Pharmaceutical composition containing FAP / 4-1BB / CD40 binding molecule, and use thereof

Abstract

A pharmaceutical composition containing a FAP / 4-1BB / CD40 binding molecule, and a use thereof. Specifically, the present invention relates to a pharmaceutical composition containing a FAP / 4-1BB / CD40 binding molecule, a lyophilized preparation, a reconstitution solution, a product, and a medical use thereof.

Description

Pharmaceutical compositions comprising fap / 4-1bb / cd40 binding molecules and uses thereof

[0001] The present disclosure claims priority from Chinese patent application CN2024112380823 filed on 2024 / 9 / 4: the aforementioned patent application is incorporated herein by reference in its entirety. TECHNICAL FIELD

[0002] The present disclosure belongs to the field of biological medicine, in particular relates to pharmaceutical compositions, freeze-dried preparations, reconstituted solutions, products and medical uses comprising FAP / 4-1BB / CD40 binding molecules. BACKGROUND

[0003] CD40 (TNFRSF5) is a transmembrane phosphorylated glycoprotein, belonging to the tumor necrosis factor receptor superfamily (TNFRS) members. It is mainly expressed on the surface of DC cells, macrophages and B cells, and also expressed on non-immune cells such as platelets and endothelial cells. Its ligand CD40L is limited to a trimer arrangement, which presents the best state of CD40 signal transmission through the receptor, thereby enhancing the aggregation of CD40 itself and the subsequent recruitment and interaction of TNF receptor-associated factors. The binding of CD40L and CD40 on DC induces DC maturation, which is manifested as up-regulation of the expression of co-stimulatory factor B7 family (CD80, CD86) and increased secretion of pro-inflammatory cytokines such as interleukin 12 (IL-12). The interaction of CD40 and CD40L provides a co-stimulatory signal for T cell activation and promotes DC cells to present antigens to T cells.

[0004] 4-1BB (CD137, TNFRSF9) is a transmembrane protein also belonging to the tumor necrosis factor receptor superfamily. It is mainly expressed on activated CD4+ and CD8+ T cells, activated B cells and natural killer (NK) cells. 4-1BB is expressed on the cell surface as a monomer or dimer, and after binding to its ligand (4-1BBL), it undergoes trimerization for signal transduction. On T cells, 4-1BB is not constitutively expressed, but is induced to express after T cell receptor (TCR) is activated, and stimulation from its natural ligand 4-1BBL or antibody agonist can further up-regulate the expression of 4-1BB. The intracellular domain of 4-1BB is believed to act by recruiting TRAF1, TRAF2, or possibly a transducer protein mediated by K63 polyubiquitination reaction through TRAF3. 4-1BBL trimerization leads to aggregation of 4-1BB receptor and activation of TRAF-mediated NF-κB and MAPK intracellular signaling pathways, resulting in T cell proliferation, maturation and prolonged survival, as well as cytokine production, etc.

[0005] Fibroblast activation protein (FAP) is a tumor-associated antigen, FAP is expressed in a low amount in normal tissues of healthy adults, and is selectively expressed in 93% of tumor tissues, of which 30% is high expression, such as colon cancer, pancreatic cancer, breast cancer, gastric cancer, prostate cancer, bladder cancer and oral squamous carcinoma. At present, there is no multi-specific antibody targeting CD40, 4-1BB and FAP on the market.

[0006] Therefore, there is an urgent need in the art to develop a multi-specific antibody capable of mediating tumor-specific CD40 and 4-1BB activation through FAP, which has good tumor treatment effect and low toxicity. The present disclosure provides a novel anti-FAP / 4-1BB / CD40 trispecific antibody, which can mediate tumor-specific CD40 and 4-1BB activation through FAP, has a promoting maturation and activation effect on APC (such as dendritic cells DC) and T cells, can remove liver toxicity, peripheral blood toxicity and other peripheral toxicities, has an excellent drug administration window and drugability, and provides a solution for clinical application.

[0007] However, due to the large molecular weight and complex structure of the trispecific antibody, it is unstable and prone to degradation and polymerization, resulting in reduced activity or even failure. In order to make the binding protein suitable for administration and maintain stability during storage and subsequent use, and to achieve better effect, the development of stable formulation research of drugs is particularly important. SUMMARY

[0008] The present disclosure provides a pharmaceutical composition comprising a FAP / 4-1BB / CD40 binding molecule, and a method for preparing the pharmaceutical composition, and a method for treating or preventing diseases or related pharmaceutical uses.

[0009] The present disclosure provides a pharmaceutical composition comprising a FAP / 4-1BB / CD40 binding molecule, and a buffer. In some embodiments, the buffer is selected from one or more of histidine salt buffer, acetate buffer, citrate buffer and succinate buffer. In some specific embodiments, the buffer is selected from one or more of histidine-acetate, citrate-citrate sodium and histidine-succinate. "One or more" in the present disclosure includes one, two or more.

[0010] In some embodiments, the concentration of the buffer is about 1 mM to about 100 mM, e.g., 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 5 mM to about 100 mM, about 5 mM to about 80 mM, about 5 mM to about 70 mM, about 5 mM to about 50 mM, about 5 mM to about 45 mM, about 5 mM to about 40 mM, about 5 mM to about 35 mM, about 5 mM to about 30 mM, about 5 mM to about 25 mM, about 5 mM to about 20 mM, about 5 mM to about 10 mM, about 10 mM to about 20 mM, about 10 mM to about 30 mM, about 10 mM to about 35 mM, about 10 mM to about 40 mM, about 10 mM to about 50 mM, about 15 mM to about 20 mM, about 15 mM to about 25 mM, about 15 mM to about 35 mM, about 15 mM to about 50 mM, about 20 mM to about 35 mM, about 1 mM to about 10 mM, about 1 mM to about 20 mM, or any range between these point values. In some embodiments, the concentration of the buffer is about 2 mM to about 50 mM. In some embodiments, the concentration of the buffer is about 5 mM to about 30 mM.

[0011] 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 40 mM, about 43 mM, about 45 mM, about 48 mM, about 50 mM, about 55 mM, about 57 mM, about 58 mM, about 60 mM, about 62 mM, about 65 mM, about 68 mM, about 70 mM, about 72 mM, about 75 mM, about 78 mM, about 80 mM, about 82 mM, about 85 mM, about 88 mM, about 90 mM, about 92 mM, about 95 mM, about 100 mM.

[0012] In some embodiments, the pH of the buffer or the pharmaceutical composition is about 3.0 to about 7.0, for example, about 3.5 to about 7.0, about 4.0 to about 7.0, about 4.0 to about 6.5, about 4.0 to about 6.0, about 4.0 to about 5.5, about 4.0 to about 5.0, about 4.0 to about 4.8, 4.0 to about 4.5, about 4.8 to about 5.0, about 5.0 to about 5.5, about 4.5 to about 4.8, or any range between these point values. In some embodiments, the pH of the buffer or the pharmaceutical composition is about 4.0 to about 6.0. In some embodiments, the pH of the buffer or the pharmaceutical composition is about 4.5 to about 5.5.

[0013] In some embodiments, the pH of the buffer or the pharmaceutical composition is about 3.0, about 3.1, about 3.2, about 3.3, about 3.4, about 3.5, about 3.6, about 3.7, about 3.8, about 3.9, about 4.0, about 4.1, about 4.2, about 4.3, about 4.4, about 4.5, about 4.6, about 4.7, about 4.8, about 4.9, about 5.0, about 5.1, about 5.2, about 5.3, about 5.4, about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, about 6.0, about 6.1, about 6.2, about 6.3, about 6.4, about 6.5, about 6.7, about 6.8, about 6.9, about 7.0.

[0014] Generally, the pH of a pharmaceutical composition obtained by substitution of a buffer is nearly identical to the buffer pH. At the same time, it is well known to those skilled in the art that, during the process of pharmaceutical formulation, there can sometimes be a pH drift, but the pH drift of a pharmaceutical formulation is generally small (e.g., within ±0.8). In some embodiments, the pH drift of a pharmaceutical formulation is within ±0.5.

