitution solution comprises:

[0638] (e-3) about 40 mg / mL to about 120 mg / mL of an immunoconjugate,

[0639] about 10 mM to about 20 mM succinate-histidine;

[0640] about 0.04% (w / v) to about 0.08% (w / v) polysorbate 80,

[0641] about 4% (w / v) to about 10% (w / v) trehalose,

[0642] The pH of the pharmaceutical composition is from about 4.0 to about 4.5; or

[0643] (e-4) about 40 mg / mL to about 80 mg / mL of an immunoconjugate,

[0644] about 20 mM succinate-histidine;

[0645] about 0.04% (w / v) to about 0.08% (w / v) polysorbate 80,

[0646] about 4.4% (w / v) to about 8.8% (w / v) trehalose,

[0647] The pH of the pharmaceutical composition is from about 4.0 to about 4.5; or

[0648] (f-3) about 40 mg / mL to about 120 mg / mL of an immunoconjugate,

[0649] about 10 mM to about 20 mM succinate-histidine;

[0650] about 0.1% (w / v) to about 0.2% (w / v) polysorbate 80,

[0651] about 4% (w / v) to about 8% (w / v) sucrose,

[0652] The pH of the pharmaceutical composition is from about 4.5 to about 5.0; or,

[0653] (f-4) about 80 mg / mL to about 120 mg / mL of an immunoconjugate,

[0654] about 10 mM succinate-histidine;

[0655] about 0.1% (w / v) to about 0.2% (w / v) polysorbate 80,

[0656] about 4% (w / v) to about 8% (w / v) sucrose,

[0657] The pH of the pharmaceutical composition is from about 4.5 to about 5.0; or,

[0658] (f-5) about 80 mg / mL to about 120 mg / mL of an immunoconjugate,

[0659] About 10 mM succinate-histidine,

[0660] About 0.2% (w / v) polysorbate 80,

[0661] About 8% (w / v) sucrose,

[0662] The pH of the pharmaceutical composition is about 4.8; or,

[0663] (f-6) about 80 mg / mL to about 120 mg / mL of an immunoconjugate,

[0664] About 10 mM succinate-histidine,

[0665] About 0.2% (w / v) polysorbate 80,

[0666] About 8% (w / v) sucrose,

[0667] The pH of the pharmaceutical composition is about 5.0.

[0668] In some embodiments, the reconstitution solution comprises any of the following:

[0669] (4) about 10 mg / mL to about 120 mg / mL of the immunoconjugate,

[0670] About 20 mM succinate-histidine,

[0671] About 0.08% (w / v) polysorbate 80,

[0672] About 8.8% (w / v) trehalose,

[0673] The pH of the pharmaceutical composition is from about 4.0 to about 4.5;

[0674] (5) about 40 mg / mL to about 120 mg / mL of the immunoconjugate,

[0675] About 20 mM succinate-histidine,

[0676] About 0.08% (w / v) polysorbate 80,

[0677] About 8.8% (w / v) trehalose,

[0678] The pH of the pharmaceutical composition is from about 4.0 to about 4.5;

[0679] (6) about 40 mg / mL to about 80 mg / mL of the immunoconjugate,

[0680] About 20 mM succinate-histidine,

[0681] About 0.08% (w / v) polysorbate 80,

[0682] About 8.8% (w / v) trehalose,

[0683] The pH of the pharmaceutical composition is about 4.5.

[0684] (13) about 80 mg / mL of immunoconjugate,

[0685] About 20 mM succinate-histidine,

[0686] About 0.08% (w / v) polysorbate 80,

[0687] About 8.8% (w / v) trehalose,

[0688] The pH of the pharmaceutical composition is about 4.5.

[0689] In some embodiments, the reconstituted solution of any of the above items, wherein the PD-1 binding domain in the immunoconjugate comprises at least one immunoglobulin single variable domain, wherein the immunoglobulin single variable domain comprises: CDR1, CDR2 and CDR3 of the amino acid sequence shown in any one of SEQ ID NOs: 2, 8-15, wherein the CDR1, CDR2 and CDR3 are defined according to the Kabat, IMGT, Chothia, AbM or Contact numbering systems.

[0690] In some embodiments, the amino acid sequences of CDR1, CDR2, and CDR3 of the immunoglobulin single variable domain are shown in SEQ ID NOs: 3, 16, and 17, respectively.

[0691] In some embodiments, the amino acid sequence of CDR1 of the immunoglobulin single variable domain in the aforementioned PD-1 binding domain is shown in SEQ ID NO: 3, the amino acid sequence of CDR2 is shown in SEQ ID NO: 4 or 18, and the amino acid sequence of CDR3 is shown in any one of SEQ ID NOs: 5, 19-20.

[0692] In some embodiments, the amino acid sequences of CDR1, CDR2, and CDR3 of the immunoglobulin single variable domain in the aforementioned PD-1 binding domain are as shown in SEQ ID NOs: 3, 18, and 19, as shown in SEQ ID NOs: 3, 4, and 5, or as shown in SEQ ID NOs: 3, 4, and 20, respectively.

[0693] In some embodiments, the cytokine is IL-2 or a variant thereof. In some specific embodiments, the IL-2 variant comprises an amino acid sequence as shown in any one of SEQ ID NOs: 23-31 or having at least 90% sequence identity thereto.

[0694] In some embodiments, the immunoconjugate comprises a combination of polypeptide chains having the following amino acid sequences:

[0695] SEQ ID NO: 42 and 43;

[0696] SEQ ID NO: 21 and 22;

[0697] SEQ ID NO: 36 and 37;

[0698] SEQ ID NO: 38 and 39; or,

[0699] SEQ ID NO: 40 and 41.

[0700] In some embodiments, the present disclosure unexpectedly discovered that the lyophilized preparations prepared with (e-1), (e-2), (f-1), (f-2), (1)-(3), (7)-(12) as the stock solution had good stability after being placed at 40°C for 4 weeks, and the SEC monomer purity and NR-CE main peak purity did not change significantly, and had a good appearance. In addition, the reconstitution time during reconstitution was short, and the reconstitution appearance, insoluble particles and moisture content of (e-3), (e-4), (f-3), (f-4), (4)-(6), and (13) after reconstitution were all good, effectively solving the problem of poor stability of immunoconjugates (e.g., fusion proteins of PD-1 binding domains and IL-2 variants) encountered during the development process under high temperature conditions of 40°C, especially the significant decrease in SEC monomer purity after the immunoconjugate concentration exceeded 50 mg / mL.

[0701] The present disclosure provides a method for preparing a liquid preparation, comprising the following steps:

[0702] 1) preparing a stock solution containing the pharmaceutical composition, and freeze-drying the stock solution to obtain a lyophilized preparation;

[0703] 2) The lyophilized preparation is reconstituted to obtain a reconstituted solution.

[0704] In some embodiments, the concentration ratio of the immunoconjugate in the reconstituted solution to the immunoconjugate in the original solution is (1-5):1, for example, 1:1, 1.5:1, 1.6:1, 1.7:1, 1.8:1, 1.9:1, 2.0:1, 2.1:1, 2.2:1, 2.3:1, 2.4:1, 2.5:1, 2.6:1, 2.7:1, 2.8:1, 2.9:1, 3.0:1, 3.5:1, 4:1, 4.5:1, 5:1, and any ranges between these points.

[0705] In some embodiments, the method of preparing a liquid formulation comprises:

[0706] 1) preparing a stock solution containing the pharmaceutical composition, and freeze-drying the stock solution to obtain a lyophilized preparation;

[0707] 2) The lyophilized preparation is reconstituted with a solvent to obtain a liquid preparation.

[0708] The present disclosure provides a liquid preparation comprising any of the following:

[0709] (e) about 10 mg / mL to about 120 mg / mL of the immunoconjugate,

[0710] about 5 mM to about 20 mM succinate-histidine;

[0711] about 0.04% (w / v) to about 0.08% (w / v) polysorbate 80,

[0712] about 1% (w / v) to about 10% (w / v) trehalose, and,

[0713] optionally about 10 mM to about 100 mM arginine hydrochloride;

[0714] The pH of the pharmaceutical composition is from about 4.0 to about 4.5; or,

[0715] (e-1) about 10 mg / mL to about 80 mg / mL of an immunoconjugate,

[0716] about 5 mM to about 10 mM succinate-histidine;

[0717] about 0.04% (w / v) to about 0.08% (w / v) polysorbate 80,

[0718] about 4% (w / v) to about 10% (w / v) trehalose, and,

[0719] optionally about 10 mM to about 100 mM arginine hydrochloride;

[0720] The pH of the pharmaceutical composition is from about 4.0 to about 4.5; or,

[0721] (e-1-1) about 10 mg / mL to about 80 mg / mL of an immunoconjugate,

[0722] about 5 mM to about 10 mM succinate-histidine;

[0723] about 0.04% (w / v) to about 0.08% (w / v) polysorbate 80,

[0724] about 4% (w / v) to about 10% (w / v) trehalose,

[0725] The pH of the pharmaceutical composition is from about 4.0 to about 4.5; or,

[0726] (e-1-2) about 10 mg / mL to about 80 mg / mL of an immunoconjugate,

[0727] about 5 mM to about 10 mM succinate-histidine;

[0728] about 0.04% (w / v) to about 0.08% (w / v) polysorbate 80,

[0729] about 4% (w / v) to about 10% (w / v) trehalose, and,

[0730] about 10 mM to about 100 mM arginine hydrochloride;

[0731] The pH of the pharmaceutical composition is from about 4.0 to about 4.5; or,

[0732] (e-2) about 10 mg / mL to about 40 mg / mL of an immunoconjugate,

[0733] about 10 mM succinate-histidine;

[0734] about 0.04% (w / v) to about 0.08% (w / v) polysorbate 80,

[0735] about 4% (w / v) to about 8.8% (w / v) trehalose,

[0736] The pH of the pharmaceutical composition is from about 4.0 to about 4.5; or,

[0737] (e-3) about 40 mg / mL to about 120 mg / mL of an immunoconjugate,

[0738] about 10 mM to about 20 mM succinate-histidine;

[0739] about 0.04% (w / v) to about 0.08% (w / v) polysorbate 80,

[0740] about 4% (w / v) to about 10% (w / v) trehalose,

[0741] The pH of the pharmaceutical composition is from about 4.0 to about 4.5; or

[0742] (e-4) about 40 mg / mL to about 80 mg / mL of an immunoconjugate,

[0743] about 20 mM succinate-histidine;

[0744] about 0.04% (w / v) to about 0.08% (w / v) polysorbate 80,

[0745] about 4.4% (w / v) to about 8.8% (w / v) trehalose,

[0746] The pH of the pharmaceutical composition is from about 4.0 to about 4.5; or

[0747] (f-1) about 10 mg / mL to about 80 mg / mL of an immunoconjugate,

[0748] about 5 mM to about 10 mM succinate-histidine;

[0749] about 0.1% (w / v) to about 0.2% (w / v) polysorbate 80,

[0750] about 4% (w / v) to about 8% (w / v) sucrose, and,

[0751] optionally about 10 mM to about 100 mM arginine hydrochloride;

[0752] The pH of the pharmaceutical composition is from about 4.5 to about 5.0; or,

[0753] (f-1-1) about 10 mg / mL to about 80 mg / mL of an immunoconjugate,

[0754] about 5 mM to about 10 mM succinate-histidine;

[0755] about 0.1% (w / v) to about 0.2% (w / v) polysorbate 80,

[0756] about 4% (w / v) to about 8% (w / v) sucrose,

[0757] The pH of the pharmaceutical composition is from about 4.5 to about 5.0; or,

[0758] (f-1-2) about 10 mg / mL to about 80 mg / mL of an immunoconjugate,

[0759] about 5 mM to about 10 mM succinate-histidine;

[0760] about 0.1% (w / v) to about 0.2% (w / v) polysorbate 80,

[0761] about 4% (w / v) to about 8% (w / v) sucrose, and,

[0762] about 10 mM to about 100 mM arginine hydrochloride;

[0763] The pH of the pharmaceutical composition is from about 4.5 to about 5.0; or,

[0764] (f-2) about 40 mg / mL to about 80 mg / mL of an immunoconjugate,

[0765] approximately 5 mM succinate-histidine;

[0766] about 0.1% (w / v) to about 0.2% (w / v) polysorbate 80,

[0767] about 4% (w / v) to about 8% (w / v) sucrose,

[0768] The pH of the pharmaceutical composition is about 4.5 to about 5.0.

[0769] (f-3) about 40 mg / mL to about 120 mg / mL of an immunoconjugate,

[0770] about 10 mM to about 20 mM succinate-histidine;

[0771] about 0.1% (w / v) to about 0.2% (w / v) polysorbate 80,

[0772] about 4% (w / v) to about 8% (w / v) sucrose,

[0773] The pH of the pharmaceutical composition is from about 4.5 to about 5.0; or,

[0774] (f-4) about 80 mg / mL to about 120 mg / mL of an immunoconjugate,

[0775] about 10 mM succinate-histidine;

[0776] about 0.1% (w / v) to about 0.2% (w / v) polysorbate 80,

[0777] about 4% (w / v) to about 8% (w / v) sucrose,

[0778] The pH of the pharmaceutical composition is from about 4.5 to about 5.0; or,

[0779] (f-5) about 80 mg / mL to about 120 mg / mL of an immunoconjugate,

[0780] About 10 mM succinate-histidine,

[0781] About 0.2% (w / v) polysorbate 80,

[0782] About 8% (w / v) sucrose,

[0783] The pH of the pharmaceutical composition is about 4.8; or,

[0784] (f-6) about 80 mg / mL to about 120 mg / mL of an immunoconjugate,

[0785] About 10 mM succinate-histidine,

[0786] About 0.2% (w / v) polysorbate 80,

[0787] About 8% (w / v) sucrose,

[0788] The pH of the pharmaceutical composition is about 5.0.

[0789] In some embodiments, the liquid formulation comprises any of the following:

[0790] (1) about 10 mg / mL to about 120 mg / mL of the immunoconjugate,

[0791] About 10 mM succinate-histidine,

[0792] About 0.04% (w / v) polysorbate 80,

[0793] About 4.4% (w / v) trehalose,

[0794] The pH of the pharmaceutical composition is from about 4.0 to about 4.5;

[0795] (2) about 10 mg / mL to about 80 mg / mL of the immunoconjugate,

[0796] About 10 mM succinate-histidine,

[0797] About 0.04% (w / v) polysorbate 80,

[0798] About 4.4% (w / v) trehalose,

[0799] The pH of the pharmaceutical composition is from about 4.0 to about 4.5;

[0800] (3) about 10 mg / mL to about 40 mg / mL of the immunoconjugate,

[0801] About 10 mM succinate-histidine,

[0802] About 0.04% (w / v) polysorbate 80,

[0803] About 4.4% (w / v) trehalose,

[0804] The pH of the pharmaceutical composition is about 4.5;

[0805] (4) about 10 mg / mL to about 120 mg / mL of the immunoconjugate,

[0806] About 20 mM succinate-histidine,

[0807] About 0.08% (w / v) polysorbate 80,

[0808] About 8.8% (w / v) trehalose,

[0809] The pH of the pharmaceutical composition is from about 4.0 to about 4.5;

[0810] (5) about 40 mg / mL to about 120 mg / mL of the immunoconjugate,

[0811] About 20 mM succinate-histidine,

[0812] About 0.08% (w / v) polysorbate 80,

[0813] About 8.8% (w / v) trehalose,

[0814] The pH of the pharmaceutical composition is from about 4.0 to about 4.5;

[0815] (6) about 40 mg / mL to about 80 mg / mL of the immunoconjugate,

[0816] About 20 mM succinate-histidine,

[0817] About 0.08% (w / v) polysorbate 80,

[0818] About 8.8% (w / v) trehalose,

[0819] The pH of the pharmaceutical composition is about 4.5.

[0820] (7) about 40 mg / mL of immunoconjugate,

[0821] About 10 mM succinate-histidine,

[0822] About 0.04% (w / v) polysorbate 80,

[0823] About 4.4% (w / v) trehalose,

[0824] The pH of the pharmaceutical composition is about 4.5.

[0825] (8) about 40 mg / mL of immunoconjugate,

[0826] About 10 mM succinate-histidine,

[0827] About 0.08% (w / v) polysorbate 80,

[0828] About 8% (w / v) trehalose,

[0829] The pH of the pharmaceutical composition is about 4.5.

[0830] (9) about 40 mg / mL of immunoconjugate,

[0831] About 10 mM succinate-histidine,

[0832] About 0.04% (w / v) polysorbate 80,

[0833] About 8% (w / v) trehalose,

[0834] The pH of the pharmaceutical composition is about 4.0.

[0835] (10) about 40 mg / mL of the immunoconjugate,

[0836] About 10 mM succinate-histidine,

[0837] About 0.08% (w / v) polysorbate 80,

[0838] About 5% (w / v) trehalose,

[0839] The pH of the pharmaceutical composition is about 4.0.

[0840] (11) about 40 mg / mL of the immunoconjugate,

[0841] About 10 mM succinate-histidine,

[0842] About 0.08% (w / v) polysorbate 80,

[0843] About 8% (w / v) trehalose,

[0844] The pH of the pharmaceutical composition is about 4.0.