[0015] In some embodiments, the FAP / 4-1BB / CD40 binding molecule is at a concentration of about 0.01 mg / mL to about 200 mg / mL, e.g., e.g., about 0.1 mg / mL to about 200 mg / mL, about 0.5 mg / mL to about 200 mg / mL, about 1 mg / mL to about 200 mg / mL, about 5 mg / mL to about 200 mg / mL, about 10 mg / mL to about 200 mg / mL, about 10 mg / mL to about 180 mg / mL, about 10 mg / mL to about 160 mg / mL, about 10 mg / mL to about 150 mg / mL, about 10 mg / mL to about 120 mg / mL, about 10 mg / mL to about 110 mg / mL, about 10 mg / mL to about 100 mg / mL, about 10 mg / mL to about 80 mg / mL, about 10 mg / mL to about 60 mg / mL, about 10 mg / mL to about 40 mg / mL, about 20 mg / mL to about 200 mg / mL, about 20 mg / mL to about 180 mg / mL, about 20 mg / mL to about 150 mg / mL, about 20 mg / mL to about 120 mg / mL, about 20 mg / mL to about 110 mg / mL, about 20 mg / mL to about 100 mg / mL, about 20 mg / mL to about 80 mg / mL, about 20 mg / mL to about 60 mg / mL, about 20 mg / mL to about 40 mg / mL, about 40 mg / mL to about 120 mg / mL, about 40 mg / mL to about 110 mg / mL, about 40 mg / mL to about 100 mg / mL, about 40 mg / mL to about 80 mg / mL, 40 mg / mL to about 60 mg / mL, about 60 mg / mL to about 80 mg / mL, about 60 mg / mL to about 120 mg / mL, about 60 mg / mL to about 150 mg / mL, about 80 mg / mL to about 120 mg / mL, or any range between these point values. In some embodiments, the IL-36R / IL-23 binding protein is at a concentration of about 0.01 mg / mL, about 0.05 mg / mL, about 0.1 mg / mL, about 0.2 mg / mL, about 0.3 mg / mL, about 0.4 mg / mL, about 0.5 mg / mL, about 0.6 mg / mL, about 0.7 mg / mL, about 0.8 mg / mL, about 0.9 mg / mL, about 1 mg / mL, about 2 mg / mL, about 3 mg / mL, about 4 mg / mL, about 5 mg / mL, about 6 mg / mL, about 7 mg / mL, about 8 mg / mL, about 9 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 65 mg / mL, about 70 mg / mL, about 75 mg / mL, about 80 mg / mL, about 85 mg / mL, about 90 mg / mL, about 95 mg / mL, about 100 mg / mL, about 110 mg / mL, about 120 mg / mL, about 130 mg / mL, about 140 mg / mL, about 150 mg / mL, about 160 mg / mL, about 170 mg / mL, about 180 mg / mL, about 190 mg / mL, or about 200 mg / mL.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, 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.

[0016] In some embodiments, the pharmaceutical composition of any one of the above, comprises a surfactant. In some embodiments, the surfactant is a non-ionic surfactant. In some embodiments, the surfactant is selected from the group consisting of poloxamer (e.g., poloxamer 188), polysorbate (e.g., polysorbate 20 (i.e., PS20), polysorbate 80 (i.e., PS80)), polyoxyl, Triton, sodium dodecylsulfonate, sodium lauryl sulfate, octyl glucoside sodium, lauryl-sulfo- betaine, myristyl-sulfo-betaine, linoleyl-sulfo-betaine, stearyl-sulfo-betaine, lauryl- sarcosine, myristyl-sarcosine, linoleyl-sarcosine, stearyl-sarcosine, linoleyl- betaine, myristyl-betaine, cetyl-betaine, lauryl-amidopropyl-betaine, cocamidopropyl- betaine, linoleamidopropyl-betaine, myristamidopropyl-betaine, palmitamidopropyl- betaine, isostearamidopropyl-betaine, myristamidopropyl-dimethylamine, palmitamidopropyl-dimethylamine, isostearamidopropyl-dimethylamine, sodium methyl cocoyl, sodium methyl oleyl taurate, polyethylene glycol, polypropylene glycol, copolymer of ethylene and propylene glycol, and the like, or any combination thereof. In some embodiments, the surfactant is sodium dodecyl sulfate (SDS) or polysorbate. In some embodiments, the surfactant is polysorbate 80.

[0017] In some embodiments, the concentration of the surfactant is about 0.01 to about 10.0 mg / mL, for example, about 0.01 to about 9.0 mg / mL, about 0.01 to about 8.0 mg / mL, about 0.01 to about 7.0 mg / mL, about 0.01 to about 6.0 mg / mL, about 0.01 to about 5.0 mg / mL, about 0.01 to about 4.0 mg / mL, about 0.01 to about 3.0 mg / mL, about 0.01 to about 2.0 mg / mL, about 0.01 to about 1.9 mg / mL, about 0.01 to about 1.8 mg / mL, about 0.01 to about 1.7 mg / mL, about 0.01 to about 1.6 mg / mL, about 0.1 to about 1.6 mg / mL, about 0.2 to about 1.6 mg / mL, about 0.2 to about 1.5 mg / mL, about 0.2 to about 1.4 mg / mL, about 0.2 to about 1.3 mg / mL, about 0.2 to about 1.2 mg / mL, and any range between these point values. In some embodiments, the concentration of the surfactant is about 0.01 mg / mL, about 0.02 mg / mL, about 0.03 mg / mL, about 0.04 mg / mL, about 0.05 mg / mL, about 0.1 mg / mL, about 0.2 mg / mL, about 0.3 mg / mL, about 0.4 mg / mL, about 0.5 mg / mL, about 0.6 mg / mL, about 0.7 mg / mL, about 0.8 mg / mL, about 0.9 mg / mL, about 1.0 mg / mL, about 1.1 mg / mL, about 1.2 mg / mL, about 1.3 mg / mL, about 1.4 mg / mL, about 1.5 mg / mL, about 1.6 mg / mL, about 1.7 mg / mL, about 1.8 mg / mL, about 1.9 mg / mL, about 2.0 mg / mL, about 3.0 mg / mL, about 4.0 mg / mL, about 5.0 mg / mL, about 6.0 mg / mL, about 7.0 mg / mL, about 8.0 mg / mL, about 9.0 mg / mL, about 10.0 mg / mL.

[0018] In some embodiments, the pharmaceutical composition comprises a sugar or alcohol, and / or an amino acid or salt thereof.

[0019] In some embodiments, the sugar or alcohol is selected from the group consisting of glucose, sucrose, trehalose, lactose, fructose, maltose, dextran, glycerol, erythritol, glycerol, arabitol, xylitol, sorbitol (also known as sorbose), mannitol, melibiose, melezitose, raffinose, manninotriose, stachyose, maltose, lactulose, maltulose, maltitol, lactitol, and isomaltulose. In some embodiments, the sugar is sucrose or trehalose, and in some particular embodiments, the sugar is sucrose.

[0020] In some embodiments, the concentration of the sugar or alcohol is about 0.1% (w / v) to about 20% (w / v), such as 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) 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), 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 range between these point values. In some embodiments, the concentration of the sugar is about 1% (w / v), 1.2% (w / v), 1.4% (w / v), about 2% (w / v), about 3% (w / v), about 4% (w / v), about 4.2% (w / v), about 4.4% (w / v), about 4.8% (w / v), about 5% (w / v), about 6% (w / v), 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).

[0021] In some embodiments, the amino acid or salt thereof is selected from arginine hydrochloride. In some embodiments, the pharmaceutical composition does not comprise an amino acid or salt thereof.

[0022] In some embodiments, the concentration of the amino acid or salt thereof is about 5 mM to about 500 mM, e.g., about 10 mM to about 150 mM, about 10 mM to about 180 mM, about 10 mM to about 120 mM, about 10 mM to about 100 mM, about 10 mM to about 80 mM, about 10 mM to about 70 mM, about 10 mM to about 50 mM, about 10 mM to about 30 mM, about 30 mM to about 50 mM, about 30 mM to about 80 mM, about 30 mM to about 100 mM, about 50 mM to about 70 mM, about 50 mM to about 100 mM, and any range between these point values. In some embodiments, the concentration of the amino acid or salt thereof 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, about 160 mM.

[0023] In some embodiments, the pharmaceutical composition comprises:

[0024] (1) about 0.01 mg / mL to about 200 mg / mL of a FAP / 4-1BB / CD40 binding molecule,

[0025] about 1 mM to about 100 mM of a buffer,

[0026] about 0.01 to about 10.0 mg / mL of a surfactant,

[0027] about 0.1% (w / v) to about 20% (w / v) of a sugar or alcohol, and

[0028] optionally about 5 mM to about 200 mM of an amino acid or salt thereof,

[0029] the pH of the pharmaceutical composition is about 3.0 to about 7.0; or

[0030] (2) about 0.01 mg / mL to about 200 mg / mL of a FAP / 4-1BB / CD40 binding molecule,

[0031] about 1 mM to about 100 mM of a histidine salt buffer, acetate buffer, citrate buffer, or succinate buffer,

[0032] about 0.01 to about 10.0 mg / mL of a polysorbate,

[0033] about 0.1% (w / v) to about 20% (w / v) of sucrose, trehalose, mannitol, or sorbitol, and

[0034] optionally about 5 mM to about 200 mM of arginine or a salt thereof,

[0035] the pH of the pharmaceutical composition is about 3.0 to about 7.0; or,

[0036] (3) about 0.01 mg / mL to about 200 mg / mL of the FAP / 4-1BB / CD40 binding molecule,

[0037] about 1 mM to about 100 mM of histidine-acetate, citrate-sodium citrate, or histidine- succinate,

[0038] about 0.01 to about 10.0 mg / mL of polysorbate 20 or polysorbate 80,

[0039] about 0.1% (w / v) to about 20% (w / v) of sucrose or trehalose, and,

[0040] optionally about 5 mM to about 200 mM of arginine hydrochloride,

[0041] the pH of the pharmaceutical composition is about 3.0 to about 7.0; or,

[0042] (4) about 0.01 mg / mL to about 200 mg / mL of the FAP / 4-1BB / CD40 binding molecule,

[0043] about 1 mM to about 100 mM of histidine-acetate,

[0044] about 0.01 to about 10.0 mg / mL of polysorbate 80,

[0045] about 0.1% (w / v) to about 20% (w / v) of sucrose, and,

[0046] optionally about 5 mM to about 200 mM of arginine hydrochloride,

[0047] the pH of the pharmaceutical composition is about 3.0 to about 7.0; or,

[0048] (5) about 10 mg / mL to about 180 mg / mL of the FAP / 4-1BB / CD40 binding molecule,

[0049] about 2 mM to about 50 mM of histidine-acetate,

[0050] about 0.1 to about 5.0 mg / mL of polysorbate 80,

[0051] about 0.2% (w / v) to about 15% (w / v) of sucrose, and,

[0052] optionally about 10 mM to about 160 mM of arginine hydrochloride,

[0053] the pH of the pharmaceutical composition is about 4.0 to about 6.0; or,

[0054] (6) about 20 mg / mL to about 160 mg / mL of the FAP / 4-1BB / CD40 binding molecule,

[0055] about 5 mM to about 30 mM histidine-acetate,

[0056] about 0.2 to about 2.0 mg / mL polysorbate 80,

[0057] about 0.5% (w / v) to about 12% (w / v) sucrose, and,

[0058] about 20 mM to about 120 mM arginine hydrochloride, optionally present,

[0059] the pH of the pharmaceutical composition is about 4.5 to about 5.5.

[0060] In some embodiments, the pharmaceutical composition comprises any one of:

[0061] (a-1) about 10 mg / mL to about 200 mg / mL of the FAP / 4-1BB / CD40 binding molecule,

[0062] about 10 mM histidine-acetate buffer,

[0063] about 8.8% w / v trehalose, and,

[0064] about 0.4 mg / mL polysorbate 80,

[0065] the pH of the pharmaceutical composition is about 3 to about 7;

[0066] (a-2) about 10 mg / mL to about 200 mg / mL of the FAP / 4-1BB / CD40 binding molecule,

[0067] about 10 mM histidine-acetate buffer,

[0068] about 8.8% w / v trehalose, and,

[0069] about 0.4 mg / mL polysorbate 80,

[0070] the pH of the pharmaceutical composition is about 4 to about 6;

[0071] (a-3) about 10 mg / mL to about 200 mg / mL of the FAP / 4-1BB / CD40 binding molecule,

[0072] about 10 mM histidine-acetate buffer,

[0073] about 8% w / v sucrose, and

[0074] about 0.4 mg / mL polysorbate 80,

[0075] the pH of the pharmaceutical composition is about 4 to about 6;

[0076] (a-5) about 10 mg / mL to about 200 mg / mL of a FAP / 4-1BB / CD40 binding molecule,

[0077] about 10 mM histidine-acetate buffer,

[0078] about 8% w / v sucrose, and

[0079] about 0.4 mg / mL polysorbate 80,

[0080] the pH of the pharmaceutical composition is about 4 to about 7;

[0081] (a-6) about 10 mg / mL to about 200 mg / mL of a FAP / 4-1BB / CD40 binding molecule,

[0082] about 10 mM histidine-acetate buffer,

[0083] about 8% w / v sucrose, and

[0084] about 0.4 mg / mL polysorbate 80,

[0085] the pH of the pharmaceutical composition is about 4 to about 6;

[0086] (a-7) about 10 mg / mL to about 200 mg / mL of a FAP / 4-1BB / CD40 binding molecule,

[0087] about 10 mM histidine-acetate buffer,

[0088] about 50 mM arginine hydrochloride,

[0089] about 6% w / v sucrose, and

[0090] about 0.8 mg / mL polysorbate 80,

[0091] the pH of the pharmaceutical composition is about 4 to about 6;

[0092] (a-7) about 10 mg / mL to about 200 mg / mL of a FAP / 4-1BB / CD40 binding molecule,

[0093] about 10 mM histidine-acetate buffer,

[0094] about 8% w / v sucrose, and

[0095] about 0.8 mg / mL polysorbate 80,

[0096] the pH of the pharmaceutical composition is about 4 to about 6;

[0097] (a-8) about 10 mg / mL to about 200 mg / mL of a FAP / 4-1BB / CD40 binding molecule,

[0098] about 10 mM citric acid-sodium citrate buffer,

[0099] about 8% w / v sucrose, and

[0100] about 0.8 mg / mL polysorbate 80,

[0101] the pH of the pharmaceutical composition is about 4 to about 6;

[0102] (b-1) about 10 mg / mL to about 200 mg / mL of a FAP / 4-1BB / CD40 binding molecule,

[0103] about 10 mM histidine-succinate buffer,

[0104] about 8% w / v sucrose, and

[0105] about 0.8 mg / mL polysorbate 80;

[0106] the pH of the pharmaceutical composition is about 4.0 to about 6.0;

[0107] (b-2) about 10 mg / mL to about 200 mg / mL of a FAP / 4-1BB / CD40 binding molecule,

[0108] about 10 mM histidine-succinate buffer,

[0109] about 4% w / v sucrose, and

[0110] about 0.4 mg / mL polysorbate 80;

[0111] the pH of the pharmaceutical composition is about 4.0 to about 6.0;

[0112] (b-3) about 10 mg / mL to about 200 mg / mL of a FAP / 4-1BB / CD40 binding molecule,

[0113] about 10 mM histidine-succinate buffer,

[0114] about 1% w / v sucrose, 2% w / v mannitol, and

[0115] about 0.4 mg / mL polysorbate 80;

[0116] the pH of the pharmaceutical composition is about 4.0 to about 6.0;

[0117] (b-4) about 10 mg / mL to about 200 mg / mL of the FAP / 4-1BB / CD40 binding molecule,

[0118] about 10 mM histidine-succinic acid buffer,

[0119] about 0.5% w / v sucrose, about 2.5% w / v mannitol, and

[0120] about 0.4 mg / mL polysorbate 80;

[0121] the pH of the pharmaceutical composition is about 4.0 to about 6.0;

[0122] (b-5) about 10 mg / mL to about 200 mg / mL of the FAP / 4-1BB / CD40 binding molecule,

[0123] about 10 mM histidine-succinic acid buffer,

[0124] about 3% w / v sucrose,

[0125] about 0.3 mg / mL polysorbate 80,

[0126] the pH of the pharmaceutical composition is about 4.0 to about 6.0;

[0127] (c-1) about 10 mg / mL to about 200 mg / mL of the FAP / 4-1BB / CD40 binding molecule,