[0845] (12) about 40 mg / mL of immunoconjugate,

[0846] About 10 mM succinate-histidine,

[0847] About 0.08% (w / v) polysorbate 80,

[0848] About 8% (w / v) trehalose,

[0849] About 50 mM arginine hydrochloride

[0850] The pH of the pharmaceutical composition is about 4.0.

[0851] (13) about 80 mg / mL of immunoconjugate,

[0852] About 20 mM succinate-histidine,

[0853] About 0.08% (w / v) polysorbate 80,

[0854] About 8.8% (w / v) trehalose,

[0855] The pH of the pharmaceutical composition is about 4.5.

[0856] The present invention adopts the method of dilute freezing and concentrated dissolving to prepare a reconstituted solution with increased protein concentration, and the reconstituted solution has a good appearance and good stability.

[0857] The present disclosure further provides a product, comprising a container containing the pharmaceutical composition, liquid preparation, lyophilized preparation or reconstituted solution as described above. In some embodiments, the container can be, but is not limited to, a neutral borosilicate glass tube injection bottle.

[0858] The present disclosure also provides use of the pharmaceutical composition, liquid preparation, lyophilized preparation, reconstituted solution or product described in any of the above items in preparing a drug for treating a disease.

[0859] The present disclosure also provides a method for treating or preventing a disease, comprising administering to a subject in need thereof an effective amount of the pharmaceutical composition, liquid preparation, lyophilized preparation, reconstituted solution, or product described above.

[0860] The present disclosure also provides the pharmaceutical composition as described in any one of the above items, the liquid preparation as described in any one of the above items, the lyophilized preparation as described in any one of the above items, the reconstituted solution as described in any one of the above items, or the product as described in any one of the above items, for use in treating a disease.

[0861] Methods of treatment and pharmaceutical uses

[0862] In some embodiments, the aforementioned disease is a proliferative disorder or any other disease or disorder characterized by uncontrolled cell growth (e.g., cancer; in this disclosure, cancer and tumor are used interchangeably), such as a disease associated with overexpression of PD-L1, or a disease that benefits from treatment with a PD-1 antibody or an antigen-binding fragment thereof, such as cancer.

[0863] In some embodiments, the aforementioned cancer is a solid tumor or a hematological tumor.

[0864] In some embodiments, the aforementioned cancer is advanced or metastatic.

[0865] In some embodiments, the aforementioned cancer is selected from the following or a combination thereof: lung cancer, prostate cancer, breast cancer, head and neck cancer, esophageal cancer, gastric cancer, colon cancer, colorectal cancer, bladder cancer, cervical cancer, uterine cancer, ovarian cancer, liver cancer, melanoma, kidney cancer, squamous cell carcinoma, hematological cancer, or any other disease or condition characterized by uncontrolled cell growth.

[0866] For example, selected from prostate cancer, liver cancer (HCC), colorectal cancer, ovarian cancer, endometrial cancer, breast cancer, triple-negative breast cancer, pancreatic cancer, stomach (stomach / gastric) cancer, cervical cancer, head and neck cancer, thyroid cancer, testicular cancer, urothelial cancer, lung cancer (small cell lung cancer, non-small cell lung cancer), melanoma, non-melanoma skin cancer (squamous and basal cell carcinoma), glioma, renal cell carcinoma (RCC), lymphoma (NHL or HL), acute myeloid leukemia (AML), T-cell acute lymphoblastic leukemia (T-ALL), diffuse large B-cell lymphoma, testicular germ cell tumor, mesothelioma, esophageal cancer, Merkel Cells cancer, MSI-high cancer, KRAS mutant tumors, adult T-cell leukemia / lymphoma and myelodysplastic syndrome (MDS).

[0867] For example, selected from basal cell carcinoma, biliary tract cancer, bladder cancer, bone cancer, brain and central nervous system cancer, breast cancer, peritoneal cancer, cervical cancer, choriocarcinoma, colorectal cancer, connective tissue cancer, digestive system cancer, endometrial cancer, esophageal cancer, eye cancer, head and neck cancer, stomach cancer, gastrointestinal cancer, glioblastoma, liver cancer, liver tumor, intraepithelial neoplasia, kidney cancer or renal cancer, laryngeal cancer, liver cancer, lung cancer, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, melanoma, myeloma, neuroblastoma, oral cancer, ovarian cancer, pancreatic cancer, prostate cancer, retinoblastoma, rhabdomyosarcoma, rectal cancer, respiratory system cancer, salivary gland cancer, sarcoma, skin cancer, squamous cell carcinoma, gastric cancer, Testicular cancer, thyroid cancer, uterine or endometrial cancer, urological cancer, vulvar cancer, lymphoma, lymphoma, Hodgkin lymphoma, non-Hodgkin lymphoma, B-cell lymphoma, low-grade / follicular non-Hodgkin lymphoma (NHL), small lymphocytic (SL) NHL, intermediate-grade / follicular NHL, intermediate-grade diffuse NHL, high-grade immunoblastic NHL, high-grade lymphoblastic NHL, high-grade small non-cleaved cell NHL, bulky NHL, mantle cell lymphoma, AIDS-related lymphoma, Waldenstrom's macroglobulinemia, chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), hairy cell leukemia, and chronic myeloblastic leukemia.

[0868] In some embodiments, the subject has a condition associated with PD-L1. In some specific embodiments, the subject's condition includes cancer that aberrantly expresses or does not aberrantly express PD-L1, and further includes non-metastatic or non-invasive and invasive or metastatic cancers.

[0869] In some embodiments, methods for activating the immune system of a subject are provided, methods for activating cytotoxic T cells (CTLs) of a subject in vivo or in vitro are provided, methods for increasing CD4 +and / or CD8 + Methods for activating T cell proliferation in vivo or in vitro, methods for activating NK cells in a subject in vivo or in vitro, methods for activating γδ T cells in a subject in vivo or in vitro, methods for activating Th1 cells in a subject in vivo or in vitro, methods for activating, reducing or eliminating the number and / or activity of at least one of regulatory T cells (Treg) in a subject, methods for increasing IFN-γ production and / or proinflammatory cytokine secretion in a subject, and methods for blocking or inhibiting the interaction between PD-1 and PD-L1 / PD-L2 in a subject, all of which comprise administering to the subject an effective amount of the aforementioned PD-1 antibody or antigen-binding fragment thereof, IL-2 variant, immunoconjugate, polynucleotide, or composition (including pharmaceutical composition) disclosed herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0870] Figure 1 shows the degree of T cell activation by A17m09_hIgG4 detected by cytokine release in DC cell:T cell mixed lymphocyte reaction, using Pembrolizumab as a positive control and hIgG4 as a negative control.

[0871] Figure 2 shows the construction of a fusion protein of an anti-PD-1 antibody and an IL-2 mutant. The anti-PD-1 nanobody A17m0902 is coupled to the N-termini of the Knob and Hole chains via the hinge region of the IgG1 heavy chain, respectively. The IL-2 mutant is linked to the C-terminus of the Knob chain of the Fc via a (G4S)3 linker, and IL-2v(V91T) in the F molecule is coupled to the N-terminus of the Knob chain via a G4S linker.

[0872] Figure 3 shows the results of proliferation activity assays of the same PD-1-IL-2v fusion protein with different IL-2 mutants against M07e and M07e-hPD-1 cell lines. Figure 3A shows the proliferation activity of the PD-1-IL-2v fusion protein against M07e cells, and Figure 3B shows the proliferation activity of the PD-1-IL-2v fusion protein against M07e-hPD-1 cells.

[0873] FIG4 shows the results of the proliferation-promoting activity detection of PD-1-IL-2v or IgG-IL-2v on M07e-hPD-1 cells.

[0874] Figure 5 shows the results of the proliferation activity test of unstimulated T cells by PD-1-IL-2v with different IL-2 mutants. + T cell proliferation results, Figure 5B is CD8 + T cell proliferation results.

[0875] Figure 6 shows the results of the detection of the proliferation activity of PD-1-IL-2v with different IL-2 mutants on T cells stimulated with anti-CD3 antibodies. + T cell proliferation results, Figure 6B is CD8 + T cell proliferation results. DETAILED DESCRIPTION

[0876] the term

[0877] In order to make the present disclosure more easily understood, certain technical and scientific terms are specifically defined below. Unless otherwise explicitly defined herein, all other technical and scientific terms used herein have the meanings commonly understood by those skilled in the art to which the present disclosure belongs.

[0878] "Buffer" refers to a buffer that tolerates changes in pH through the action of its acid-base conjugate components. Examples of buffers that control pH within an appropriate range include acetate, succinate, gluconate, histidine, oxalate, lactate, phosphate, citrate, tartrate, fumarate, glycylglycine, and other organic acid buffers.

[0879] A "histidine buffer" is a buffer containing histidine ions. Examples of histidine buffers include acetate-histidine, succinate-histidine, histidine-histidine hydrochloride, hydrochloride-histidine, and sulfate-histidine. For example, a histidine-hydrochloride buffer is a mixture of histidine and hydrochloric acid, or histidine and histidine hydrochloride.

[0880] A "pharmaceutical composition" refers to a mixture containing one or more compounds described herein, or their physiologically / pharmaceutically acceptable salts or prodrugs, together with other chemical components, such as physiologically / pharmaceutically acceptable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration to an organism, facilitating absorption of the active ingredient and thereby exerting its biological activity. As used herein, the terms "pharmaceutical composition" and "preparation" are not mutually exclusive.

[0881] Unless otherwise specified, the solvent in the solution form of the pharmaceutical composition described in the present disclosure is water.

[0882] As used herein, the term "about" refers to a value that is within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which value depends in part on how the value is measured or determined (i.e., the limitations of the measurement system). For example, "about" can mean within 1 or more than 1 standard deviation per practice in the art. Alternatively, "about" or "substantially comprising" can mean a range of up to 20%. Furthermore, particularly with respect to biological systems or processes, the term can mean up to an order of magnitude or up to 5 times the value. Unless otherwise indicated, when a specific value appears in the application and claims, the meaning of "about" or "substantially comprising" should be assumed to be within an acceptable error range for that specific value.

[0883] The pharmaceutical compositions disclosed herein can achieve a stable effect: the antibodies therein substantially retain their physical stability and / or chemical stability and / or biological activity after storage. Preferably, the pharmaceutical compositions substantially retain their physical and chemical stability as well as their biological activity after storage. The storage period is generally selected based on the intended shelf life of the pharmaceutical composition. Currently, there are various analytical techniques for measuring protein stability after storage at a selected temperature for a selected period of time.

[0884] A stable pharmaceutical antibody formulation is one in which no significant change is observed after storage 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 a period of time, including 1 month, 3 months, 6 months, or 40°C for 1 month. Typical acceptable standards for stability are as follows: typically no more than about 10%, preferably no more than about 5%, of the antibody monomers degrade as measured by SEC-HPLC. By visual analysis, the pharmaceutical antibody formulation is colorless or yellow, clear to slightly opalescent. The concentration, pH, and weight-gram molecular osmotic pressure concentration of the formulation vary by no more than ±10%. Typically, no more than about 10%, preferably no more than about 5%, of truncation is observed, and typically no more than about 10%, preferably no more than about 5%, of aggregation is formed.

[0885] An antibody "retains its physical stability" in a pharmaceutical formulation if it shows no significant increase in aggregation, precipitation, and / or denaturation as measured by visual inspection of color and / or clarity, or by UV light scattering, size exclusion chromatography (SEC), and dynamic light scattering (DLS). Changes in protein conformation can be assessed by fluorescence spectroscopy (which determines protein tertiary structure) and by FTIR spectroscopy (which determines protein secondary structure).

[0886] An antibody "retains its chemical stability" in a pharmaceutical formulation if it shows no significant chemical changes. Chemical stability can be assessed by detecting and quantifying chemically altered forms of the protein. Degradation processes that often change the chemical structure of a protein include hydrolysis or truncation (assessed by methods such as size exclusion chromatography and SDS-PAGE), oxidation (assessed by methods such as peptide mapping in combination with mass spectrometry or MALDI / TOF / MS), deamidation (assessed by methods such as ion exchange chromatography, capillary isoelectric focusing, peptide mapping, isoaspartate measurement), and isomerization (assessed by measuring isoaspartate content, peptide mapping, etc.).

[0887] An antibody "retains its biological activity" in a pharmaceutical formulation if its biological activity at a given time is within a predetermined range of the biological activity exhibited when the pharmaceutical formulation is prepared. The biological activity of an antibody can be determined, for example, by antigen binding assays.

[0888] The three letter and one letter codes for amino acids used in this disclosure are as described in J. biol. chem, 243, p3558 (1968).

[0889] "Programmed death 1," "programmed cell death 1," "protein PD-1," "PD-1," "PDCD1," and "hPD-1" are used interchangeably and include variants, isoforms, species homologs, and analogs of human PD-1 that share at least one common epitope with PD-1. The complete PD-1 sequence can be found at GenBank Accession No. U64863. "Programmed death ligand-1 (PD-L1)" is one of the two cell surface glycoprotein ligands of PD-1 (the other being PD-L2), which downregulates T cell activation and cytokine secretion when bound to PD-1. "PD-L1," as used herein, includes human PD-L1 (hPD-L1), variants, isoforms, and species homologs of hPD-L1, and analogs that share at least one common epitope with hPD-L1. The complete hPD-L1 sequence can be found at GenBank Accession No. Q9NZQ7.

[0890] "PD-1 binding protein" means any protein that can specifically bind to PD-1 or any molecule comprising the protein. PD-1 binding proteins may include antibodies, antigen-binding fragments thereof, or conjugates thereof as defined in the present disclosure directed against PD-1. PD-1 binding proteins also encompass immunoglobulin superfamily antibodies (IgSF) or CDR-grafted molecules. The "PD-1 binding protein" of the present disclosure may comprise at least one immunoglobulin single variable domain (such as VHH) that binds to PD-1. In some embodiments, the "PD-1 binding protein" may comprise 2, 3, 4, or more immunoglobulin single variable domains (such as VHH) that bind to PD-1. In addition to comprising an immunoglobulin single variable domain of PD-1, the PD-1 binding protein of the present disclosure may also comprise a linker and / or a portion having effector function, such as a half-life extending portion (such as an immunoglobulin single variable domain that binds serum albumin), and / or a fusion partner (such as serum albumin) and / or a conjugated polymer (such as PEG) and / or an Fc region. In some embodiments, the “PD-1 binding protein” of the present disclosure also encompasses bispecific / multispecific antibodies, which contain immunoglobulins that bind to different antigens (e.g., a first antibody that binds to a first antigen (e.g., PD-1) and a second antibody that binds to a second antigen (e.g., 4-1BB), optionally, a third antibody that binds to a third antigen, and further optionally, a fourth antibody that binds to a fourth antigen).

[0891] The "PD-1 binding protein" or "PD-1 antibody or antigen-binding fragment thereof" of the present disclosure may contain one or more effector molecules, for example, in a conjugated manner. The "effector molecules" include, for example, anti-tumor agents, drugs, toxins, biologically active proteins (e.g., enzymes), other antibodies or antibody fragments, synthetic or naturally occurring polymers, nucleic acids and fragments thereof (e.g., DNA, RNA and fragments thereof), radionuclides, in particular radioiodides, radioisotopes, chelated metals, nanoparticles, and reporter groups such as fluorescent compounds or compounds detectable by NMR or ESR spectroscopy. In some embodiments, the effector molecule is a cytokine (e.g., IL-2, IL-15, or a variant thereof). In other embodiments, the effector molecule is a polymer, which may generally be a synthetic or naturally occurring polymer. Specific examples of synthetic polymers include optionally substituted linear or branched poly(ethylene glycol), poly(propylene glycol), poly(vinyl alcohol), or derivatives thereof. Specific naturally occurring polymers include lactose, amylose, dextran, glycogen, or derivatives thereof. In other embodiments, the polymer is albumin or a fragment thereof, such as human serum albumin or a fragment thereof. The conjugation of the polymer to the PD-1 binding protein or PD-1 antibody can be achieved by conventional methods.

[0892] "Cytokines" are proteins released by a cell population that act as intercellular mediators on other cells. Examples of such cytokines include lymphokines, monokines, chemokines, and traditional polypeptide hormones. Exemplary cytokines include human IL-2, IL-15, IFN-γ, IL-6, TNFα, IL-17, and IL-5, and variants thereof.

[0893] "Interleukin-2" or "IL-2" refers to any natural IL-2 from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats). The term encompasses unprocessed IL-2 as well as any form of IL-2 derived from processing in cells. The term also encompasses naturally occurring IL-2 variants, such as splice variants or allelic variants. The amino acid sequence of an exemplary wild-type human IL-2 is shown in SEQ ID NO: 87. Unprocessed human IL-2 additionally comprises an N-terminal 20 amino acid signal peptide (as shown in SEQ ID NO.272 in WO2012107417), which is absent in mature IL-2 molecules.

[0894] "Mutated IL-2" or "IL-2 variant" refers to a polypeptide that differs from the wild-type IL-2 amino acid sequence by a mutation (eg, substitution, deletion, addition) at at least one amino acid position.

[0895] In this disclosure, when referring to IL-2 variants, single amino acid substitutions are described as follows: [original amino acid residue / position / substituted amino acid residue]. For example, a substitution of phenylalanine at position 42 with alanine can be represented as F42A. Single amino acid substitutions can be linked by " / " to represent combined mutations at multiple given positions. For example, a combined mutation at positions F42A, L72G, and N88R can be represented as: F42A / L72G / N88R.