[0128] about 10 mM histidine-succinic acid buffer,

[0129] about 8% w / v sucrose,

[0130] about 0.8 mg / mL polysorbate 80;

[0131] the pH of the pharmaceutical composition is about 4.0 to about 6.0;

[0132] (c-2) about 10 mg / mL to about 200 mg / mL of the FAP / 4-1BB / CD40 binding molecule,

[0133] about 20 mM histidine-succinic acid buffer,

[0134] about 8% w / v sucrose,

[0135] about 0.8 mg / mL polysorbate 80;

[0136] the pH of the pharmaceutical composition is about 4.0 to about 6.0;

[0137] (c-3) about 10 mg / mL to about 200 mg / mL of the FAP / 4-1BB / CD40 binding molecule,

[0138] about 20 mM histidine-succinate buffer,

[0139] about 8% w / v sucrose,

[0140] about 0.8 mg / mL polysorbate 80;

[0141] the pH of the pharmaceutical composition is about 4.0 to about 6.0;

[0142] (c-4) about 10 mg / mL to about 200 mg / mL of the FAP / 4-1BB / CD40 binding molecule,

[0143] about 20 mM histidine-succinate buffer,

[0144] about 2% w / v sucrose, about 4% w / v mannitol,

[0145] about 0.8 mg / mL polysorbate 80;

[0146] the pH of the pharmaceutical composition is about 4.0 to about 6.0;

[0147] (c-5) about 10 mg / mL to about 200 mg / mL of the FAP / 4-1BB / CD40 binding molecule,

[0148] about 20 mM histidine-succinate buffer,

[0149] about 1.0% w / v sucrose, about 5% w / v mannitol,

[0150] about 0.8 mg / mL polysorbate 80;

[0151] the pH of the pharmaceutical composition is about 4.0 to about 6.0;

[0152] (c-6) about 10 mg / mL to about 200 mg / mL of the FAP / 4-1BB / CD40 binding molecule,

[0153] about 20 mM histidine-succinate buffer,

[0154] about 8% w / v sucrose,

[0155] about 0.8 mg / mL polysorbate 80;

[0156] the pH of the pharmaceutical composition is about 4.0 to about 6.0;

[0157] (c-7) about 10 mg / mL to about 200 mg / mL of the FAP / 4-1BB / CD40 binding molecule,

[0158] about 30 mM histidine-succinate buffer,

[0159] about 9% w / v sucrose,

[0160] about 0.9 mg / mL polysorbate 80;

[0161] the pH of the pharmaceutical composition is about 4.0 to about 6.0;

[0162] (c-8) about 10 mg / mL to about 200 mg / mL of the FAP / 4-1BB / CD40 binding molecule,

[0163] about 25 mM histidine-succinate buffer,

[0164] about 7.5% w / v sucrose,

[0165] about 0.75 mg / mL polysorbate 80;

[0166] the pH of the pharmaceutical composition is about 4.0 to about 6.0.

[0167] FAP binding molecule

[0168] In some embodiments, the present disclosure provides a FAP binding molecule comprising a first antigen binding domain that specifically binds FAP. In some embodiments, the first antigen binding domain comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein: the VH comprises HCDR1, HCDR2, and HCDR3 in the amino acid sequence set forth in SEQ ID NO: 1, and / or the VL comprises LCDR1, LCDR2, and LCDR3 in the amino acid sequence set forth in SEQ ID NO: 2.

[0169] The CDRs are defined according to the Kabat, IMGT, Chothia, AbM, or Contact numbering system. In some particular embodiments, the CDRs are defined according to the Kabat numbering system.

[0170] In some embodiments, the FAP binding molecule comprises a VH comprising HCDR1, HCDR2, and HCDR3 as set forth in SEQ ID NOs: 3, 4, and 5, respectively, and / or a VL comprising LCDR1, LCDR2, and LCDR3 as set forth in SEQ ID NOs: 6, 7, and 8, respectively.

[0171] In some embodiments, the FAP binding molecule comprises a VH and a VL, wherein: any one of the HCDRs comprised by the VH has 0, 1, 2, 3, 4, or 5 amino acid mutations compared to any of the foregoing HCDRs; and / or, any one of the LCDRs comprised by the VL has 0, 1, 2, 3, 4, or 5 amino acid mutations compared to any of the foregoing LCDRs.

[0172] In some specific embodiments, the above amino acid mutations are conservative substitutions, replacements, or modifications, and / or deletions, additions that do not affect function.

[0173] In some embodiments, the present disclosure provides a FAP binding molecule comprising any one or any combination of 2, 3, 4, 5, or 6 of the foregoing HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3.

[0174] In some embodiments, the FAP binding molecule comprises a VH and a VL, wherein:

[0175] the VH comprises an amino acid sequence as set forth in SEQ ID NO: 1, or an amino acid sequence having at least 80% sequence identity thereto; and / or,

[0176] the VL comprises an amino acid sequence as set forth in SEQ ID NO: 2, or an amino acid sequence having at least 80% sequence identity thereto.

[0177] In some embodiments, the present disclosure provides a FAP binding molecule comprising any one or a combination of 2 of the foregoing VH, VL.

[0178] In some embodiments, the FAP binding molecule further comprises a light chain constant region, and / or a heavy chain constant region.

[0179] In some embodiments, the light chain constant region is derived from a kappa light chain, a lambda light chain, or a variant of any of the foregoing. For example, derived from a human kappa light chain, a human lambda light chain, or a variant of any of the foregoing. In some specific embodiments, the light chain constant region is derived from a human kappa light chain or a variant thereof.

[0180] In some embodiments, the heavy chain constant region is derived from an IgGl, IgG2, IgG3, IgG4, or a variant of any of the foregoing. For example, derived from a human IgGl, a human IgG2, a human IgG3, a human IgG4, or a variant of any of the foregoing.

[0181] In some embodiments, the FAP binding molecule comprises a Fab heavy chain and a Fab light chain, wherein the Fab heavy chain comprises a heavy chain variable region (VH) and a heavy chain constant region (CH1), and the Fab light chain comprises a light chain variable region (VL) and a light chain constant region (CL).

[0182] In some specific embodiments, the FAP binding molecule comprises a Fab heavy chain and a Fab light chain, wherein:

[0183] the Fab heavy chain comprises an amino acid sequence as set forth in SEQ ID NO: 66, or an amino acid sequence having at least 80% sequence identity thereto; and / or, the Fab light chain comprises an amino acid sequence as set forth in SEQ ID NO: 67, or an amino acid sequence having at least 80% sequence identity thereto.

[0184] In the context of the present disclosure, “at least 80% (sequence) identity” encompasses at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% (sequence) identity; “at least 90% (sequence) identity” encompasses at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% (sequence) identity.

[0185] In some embodiments, the FAP binding molecule further comprises a human immunoglobulin Fc region. In some specific embodiments, the Fc region is a Fc region of human IgGl, IgG2, IgG4.

[0186] In some embodiments, the foregoing FAP binding molecules further comprise a human immunoglobulin Fc region; for example, the Fc region is that of human IgGl, IgG2, or IgG4. In some embodiments, the Fc region can have mutations, exemplary mutations include L234A / L235A, L234A / L235A / P329G, L234E, L234F, L234E / L235F, L234E / L235F / P329G on IgGl, V234A / G237A / P238S / H268A / V309L / A330S / P331S on IgG2, F234A / L235A on IgG4, S228P / F234A / L235A on IgG4, N297A on IgG2 or IgG4, V234A / G237A on IgG2, K214T / E233P / L234V / L235A / G236 deletion / A327G / P331A / D365E / L358M on IgGl, H268Q / V309L / A330S / P331S on IgG2, S267E / L328F on IgGl, L234F / L235E / D265A on IgGl, L234A / L235A / G237A / P238S / H268A / A330S / P331S on IgGl, S228P / F234A / L235A / G237A / P238S on IgG4. Hybrid IgG2 / 4 Fc domains can also be used, for example, an Fc having residues 117-260 from IgG2 and residues 261-447 from IgG4. In some embodiments, mutations are introduced into the Fc region to reduce or eliminate effector function of the Fc region (e.g., to reduce or eliminate ADCC effects).

[0187] In some specific embodiments, the foregoing FAP binding molecules comprise an Fc region of human IgGl having at least one of the following mutations: 234A, 235A, 220A, 297A or 297Q, 267E, 328F.

[0188] Exemplary, the Fc region of human IgGl comprises: 234A / 235A, 234A / 235A / 297A, 220A / 234A / 235A, 220A / 267E / 328F, 220A / 234A / 235A / 297A, 267E / 328F, or 234A / 235A / 297A mutations. In some specific embodiments, the Fc region of human IgGl comprises: L234A / L235A, L234A / L235A / N297A, C220A / L234A / L235A, C220A / 267E / 328F, C220A / L234A / L235A / N297A, S267E / L328F, or L234A / L235A / N297A mutations.

[0189] In some specific embodiments, the aforementioned FAP binding molecule comprises an Fc region of human IgG4, which has at least one mutation as shown below: 228P, 234A, 235A, 447A. Exemplary, the Fc region of human IgG4 comprises S228P mutation.

[0190] In the context of the mutations comprised by the Fc region in the present disclosure, " / " means "and", for example, "L234A / L235A" means "L234A and L235A, i.e., the Fc comprises L234A and L235A mutations; the mutated amino acid positions are numbered according to the EU numbering system.

[0191] In some embodiments, the FAP binding molecule comprises a heavy chain (HC) and a light chain (LC), wherein:

[0192] the heavy chain comprises an amino acid sequence as set forth in SEQ ID NO: 9, or an amino acid sequence having at least 80% sequence identity thereto; and / or,

[0193] the light chain comprises an amino acid sequence as set forth in SEQ ID NO: 10, or an amino acid sequence having at least 80% sequence identity thereto.

[0194] In some embodiments, the present disclosure provides a FAP binding molecule, which comprises a combination of any 1 or 2 of the aforementioned heavy chain (HC) and light chain (LC).

[0195] In some embodiments, the aforementioned FAP binding molecule binds to human FAP or an epitope thereof with a KD value of < 10"7; the aforementioned FAP binding molecule binds to FAP (e.g., human FAP) or a fragment thereof with a KD of 10"7M, 10"8M, 10"9M, 10"10M, 10"11M, or lower.

[0196] In some embodiments, the FAP binding molecule is an anti-FAP antibody or antigen binding fragment thereof; illustratively, the anti-FAP antibody is a murine, chimeric, humanized, or human antibody.

[0197] In some embodiments, the FAP binding molecule is a human antibody or antigen binding fragment thereof, or a humanized antibody or antigen binding fragment thereof.

[0198] In some specific embodiments, the anti-FAP antibody or antigen binding fragment thereof includes, but is not limited to, Fab, Fv, sFv, Fab', F(ab')2, linear antibodies, single chain antibodies, scFv, sdAb, sdFv, nanobodies, peptibody, domain antibodies, and multispecific antibodies (bispecific antibodies, diabodies, triabodies and tetrabodies, tandem di-scFv, tandem tri-scFv), e.g., the anti-FAP antigen binding fragment is a scFv, Fv, Fab, or Fab' fragment.

[0199] In some embodiments, there is provided an anti-FAP antibody or antigen binding fragment thereof that binds to or competes for binding to the same FAP (e.g., human FAP) epitope as a preceding anti-FAP antibody or antigen binding fragment thereof.

[0200] In some embodiments, there is provided an anti-FAP antibody or antigen binding fragment thereof that blocks the binding of a preceding anti-FAP antibody or antigen binding fragment thereof to FAP (e.g., human FAP).

[0201] In some embodiments, there is provided an anti-FAP antibody or antigen binding fragment thereof whose binding to FAP (e.g., human FAP) is blocked by a preceding anti-FAP antibody or antigen binding fragment thereof.

[0202] FAP / 4-1BB / CD40 binding molecules

[0203] In some embodiments, a preceding FAP / 4-1BB / CD40 binding molecule, comprising a first antigen binding domain that specifically binds FAP, a second antigen binding domain that specifically binds CD40, and a third antigen binding domain that specifically binds 4-1BB; capable of simultaneously or separately specifically binding FAP, 4-1BB, and CD40.

[0204] In some embodiments, the third antigen binding domain comprises at least one immunoglobulin single variable domain that specifically binds to 4-1BB, comprising a CDR1, a CDR2 and a CDR3 as shown in any one of SEQ ID NOs: 28, 23-26, 18 and 33. The CDR1, CDR2 and CDR3 are defined according to the Kabat, IMGT, Chothia, AbM or Contact numbering system. In some particular embodiments, the CDR1, CDR2 and CDR3 are defined according to the Kabat numbering system.

[0205] Exemplarily, in some particular embodiments, the immunoglobulin single variable domain in the third antigen binding domain comprises a CDR1, a CDR2 and a CDR3 as set forth in SEQ ID NOs: 19, 30 and 31, respectively.

[0206] Exemplarily, in some particular embodiments, the immunoglobulin single variable domain in the third antigen binding domain, comprises:

[0207] a CDR1 as set forth in SEQ ID NO: 19,

[0208] a CDR2 as set forth in SEQ ID NO: 20 or 29, and

[0209] a CDR3 as set forth in SEQ ID NO: 21 or 27.

[0210] Exemplarily, in some particular embodiments, the immunoglobulin single variable domain in the third antigen binding domain, comprises a CDR1, a CDR2 and a CDR3 as set forth in any one of:

[0211] a-1) a CDR1, a CDR2 and a CDR3 as set forth in SEQ ID NOs: 19, 29 and 27, respectively;

[0212] a-2) a CDR1, a CDR2 and a CDR3 as set forth in SEQ ID NOs: 19, 20 and 27, respectively;

[0213] a-3) a CDR1, a CDR2 and a CDR3 as set forth in SEQ ID NOs: 19, 20 and 21, respectively.

[0214] Exemplarily, in some particular embodiments, the immunoglobulin single variable domain in the third antigen binding domain is humanized, affinity matured, T-cell epitope- removed, antibody deamidation-reduced, antibody aggregation-reduced, antibody isomerization- reduced, pre-existing anti-drug antibody (pre-ADA) binding-reduced, and / or anti-drug antibody (ADA) binding-reduced.

[0215] Exemplarily, the immunoglobulin single variable domain in the third antigen binding domain is humanized, wherein the framework region templates used in the humanization are derived from IGHV3-64*04, IGHV3-23*03, and / or IGHV3-74*01. For example, FR1 is from IGHV3-64*04, FR2 is from IGHV3-23*03, and FR3 is from IGHV3-74*01.

[0216] Exemplarily, the immunoglobulin single variable domain in the third antigen binding domain is T-cell epitope (TCE)-removed and / or reduced. For example, the T-cell epitope-removed immunoglobulin single variable domain comprises the following amino acid mutation: 99Y, numbered according to the Kabat numbering convention.

[0217] Exemplarily, the immunoglobulin single variable domain in the third antigen binding domain is pre-existing anti-drug antibody (pre-ADA) binding-reduced and / or anti-drug antibody (ADA) binding-reduced. For example, the C-terminal sequence of the immunoglobulin single variable domain is mutated from “TVSS” (SEQ ID NO: 68) to “TVSAA” (SEQ ID NO: 69).

[0218] Exemplarily, in some particular embodiments, the immunoglobulin single variable domain in the third antigen binding domain is antibody aggregation-reduced. For example, the C-terminal sequence of the immunoglobulin single variable domain is mutated from “TLVTVSS” (SEQ ID NO: 70) to “TQVTVSS” (SEQ ID NO: 71).

[0219] Exemplarily, in some particular embodiments, the immunoglobulin single variable domain in the third antigen binding domain comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 18, 23-26, 28, and 33, or has at least 80% sequence identity thereto.

[0220] Exemplarily, in some particular embodiments, the third antigen binding domain comprises one or more (e.g. 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.) of the aforementioned immunoglobulin single variable domains, any two of which can be the same or different, and any two of which can be directly connected or connected via a linker.

[0221] In some embodiments, the second antigen binding domain comprises at least one immunoglobulin single variable domain that specifically binds to CD40, which comprises a CDR1, a CDR2 and a CDR3 as shown in any one of the following:

[0222] 1) CDR1, CDR2 and CDR3 in the amino acid sequences shown in SEQ ID NO: 11, 15 or 32, respectively;

[0223] 2) CDR1 comprises an amino acid sequence shown in SEQ ID NO: 12, and / or, CDR2 comprises an amino acid sequence shown in SEQ ID NO: 13, and / or, CDR3 comprises an amino acid sequence shown in SEQ ID NO: 14;

[0224] 3) CDR1, CDR2 and CDR3 shown in SEQ ID NO: 12, 13 and 14, respectively.