[0896] In this disclosure, when referring to amino acid positions in an IL-2 protein or IL-2 sequence segment, the amino acid positions are determined by reference to the amino acid sequence of wild-type human IL-2 protein (also referred to as wt IL-2), SEQ ID NO: 87. The corresponding amino acid positions on other IL-2 proteins or polypeptides (including full-length sequences or truncated fragments) can be identified by amino acid sequence alignment with SEQ ID NO: 87. Therefore, in this disclosure, unless otherwise indicated, the amino acid positions of IL-2 proteins or polypeptides are the amino acid positions numbered according to SEQ ID NO: 87. For example, when referring to "F42", it refers to the phenylalanine residue F at position 42 of SEQ ID NO: 40, or the amino acid residue at the corresponding position on another IL-2 polypeptide sequence after alignment. Sequence alignment for amino acid position determination can be performed using the Basic Local Alignment Search Tool available at https: / / blast.ncbi.nlm.nih.gov / Blast.cgi using default parameters.

[0897] "CD25" or "alpha subunit of the IL-2 receptor" refers to any native CD25 from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats), including "full-length," unprocessed CD25 and any form of CD25 derived from processing in cells, and also includes naturally occurring CD25 variants, such as splice variants or allelic variants. In certain embodiments, CD25 is human CD25.

[0898] "High-affinity IL-2 receptor" refers to the heterotrimeric form of the IL-2 receptor, which is composed of a receptor gamma subunit (also known as the universal cytokine receptor gamma subunit, gamma c or CD132), a receptor beta subunit (also known as CD122 or p70), and a receptor alpha subunit (also known as CD25 or p55). In contrast, a "medium-affinity IL-2 receptor" refers to an IL-2 receptor that contains only gamma and beta subunits, without an alpha subunit (see, e.g., Olejniczak and Kasprzak, MedSci Monit 14, RA179-189 (2008)).

[0899] "Antibody" is used in the broadest sense to encompass 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. An antibody may refer to an immunoglobulin, a tetrapeptide chain structure composed of two identical heavy chains and two identical light chains connected by interchain disulfide bonds. Immunoglobulins differ in their antigenicity due to the amino acid composition and arrangement order of their heavy chain constant regions. Consequently, immunoglobulins can be divided into five classes, or isotypes, namely IgM, IgD, IgG, IgA, and IgE, corresponding to μ, δ, γ, α, and ε chains, respectively. Within the same class of Ig, different subclasses can be formed based on the amino acid composition of the hinge region and the number and location of heavy chain disulfide bonds. For example, IgG can be divided into IgG1, IgG2, IgG3, and IgG4. Light chains are classified as either κ or λ chains based on differences in their constant regions. Each of the five classes of Ig can have either kappa or lambda chains. The approximately 110 amino acids near the N-terminus of both the heavy and light chains of antibodies vary greatly in sequence and constitute the variable region (V region); the remaining amino acid sequences near the C-terminus are relatively stable and constitute the constant region (C region). The variable region consists of three hypervariable regions (CDRs) and four framework regions (FRs) whose sequences are relatively conserved. These three hypervariable regions determine the specificity of the antibody and are also known as complementarity-determining regions (CDRs). Each light chain variable region (VL) and heavy chain variable region (VH) consists of three CDRs and four FRs, arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The three CDRs of the light chain are referred to as LCDR1, LCDR2, and LCDR3; the three CDRs of the heavy chain are referred to as HCDR1, HCDR2, and HCDR3.

[0900] "Antigen-binding fragment" encompasses 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 or VL or VHH), scFv, bivalent or trivalent or tetravalent antibodies, Bis-scFv, diabody, tribody, triabody, tetrabody, and epitope-binding fragments of any of the above (see, e.g., Holliger and Hudson, 2005, Nature Biotech. 23(9): 1126-1136; Adair and Lawson, 2005, Drug Design Reviews-Online 2(3), 209-217). In some embodiments, the antigen-binding fragment is VHH. Methods for producing 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). The Fab-Fv format was first disclosed in WO2009 / 040562, and its disulfide-stabilized form, Fab-dsFv, was first disclosed in WO2010 / 035012. Antigen-binding fragments of the present disclosure also include Fab and Fab' fragments described in WO2005 / 003169, WO2005 / 003170, and WO2005 / 003171. Multivalent antibodies may comprise multispecifics, such as bispecifics, or may be monospecifics (see, for example, WO92 / 22583 and WO05 / 113605), an example of which is Tri-Fab (or TFM) described in WO 92 / 22583.

[0901] "Fc domain" or "Fc region" is used to define the C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region. It includes native sequence Fc regions and variant Fc regions. Although the boundaries of the Fc region of an IgG heavy chain can vary slightly, the human IgG heavy chain Fc region is generally defined as extending from Cys226 or Pro230 to the carboxyl terminus of the heavy chain. However, antibodies produced by host cells may undergo post-translational cleavage, removing one or more, particularly one or two, amino acids from the C-terminus of the heavy chain. Therefore, antibodies produced by host cells by expressing a specific nucleic acid molecule encoding a full-length heavy chain may include a full-length heavy chain, or it may include a cleavage variant of the full-length heavy chain (also referred to in this disclosure as a "cleavage variant heavy chain"). This may be the case when the final two C-terminal amino acids of the heavy chain are glycine (G446) and lysine (K447, numbering according to the Kabat EU index). Therefore, the C-terminal lysine (K447) or the C-terminal glycine (G446) and lysine (K447) of the Fc region may be present or absent. If not otherwise specified, the amino acid sequence of the heavy chain comprising the Fc region (or the subunit of the Fc region defined in the present disclosure) is represented in the present disclosure as being free of a C-terminal glycine-lysine dipeptide. In one embodiment, the heavy chain of a subunit of the Fc region included in the immunoconjugate comprises an additional C-terminal glycine-lysine dipeptide (G446 and K447, numbering is according to the EU index of Kabat). In one embodiment, the heavy chain of a subunit of the Fc region included in the immunoconjugate comprises an additional C-terminal glycine residue (G446, numbering is according to the EU index of Kabat). Compositions of the present disclosure (e.g., pharmaceutical compositions) comprise a population of immunoconjugates of the present disclosure. A population of immunoconjugates may comprise molecules with full-length heavy chains and molecules with cleavage variant heavy chains. The population of immunoconjugates can be composed of a mixture of molecules with full-length heavy chains and molecules with cleaved variant heavy chains, wherein at least 50%, at least 60%, at least 70%, at least 80% or at least 90% of the immunoconjugates have cleaved variant heavy chains, which can be C-terminal lysine (K447) of the Fc region, or C-terminal glycine (G446) and lysine (K447) are absent. Unless otherwise specified in the present disclosure, the numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also known as the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

[0902] A "subunit" of an Fc region refers to one of the two polypeptides that form the Fc region dimer, i.e., the polypeptide comprising the C-terminal constant region of an immunoglobulin heavy chain that stabilizes its association. For example, a subunit of an IgG Fc region comprises the IgG CH2 and IgG CH3 constant domains. Throughout this disclosure, the terms "first subunit" or Fc1 and "second subunit" or Fc2 are not intended to confer a particular order or orientation and are used for ease of distinction.

[0903] “Modification (or mutation) that promotes the association of the first subunit and the second subunit of the Fc domains” is a peptide backbone operation or post-translational modification of the Fc domain subunits that reduces or prevents the polypeptide comprising the Fc domain subunit from being associated with the same polypeptide to form a homodimer. As used herein, specifically, the modification that promotes association includes separate modifications performed on each of the two Fc domain subunits (i.e., the first subunit and the second subunit of the Fc domain) that are desired to be associated, wherein the modifications are complementary to each other, thereby promoting the association of the two Fc domain subunits. For example, the modification that promotes association can change the structure or charge of one or both Fc domain subunits, thereby promoting their association in three dimensions or electrostatically, respectively. In this way, (hetero) dimerization occurs between a polypeptide comprising the first Fc domain subunit and a polypeptide comprising the second Fc domain subunit, which may be different in the sense that the other components (e.g., antigen binding moieties) fused to each subunit are different. In some embodiments, the modification that promotes association is included in an amino acid mutation in the Fc domains, specifically an amino acid substitution.

[0904] "Complementarity" refers to the combination of interactions that influence heavy chain / light chain pairing at the interface of, for example, CH1 and CL (or CH3 and CH3) of an antigen binding protein described herein. "Steric complementarity" or "conformational complementarity" refers to the compatibility of the three-dimensional structures at the interacting surfaces of, for example, CH1 and CL (or CH3 and CH3). "Electrostatic complementarity" refers to the compatibility of the placement of negatively and / or positively charged atoms at the interacting surfaces of, for example, CH1 and CL (or CH3 and CH3). Methods for measuring electrostatic complementarity at protein / protein interfaces are known in the art and are described, for example, in McCoy et al. (1997) J Mol Biol 268, 570-584; Lee et al., (2001) Protein Sci. 10, 362-377; and Chau et al. (1994) J Comp Mol Des 8, 51325. Methods of measuring steric complementarity at protein / protein interfaces are known in the art and are described, for example, in Lawrence et al. (1993) J Mol Biol 234, 946-950; Walls et al. (1992) J Mol Biol 228, 277-297; and Schueler-Furman et al. (2005) Proteins 60, 187-194.

[0905] "Effector function" when referring to antibody use refers to those biological activities that can be attributed to the Fc region of the antibody and vary with the antibody isotype. Examples of antibody effector functions include: C1q binding and complement dependent cytotoxicity (CDC), Fc receptor (FcR) binding, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), cytokine secretion, immune complex-mediated antigen uptake by antigen-presenting cells, cell surface receptors (e.g., B cell receptor) downregulation and B cell activation. ADCC is an immune mechanism that leads to the lysis of antibody-coated target cells by immune effector cells. Target cells are cells to which antibodies or derivatives thereof comprising the Fc region are generally specifically bound via the protein portion of the N-terminus of the Fc region. "Reduced ADCC" is defined as a reduction in the number of target cells that are lysed within a given time at a given concentration of antibody in the medium surrounding the target cells by the ADCC mechanism defined above, and / or an increase in the antibody concentration in the medium surrounding the target cells required for the lysis of a given number of target cells within a given time by the ADCC mechanism. A reduction in ADCC is relative to ADCC mediated by the same antibody produced by the same type of host cell but not engineered using the same standard production, purification, formulation, and storage methods known to those skilled in the art. For example, a reduction in ADCC mediated by an antibody comprising an amino acid substitution in its Fc domain that reduces ADCC is relative to ADCC mediated by the same antibody without the amino acid substitution in the Fc domain. Suitable assays for measuring ADCC are well known in the art (see, for example, WO2006 / 082515 or WO2012 / 130831).

[0906] An "activating Fc receptor" is an Fc receptor that, upon engagement by the Fc domain of an antibody, triggers signaling events that stimulate cells bearing the receptor to carry out effector functions. Human activating Fc receptors include FcγRIIIa (CD16a), FcγRI (CD64), FcγRIIa (CD32), and FcαRI (CD89).

[0907] An "immunoconjugate" refers to a polypeptide molecule comprising at least one IL-2 molecule and at least one antibody. As described herein, the IL-2 molecule can be linked to the antibody through a variety of interactions and in a variety of configurations. In certain embodiments, the IL-2 molecule is fused to the antibody via a peptide linker. Certain immunoconjugates according to the present invention essentially consist of an IL-2 molecule and an antibody connected by one or more linker sequences.

[0908] "Decreased affinity" or "decreased binding," e.g., decreased binding to IL-2Rα, refers to a decrease in the affinity of the corresponding interaction, e.g., as measured by SPR. The term also encompasses a decrease in affinity to zero or below the detection limit of the analytical method, i.e., complete abolition of the interaction. Conversely, "decreased affinity" or "increased binding" refers to an increase in the binding affinity of the corresponding interaction.

[0909] For the determination or definition of CDRs, the definitive delineation of CDRs and the identification of residues comprising the binding site of the antibody can be accomplished by resolving the structure of the antibody and / or resolving the structure of the antibody-ligand complex. This can be accomplished by 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 definitions, and conformational definitions.

[0910] The Kabat numbering system is a standard for numbering residues in antibodies and is commonly used to identify CDR regions (see, e.g., Johnson & Wu, 2000, Nucleic Acids Res., 28:214-8). The Chothia numbering system is similar to the Kabat numbering system, but takes into account the positions of certain structural loop regions. (See, e.g., Chothia et al., 1986, J. Mol. Biol., 196:901-17; Chothia et al., 1989, Nature, 342:877-83). The AbM numbering system uses an integrated suite of computer programs produced by the Oxford Molecular Group for modeling antibody structure (see, e.g., Martin et al., 1989, Proc Natl Acad Sci (USA), 86:9268-9272; "AbM™, 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 ab initio methods to model the tertiary structure of antibodies from the primary sequence (see Samudrala et al., 1999, "Ab Initio Protein Structure Prediction Using a Combined Hierarchical Approach," PROTEINS, Structure, Function and Genetics Suppl., 3: 194-198). Contact definitions are based on analysis of available complex crystal structures (see, e.g., MacCallum et al., 1996, J. Mol. Biol., 5: 732-45). In conformational definitions, CDR positions can be identified as residues that make enthalpic contributions to antigen binding (see, e.g., Makabe et al., 2008, Journal of Biological Chemistry, 283: 1156-1166). Other CDR boundary definitions may not strictly follow one of the above methods, but still overlap with at least a portion of the Kabat CDRs, although they may be shortened or lengthened based on predictions or experimental results that a particular residue or group of residues does not significantly affect antigen binding. As used herein, CDR may refer to a CDR defined by any method known in the art (including a combination of methods). The correspondence between various numbering systems is well known to those skilled in the art, and is exemplified by the following Table 1.

[0911] Table 1. Relationships between CDR numbering systems

[0912] The CDR amino acid residues of the VL and VH regions of the antibodies of the present disclosure conform in number and position to the well-known Kabat numbering system.

[0913] The antibodies of the present disclosure may be polyclonal, monoclonal, xenogeneic, allogeneic, isogenic, or modified forms thereof, with monoclonal antibodies being particularly suitable for use in a number of embodiments. In general, the antibodies of the present disclosure are recombinant antibodies. As used herein, "recombinant" refers generally to products such as cells or nucleic acids, proteins, or vectors, indicating that the cells, nucleic acids, proteins, or vectors have been modified by the introduction of heterologous nucleic acids or proteins or by altering native nucleic acids or proteins, or that the cells are derived from cells so modified. For example, recombinant cells express genes that are not present in the native (non-recombinant) cell form or express native genes that are abnormally expressed, underexpressed, or not expressed at all.

[0914] A "domain" of a polypeptide or protein refers to a folded protein structure that is capable of maintaining its tertiary structure independently of the rest of the protein. In general, a domain is responsible for a single functional property of a protein and in many cases can be added, removed, or transferred to other proteins without loss of function of the rest of the protein and / or the domain.

[0915] "Immunoglobulin domain" refers to a globular region of an antibody chain (e.g., a chain of a conventional tetrapeptide chain structure antibody or a heavy chain antibody chain), or a polypeptide consisting essentially of such a globular region. An immunoglobulin domain is characterized in that it maintains the immunoglobulin fold characteristic of an antibody molecule, consisting of a two-layer sandwich of approximately seven antiparallel beta-sheet strands arranged in two beta-sheets, optionally stabilized by conserved disulfide bonds.

[0916] An "immunoglobulin variable domain" is an immunoglobulin domain that essentially consists of four "framework regions," referred to in the art and hereinafter as "framework region 1" or "FR1," "framework region 2" or "FR2," "framework region 3" or "FR3," and "framework region 4" or "FR4," respectively, wherein the framework regions are separated by three "complementarity determining regions" or "CDRs," referred to in the art and hereinafter as "complementarity determining region 1" or "CDR1," "complementarity determining region 2" or "CDR2," and "complementarity determining region 3" or "CDR3," respectively. Thus, the general structure or sequence of an immunoglobulin variable domain can be represented as follows: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. An immunoglobulin variable domain confers specificity for an antigen by having an antigen-binding site.

[0917] "Immunoglobulin single variable domain" is generally used to refer to an immunoglobulin variable domain (which can be a heavy chain or light chain domain, including a VH, VHH or VL domain) that can form a functional antigen binding site without interacting with other variable domains (e.g., without the VH / VL interaction required between the VH and VL domains of conventional four-chain monoclonal antibodies). Examples of "immunoglobulin single variable domains" include nanobodies (including VHH, humanized VHH and / or camelized VH, such as camelized human VH), IgNAR, domains, (single domain) antibodies (e.g., dAbs™) that are VH domains or derived from VH domains, and (single domain) antibodies (e.g., dAbs™) that are VL domains or derived from VL domains. Immunoglobulin single variable domains based on and / or derived from heavy chain variable domains (e.g., VH or VHH domains) are generally preferred. A specific example of an immunoglobulin single variable domain is a "VHH domain" (or simply "VHH") as defined below.