[0225] The aforementioned CDR1, CDR2 and CDR3 are defined according to the Kabat, IMGT, Chothia, AbM or Contact numbering system. In some particular embodiments, the CDR1, CDR2 and CDR3 are defined according to the Kabat numbering system.

[0226] Exemplarily, in some particular embodiments, the immunoglobulin single variable domain in the second antigen binding domain is humanized, affinity matured, T-cell epitope removed, antibody deamidation reduced, antibody aggregation reduced, antibody isomerization reduced, binding to pre-existing anti-drug antibodies (pre-ADAs) reduced, and / or binding to anti-drug antibodies (ADAs) reduced.

[0227] Exemplarily, in some particular embodiments, the immunoglobulin single variable domain in the second antigen binding domain is humanized, which uses a framework region template of human germline genes derived from IGHV3-48*03.

[0228] Exemplarily, in some particular embodiments, the immunoglobulin single variable domain in the second antigen binding domain is engineered to reduce and / or abrogate binding to pre-existing anti-drug antibodies (pre-ADAs) and / or reduce binding to anti-drug antibodies (ADAs). For example, the C-terminal sequence of the immunoglobulin single variable domain is mutated from "TVSS" (SEQ ID NO: 68) to "TVSAA" (SEQ ID NO: 69).

[0229] Exemplarily, in some particular embodiments, the immunoglobulin single variable domain in the second antigen binding domain comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 11, 15, or 32, or an amino acid sequence having at least 80% sequence identity thereto.

[0230] Exemplarily, in some particular embodiments, the second antigen binding domain comprises one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.) of the aforementioned immunoglobulin single variable domains, any two of which can be the same or different, and any two of which can be directly connected or connected via a linker.

[0231] In some embodiments, the first antigen binding domain comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein: the VH comprises HCDR1, HCDR2, and HCDR3 in the amino acid sequence set forth in SEQ ID NO: 1, and / or the VL comprises LCDR1, LCDR2, and LCDR3 in the amino acid sequence set forth in SEQ ID NO: 2. The CDRs are defined according to the Kabat, IMGT, Chothia, AbM, or Contact numbering system. In some particular embodiments, the CDRs are defined according to the Kabat numbering system.

[0232] Exemplarily, in some particular embodiments, the first antigen binding domain comprises a VH comprising HCDR1, HCDR2, and HCDR3 as set forth in SEQ ID NOs: 3, 4, and 5, respectively, and / or a VL comprising LCDR1, LCDR2, and LCDR3 as set forth in SEQ ID NOs: 6, 7, and 8, respectively.

[0233] Exemplarily, in some particular embodiments, wherein: the VH comprises an amino acid sequence as set forth in SEQ ID NO: 1, or an amino acid sequence having at least 80% sequence identity thereto; and / or, the VL comprises an amino acid sequence as set forth in SEQ ID NO: 2, or an amino acid sequence having at least 80% sequence identity thereto.

[0234] In some embodiments, the first antigen binding domain further comprises a heavy chain constant region. In some embodiments, the heavy chain constant region is derived from human IgGl, human IgG2, human IgG3, human IgG4, or a variant of any of the foregoing. In some embodiments, the heavy chain constant region is derived from human IgGl or a variant thereof. In some embodiments, the heavy chain constant region is derived from human IgG4 or a variant thereof.

[0235] In some embodiments, the first antigen binding domain comprises a Fab heavy chain and a Fab light chain, wherein the Fab heavy chain comprises a heavy chain variable region (VH) and a heavy chain constant region (CH1), and the Fab light chain comprises a light chain variable region (VL) and a light chain constant region (CL).

[0236] In some embodiments, the Fab heavy chain comprises an amino acid sequence as set forth in SEQ ID NO: 66, or an amino acid sequence having at least 80% sequence identity thereto; and / or the Fab light chain comprises an amino acid sequence as set forth in SEQ ID NO: 67, or an amino acid sequence having at least 80% sequence identity thereto.

[0237] In some embodiments, the FAP / 4-1BB / CD40 binding molecule further comprises a Fc region. In some embodiments, the Fc region is a Fc region of human IgGl, IgG2, IgG4.

[0238] In some embodiments, the Fc region comprises an amino acid mutation, exemplary mutations include L234A / L235A, L234A / L235A / P329G, L234E, L234F, L234E / L235F, L234E / L235F / P329G on IgGl, V234A / G237A / P238S / H268A / V309L / A330S / P331S on IgG2, F234A / L235A on IgG4, S228P / F234A / L235A on IgG4, N297A on IgG2 or IgG4, V234A / G237A on IgG2, K214T / E233P / L234V / L235A / G236 deletion / A327G / P331A / D365E / L358M on IgGl, H268Q / V309L / A330S / P331S on IgG2, S267E / L328F on IgGl, L234F / L235E / D265A on IgGl, L234A / L235A / G237A / P238S / H268A / A330S / P331S on IgGl, S228P / F234A / L235A / G237A / P238S on IgG4. Hybrid IgG2 / 4 Fc domains can also be used, for example, an Fc having residues 117-260 from IgG2 and residues 261-447 from IgG4.

[0239] In some specific embodiments, the FAP / 4-1BB / CD40 binding molecule comprises an Fc region of human IgGl having at least one mutation as shown below: 234A, 235A, 220A, 297A or 297Q, 267E, 328F.

[0240] Exemplary, the Fc region of human IgGl comprises: 234A / 235A, 234A / 235A / 297A, 220A / 234A / 235A, 220A / 267E / 328F, 220A / 234A / 235A / 297A, 267E / 328F, or 234A / 235A / 297A mutations. In some specific embodiments, the Fc region of human IgGl comprises: L234A / L235A, L234A / L235A / N297A, C220A / L234A / L235A, C220A / 267E / 328F, C220A / L234A / L235A / N297A, S267E / L328F, or L234A / L235A / N297A mutations.

[0241] In some embodiments, the FAP / 4-1BB / CD40 binding molecules comprise a Fc region of human IgG4 having at least one mutation as shown below: 228P, 234A, 235A, 447A. Exemplarily, the Fc region of human IgG4 comprises S228P mutation.

[0242] In some embodiments, mutations are introduced into the Fc region to reduce or eliminate effector functions of the Fc region (e.g., to reduce or eliminate ADCC effect).

[0243] In some embodiments, mutations can also be introduced into the Fc region to pair the two subunits (Fc1, Fc2) of the Fc region to form a dimer. For example, 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 to create a protuberance (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 to create 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 input residues having a larger side chain volume are phenylalanine (F), tyrosine (Y), arginine (R), or tryptophan (W). In some embodiments, the input residues having a smaller side chain volume are serine (S), alanine (A), valine (V), or threonine (T). Exemplarily, the Knob mutation comprises T366W substitution, and the Hole mutation is selected from at least one, at least two, or three mutations in T366S, L368A, and Y407V.

[0244] In some embodiments, the Fc region can allow the binding molecule to form a dimeric molecule while prolonging the in vivo half-life of the binding molecule.

[0245] In some embodiments, in the FAP / 4-1BB / CD40 binding molecule, the first antigen binding domain comprises a Fab heavy chain and a Fab light chain, wherein:

[0246] the Fab heavy chain is located at the N-terminus of the Fc region, the third antigen binding domain is located at the C-terminus of the Fc region, and the second antigen binding domain is located at the C-terminus of the third antigen binding domain;

[0247] the Fab heavy chain is located at the N-terminus of the Fc region, the third antigen binding domain is located at the C-terminus of the Fc region, and the second antigen binding domain is located at the C-terminus of the Fab light chain;

[0248] the Fab heavy chain is located at the N-terminus of the Fc region, the second antigen binding domain is located at the C-terminus of the Fc region, and the third antigen binding domain is located at the C-terminus of the Fab light chain.

[0249] Alternatively,

[0250] the Fab heavy chain is located at the N-terminus of the Fc region, the second antigen binding domain is located at the C-terminus of the Fc region, and the third antigen binding domain is located at the C-terminus of the Fab light chain.