[0918] "VHH domain", also known as heavy chain single domain antibody, VHH, VHH antibody fragment, VHH antibody, nanobody, is the variable domain of the antigen-binding immunoglobulin called "heavy chain antibody" (i.e., "antibody lacking light chain") (Hamers-Casterman C, Atarhouch T, Muyldermans S, Robinson G, Hamers C, Songa EB, Bendahman N, Hamers R.: "Naturally occurring antibodies devoid of light chains"; Nature 363, 446-448 (1993)). "VHH domain" is used to distinguish the variable domain from the heavy chain variable domain (referred to as "VH domain" in the present disclosure) and the light chain variable domain (referred to as "VL domain" in the present disclosure) present in conventional tetrapeptide chain structure antibodies. The VHH domain specifically binds to an epitope without the need for additional antigen-binding domains (in contrast to the VH or VL domains in conventional tetrapeptide antibodies, in which the epitope is recognized by both the VL and VH domains). A VHH domain is a small, stable, and highly efficient antigen recognition unit formed by a single immunoglobulin domain. The terms "heavy chain single domain antibody," "VHH domain," "VHH," "VHH antibody fragment," "VHH antibody," and "domain" ("Nanobody" is a trademark of Ablynx NV, Ghent, Belgium) are used interchangeably. A "VHH domain" includes, but is not limited to, a natural antibody produced by a camelid, an antibody produced by a camelid that has been humanized, or a VHH domain obtained through phage display screening. In some embodiments, a VHH domain comprises three CDRs and four framework regions, designated FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. In some embodiments, the VHH domain can be truncated at the N-terminus or C-terminus so that it comprises only part of FR1 and / or FR4, or lacks one or both of those framework regions, as long as the VHH substantially retains antigen binding and specificity. The total number of amino acid residues in a VHH domain will typically be in the range of 110 to 120, often between 112 and 115. However, it should be noted that smaller and longer sequences may also be suitable for the purposes described in the present disclosure.

[0919] As is well known in the art for VH and VHH domains, the total number of amino acid residues in each CDR may vary and may not correspond to the total number of amino acid residues indicated by the Kabat numbering (i.e., one or more positions numbered according to Kabat may not be occupied in the actual sequence, or the actual sequence may contain more amino acid residues than allowed by Kabat numbering). This means that, in general, the numbering according to Kabat may or may not correspond to the actual numbering of amino acid residues in the actual sequence. Other numbering systems or coding conventions include Chothia, IMGT, and AbM.

[0920] "Humanized antibodies," also known as CDR-grafted antibodies, are antibodies produced by transplanting non-human CDR sequences into the human variable region framework. This can overcome the strong immune response induced by chimeric antibodies due to the presence of a large number of non-human protein components. To avoid a simultaneous decrease in immunogenicity and activity, minimal reverse mutations can be performed on the fully human variable region to maintain activity. Examples of "humanization" include "humanizing" a Camelidae-derived VHH domain by replacing one or more amino acid residues in the amino acid sequence of the original VHH sequence with one or more amino acid residues present at corresponding positions in a VH domain of a conventional human tetrapeptide antibody (also referred to as "sequence optimization" in this disclosure; in addition to humanization, "sequence optimization" may also encompass other modifications to the sequence by one or more mutations that provide improved VHH properties, such as removal of potential post-translational modification sites). A humanized VHH domain may contain one or more fully human framework region sequences, and in some embodiments, may contain human framework region sequences from IGHV3. Humanization methods such as protein surface amino acid humanization (resurfacing) and antibody humanization universal framework transplantation (CDR grafting to a universal framework), i.e., CDR "grafting" onto other "scaffolds" (including but not limited to human scaffolds or non-immunoglobulin scaffolds). Scaffolds and techniques suitable for the CDR transplantation are known in the art. For example, the germline DNA sequences of human heavy and light chain variable region genes can be found in the "VBase" human germline sequence database, as well as in Kabat, EA et al., 1991 Sequences of Proteins of Immunological Interest, 5th edition. The humanized antibodies disclosed herein also include humanized antibodies that are further subjected to affinity maturation of CDRs by phage display. In addition, in order to avoid a decrease in immunogenicity and the resulting decrease in activity, the human antibody variable region framework sequences can be subjected to minimal reverse mutation or back mutation to maintain activity.

[0921] An "affinity matured" antibody is one that has one or more alterations in one or more hypervariable regions (HVRs) that result in improved affinity for the antigen compared to a parent antibody that does not have such alterations. For example, an "affinity matured" PD-1 antibody has one or more alterations in one or more CDRs that result in increased affinity for the antigen compared to its parent antibody. Affinity matured antibodies can be prepared by methods known in the art, for example, as described in 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.

[0922] Typically, the PD-1 antibodies, immunoconjugates of the present disclosure will be expressed as preferably 10 -7 to 10 -10 Mole / liter (M), more preferably 10 -8 to 10 -10 mol / L, even more preferably 10 -9 to 10 -10 or lower dissociation constant (KD), and / or with a dissociation constant of 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 to be bound (i.e. PD-1). Any value greater than 10 -4A KD value of M is generally considered to be indicative of nonspecific binding. Specific binding of an antigen-binding protein to an antigen or epitope can be determined in any suitable manner known in the art, including, for example, surface plasmon resonance (SPR) assays, Scatchard assays, and / or competitive binding assays (e.g., radioimmunoassays (RIA), enzyme immunoassays (EIA), and sandwich competition assays) as described herein.

[0923] "Antigen" refers to a molecule of an immunocompetent vertebrate used for immunization to produce antibodies that recognize the antigen, or to screen an expression library (e.g., a phage, yeast, or ribosome display library). In the present disclosure, antigens are defined more broadly to include target molecules specifically recognized by antibodies, as well as a portion or mimetic of a molecule used in an immunization process for producing antibodies or in a library screening for selecting antibodies. For example, for antibodies that bind to human PD-1 of the present disclosure, monomers and multimers (e.g., dimers, trimers, etc.) of human PD-1, as well as truncated variants and other variants of human PD-1 are referred to as PD-1 antigens.

[0924] "Epitope" refers to a site on an antigen that binds to an immunoglobulin or antibody. An epitope can be formed by adjacent amino acids, or non-adjacent amino acids juxtaposed by tertiary folding of the protein. Epitopes formed by adjacent amino acids are generally retained after exposure to denaturing solvents, while epitopes formed by tertiary folding are generally lost after treatment with denaturing solvents. An epitope generally comprises at least 3-15 amino acids in a unique spatial conformation. Methods for determining which epitope is bound by a given antibody are well known in the art and include immunoblotting and immunoprecipitation assays. Methods for determining the spatial conformation of an epitope include techniques in the art and the techniques described herein, such as X-ray crystallography and two-dimensional nuclear magnetic resonance.

[0925] "Specific binding" and "selective binding" refer to the binding of an antibody to an epitope on a predetermined antigen. For example, when human PD-1 or its epitope is used as an analyte and an antibody is used as a ligand, the antibody binds to the epitope at a specific binding rate of less than 10 -7 M or even smaller equilibrium dissociation constant (K D ) binds to a predetermined antigen or an epitope thereof, and its affinity for binding to the predetermined antigen or an epitope thereof is at least twice that of its affinity for binding to a nonspecific antigen (such as BSA, etc.) other than the predetermined antigen (or its epitope) or a closely related antigen. "Antibody that recognizes an antigen" may be used interchangeably with "specifically binding antibody" in this disclosure.

[0926] "Binding affinity" or "avidity" is used in the present disclosure as a measure of the strength of a non-covalent interaction between two molecules (e.g., an antibody or portion thereof and an antigen). The binding affinity between two molecules can be quantified by determining the dissociation constant (KD). KD can be determined by measuring the kinetics of complex formation and dissociation using, for example, a surface plasmon resonance (SPR) method (Biacore). The rate constants corresponding to the association and dissociation of a monovalent complex are referred to as the association rate constant ka (or kon) and the dissociation rate constant kd (or koff), respectively. K 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 can be calculated even for complex mixtures by methods such as those described in Caceci et al. (1984, Byte 9:340-362). For example, K can be determined using a double filtration nitrocellulose filter binding assay such as that disclosed in Wong & Lohman (1993, Proc. Natl. Acad. Sci. USA 90:5428-5432). D Other standard assays for assessing the binding ability of an antibody to a target antigen are known in the art and include, for example, ELISA, Western blot, RIA, and flow cytometry analysis, as well as other assays exemplified elsewhere in this disclosure. The binding kinetics and binding affinity of an antibody can also be determined by standard assays known in the art, such as surface plasmon resonance (SPR), for example, by using Biacore TM The K of each antibody / antigen complex can be compared by comparing the K D The K values ​​can be used to compare the binding affinities associated with different molecular interactions, for example, the binding affinities of different antibodies for a given antigen. Similarly, the specificity of an interaction can be determined and compared by determining and comparing the K values ​​for the interaction of interest (e.g., a specific interaction between an antibody and an antigen). D The K values ​​were compared with those of non-target interactions (e.g., control antibodies known not to bind CD40). D The value is evaluated.

[0927] A "conservative substitution" refers to a substitution with another amino acid residue having 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. In addition, 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 non-polar side chains. In addition, 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 substituting an amino acid residue in a group that exhibits similar properties as described above, it will not exhibit specific changes in properties.

[0928] "Homology," "identity," or "sequence identity" refers to the sequence similarity between two polynucleotide sequences or between two polypeptides. When a position in the two compared sequences is 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 percent 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 × 100%. For example, if 6 out of 10 positions in the two sequences match or are homologous when the sequences are optimally aligned, then the two sequences are 60% homologous. Generally, a comparison is made when the two sequences are aligned to achieve the maximum percent homology.

[0929] "Administering" and "treating" as applied to an animal, a human, a laboratory subject, a cell, a tissue, an organ or a biological fluid, refers to the contacting of an exogenous drug, therapeutic agent, diagnostic agent or composition with an animal, a human, a subject, a cell, a tissue, an organ or a biological fluid. "Administering" and "treating" can refer to, for example, therapeutic, pharmacokinetics, diagnostics, research and experimental procedures. Treatment of cells includes contacting an agent with a cell, and contacting an agent with a fluid, wherein the fluid is in contact with the cell. "Administering" and "treating" also mean treating, for example, a cell in vitro and ex vivo, by an agent, a diagnostic, a binding composition or by another cell. "Treatment" as applied to a human, veterinary or research subject, refers to therapeutic treatment, prophylactic or preventative measures, research and diagnostic applications.

[0930] "Treatment" means administering an internal or external therapeutic agent, such as a composition comprising any of the binding compounds of the present disclosure, to a patient who has one or more symptoms of a disease for which the therapeutic agent is known to have a therapeutic effect. Typically, the therapeutic agent is administered in an amount effective to alleviate one or more symptoms of the disease in the patient or population being treated, to induce regression of such symptoms or to inhibit the progression of such symptoms to any clinically measurable degree. The amount of a therapeutic agent effective to alleviate any specific disease symptom (also referred to as a "therapeutically effective amount") can vary according to a variety of factors, such as the patient's disease state, age, and weight, and the ability of the drug to produce the desired therapeutic effect in the patient. Whether the symptoms of the disease have been alleviated can be assessed by any clinical test method commonly used by a physician or other health care professional to assess the severity or progression of the symptoms. Although embodiments of the present disclosure (e.g., methods of treatment or articles of manufacture) may not be effective in alleviating every symptom of the target disease, they should alleviate the target disease symptoms in a statistically significant number of patients as determined by any statistical test known in the art, such as Student's t-test, chi-square test, U test according to Mann and Whitney, Kruskal-Wallis test (H test), Jonckheere-Terpstra test, and Wilcoxon test.

[0931] An "effective amount" encompasses an amount sufficient to ameliorate or prevent the symptoms or conditions of a medical condition. An effective amount also means an amount sufficient to permit or facilitate diagnosis. The effective amount for a particular patient or veterinary subject may vary depending on factors such as the condition to be treated, the patient's overall health, the route and dosage of administration, and the severity of side effects. An effective amount can be the maximum dose or dosage regimen that avoids significant side effects or toxic effects.

[0932] "Tm value" refers to the thermal denaturation temperature of protein, that is, the temperature at which half of the protein unfolds. At this time, the spatial structure of the protein is destroyed. Therefore, the higher the Tm value, the higher the thermal stability of the protein.

[0933] Example

[0934] The present disclosure is further described below with reference to the following examples, but these examples are not intended to limit the scope of this disclosure. Experimental methods in the examples herein where specific conditions are not specified generally follow conventional conditions, such as those in the Cold Spring Harbor Laboratory Manual of Antibody Techniques and the Molecular Cloning Manual, or the conditions recommended by the raw material or product manufacturer. Reagents where the specific source is not specified are commercially available.

[0935] Example 1. Screening, preparation and modification of anti-PD-1 nanobodies

[0936] 1. Antibody Screening

[0937] This study used His-tagged human PD-1 as an immune antigen (AcroBiosystems, PD1-H5221) to immunize alpacas and construct a yeast display library of nanobodies (VHHs) for antibody screening. Biotin-labeled human PD-1 and cynomolgus monkey PD-1 were used as screening antigens (AcroBiosystems, PD1-H82E4; PD1-C82E6). Through monoclonal identification, sequencing, and sequence analysis, 18 unique VHH sequences that cross-bind with human and monkey PD-1 antigens were obtained.

[0938] The sequence of human PD-1 protein (NP_005009.2) is as follows, where the underlined portion is the extracellular region sequence, with amino acids starting and ending at Leu25-Gln 167.

[0939] >Human PD-1 amino acid sequence

[0940] The A17 sequence is shown below, and the CDRs use the Kabat numbering convention.

[0941] >A17 variable region

[0942] >A17 CDR1

[0943] >A17 CDR2

[0944] >A17 CDR3

[0945] A17 was linked to a human IgG4 Fc fragment (including the hinge region and with S228P according to the EU numbering system) to construct a VHH-Fc antibody. The plasmid was transiently transfected into 293 cells for expression and purification using a protein A column. The column was washed with PBS and eluted with 0.1 M glycine buffer, pH 2.5. The column was dialyzed into PBS, pH 7.4. The target protein was detected.

[0946] >hIgG4

[0947] >A17_hIgG4

[0948] 2. Humanization and backmutation

[0949] Antibody humanization was performed using the CDR-grafting method. The parental PD-1 antibody was aligned with fully human germline genes in the IMGT or NCBI / igblast databases. Human germline genes IGHV 3-23 (IGHV3-23*01; IGHV3-23*04), which are highly homologous to the PD-1 nanobody, were selected as humanization templates. CDRs were grafted, retaining four key residues in FR2 of the nanobody (Y37, E44, R45, and L47). Key core residues near the CDR regions and Vernier position residues involved in CDR interactions were backmutated. Humanized antibodies A17h1, A17h2, and A17h3 were obtained.

[0950] >A17h1 variable region

[0951] >A17h2 variable region

[0952] >A17h3 variable region

[0953] 3. Remove TCE (T cell epitope), reduce antibody deamidation, and reduce antibody isomerization modification

[0954] The following sequence is obtained:

[0955] >A17m09 variable region

[0956] A17m09 was further selected to germline the antibody sequence to improve the degree of humanization, and the following sequence was obtained:

[0957] >A17m0901 variable region

[0958] >A17m0902 variable region

[0959] >A17m0903 variable region

[0960] >A17m0905 variable region

[0961] That is, A17 of the present disclosure has the following general sequence:

[0962] CDR1: DYSMS (SEQ ID NO: 3)

[0963] CDR2: IISGSGVIX1HYVDSVKG, wherein X1 is selected from T or A (SEQ ID NO: 16)

[0964] CDR3: VSDWX4X5Y, wherein X4 is selected from D or E, and X5 is selected from D or E (SEQ ID NO: 17)

[0965] Specifically, CDR2 can be:

[0966] CDR3 can be:

[0967] Example 2. Functional detection of anti-PD-1 nanobody

[0968] 1.ELISA

[0969] Use PBS to dissolve human PD-1 protein (his tag) to 1μg / mL, add 100uL / well to a 96-well plate, and incubate at 4°C overnight to coat the antigen. Wash three times with PBST (PBS+0.05% tween20) solution. Add PBST / 1% BSA solution and incubate at 37°C for 1h for blocking. After washing three times with PBST solution, add different concentrations of PD-1 candidate VHH-Fc antibodies (diluted with PBST / 1% BSA solution) and incubate at 37°C for 1.5 hours. Wash three times with PBST solution. Add 100μL HRP-conjugated anti-human IgG4 Fc secondary antibody (Thermo) solution and incubate at 37°C for 1 hour. After washing three times with PBST solution, add 100μL / well TMB solution, react at room temperature for 5min, add 50μL stop solution, and then use an enzyme reader to read the 450nM value and calculate the EC 50 ,Table 4 shows that A17_hIgG4 has a strong binding affinity with PD-1 antigen.

[0970] Table 4. EC of anti-PD-1 nanobody detected by ELISA 50 value

[0971] 2.FACS

[0972] To detect the binding ability of anti-PD-1 nanobody to PD-1 expressed on cell membrane, different concentrations of candidate anti-PD-1 VHH-Fc antibody were added to 10 5 100 μL of anti-human IgG Fc fluorescent secondary antibody (Biolegend) was added to Jurkat cells (Cat: J1250, Promega) overexpressing human PD-1 at room temperature for 20 min and washed twice with PBS. The cells were then incubated at room temperature for 20 min and washed twice with PBS. The cells were resuspended in 250 μL of PBS and the fluorescence signal was detected by FACS. EC 50 Pembrolizumab (purchased from Baiying Bio) was used as a control. Table 5 shows the EC values ​​of A17_hIgG4.50 The value was 0.3568 nM, which was significantly better than other antibodies obtained in the simultaneous screening (results not shown).

[0973] Table 5. ECs of anti-PD-1 nanobody binding to cell surface PD-1 detected by FACS 50 value

[0974] 3. PD-1 / PD-L1 reporter gene system to detect the blocking effect of anti-PD-1 nanoantibodies on PD-1

[0975] CHO-K1 cells overexpressing PD-L1 were diluted to 4 × 10 5 / mL, add 100 μL to each well of a 96-well plate and culture overnight. After aspirating the culture medium, quickly add 40 μL of 1.25×10 6 Jurkat cells with PD-1 / mL and 40 μL of anti-PD-1 nanobody solution (diluted with 1640+2% FBS solution) at different concentrations were incubated at 37°C for 6 hours, returned to room temperature, and 40 μL of Bright-glo reagent (Cat: E2620, Promega) was added to each well. The cells were shaken at 350 rpm in the dark for 5 minutes. After standing at room temperature in the dark for 5 minutes, the fluorescence value was read using a microplate reader and the IC was calculated. 50 Pembrolizumab and hIgG4 isotype antibodies were used as controls. As shown in Table 6, the biological functional activity IC 50 The value was 1.199 nM, which was significantly better than other antibodies screened simultaneously in the present disclosure (e.g., A3_hIgG4 was 7.62, A13_hIgG4 was 3.208; the sequences of A3_hIgG4 and A13_hIgG4 are not shown).

[0976] Table 6. IC values ​​of anti-PD-1 nanoantibodies detected using the PD-1 / PD-L1 NFAT reporter gene system 50 value

[0977] The above method was used to functionally verify the anti-PD-1 nanobody. See Table 7 for the IC of A17h1_hIgG4. 50 The activity is similar to that of the positive antibody Pembrolizumab. Among the candidate molecules after sequence modification, the biological activity IC 50 The values ​​were 0.5307nM and 0.4352nM, respectively, which were better than Pembrolizumab.

[0978] Table 7. IC values ​​of modified anti-PD-1 nanobodies detected by PD-1 / PD-L1 NFAT reporter gene system 50

[0979] 4. Identification of the binding affinity of the modified anti-PD-1 nanobody to the antigen PD-1

[0980] Surface plasmon resonance (SPR) was used to measure the affinity of anti-PD-1 nanobodies for the PD-1 antigen using a Biacore 8k (GE Healthcare). A CM5 sensor chip was used, and the mobile phase consisted of HBS-EP+ buffer (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.05% surfactant P20). Anti-human IgG (Fc) antibody was prepared at a concentration of 30 μg / mL in 10 mM sodium acetate buffer (pH 5.0). The immobilization program was selected for automated amino-coupled immobilization of the anti-human IgG (Fc) antibody channel. Each test antibody was prepared as a ligand in HBS-EP+ buffer and captured using the anti-human IgG (Fc) antibody on the chip channel. Human antigen protein (Sino Biological, 10377-H08H; 90311-C08H) was used as the analyte and prepared with HBS-EP+ buffer solution. The analyte was diluted 2-fold and flowed through the experimental channel and reference channel at a flow rate of 30 μL / min. The binding time was 1 minute and the dissociation time was 15 minutes. The regeneration buffer 10mM Glycine pH 1.5 (GE Healthcare, BR-1003-54) was run at a flow rate of 10 μL / min for 30 seconds. The association rate Ka and dissociation rate Kd, as well as the dissociation constant (i.e., affinity K) were calculated. D ). See Table 8 for the results.

[0981] Table 8. SPR affinity data of anti-PD-1 nanobody binding to human PD-1 antigen

[0982] The results showed that the humanized antibody A17h1_hIgG4 and the subsequently modified and optimized antibodies A17m09_hIgG4, A17m0901_hIgG4, and A17m0902_hIgG4 had similar K values ​​to Pembrolizumab. D The dissociation times of A17h1_hIgG4, A17m09_hIgG4, A17m0901_hIgG4, and A17m0902_hIgG4 were all slower than those of Pembrolizumab.

[0983] 5. DC:T mixed lymphocyte reaction (MLR) to test the in vitro efficacy of the modified anti-PD-1 nanobody

[0984] Co-culturing mature dendritic cells (DCs) with allogeneic T cells can activate T cells and promote their cytokine secretion. This process is inhibited by PD-1 / PD-L1 signaling, and the use of PD-1 antibodies can relieve this inhibition and promote T cell activation. Therefore, the DC:T mixed lymphocyte reaction assay can be used to test the in vitro efficacy of PD-1 antibodies.

[0985] Monocytes were isolated from fresh peripheral blood (PBMC) of a healthy individual using a sorting kit (cat: 19359, stemcell). Monocytes were induced to differentiate into mature DC cells after seven days of culture using a DC cell induction kit (cat: 10985, stemcell). Allogeneic T cells were then isolated from fresh PBMC of another healthy individual using a sorting kit (cat: 17951, stemcell). 5,000 mature DC cells were co-cultured with 50,000 allogeneic T cells, and candidate antibodies of corresponding concentrations were added. After six days of co-culture, the cell supernatant was aspirated, and the IFN-γ concentration was detected using a Cisbio-HTRF IFN-γ detection kit (Cat: 62HIFNGPEH, PerkinElmer), and the EC was calculated. 50 .

[0986] As shown in Figure 1, A17m09_hIgG4 showed in vitro efficacy that was essentially the same as that of Pembrolizumab in the MLR experiment. 50 The EC of pembrolizumab is 0.2931 nM. 50 It is 0.3271nM.

[0987] Example 3. Construction and activity detection of fusion protein of anti-PD-1 antibody and IL-2 mutant (PD-1-IL-2v)

[0988] 1. Molecular Construction

[0989] Fusion proteins of anti-PD-1 antibodies and IL-2 mutants were constructed according to the molecular format schematic diagram shown in Figure 2. The Fc of the molecules in Figure 2 all used Knob-in-Hole (KIH) technology, that is, S354C, T366W (constituting the Knob chain) and Y349C, T366S, L368A, Y407V (Eu numbering system) (constituting the Hole chain) mutations were introduced into the two Fcs to promote heterodimer formation; to facilitate purification, H435R and Y436F (Eu numbering system) were additionally introduced into the Hole chain; in addition, L234A, L235A, and P329G mutations were introduced into both the Knob and Hole chains to eliminate ADCC effects; for the heavy chain without a light chain pairing, a C220S mutation was introduced into the hinge region to remove a free, unpaired cysteine. IL-2v (V91T) containing T3A / R38E / F42A / V91T / C125A mutations was used.

[0990] In the molecule A-V91T with the structure shown in Figure 2, the anti-PD-1 nanoantibody A17m0902 is coupled to the N-terminus of the Knob and Hole chains respectively through the hinge region of the IgG1 heavy chain; IL-2v(V91T) is connected to the C-terminus of the Knob chain of Fc through a (G4S)3 linker.

[0991] The sequences of each molecule are as follows, with the Fc region underlined and the linker in italics.

[0992] >A-V91T Knob chain

[0993] >A-V91T Hole Chain

[0994] >IL-2 (including T3A / R38E / F42A / V91T / C125A mutations)

[0995] The above fusion protein was expressed in HEK293 cells and purified by Protein A. The molecular weight of the purified product was confirmed by SDS-PAGE to be consistent with the expectation, proving that the target protein was prepared.

[0996] 2. Detection of PD-1-IL-2v-promoted M07e cell proliferation activity

[0997] M07e is a human cell line that expresses only IL-2Rβ and IL-2Rγ, but not IL-2Rα. To test the activity of the different forms of the PD-1-IL2v(V91T) fusion protein and their dependence on PD-1 expression, we overexpressed human PD-1 (SEQ ID NO: 1) in the M07e cell line to generate the M07e-hPD-1 cell line. The effects of the fusion proteins on cell proliferation were then tested in both M07e and M07e-hPD-1 cell lines.

[0998] Complete culture medium: RPMI 1640 + 2 mM L-glutamine + 1 mM sodium pyruvate + 10% fetal bovine serum + 15 ng / mL GM-CSF; basal medium: RPMI 1640 + 2 mM L-glutamine + 1 mM sodium pyruvate + 10% fetal bovine serum.

[0999] M07e cell proliferation assay: M07e cells or M07e-hPD1 cells were cultured in complete culture medium at 37°C and 5% CO2. The cells were passaged and collected by centrifugation after 1-2 days. The cells were washed three times with PBS and resuspended in basal culture medium to a concentration of 6.0×10 5 A cell suspension of 100 cells was placed in a 96-well plate, with a volume of 50 μL per well. 50 μL of the test drug at a 2× concentration, prepared in basal culture medium, was added. After 72 hours of cell culture, 100 μL of lysis buffer (CELLTITER-Glo reagent (Promega)) was added to each well. The cells were mixed thoroughly, and the absorbance was measured at 570 nm using a microplate reader with a reference wavelength of 630 nm.

[1000] The data on the proliferative activity of the PD-1-IL-2v(V91T) fusion protein on M07e and M07e-hPD-1 cells are shown in Table 12. A PEGylated IL-2 mutant (PEG-IL-2v) was used as a positive control. The IL-2 mutant contained T3A, F42A, L72G, N26Q, N29S, N71Q, and C125A mutations, which did not bind to IL-2Rα but could bind to the affinity receptor IL-2Rβγ in IL-2.

[1001] The results showed that PEG-IL-2v had comparable activity against M07e and M07e-hPD-1 cell proliferation. The various PD-1-IL-2(V91T) variants exhibited significantly greater activity against M07e-hPD-1 cells than against M07e cells, suggesting that anti-PD-1 antibodies can increase the selectivity of IL-2 mutants for PD-1-positive cells. Furthermore, the various PD-1-IL-2(V91T) variants exhibited comparable activity against M07e cells. However, C-V91T exhibited slightly greater activity against M07e-hPD-1 cells than A-V91T, which in turn exhibited greater activity than B-V91T and E-V91T.

[1002] Table 9. Proliferative activity of PD-1-IL-2v(V91T) on M07e and M07e-hPD-1 cells

[1003] Example 4. Construction of PD-1-IL-2v carrying different IL-2 mutants

[1004] Although PD-1-IL-2v(V91T) is less active than PEG-IL-2v in M07e cells without PD-1 expression, it can still stimulate cell proliferation, indicating that IL-2v(V91T) can still strongly bind to the IL-2 medium-affinity receptor IL-2Rβγ, and PD-1-IL-2(V91T) may still systemically activate NK and CD8 + T cell toxicity. To further reduce the toxicity of PD-1-IL-2v and make IL-2 activity more dependent on PD-1 expression, we constructed PD-1-IL-2v with different IL-2 mutants based on the A-V91T molecular form. All IL-2 mutants carry the F42A and L72G mutations to eliminate IL-2 binding to IL-2Rα. In addition, for stability considerations, N26Q, N29S, and N71Q were added to the IL-2 mutants to remove possible deamidation modifications in IL-2 and enhance IL-2 stability. Furthermore, each of these IL-2 mutants carries unique mutations, as shown in Table 10, which, to varying degrees, reduce the affinity of IL-2 for the intermediate-affinity receptor IL-2Rβγ.

[1005] Table 10. IL-2 mutations in PD-1-IL-2v

[1006] >IL-2 sequence in A-PC

[1007] >IL-2 sequence in A-V91T-02

[1008] >IL-2 sequence in A-D20A

[1009] >IL-2 sequence in A-D20N

[1010] >IL-2 sequence in A-N88D

[1011] >IL-2 sequence in A-N88R

[1012] >IL-2 sequence in A-Q126D

[1013] >IL-2 sequence in A-H16A / D84S

[1014] L234A and L235A mutations were introduced into the Fc region to eliminate the ADCC effect; S354C, T366W (constituting the Knob chain) and Y349C, T366S, L368A, Y407V (Eu numbering system) (constituting the Hole chain) mutations were introduced to promote heterodimer formation; and a C220S mutation was introduced into the hinge region. To facilitate purification, the antibody may contain H435R, Y436F (Eu numbering system) mutations in the Hole chain. In addition, the Hole chain of the Fc region of these antibodies may be D356, L358 or E356, M358 (Eu numbering system) at amino acids 356 and 358, respectively. They appear in different alleles and do not affect the function of the antibody.

[1015] >Fc Knob chain (D356, L358)

[1016] >Fc Hole chain (D356, L358)

[1017] >Fc Knob chain (E356, M358)

[1018] >Fc Hole chain (E356, M358)

[1019] As an example, the full-length sequences of A-D20A and A-N88D are given, with the Fc underlined and the linker in italics.

[1020] >A-D20A Knob chain (D356, L358)

[1021] >A-D20A Hole Chain (D356, L358)

[1022] >A-D20A Knob Chain (E356, M358)

[1023] >A-D20A Hole Chain (E356, M358)

[1024] >Knob chain for A-N88D (D356, L358)

[1025] >A-N88D Hole Chain (D356, L358)

[1026] >Knob chain for A-N88D (E356, M358)

[1027] >A-N88D Hole Chain (E356, M358)

[1028] Furthermore, to demonstrate the dependence of PD-1-IL-2v on PD-1, we partially replaced the anti-PD-1 antibody in PD-1-IL-2v with an antibody targeting an unrelated antigen, while retaining the mutation in the IL-2 portion. This resulted in IgG-PC, the corresponding IgG-V91T containing V91T on IL-2, the corresponding IgG-D20A containing D20A on IL-2, and the corresponding IgG-N88D containing N88D on IL-2. The only difference between IgG-PC and IgG-V91T, IgG-D20A, and IgG-N88D is the IL-2 portion. The sequences are as follows:

[1029] >IgG-PC Knob chain

[1030] > Knob chain of IgG-V91T

[1031] > Knob chain of IgG-N88D

[1032] > Knob chain of IgG-D20A

[1033] >Hole chain of IgG-PC, IgG-V91T, IgG-D20A, or IgG-N88D

[1034] >Light chain of IgG-PC, IgG-V91T, IgG-D20A, or IgG-N88D

[1035] The above fusion protein was expressed in HEK293 cells and purified by Protein A. The molecular weight of the purified product was confirmed by SDS-PAGE to be consistent with the expectation, indicating that the target protein was obtained.

[1036] In addition, the wild-type IL-2 sequence of the present disclosure is:

[1037] >wt IL-2

[1038] Example 5. Detection of PD-1-IL-2v-promoting M07e cell proliferation activity

[1039] To examine the activity of the PD-1-IL-2v fusion proteins with different IL-2 mutants and their dependence on PD-1 expression, we tested the effects of the fusion proteins described in Example 4 on cell proliferation in M07e and M07e-hPD-1 cell lines. The experimental methods are described in Example 3. The relative cell proliferation activity of PD-1-IL-2v was calculated as follows:

[1040] in, Indicates the activity of PD-1-IL-2v at the concentration of conc, Represents the luminescence intensity of cell lysate when stimulated by PD-1-IL-2v at the concentration of conc, Luminescence intensity of cell lysate after stimulation of M07e or M07e-hPD-1 cell lines with the positive control molecule A-PC at a concentration of 1000 nM, Lumin Medium The background luminescence intensity is shown when only culture medium is added to M07e or M07e-hPD-1 cell lines without any PD-1-IL2v or PEG-IL-2v stimulation.

[1041] The experimental results are shown in Table 11 and Figures 3A and 3B. The results showed that 1) in M07e cells, A-PC and PEG-IL-2v had comparable activity, indicating that in the absence of PD-1 overexpression, the activity of antibody-bound IL-2 and PEG-conjugated IL-2 was consistent; A-V91T and IgG-V91T had comparable activity, but weaker than A-PC, indicating that V91T did reduce IL-2 activity but still stimulated M07e proliferation; other mutants, including D20A, D20N, N88R, N88D, and Q126D, essentially failed to activate M07e cells, with only A-N88D showing weak activity at a concentration of 1000 nM. 2) In M07e-hPD-1 cells, due to the overexpression of PD-1 on the cells, the activity of each PD-1-IL-2v at lower concentrations far exceeded that of IgG-V91T and PEG-IL-2v, indicating that anti-PD-1 antibodies significantly increased the dependence of the PD-1-IL-2v cell proliferation-promoting activity on PD-1 expression; compared with A-PC and A-V91T, the maximum activity of other PD-1-IL-2v on M07e-hPD-1 cell proliferation was slightly lower. 3) Comparison of the activity of each PD-1-IL-2v in M07e and M07e-hPD-1 cell proliferation assays revealed that PD-1-IL2v with D20A, D20N, N88R, N88D, or Q126D mutations essentially did not activate M07e cells but strongly stimulated the proliferation of M07e-hPD-1 cells, demonstrating that PD-1-IL-2v reduced activation of PD-1-negative cells while retaining selective activation of PD-1-positive cells. A-PC and A-V91T, while selectively activating PD-1-positive cells, could also activate PD-1-negative cells.

[1042] Table 11. Proliferative activity of PD-1-IL-2v with different IL-2 mutations on M07e and M07e-hPD-1 cells

[1043] To further demonstrate the PD-1 dependence of PD-1-IL-2v(N88D) and PD-1-IL-2v(D20A), we compared the effects of A-N88D with IgG-N88D, and A-D20A with IgG-D20 on cell proliferation in the M07e-hPD-1 cell line. The results are shown in Table 12 and Figure 4. The results showed that at a higher concentration (1000 nM), IgG-N88D and IgG-D20A were unable to activate M07e-hPD-1 cell proliferation, whereas A-N88D and A-D20A activated M07e-hPD-1 cell proliferation at lower concentrations.