[0251] In some embodiments, the third antigen binding domain that specifically binds to 4-1BB and the second antigen binding domain that specifically binds to CD40 can be directly connected or connected through a linker.

[0252] In some embodiments, the third antigen binding domain that specifically binds to 4-1BB and the Fc region can be directly connected or connected through a linker.

[0253] In some embodiments, the second antigen binding domain that specifically binds to CD40 and the Fc region can be directly connected or connected through a linker.

[0254] In some embodiments, the third antigen binding domain that specifically binds to 4-1BB and the Fab light chain of the first antigen binding domain that specifically binds to FAP can be directly connected or connected through a linker.

[0255] In some embodiments, the second antigen binding domain that specifically binds to CD40 and the Fab light chain of the first antigen binding domain that specifically binds to FAP can be directly connected or connected through a linker.

[0256] In some embodiments, the third antigen binding domain that specifically binds to 4-1BB comprises 2, 3, 4, 5, 6, 7, 8, etc. immunoglobulin single variable domains. Among them, any two immunoglobulin single variable domains can be directly connected or connected through a linker.

[0257] In some embodiments, the second antigen binding domain that specifically binds to CD40 comprises 2, 3, 4, 5, 6, 7, 8, etc. immunoglobulin single variable domains. Among them, any two immunoglobulin single variable domains can be directly connected or connected through a linker.

[0258] Exemplarily, the linker comprises, but is not limited to, an amino acid sequence as set forth in (GmSn)h or (GGNGT)h (SEQ ID NO: 72) or (YGNGT)h (SEQ ID NO: 73) or (EPKSS)h (SEQ ID NO: 74), wherein m, 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).

[0259] In some embodiments, the linker is a (GxS)y linker, wherein x is selected from an integer of 1-5 (e.g., 1, 2, 3, 4, or 5), and y is selected from an integer of 1-6 (e.g., 1, 2, 3, 4, 5, or 6); in some specific embodiments, the linker is selected from G4S (SEQ ID NO: 75), GS, GAP, (G4S)2(SEQ ID NO: 76), (G4S)3(SEQ ID NO: 77), (G4S)4(SEQ ID NO: 78), (G4S)5(SEQ ID NO: 79), ASGS (SEQ ID NO: 80), e.g., (G4S)2.

[0260] In some embodiments, when the number of linkers comprised in the FAP / 4-1BB / CD40 binding molecule is 2 or more, any two linkers can be the same or different.

[0261] In some specific embodiments, the FAP / 4-1BB / CD40 binding molecule comprises an anti-FAP antibody, which comprises two heavy chains (HC) and two light chains (LC). The VH of one HC forms an antigen binding site with the VL of one LC, and the VH of the other HC forms an antigen binding site with the VL of the other LC.

[0262] In some specific embodiments, the FAP / 4-1BB / CD40 binding molecule comprises at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, etc.) VHH that specifically binds to CD40, and comprises at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, etc.) VHH that specifically binds to 4-1BB.

[0263] In some specific embodiments, any one of the VHHs that specifically binds to CD40 can be linked to the C-terminus of the heavy chain of the anti-FAP antibody, and / or, to the C-terminus of the light chain of the anti-FAP antibody. The C-terminus of the VHH that specifically binds to CD40 can be further linked to one or more VHHs that specifically binds to 4-1BB, and / or, one or more VHHs that specifically binds to CD40.

[0264] In some specific embodiments, any one of the VHHs that specifically binds to 4-1BB can be linked to the C-terminus of the heavy chain of the anti-FAP antibody, and / or, to the C-terminus of the light chain of the anti-FAP antibody. The C-terminus of the VHH that specifically binds to 4-1BB can be further linked to one or more VHHs that specifically binds to 4-1BB, and / or, one or more VHHs that specifically binds to CD40.

[0265] In some embodiments, the valency ratio of the first, the second and the third antigen binding domain in the FAP / 4-1BB / CD40 binding molecule is (1-2):(1-5):(1-5); in some embodiments, the valency ratio of the first, the second and the third antigen binding domain is 1:(1-4):(1-4). In some specific embodiments, the valency ratio is 1:2:2, 1:2:4, 1:1:1 or 1:1:2.

[0266] In the present disclosure, the "valency" is described as the number of antigen binding moieties present in an antigen binding molecule (e.g., an antigen binding domain). Thus, a single binding molecule can bind to more than one binding site on a target molecule.

[0267] In some embodiments, the FAP / 4-1BB / CD40 binding molecule comprises a first polypeptide chain and a second polypeptide chain, wherein, from N-terminus to C-terminus direction, the FAP / 4-1BB / CD40 binding molecule comprises any one of the following groups or combinations:

[0268] (I) the first polypeptide chain: [Fab heavy chain of the first antigen binding domain that specifically binds to FAP]-[Fc region]-[linker]a-[the third antigen binding domain that specifically binds to 4-1BB]-[linker]b-[the second antigen binding domain that specifically binds to CD40]; and,

[0269] the second polypeptide chain: [Fab light chain of the first antigen binding domain that specifically binds to FAP];

[0270] (I) first polypeptide chain: [Fab heavy chain of a first antigen binding domain specifically binding to FAP]-[Fc region]-[linker]a-[second antigen binding domain specifically binding to CD40]; and,

[0271] second polypeptide chain: [Fab light chain of a first antigen binding domain specifically binding to FAP]-[linker]b-[second antigen binding domain specifically binding to 4-1BB];

[0272] (III) first polypeptide chain: [Fab heavy chain of a first antigen binding domain specifically binding to FAP]-[Fc region]-[linker]a-[third antigen binding domain specifically binding to 4-1BB]-[linker]b-[third antigen binding domain specifically binding to 4-1BB]; and,

[0273] second polypeptide chain: [Fab light chain of a first antigen binding domain specifically binding to FAP]-[linker]c-[second antigen binding domain specifically binding to CD40];

[0274] (IV) first polypeptide chain: [Fab heavy chain of a first antigen binding domain specifically binding to FAP]-[Fc region]-[linker]a-[second antigen binding domain specifically binding to CD40]-[linker]b-[third antigen binding domain specifically binding to 4-1BB]; and,

[0275] second polypeptide chain: [Fab light chain of a first antigen binding domain specifically binding to FAP];

[0276] (V) first polypeptide chain: [Fab heavy chain of a first antigen binding domain specifically binding to FAP]-[Fc region]-[linker]a-[second antigen binding domain specifically binding to CD40]; and,

[0277] second polypeptide chain: [Fab light chain of a first antigen binding domain specifically binding to FAP]-[linker]b-[third antigen binding domain specifically binding to 4-1BB];

[0278] wherein - represents a peptide bond, any two linkers can be the same or different; a, b, c are independently selected from 0 or 1.

[0279] Exemplarily, the linker comprises, but is not limited to, an amino acid sequence as set forth in (GmSn)h or (GGNGT)h (SEQ ID NO: 72) or (YGNGT)h (SEQ ID NO: 73) or (EPKSS)h (SEQ ID NO: 74), wherein m, 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).

[0280] In some embodiments, the linker is a (GxS)y linker, wherein x is selected from an integer of 1-5 (e.g., 1, 2, 3, 4, or 5), and y is selected from an integer of 1-6 (e.g., 1, 2, 3, 4, 5, or 6); in some specific embodiments, the linker is selected from G4S, GS, GAP, (G4S)2, (G4S)3, (G4S)4, (G4S)5, ASGS, for example, (G4S)2.

[0281] In some embodiments, the aforementioned FAP / 4-1BB / CD40 binding molecules comprise two or more first antigen binding domains specifically binding to FAP, any two of which can be the same or different, selected from any of the first antigen binding domains provided in the present disclosure;

[0282] In some embodiments, the aforementioned FAP / 4-1BB / CD40 binding molecules comprise two or more second antigen binding domains specifically binding to CD40, any two of which can be the same or different, selected from any of the second antigen binding domains provided in the present disclosure;

[0283] In some embodiments, the aforementioned FAP / 4-1BB / CD40 binding molecules comprise two or more third antigen binding domains specifically binding to 4-1BB, any two of which can be the same or different, selected from any of the third antigen binding domains provided in the present disclosure.