[1044] Table 12. Proliferative activity of PD-1-IL-2v or IgG-IL-2v on M07e-hPD-1 cells

[1045] Example 6. Detection of PD-1-IL-2v T cell proliferation activity

[1046] Unstimulated T cells do not express or low-express PD-1, while T cells stimulated with CD3 antibodies highly express PD-1. To test whether the PD-1-IL-2v fusion proteins with different IL-2 mutants can activate T cells from human peripheral blood cells (PBMCs) with low and high PD-1 expression, we isolated T cells from PBMCs and stimulated the isolated T cells with CD3 antibodies to examine the effects of the fusion proteins on the proliferation of unstimulated and stimulated T cells.

[1047] T cell proliferation assay stimulated by CD3 antibody: After cryopreserved human PBMCs were revived, T cells were isolated using the Pan T Cell Isolation Kit (Miltenyi, 130-096-535). CD3 antibody was diluted to 0.1 μg / mL with PBS and added to a 10 cm culture dish. The dish was incubated at 37°C for 2 hours for coating, and then washed with PBS for later use. T cells were plated at 2×10 6 Cells / mL were plated in a 10 cm culture dish pre-coated with CD3 antibody and stimulated for 24 h at 37°C and 5% CO2. The stimulated T cells were collected, washed, counted, and the cell density was adjusted to 5×10 5 Cells were plated at 500 μL per well in a 96-well plate. The test samples were serially diluted with culture medium to prepare a 5× working solution. 50 μL of each test sample was added to the stimulated T cells and mixed thoroughly. The cells were cultured for 4 days. Following drug treatment, the cells were washed twice with FACS buffer (PBS + 2% FBS) and labeled for viability using Fixable Viability Dye (eBioscience, 65-0865-14). Following flow cytometry staining with antibodies against CD3 (BD, 565491), CD4 (BD, 558116), CD8 (BD, 565310), and Ki-67 (eBioscience, 12-5698-82), cell proliferation was assessed by FACS.

[1048] Unstimulated T cell proliferation assay: After separation, the T cells were counted and counted as 5×10 5Cells / mL were plated in a 96-well plate, with 200 μL per well. The test sample was serially diluted with culture medium to prepare a 5× working solution. 50 μL of each test sample concentration was added to the stimulated T cells, mixed thoroughly, and cultured for 4 days. Following drug treatment, the cells were washed twice with FACS buffer, labeled for viability with FVD780, and stained with flow cytometry antibodies before cell proliferation was assessed using a FACS analyzer.

[1049] The experimental results are shown in Table 13, Table 14 and Figures 5A, 5B, 6A and 6B. The results showed that 1) in unstimulated T cells, A-PC showed the activity of activating T proliferation (CD8 + T cell proliferation EC 50 =21.46nM, CD4 + T cell proliferation EC 50 =8.61nM); A-V91T showed weaker activity than A-PC, indicating that V91T does reduce IL-2 activity but can still promote the proliferation of unstimulated T cells; other mutants, including D20A, D20N, N88R, N88D, and Q126D, showed no T cell activation at concentrations up to 100nM. 2) In T cells stimulated with CD3 antibodies, each PD-1-IL-2v exhibited significant T cell proliferation-promoting activity at a relatively low concentration (0.1nM). Compared to A-PC and A-V91T, PD-1-IL-2v carrying other mutations had slightly lower maximal activity on pan T cell proliferation stimulated with CD3 antibodies. 3) Comparison of the activity of each fusion protein in unstimulated and CD3 antibody-stimulated T cell proliferation assays revealed that PD-1-IL-2v with D20A, D20N, N88R, N88D, or Q126D mutations did not activate the proliferation of unstimulated T cells, but strongly stimulated the proliferation of T cells stimulated with CD3 antibodies. Furthermore, A-PC and A-V91T, while more strongly activating the proliferation of T cells stimulated with CD3 antibodies, also strongly activated T cells not stimulated with CD3 antibodies.

[1050] Table 13. Proliferative activity of PD-1-IL-2v carrying different IL-2 mutations on unstimulated T cells

[1051] Table 14. Proliferative activity of PD-1-IL-2v with different IL-2 mutations on T cells stimulated with CD3 antibodies

[1052] Example 7. Identification of the binding affinity of PD-1-IL2v to the antigen PD-1 and IL-2Rα or IL-2Rβ / γ

[1053] Surface plasmon resonance (SPR) was used to determine the binding affinity of the PD-1-IL-2v fusion proteins carrying different IL-2 mutants for PD-1 and human IL-2Rα or IL-2Rβ / γ. This assay was performed using a Biacore 8K instrument (GE Healthcare). A CM5 sensor chip was used, and the mobile phase consisted of HBS-EP+ buffer (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.05% surfactant P20). Anti-human IgG (Fc) antibody was prepared at a concentration of 25 μg / mL in 10 mM sodium acetate buffer (pH 5.0). The immobilization program was selected for automated amino-coupled immobilization of the anti-human IgG (Fc) antibody. Each PD-1-IL2v ligand was prepared in HBS-EP+ buffer and captured using the anti-human IgG (Fc) antibody on the chip channel. Human PD-1 antigen protein (Sino Biological, 10377-H08H), human IL-2Rα receptor protein (Sino Biological, 10165-H08H) or human IL-2Rβ / γ receptor heterodimer protein (Acrosystem, ILG-H5283) were used as analytes and prepared with HBS-EP+ buffer solution. The analytes were diluted 2-fold from 200nM, with a total of 7 concentration points. The analytes flowed through the experimental channel and the reference channel at a flow rate of 30μL / min, with an association time of 140s and a dissociation time of 600s. The regeneration buffer 10mM Glycine pH 1.5 (GE Healthcare, 29238268-AA) was run at a flow rate of 10μL / min for 30s. The association rate Ka and dissociation rate Kd, as well as the dissociation constant (i.e., affinity K) were calculated. D value). The result is as follows.

[1054] Table 15. SPR affinity of PD-1-IL2v binding to human PD-1 antigen

[1055] Table 16. SPR binding affinity of anti-PD-1 antibody and IL-2 mutant fusion proteins to human IL-2Rα

[1056] (NB: not combined)

[1057] Table 17. SPR binding affinity of anti-PD-1 antibody and IL-2 mutant fusion proteins to human IL-2Rβ / γ

[1058] Results from binding to human PD-1 antigen showed that A-PC, A-N88D, and A-D20A had similar binding affinities to human PD-1, and similar binding affinities to A17m0902_hIgG4 (Table 8). Results from binding to human IL-2Rα (Table 16) showed that A-PC, A-N88D, and A-D20A did not bind to human IL-2Rα. Results from binding to human IL-2Rβ / γ (Table 17) showed that A-PC had the strongest affinity for human IL-2Rβ / γ, while A-N88D and A-D20A had approximately 13-fold and 63-fold lower affinities for human IL-2Rβ / γ, respectively.

[1059] The following formulation development is performed for PD-1-IL2v (A-N88D, the Knob chain is shown in SEQ ID NO: 42, and the Hole chain is shown in SEQ ID NO: 43).

[1060] Example 8. Screening of pH Values ​​of PD-1-IL2v Formulations

[1061] A-N88D (i.e., "protein" in the following formulations) was prepared as a liquid formulation according to the formulation information shown in Table 18. After aseptic filling, the samples were stored at 40°C for 1 week, 2 weeks, and 4 weeks, freeze-thawed (-35°C / room temperature) for 5 cycles, and shaken (300 rpm, 25°C) for 3 days. The samples were then evaluated for appearance, SEC, NR-CE, and iCIEF. The screening results are shown in Tables 18 and 19.

[1062] Table 18. pH screening formulation

[1063] Table 19. pH Screening - Appearance and Purity Results

[1064] The pH and protein concentration results at T0 (Table 18) were both within the target range. The particle size results showed that the particle sizes of the two formulations F1 and F2 were smaller, indicating that the immunoconjugates were more stable under these formulation conditions.

[1065] Appearance results (Table 19) show that after shaking for 3 days and five freeze-thaw cycles, particles appeared in both formulations F3 (pH 5.5) and F4 (pH 5.0, containing 30 mM arginine), while no visible particles were observed in the other formulations. Similarly, after 4 weeks at 40°C, a small amount of particles appeared in formulations F3 (pH 5.5) and F4 (pH 5.0, containing 30 mM arginine), while very small amounts appeared in formulations F1 (pH 4.5) and F2 (pH 5.0). This indicates that in the 20 mM acetate-histidine buffer system, the stability of the sample at pH 5.5 is poor, and the addition of 30 mM arginine at pH 5.0 reduces the stability of the sample.

[1066] SEC results (Table 19) show that after three days of shaking, the monomer purity of each formulation decreased slightly, and the decrease in monomer purity became more pronounced with increasing pH. After five freeze-thaw cycles, no significant decrease in monomer purity was observed in each formulation. NR-CE results (Table 19) show that after three days of shaking and five freeze-thaw cycles, no significant decrease in the purity of the main NR-CE peak was observed in each formulation. iCIEF results (Table 19) show that after three days of shaking and five freeze-thaw cycles, no significant decrease in the purity of the main iCIEF peak was observed in each formulation.

[1067] In summary, PD-1-IL2v has good stability at pH 4.5 to pH 5.0.

[1068] Example 9. Screening of buffer system and protein concentration in PD-1-IL2v preparation

[1069] The stability of formulations with different protein concentrations was investigated at pH 5.0. The effects of three different buffer systems (20 mM acetate-histidine, pH 5.0; 35 mM acetic acid, pH 5.5; and 20 mM histidine-histidine hydrochloride, pH 5.0) on protein stability were also compared. Considering that the SEC monomer purity of formulations F1-F4 decreased slightly after shaking for three days, the PS80 concentration was increased in this round of screening. Samples were prepared according to the formulations shown in Table 20 and sterilely filtered through a 0.22 μm PES filter. The prepared samples were then placed at 40°C for 1 week, 2 weeks, 3 weeks, and 4 weeks, followed by freeze-thaw (-35°C / room temperature) for 5 cycles and shaking for 3 days (300 rpm, 25°C). Appearance, SEC, and NR-CE were evaluated. The results are shown in Table 21.

[1070] Table 20. Buffer system and protein concentration screening recipes

[1071] Table 21. Buffer system and protein concentration screening appearance and purity results

[1072] Appearance results (Table 21) show that after shaking for 3 days and 5 freeze-thaw cycles, the appearance of each formulation remained unchanged. However, after 1 week at 40°C, a large number of particles appeared in formulation F9 (pH 5.5), indicating poor protein stability at pH 5.5.

[1073] SEC (Table 21) results showed that after shaking for 3 days and five freeze-thaw cycles, no significant change in monomer purity was observed in all formulations. NR-CE (Table 21) results also showed that after shaking for 3 days and five freeze-thaw cycles, no significant change in the main NR-CE peak content was observed in all formulations.

[1074] Example 10. PD-1-IL2v freeze-dried formulation concentration and excipient screening

[1075] To further improve stability and protein concentration, a lyophilized formulation of PD-1-IL2v was developed. Samples were prepared according to the formulation in Table 22. The prepared samples were filtered through a 0.22 μm PES filter, aseptically filled into vials, and lyophilized.

[1076] Table 22. Lyophilized Prescription Information

[1077] After lyophilization, the product was reconstituted in 1.5 mL of water. The appearance of the lyophilized product, reconstitution time, and appearance after reconstitution are shown in Table 23.

[1078] Table 23. Lyophilization results

[1079] *Note: “ / ” means not tested.

[1080] Reconstitution time results showed that high-concentration formulations F11 to F13 all took longer to reconstitute, while formulation F14 took shorter to reconstitute. The formulation F14 contained the following components after reconstitution: 10 mM succinate-histidine, pH 5.0, 80 mg / mL PD-1-IL2v, 8% (w / v) sucrose, and 0.2% (w / v) PS80.

[1081] Example 11. Concentration screening of PD-1-IL2v lyophilized formulations

[1082] In order to study the effect of different protein concentrations on the reconstitution time of freeze-dried products, samples were prepared according to the prescription in Table 24-1, filtered, aseptically filled, and freeze-dried.

[1083] Table 24-1 Freeze-dried formula

[1084] After lyophilization, add 1.5 mL of water for reconstitution. See Table 24-2 for reconstitution solution information.

[1085] Table 24-2. Reconstitution solution

[1086] The results of reconstitution time showed that F15 had a shorter reconstitution time, while F16 and F17 had longer reconstitution times. The appearance of the freeze-dried product met the quality standards (white loose body).

[1087] Example 12. pH and excipient screening of PD-1-IL2v freeze-dried formulation

[1088] The appearance of the reconstituted formulations with different pH values ​​and excipients was further investigated. Samples were prepared according to the formulation in Table 25, filtered through a 0.22 μm PES filter, aseptically filled, and lyophilized. After lyophilization, all samples were reconstituted in 1.0 mL of water. The reconstitution results are shown in Table 25. Both sucrose and trehalose formulations were used to prepare lyophilized formulations, and both achieved good appearance after reconstitution.

[1089] Table 25. Freeze-dried formula and appearance after reconstitution

[1090] As shown in the appearance results after reconstitution in Table 25, a small amount of particles were still found in formulation F23 (pH 5.0) after reconstitution, while no particles were found in other formulations. This shows that the formulation pH has a great influence on the appearance after reconstitution, and different protective agent concentrations have little effect on the appearance of the lyophilized product after reconstitution.

[1091] Example 13. Stability Study of Lyophilized Formulation of PD-1-IL-2v

[1092] The sample was prepared as follows: 10 mM succinate-histidine, pH 4.5, 40 mg / mL PD-1-IL-2v, 4.4% (w / v) trehalose, and 0.04% (w / v) PS80. The volume was 3.64 mL.

[1093] The prepared sample was filtered through a 0.22 μm PES filter, aseptically filled, lyophilized, and then reconstituted in a 1.6 mL volume. The reconstitution solution contained: 20 mM succinate-histidine, pH 4.5, 80 mg / mL PD-1-IL-2v, 8.8% (w / v) trehalose, and 0.08% (w / v) PS80.

[1094] The results showed that the appearance, reconstitution time, appearance after reconstitution, insoluble particles after reconstitution, and moisture content of the lyophilized product were all good and in line with expectations. The lyophilized product showed good stability after 4 weeks at 40°C, with no significant changes in SEC monomer purity or NR-CE main peak purity.

[1095] Example 14. PD-1-IL2v formulation prescription

[1096] This embodiment provides the following prescription:

[1097] 1) 5 mM succinate-histidine, pH 4.5, 50 mg / mL PD-1-IL-2v, 4% (w / v) trehalose, and 0.04% (w / v) PS80;

[1098] 2) 8 mM succinate-histidine, pH 4.5, 40 mg / mL PD-1-IL-2v, 4.4% (w / v) trehalose, and 0.04% (w / v) PS80;

[1099] 3) 10 mM succinate-histidine, pH 4.5, 40 mg / mL PD-1-IL-2v, 5% (w / v) trehalose, and 0.04% (w / v) PS80;

[1100] 4) 10 mM succinate-histidine, pH 4.0, 40 mg / mL PD-1-IL-2v, 4.4% (w / v) trehalose, and 0.04% (w / v) PS80;

[1101] 5) 10 mM succinate-histidine, pH 4.0, 40 mg / mL PD-1-IL-2v, 2% (w / v) trehalose, and 0.05% (w / v) PS80;

[1102] 6) 10 mM succinate-histidine, pH 4.5, 40 mg / mL PD-1-IL-2v, 4.4% (w / v) trehalose, and 0.08% (w / v) PS80;

[1103] 7) 10 mM succinate-histidine, pH 4.5, 30 mg / mL PD-1-IL-2v, 5% (w / v) trehalose, and 0.04% (w / v) PS80;

[1104] 8) 10 mM succinate-histidine, pH 4.5, 40 mg / mL PD-1-IL-2v, 5% (w / v) trehalose, and 0.04% (w / v) PS80;

[1105] 9) 10 mM succinate-histidine, pH 4.5, 50 mg / mL PD-1-IL-2v, 5% (w / v) trehalose, and 0.04% (w / v) PS80;

[1106] 10) 10 mM succinate-histidine, pH 4.5, 30 mg / mL PD-1-IL-2v, 4.4% (w / v) trehalose, and 0.04% (w / v) PS80;

[1107] 11) 10 mM succinate-histidine, pH 4.5, 50 mg / mL PD-1-IL-2v, 4.4% (w / v) trehalose, and 0.04% (w / v) PS80;

[1108] 12) 8 mM succinate-histidine, pH 4.5, 30 mg / mL PD-1-IL-2v, 5% (w / v) trehalose, and 0.02% (w / v) PS80;

[1109] 13) 10 mM succinate-histidine, pH 5.0, 40 mg / mL PD-1-IL-2v, 4.4% (w / v) trehalose, and 0.04% (w / v) PS80.

[1110] The above samples were freeze-dried and then redissolved to obtain the following formula:

[1111] 14) 10 mM succinate-histidine, pH 4.5, 100 mg / mL PD-1-IL-2v, 8% (w / v) trehalose, and 0.08% (w / v) PS80;

[1112] 15) 16 mM succinate-histidine, pH 4.5, 80 mg / mL PD-1-IL-2v, 8.8% (w / v) trehalose, and 0.08% (w / v) PS80;

[1113] 16) 20 mM succinate-histidine, pH 4.5, 80 mg / mL PD-1-IL-2v, 10% (w / v) trehalose, and 0.08% (w / v) PS80;

[1114] 17) 20 mM succinate-histidine, pH 4.0, 80 mg / mL PD-1-IL-2v, 8.8% (w / v) trehalose, and 0.08% (w / v) PS80;

[1115] 18) 20 mM succinate-histidine, pH 4.0, 80 mg / mL PD-1-IL-2v, 4% (w / v) trehalose, and 0.1% (w / v) PS80;

[1116] 19) 20 mM succinate-histidine, pH 4.5, 80 mg / mL PD-1-IL-2v, 8.8% (w / v) trehalose, and 0.08% (w / v) PS80;

[1117] 20) 20 mM succinate-histidine, pH 4.5, 80 mg / mL PD-1-IL-2v, 10% (w / v) trehalose, and 0.08% (w / v) PS80;

[1118] 21) 20 mM succinate-histidine, pH 4.5, 80 mg / mL PD-1-IL-2v, 10% (w / v) trehalose, and 0.08% (w / v) PS80;

[1119] 22) 20 mM succinate-histidine, pH 4.5, 100 mg / mL PD-1-IL-2v, 10% (w / v) trehalose, and 0.08% (w / v) PS80;

[1120] 23) 20 mM succinate-histidine, pH 4.5, 60 mg / mL PD-1-IL-2v, 8.8% (w / v) trehalose, and 0.08% (w / v) PS80;

[1121] 24) 20 mM succinate-histidine, pH 4.5, 100 mg / mL PD-1-IL-2v, 8.8% (w / v) trehalose, and 0.08% (w / v) PS80;

[1122] 25) 16 mM succinate-histidine, pH 4.5, 60 mg / mL PD-1-IL-2v, 10% (w / v) trehalose, and 0.04% (w / v) PS80;

[1123] 26) 20 mM succinate-histidine, pH 5.0, PD-1-IL-2v at a concentration of 80 mg / mL, 8.8% (w / v) trehalose, and 0.08% (w / v) PS80.

Claims

1. A pharmaceutical composition comprising an immunoconjugate and a buffer; the immunoconjugate contains a PD-1 binding domain and a cytokine, and the buffer is a histidine salt buffer; Preferably, the buffer is selected from one or more of acetate-histidine, succinate-histidine, and histidine-histidine hydrochloride; in, The PD-1 binding domain comprises an immunoglobulin single variable domain, wherein the immunoglobulin single variable domain comprises CDR1, CDR2 and CDR3 in the amino acid sequence shown in any one of SEQ ID NOs: 2, 8-15, or the amino acid sequences of CDR1, CDR2 and CDR3 of the immunoglobulin single variable domain are shown in SEQ ID NOs: 3, 16 and 17, respectively, and the CDRs are defined according to the Kabat, IMGT, Chothia, AbM or Contact numbering systems; Preferably, the CDR1, CDR2 and CDR3 of the immunoglobulin single variable domain are As shown in SEQ ID NO:3, 18, 19, As shown in SEQ ID NO:3, 4, 5, or As shown in SEQ ID NO:3, 4, 20.

2. The pharmaceutical composition of claim 1, wherein the pH of the buffer or the pharmaceutical composition is about 4.0 to about 6.5, preferably about 4.0 to about 5.0, and more preferably about 4.0 to about 4.

5.

3. The pharmaceutical composition according to claim 1 or 2, wherein the concentration of the buffer is about 1 mM to about 100 mM, preferably about 5 mM to about 35 mM, and more preferably about 10 mM to about 20 mM.

4. The pharmaceutical composition of any one of claims 1 to 3, wherein the concentration of the immunoconjugate is about 0.01 mg / mL to about 200 mg / mL, preferably about 10 mg / mL to about 120 mg / mL, and more preferably about 40 mg / mL to about 80 mg / mL.

5. The pharmaceutical composition according to any one of claims 1 to 4, further comprising a surfactant; Preferably, the surfactant is sodium dodecyl sulfate (SDS), poloxamer, or polysorbate, more preferably polysorbate 80.

6. The pharmaceutical composition of claim 5, wherein the concentration of the surfactant is from about 0.001% (w / v) to about 1.0% (w / v), preferably from about 0.04% (w / v) to about 0.2% (w / v), preferably from about 0.04% (w / v) to about 0.08% (w / v).

7. The pharmaceutical composition according to any one of claims 1 to 6, further comprising an osmotic pressure regulator; Preferably, the osmotic pressure regulator is selected from one or more of sucrose, trehalose, sorbitol, arginine, proline, glycine, mannitol and sodium chloride; Preferably, the osmotic pressure regulator is selected from sucrose and / or trehalose, more preferably sucrose or trehalose; Optionally, the pharmaceutical composition further comprises a stabilizer, and the stabilizer is selected from arginine or a salt thereof, preferably arginine hydrochloride.

8. The pharmaceutical composition of claim 7, wherein the concentration of the osmotic pressure regulator is about 1% (w / v) to about 20% (w / v), preferably about 1% (w / v) to about 10% (w / v), preferably about 4% (w / v) to about 8.8% (w / v); Optionally, the concentration of the stabilizer is about 5 mM to about 200 mM, preferably about 30 mM to about 100 mM.

9. The pharmaceutical composition according to any one of claims 1 to 8, comprising: (a) about 10 mg / mL to about 200 mg / mL of the immunoconjugate, about 1 mM to about 50 mM histidine salt buffer, about 0.01% (w / v) to about 0.2% (w / v) of a surfactant, about 1% (w / v) to about 20% (w / v) of an osmotic pressure regulating agent, and, optionally, about 10 mM to about 100 mM of a stabilizer; The pH of the pharmaceutical composition is about 4.0 to about 5.5; or, (b) about 10 mg / mL to about 120 mg / mL of the immunoconjugate, about 5 mM to about 35 mM histidine salt buffer; about 0.04% (w / v) to about 0.2% (w / v) polysorbate 80, about 4% (w / v) to about 10% (w / v) sucrose or trehalose, and, about 30 mM to about 100 mM arginine or arginine hydrochloride, optionally; The pH of the pharmaceutical composition is about 4.0 to about 5.5; or, (c) about 10 mg / mL to about 120 mg / mL of the immunoconjugate, From about 5 mM to about 35 mM histidine-histidine hydrochloride, histidine acetate, or histidine succinate; about 0.04% (w / v) to about 0.2% (w / v) polysorbate 80, about 4% (w / v) to about 10% (w / v) sucrose or trehalose, The pH of the pharmaceutical composition is about 4.0 to about 5.0; or, (d) about 10 mg / mL to about 120 mg / mL of the immunoconjugate, about 5 mM to about 20 mM of histidine acetate or histidine succinate, about 0.04% (w / v) to about 0.2% (w / v) polysorbate 80, about 1% (w / v) to about 10% (w / v) sucrose or trehalose, The pH of the pharmaceutical composition is about 4.0 to about 5.0; or, (e) about 10 mg / mL to about 120 mg / mL of the immunoconjugate, about 5 mM to about 20 mM succinate-histidine; about 0.04% (w / v) to about 0.08% (w / v) polysorbate 80, about 1% (w / v) to about 10% (w / v) trehalose, and, optionally about 10 mM to about 100 mM arginine hydrochloride; The pH of the pharmaceutical composition is about 4.0 to about 4.5; or, (e-1) about 10 mg / mL to about 80 mg / mL of an immunoconjugate, about 5 mM to about 10 mM succinate-histidine; about 0.04% (w / v) to about 0.08% (w / v) polysorbate 80, about 4% (w / v) to about 10% (w / v) trehalose, and, optionally about 10 mM to about 100 mM arginine hydrochloride; The pH of the pharmaceutical composition is about 4.0 to about 4.5; or, (e-2) about 10 mg / mL to about 40 mg / mL of an immunoconjugate, about 10 mM succinate-histidine; about 0.04% (w / v) to about 0.08% (w / v) polysorbate 80, about 4% (w / v) to about 8.8% (w / v) trehalose, The pH of the pharmaceutical composition is about 4.0 to about 4.5; or, (e-3) about 40 mg / mL to about 120 mg / mL of an immunoconjugate, about 10 mM to about 20 mM succinate-histidine; about 0.04% (w / v) to about 0.08% (w / v) polysorbate 80, about 4% (w / v) to about 10% (w / v) trehalose, The pH of the pharmaceutical composition is about 4.0 to about 4.5; or (e-4) about 40 mg / mL to about 80 mg / mL of an immunoconjugate, about 20 mM succinate-histidine; about 0.04% (w / v) to about 0.08% (w / v) polysorbate 80, about 4.4% (w / v) to about 8.8% (w / v) trehalose, The pH of the pharmaceutical composition is about 4.0 to about 4.5; or (f) about 10 mg / mL to about 120 mg / mL of the immunoconjugate, about 5 mM to about 20 mM succinate-histidine; about 0.1% (w / v) to about 0.2% (w / v) polysorbate 80, about 4% (w / v) to about 8% (w / v) sucrose, and optionally about 10 mM to about 100 mM arginine hydrochloride; The pH of the pharmaceutical composition is about 4.5 to about 5.0; or, (f-1) about 10 mg / mL to about 80 mg / mL of an immunoconjugate, about 5 mM to about 10 mM succinate-histidine, about 0.1% (w / v) to about 0.2% (w / v) polysorbate 80, about 4% (w / v) to about 8% (w / v) sucrose, and optionally about 10 mM to about 100 mM arginine hydrochloride; The pH of the pharmaceutical composition is about 4.5 to about 5.0; or, (f-2) about 40 mg / mL to about 80 mg / mL of an immunoconjugate, About 5 mM succinate-histidine, about 0.1% (w / v) to about 0.2% (w / v) polysorbate 80, about 4% (w / v) to about 8% (w / v) sucrose, The pH of the pharmaceutical composition is about 4.5 to about 5.0; or, (f-3) about 40 mg / mL to about 120 mg / mL of an immunoconjugate, about 10 mM to about 20 mM succinate-histidine; about 0.1% (w / v) to about 0.2% (w / v) polysorbate 80, about 4% (w / v) to about 8% (w / v) sucrose, The pH of the pharmaceutical composition is about 4.5 to about 5.0; or, (f-4) about 80 mg / mL to about 120 mg / mL of an immunoconjugate, about 10 mM succinate-histidine; about 0.1% (w / v) to about 0.2% (w / v) polysorbate 80, about 4% (w / v) to about 8% (w / v) sucrose, The pH of the pharmaceutical composition is about 4.5 to about 5.0; or, (g) about 10 mg / mL to about 120 mg / mL of the immunoconjugate, about 5 mM to about 20 mM of acetate-histidine or histidine-histidine hydrochloride; about 0.04% (w / v) to about 0.08% (w / v) polysorbate 80, about 4% (w / v) to about 8% (w / v) sucrose; The pH of the pharmaceutical composition is about 4.5 to about 5.

0.

10. The pharmaceutical composition according to any one of claims 1 to 9, comprising any one of the following: (1) about 10 mg / mL to about 120 mg / mL of an immunoconjugate, About 10 mM succinate-histidine, About 0.04% (w / v) polysorbate 80, About 4.4% (w / v) trehalose, The pH of the pharmaceutical composition is about 4.0 to about 4.5; (2) about 10 mg / mL to about 80 mg / mL of the immunoconjugate, About 10 mM succinate-histidine, About 0.04% (w / v) polysorbate 80, About 4.4% (w / v) trehalose, The pH of the pharmaceutical composition is about 4.0 to about 4.5; (3) about 10 mg / mL to about 40 mg / mL of the immunoconjugate, About 10 mM succinate-histidine, About 0.04% (w / v) polysorbate 80, About 4.4% (w / v) trehalose, The pH of the pharmaceutical composition is about 4.5; (4) about 10 mg / mL to about 120 mg / mL of an immunoconjugate, About 20 mM succinate-histidine, About 0.08% (w / v) polysorbate 80, About 8.8% (w / v) trehalose, The pH of the pharmaceutical composition is about 4.0 to about 4.5; (5) about 40 mg / mL to about 120 mg / mL of an immunoconjugate, About 20 mM succinate-histidine, About 0.08% (w / v) polysorbate 80, About 8.8% (w / v) trehalose, The pH of the pharmaceutical composition is about 4.0 to about 4.5; (6) about 40 mg / mL to about 80 mg / mL of an immunoconjugate, About 20 mM succinate-histidine, About 0.08% (w / v) polysorbate 80, About 8.8% (w / v) trehalose, The pH of the pharmaceutical composition is about 4.

5.

11. The pharmaceutical composition according to any one of claims 1 to 10, wherein The PD-1 binding domain comprises an amino acid sequence as shown in any one of SEQ ID NOs: 2, 8-15, or having at least 90% sequence identity thereto; Preferably, the PD-1 binding domain comprises an amino acid sequence as set forth in SEQ ID NO: 13 or having at least 90% sequence identity thereto.

12. The pharmaceutical composition according to any one of claims 1 to 11, wherein The cytokine is wild-type IL-2 or a variant thereof; preferably, the IL-2 variant contains the following mutations: 42A / 72G / 88D, 42A / 72G / 88R, 42A / 72G / 20A, 42A / 72G / 20N, 42A / 72G / 91T, 42A / 72G / 126D, 42A / 72G / 16A / 84S, 42A / 72G / 26Q / 29S / 71Q, 38E / 42A / 91T, 42A / 72G / 88D / 26Q / 29S / 71Q, 42A / 72G / 88R / 26Q / 29S / 71Q, 42A / 72G / 20A / 26Q / 29S / 71Q, 42A / 72G / 20N / 26Q / 29S / 71Q, 42A / 72G / 91T / 26Q / 29S / 71Q, 42A / 72G / 126D / 26Q / 29S / 71Q, or 42A / 72G / 16A / 84S / 26Q / 29S / 71Q; Preferably, the mutations shown below are included: F42A / L72G / N88D, F42A / L72G / N88R, F42A / L72G / D20A, F42A / L72G / D20N, F42A / L72G / V91T, F42A / L72G / Q126D, F42A / L72G / H16A / D84S, F42A / L72G / N26Q / N29S / N71Q, R38E / F42A / V91T, F42A / L72G / N88D / N26Q / N29S / N71Q, F42A / L72G / N88R / N26Q / N29S / N71Q, F42A / L72G / D20A / N26Q / N29S / N71Q, F42A / L72G / D20N / N26Q / N29S / N71Q, F42A / L72G / V91T / N26Q / N29S / N71Q, F42A / L72G / Q126D / N26Q / N29S / N71Q, or F42A / L72G / H16A / D84S / N26Q / N29S / N71Q.

13. The pharmaceutical composition according to claim 12, wherein The IL-2 variant contains mutations at position 3 and / or 125, wherein the mutation at position 3 is preferably T3A; and the mutation at position 125 is preferably C125A or C125S.

14. The pharmaceutical composition according to claim 12 or 13, wherein The IL-2 variant contains or has an amino acid sequence as shown in any one of SEQ ID NOs: 23-31, or a sequence having at least 90% identity thereto.

15. The pharmaceutical composition according to any one of claims 1 to 14, wherein The immunoconjugate further comprises an immunoglobulin Fc region; preferably, the Fc region is the Fc region of human IgG1, IgG2 or IgG4; Preferably, the Fc region of human IgG1 contains one or more amino acid mutations that reduce binding to Fcγ receptor (FcγR) and / or reduce ADCC effect; More preferably, the Fc region of human IgG1 contains mutations of L234, L235, P329 or any combination thereof; Most preferably, the Fc region of the human IgG1 contains L234A / L235A, L234A / L235A / P329G mutations; The mutations in the Fc region are numbered according to the EU numbering system.

16. The pharmaceutical composition according to claim 15, wherein The Fc region comprises a mutation that promotes association of the first and second subunits of the Fc region; Preferably, amino acid residues in the CH3 region of the first subunit of the Fc region are replaced with amino acid residues having a larger side chain volume to generate a protuberance in the CH3 region of the first subunit, which protuberance can be placed in a cavity in the CH3 region of the second subunit, and amino acid residues in the CH3 region of the second subunit of the Fc region are replaced with amino acid residues having a smaller side chain volume to generate a cavity in the CH3 region of the second subunit in which the protuberance in the CH3 region of the first subunit can be placed; Preferably, the first subunit of the Fc region contains a T366W mutation, and the second subunit contains a mutation selected from T366S, L368A, Y407V or any combination thereof; the first subunit of the Fc region contains a S354C or E356C mutation, and the second subunit contains a mutation selected from Y349C; or, the first subunit of the Fc region contains a S354C / T366W mutation, and the second subunit contains a Y349C / T366S / L368A / Y407V mutation; Preferably, the second subunit of the Fc region further contains H435R / Y436F, H435R, or H435K mutation.

17. The pharmaceutical composition according to any one of claims 15 to 16, wherein The PD-1 binding domain is located at the N-terminus of the Fc region, and the cytokine is located at the N-terminus or C-terminus of the Fc region; Preferably, the cytokine is located in the polypeptide chain where the first subunit of the Fc region is located.

18. The pharmaceutical composition according to any one of claims 1 to 17, wherein The immunoconjugate comprises the following structure: 1) The first polypeptide chain comprises, from N-terminus to C-terminus: [PD-1 binding domain 1]-[Fc region first subunit]-[linker 1]a-[IL-2 or its variant], 2) The second polypeptide chain comprises from the N-terminus to the C-terminus: [PD-1 binding domain 1]-[Fc region second subunit]; Wherein, a is selected from 0 or 1, and the structure of the linker is (G x S) y , wherein x is selected from an integer of 1-5, and y is selected from an integer of 1-6; Preferably, x is 4 and y is 1, 2 or 3.

19. The pharmaceutical composition according to any one of claims 1 to 18, wherein The immunoconjugate comprises a polypeptide chain combination of the following amino acid sequence: SEQ ID NO:42 and 43; SEQ ID NO: 21 and 22; SEQ ID NO:36 and 37; SEQ ID NO:38 and 39; or, SEQ ID NO:40 and 41.

20. A lyophilized preparation, wherein the lyophilized preparation is obtained by freeze-drying the pharmaceutical composition according to any one of claims 1 to 19, or the lyophilized preparation can be reconstituted to form the pharmaceutical composition according to any one of claims 1 to 19.

21. A lyophilized preparation obtained by freeze-drying a liquid preparation containing a pharmaceutical composition; wherein: The liquid preparation comprises: (e-1) about 10 mg / mL to about 80 mg / mL of an immunoconjugate, about 5 mM to about 10 mM succinate-histidine, about 0.04% (w / v) to about 0.08% (w / v) polysorbate 80, about 4% (w / v) to about 10% (w / v) trehalose, and, optionally about 10 mM to about 100 mM arginine hydrochloride; The pH of the pharmaceutical composition is about 4.0 to about 4.5; or, (e-2) about 10 mg / mL to about 40 mg / mL of an immunoconjugate, About 10 mM succinate-histidine, about 0.04% (w / v) to about 0.08% (w / v) polysorbate 80, about 4% (w / v) to about 8.8% (w / v) trehalose, The pH of the pharmaceutical composition is about 4.0 to about 4.5; or, (f-1) about 10 mg / mL to about 80 mg / mL of an immunoconjugate, about 5 mM to about 10 mM succinate-histidine, about 0.1% (w / v) to about 0.2% (w / v) polysorbate 80, about 4% (w / v) to about 8% (w / v) sucrose, and optionally about 10 mM to about 100 mM arginine hydrochloride; The pH of the pharmaceutical composition is about 4.5 to about 5.0; or, (f-2) about 40 mg / mL to about 80 mg / mL of an immunoconjugate, about 5 mM succinate-histidine; about 0.1% (w / v) to about 0.2% (w / v) polysorbate 80, about 4% (w / v) to about 8% (w / v) sucrose, The pH of the pharmaceutical composition is about 4.5 to about 5.0; or, Preferably, it comprises any of the following: (1) about 10 mg / mL to about 120 mg / mL of an immunoconjugate, About 10 mM succinate-histidine, About 0.04% (w / v) polysorbate 80, About 4.4% (w / v) trehalose, The pH of the pharmaceutical composition is about 4.0 to about 4.5; (2) about 10 mg / mL to about 80 mg / mL of the immunoconjugate, About 10 mM succinate-histidine, About 0.04% (w / v) polysorbate 80, About 4.4% (w / v) trehalose, The pH of the pharmaceutical composition is about 4.0 to about 4.5; (3) about 10 mg / mL to about 40 mg / mL of the immunoconjugate, About 10 mM succinate-histidine, About 0.04% (w / v) polysorbate 80, About 4.4% (w / v) trehalose, The pH of the pharmaceutical composition is about 4.5; Preferably, the immunoconjugate contains a PD-1 binding domain and a cytokine, wherein the PD-1 binding domain comprises an immunoglobulin single variable domain, wherein the immunoglobulin single variable domain comprises CDR1, CDR2 and CDR3 in the amino acid sequence shown in any one of SEQ ID NOs: 2, 8-15, or the amino acid sequences of CDR1, CDR2 and CDR3 of the immunoglobulin single variable domain are shown in SEQ ID NOs: 3, 16 and 17, respectively, and the CDRs are defined according to the Kabat, IMGT, Chothia, AbM or Contact numbering systems; Preferably, the CDR1, CDR2 and CDR3 of the immunoglobulin single variable domain are As shown in SEQ ID NO:3, 18, 19, As shown in SEQ ID NO:3, 4, 5, or As shown in SEQ ID NO:3, 4, 20; More preferably, the immunoconjugate comprises a polypeptide chain combination of the following amino acid sequence: SEQ ID NO:42 and 43; SEQ ID NO: 21 and 22; SEQ ID NO:36 and 37; SEQ ID NO:38 and 39; or, SEQ ID NO:40 and 41.

22. A reconstituted solution, which is prepared by reconstituted the lyophilized preparation according to claim 20 or 21; Preferably, the concentration of the immunoconjugate in the reconstituted solution is greater than the concentration of the immunoconjugate in the stock solution used to prepare the lyophilized formulation; Preferably, the concentration ratio of the immunoconjugate in the reconstituted solution to the immunoconjugate in the original solution is (1-5):1, more preferably (1-2):

1.

23. The reconstituted solution according to claim 22, which is obtained by reconstituted the lyophilized preparation according to claim 20 or 21 via a solvent; preferably, the solvent is water, physiological saline or glucose.

24. The reconstitution solution according to claim 22 or 23, comprising: (e-3) about 40 mg / mL to about 120 mg / mL of an immunoconjugate, about 10 mM to about 20 mM succinate-histidine; about 0.04% (w / v) to about 0.08% (w / v) polysorbate 80, about 4% (w / v) to about 10% (w / v) trehalose, The pH of the pharmaceutical composition is about 4.0 to about 4.5; or (e-4) about 40 mg / mL to about 80 mg / mL of an immunoconjugate, about 20 mM succinate-histidine; about 0.04% (w / v) to about 0.08% (w / v) polysorbate 80, about 4.4% (w / v) to about 8.8% (w / v) trehalose, The pH of the pharmaceutical composition is about 4.0 to about 4.5; or (f-3) about 40 mg / mL to about 120 mg / mL of an immunoconjugate, about 10 mM to about 20 mM succinate-histidine; about 0.1% (w / v) to about 0.2% (w / v) polysorbate 80, about 4% (w / v) to about 8% (w / v) sucrose, The pH of the pharmaceutical composition is about 4.5 to about 5.0; or, (f-4) about 80 mg / mL to about 120 mg / mL of an immunoconjugate, about 10 mM succinate-histidine; about 0.1% (w / v) to about 0.2% (w / v) polysorbate 80, about 4% (w / v) to about 8% (w / v) sucrose, The pH of the pharmaceutical composition is from about 4.5 to about 5.0; Preferably, it comprises any of the following: (4) about 10 mg / mL to about 120 mg / mL of an immunoconjugate, About 20 mM succinate-histidine, About 0.08% (w / v) polysorbate 80, About 8.8% (w / v) trehalose, The pH of the pharmaceutical composition is about 4.0 to about 4.5; (5) about 40 mg / mL to about 120 mg / mL of an immunoconjugate, About 20 mM succinate-histidine, About 0.08% (w / v) polysorbate 80, About 8.8% (w / v) trehalose, The pH of the pharmaceutical composition is about 4.0 to about 4.5; (6) about 40 mg / mL to about 80 mg / mL of an immunoconjugate, About 20 mM succinate-histidine, About 0.08% (w / v) polysorbate 80, About 8.8% (w / v) trehalose, The pH of the pharmaceutical composition is about 4.5; Preferably, the PD-1 binding domain comprises at least one immunoglobulin single variable domain, the immunoglobulin single variable domain comprises: CDR1, CDR2 and CDR3 in the amino acid sequence shown in any one of SEQ ID NOs: 2, 8-15, or the amino acid sequences of CDR1, CDR2 and CDR3 of the immunoglobulin single variable domain are shown in SEQ ID NOs: 3, 16 and 17, respectively; the CDR1, CDR2 and CDR3 are defined according to the Kabat, IMGT, Chothia, AbM or Contact numbering systems; Preferably, the CDR1, CDR2 and CDR3 of the immunoglobulin single variable domain are As shown in SEQ ID NO:3, 18, 19, As shown in SEQ ID NO:3, 4, 5, or As shown in SEQ ID NO:3, 4, 20; Preferably, the cytokine is IL-2 or a variant thereof, more preferably comprising a sequence shown in any one of SEQ ID NOs: 23-31; More preferably, the immunoconjugate comprises a polypeptide chain combination of the following amino acid sequence: SEQ ID NO:42 and 43; SEQ ID NO: 21 and 22; SEQ ID NO:36 and 37; SEQ ID NO:38 and 39; or, SEQ ID NO:40 and 41.

25. A liquid preparation comprising any of the following: (e) about 10 mg / mL to about 120 mg / mL of the immunoconjugate, about 5 mM to about 20 mM succinate-histidine; about 0.04% (w / v) to about 0.08% (w / v) polysorbate 80, about 1% (w / v) to about 10% (w / v) trehalose, and, optionally about 10 mM to about 100 mM arginine hydrochloride; The pH of the pharmaceutical composition is about 4.0 to about 4.5; or, (e-1) about 10 mg / mL to about 80 mg / mL of an immunoconjugate, about 5 mM to about 10 mM succinate-histidine; about 0.04% (w / v) to about 0.08% (w / v) polysorbate 80, about 4% (w / v) to about 10% (w / v) trehalose, and, optionally about 10 mM to about 100 mM arginine hydrochloride; The pH of the pharmaceutical composition is about 4.0 to about 4.5; or, (e-2) about 10 mg / mL to about 40 mg / mL of an immunoconjugate, about 10 mM succinate-histidine; about 0.04% (w / v) to about 0.08% (w / v) polysorbate 80, about 4% (w / v) to about 8.8% (w / v) trehalose, The pH of the pharmaceutical composition is about 4.0 to about 4.5; or, (e-3) about 40 mg / mL to about 120 mg / mL of an immunoconjugate, about 10 mM to about 20 mM succinate-histidine; about 0.04% (w / v) to about 0.08% (w / v) polysorbate 80, about 4% (w / v) to about 10% (w / v) trehalose, The pH of the pharmaceutical composition is about 4.0 to about 4.5; or (e-4) about 40 mg / mL to about 80 mg / mL of an immunoconjugate, about 20 mM succinate-histidine; about 0.04% (w / v) to about 0.08% (w / v) polysorbate 80, about 4.4% (w / v) to about 8.8% (w / v) trehalose, The pH of the pharmaceutical composition is about 4.0 to about 4.5; or (f) about 10 mg / mL to about 120 mg / mL of the immunoconjugate, about 5 mM to about 20 mM succinate-histidine; about 0.1% (w / v) to about 0.2% (w / v) polysorbate 80, about 4% (w / v) to about 8% (w / v) sucrose, and optionally about 10 mM to about 100 mM arginine hydrochloride; The pH of the pharmaceutical composition is about 4.5 to about 5.0; or, (f-1) about 10 mg / mL to about 80 mg / mL of an immunoconjugate, about 5 mM to about 10 mM succinate-histidine, about 0.1% (w / v) to about 0.2% (w / v) polysorbate 80, about 4% (w / v) to about 8% (w / v) sucrose, and optionally about 10 mM to about 100 mM arginine hydrochloride; The pH of the pharmaceutical composition is about 4.5 to about 5.0; or, (f-2) about 40 mg / mL to about 80 mg / mL of an immunoconjugate, About 5 mM succinate-histidine, about 0.1% (w / v) to about 0.2% (w / v) polysorbate 80, about 4% (w / v) to about 8% (w / v) sucrose, The pH of the pharmaceutical composition is about 4.5 to about 5.0; or, (f-3) about 40 mg / mL to about 120 mg / mL of an immunoconjugate, about 10 mM to about 20 mM succinate-histidine; about 0.1% (w / v) to about 0.2% (w / v) polysorbate 80, about 4% (w / v) to about 8% (w / v) sucrose, The pH of the pharmaceutical composition is about 4.5 to about 5.0; or, (f-4) about 80 mg / mL to about 120 mg / mL of an immunoconjugate, about 10 mM succinate-histidine; about 0.1% (w / v) to about 0.2% (w / v) polysorbate 80, about 4% (w / v) to about 8% (w / v) sucrose, The pH of the pharmaceutical composition is about 4.5 to about 5.0; or, Preferably, the liquid preparation comprises any of the following: (1) about 10 mg / mL to about 120 mg / mL of an immunoconjugate, About 10 mM succinate-histidine, About 0.04% (w / v) polysorbate 80, About 4.4% (w / v) trehalose, The pH of the pharmaceutical composition is about 4.0 to about 4.5; (2) about 10 mg / mL to about 80 mg / mL of the immunoconjugate, About 10 mM succinate-histidine, About 0.04% (w / v) polysorbate 80, About 4.4% (w / v) trehalose, The pH of the pharmaceutical composition is about 4.0 to about 4.5; (3) about 10 mg / mL to about 40 mg / mL of the immunoconjugate, About 10 mM succinate-histidine, About 0.04% (w / v) polysorbate 80, About 4.4% (w / v) trehalose, The pH of the pharmaceutical composition is about 4.5; (4) about 10 mg / mL to about 120 mg / mL of an immunoconjugate, About 20 mM succinate-histidine, About 0.08% (w / v) polysorbate 80, About 8.8% (w / v) trehalose, The pH of the pharmaceutical composition is about 4.0 to about 4.5; (5) about 40 mg / mL to about 120 mg / mL of an immunoconjugate, About 20 mM succinate-histidine, About 0.08% (w / v) polysorbate 80, About 8.8% (w / v) trehalose, The pH of the pharmaceutical composition is about 4.0 to about 4.5; (6) about 40 mg / mL to about 80 mg / mL of an immunoconjugate, About 20 mM succinate-histidine, About 0.08% (w / v) polysorbate 80, About 8.8% (w / v) trehalose, The pH of the pharmaceutical composition is about 4.5; Preferably, the PD-1 binding domain comprises an immunoglobulin single variable domain, the immunoglobulin single variable domain comprises CDR1, CDR2 and CDR3 in the amino acid sequence shown in any one of SEQ ID NOs: 2, 8-15, or the amino acid sequences of CDR1, CDR2 and CDR3 of the immunoglobulin single variable domain are shown in SEQ ID NOs: 3, 16 and 17, respectively, and the CDRs are defined according to the Kabat, IMGT, Chothia, AbM or Contact numbering systems; Preferably, the CDR1, CDR2 and CDR3 of the immunoglobulin single variable domain are As shown in SEQ ID NO:3, 18, 19, As shown in SEQ ID NO:3, 4, 5, or As shown in SEQ ID NO:3, 4, 20; More preferably, the immunoconjugate comprises a polypeptide chain combination of the following amino acid sequence: SEQ ID NO:42 and 43; SEQ ID NO: 21 and 22; SEQ ID NO:36 and 37; SEQ ID NO:38 and 39; or, SEQ ID NO:40 and 41.

26. The pharmaceutical composition according to any one of claims 1 to 19, the reconstituted solution according to any one of claims 22 to 24, or the liquid preparation according to claim 25; which is an intravenous injection, a subcutaneous injection, an intraperitoneal injection or an intramuscular injection; preferably an intravenous or subcutaneous injection.

27. A product comprising a container containing the pharmaceutical composition of any one of claims 1 to 19, the lyophilized preparation of claim 20 or 21, the reconstituted solution of any one of claims 22-24, or the liquid preparation of claim 25.

28. A method for treating or preventing a disease, comprising administering to a subject in need thereof a therapeutically or preventively effective amount of the pharmaceutical composition of any one of claims 1 to 19, the lyophilized preparation of claim 20 or 21, the reconstituted solution of any one of claims 22 to 24, the liquid preparation of claim 25, or the product of claim 27, wherein: Preferably, the disease is cancer; More preferably, the cancer is selected from lung cancer, prostate cancer, breast cancer, head and neck cancer, esophageal cancer, gastric cancer, colon cancer, Colorectal cancer, bladder cancer, cervical cancer, uterine cancer, ovarian cancer, liver cancer, melanoma, kidney cancer, squamous cell carcinoma, blood system cancer.

29. A method for preparing a liquid preparation, comprising the steps of: Preparing a stock solution comprising the pharmaceutical composition according to any one of claims 1 to 19, and subjecting the stock solution to freeze-drying treatment to obtain a lyophilized preparation; and, The lyophilized preparation is reconstituted to obtain a liquid preparation; in, The concentration of the immunoconjugate in the liquid preparation is greater than the concentration of the immunoconjugate in the stock solution; Preferably, the concentration ratio of the immunoconjugate in the liquid preparation to the immunoconjugate in the stock solution is (1-5):1, preferably (1-2):

1.

30. A method for preparing a pharmaceutical composition, comprising: Preparing a stock solution comprising the pharmaceutical composition according to any one of claims 1 to 19, and subjecting the stock solution to freeze-drying treatment to obtain a freeze-dried preparation; as well as, The lyophilized preparation is reconstituted with a solvent to obtain a liquid preparation; Wherein, the concentration of the immunoconjugate in the liquid preparation is greater than the concentration of the immunoconjugate in the stock solution, and the solvent is preferably water; Preferably, the concentration ratio of the immunoconjugate in the liquid preparation to the immunoconjugate in the stock solution is (1-5):1, preferably (1-2):1.

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