[0284] In some embodiments, the FAP / 4-1BB / CD40 binding molecules comprise a first polypeptide chain and a second polypeptide chain as set forth in any of the following:

[0285] 1) the first polypeptide chain comprises an amino acid sequence as set forth in SEQ ID NO: 49 or an amino acid sequence having 80% sequence identity thereto, and the second polypeptide chain comprises an amino acid sequence as set forth in SEQ ID NO: 50 or an amino acid sequence having 80% sequence identity thereto.

[0286] 2) the first polypeptide chain comprises an amino acid sequence as shown in SEQ ID NO: 34 or an amino acid sequence having 80% sequence identity thereto, and the second polypeptide chain comprises an amino acid sequence as shown in SEQ ID NO: 35 or an amino acid sequence having 80% sequence identity thereto.

[0287] 3) the first polypeptide chain comprises an amino acid sequence as shown in SEQ ID NO: 36 or an amino acid sequence having 80% sequence identity thereto, and the second polypeptide chain comprises an amino acid sequence as shown in SEQ ID NO: 37 or an amino acid sequence having 80% sequence identity thereto.

[0288] 4) the first polypeptide chain comprises an amino acid sequence as shown in SEQ ID NO: 38 or an amino acid sequence having 80% sequence identity thereto, and the second polypeptide chain comprises an amino acid sequence as shown in SEQ ID NO: 39 or an amino acid sequence having 80% sequence identity thereto.

[0289] 5) the first polypeptide chain comprises an amino acid sequence as shown in SEQ ID NO: 40 or an amino acid sequence having 80% sequence identity thereto, and the second polypeptide chain comprises an amino acid sequence as shown in SEQ ID NO: 41 or an amino acid sequence having 80% sequence identity thereto.

[0290] 6) the first polypeptide chain comprises an amino acid sequence as shown in SEQ ID NO: 42 or an amino acid sequence having 80% sequence identity thereto, and the second polypeptide chain comprises an amino acid sequence as shown in SEQ ID NO: 43 or an amino acid sequence having 80% sequence identity thereto.

[0291] 7) the first polypeptide chain comprises an amino acid sequence as shown in SEQ ID NO: 45 or an amino acid sequence having 80% sequence identity thereto, and the second polypeptide chain comprises an amino acid sequence as shown in SEQ ID NO: 46 or an amino acid sequence having 80% sequence identity thereto.

[0292] 8) the first polypeptide chain comprises an amino acid sequence as shown in SEQ ID NO: 47 or an amino acid sequence having 80% sequence identity thereto, and the second polypeptide chain comprises an amino acid sequence as shown in SEQ ID NO: 48 or an amino acid sequence having 80% sequence identity thereto.

[0293] In some embodiments, the FAP / 4-1BB / CD40 binding molecules comprise two first polypeptide chains and two second polypeptide chains. In some specific embodiments, the aforementioned FAP / 4-1BB / CD40 binding molecules have two identical first polypeptide chains and two identical second polypeptide chains.

[0294] In some embodiments, the aforementioned FAP / 4-1BB / CD40 binding molecules have at least one of the following properties:

[0295] a) specifically binds to human FAP or an epitope thereof;

[0296] b) specifically binds to human 4-1BB or an epitope thereof;

[0297] c) specifically binds to human CD40 or an epitope thereof;

[0298] d) weakly activates or does not activate 4-1BB signaling pathway without FAP and CD40 crosslinking;

[0299] e) strongly activates or strongly activates 4-1BB signaling pathway after FAP and / or CD40 crosslinking; for example, EC50≤1 nM. Illustratively, EC50≤0.9 nM, EC50≤0.8 nM, EC50≤0.7 nM, EC50≤0.6 nM, EC50≤0.5 nM, EC50≤0.4 nM, EC50≤0.3 nM, EC50≤0.2 nM, EC50≤0.1 nM, EC50≤0.09 nM, EC50≤0.08 nM, EC50≤0.07 nM, EC50≤0.06 nM, EC50≤0.05 nM, EC50≤0.04 nM, EC50≤0.03 nM, EC50≤0.02 nM, or lower;

[0300] f) weakly activates or does not activate CD40 signaling pathway without FAP and 4-1BB crosslinking;

[0301] g) strongly activates or strongly activates CD40 signaling pathway after FAP and / or 4-1BB crosslinking; for example, EC50≤1 nM. Illustratively, EC50≤0.9 nM, EC50≤0.8 nM, EC50≤0.7 nM, EC50≤0.6 nM, EC50≤0.5 nM, EC50≤0.4 nM, EC50≤0.3 nM, EC50≤0.2 nM, EC50≤0.1 nM, EC50≤0.09 nM, EC50≤0.08 nM, EC50≤0.07 nM, EC50≤0.06 nM, EC50≤0.05 nM, EC50≤0.04 nM, EC50≤0.03 nM, EC50≤0.02 nM, or lower;

[0302] h) activating T cells and / or promoting T cell proliferation;

[0303] I) promoting APC (e.g., dendritic cell DC) activation and / or promoting APC (e.g., dendritic cell DC) proliferation;

[0304] j) having low immunogenicity, and / or having low binding level to pre-existing anti-drug antibodies (pre-ADAs);

[0305] k) inhibiting tumor growth.

[0306] In some embodiments, the foregoing FAP / 4-1BB / CD40 binding molecules of the present disclosure are capable of inhibiting tumor growth by at least about 10%, for example, at least about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%.

[0307] In some embodiments, the foregoing FAP / 4-1BB / CD40 binding molecules are anti-FAP / 4-1BB / CD40 multispecific antibodies or antigen-binding fragments thereof, including but not limited to: Fab, Fv, sFv, Fab', F(ab')2, linear antibodies, single-chain antibodies, scFv, sdAb, sdFv, nanobodies, peptibodies, domain antibodies, and multispecific antibodies (bispecific antibodies, diabodies, triabodies and tetrabodies, tandem di-scFv, tandem tri-scFv).

[0308] In some embodiments, there are provided anti-FAP / 4-1BB / CD40 multispecific antibodies or antigen-binding fragments thereof that bind to or compete with the foregoing FAP / 4-1BB / CD40 binding molecules of the present disclosure for binding to FAP, 4-1BB and / or CD40, or to the same epitope of FAP, 4-1BB and / or CD40.

[0309] In some embodiments, there are provided anti-FAP / 4-1BB / CD40 multispecific antibodies or antigen-binding fragments thereof that block the binding of the foregoing FAP / 4-1BB / CD40 binding molecules of the present disclosure to FAP, 4-1BB and / or CD40.

[0310] The anti-FAP / 4-1BB / CD40 multispecific antibodies of the present disclosure can mediate tumor-specific CD40 and 4-1BB activation through FAP, have promoting maturation and activation effects on APC (e.g., dendritic cells DC) and T cells, have low or no liver toxicity, peripheral blood toxicity and other peripheral toxicities, have excellent drug administration windows and drug properties, and provide a solution for clinical treatment of tumors.

[0311] The present disclosure also provides a lyophilized preparation obtained by lyophilizing the aforementioned pharmaceutical composition, or the lyophilized preparation can form the aforementioned pharmaceutical composition after reconstitution.

[0312] In some embodiments, the lyophilized preparation is obtained by lyophilizing a liquid preparation comprising the pharmaceutical composition; wherein the liquid preparation comprises:

[0313] (1) about 0.01 mg / mL to about 200 mg / mL of the FAP / 4-1BB / CD40 binding molecule,

[0314] about 1 mM to about 100 mM of a buffer,

[0315] about 0.01 to about 10.0 mg / mL of a surfactant,

[0316] about 0.1% (w / v) to about 20% (w / v) of a sugar or an alcohol, and

[0317] optionally about 5 mM to about 200 mM of an amino acid or a salt thereof,

[0318] the pH of the pharmaceutical composition is about 3.0 to about 7.0; or

[0319] (2) about 0.01 mg / mL to about 200 mg / mL of the FAP / 4-1BB / CD40 binding molecule,

[0320] about 1 mM to about 100 mM of a histidine salt buffer, a citrate buffer, or a succinate buffer,

[0321] about 0.01 to about 10.0 mg / mL of a polysorbate,

[0322] about 0.1% (w / v) to about 20% (w / v) of sucrose, trehalose, mannitol, or sorbitol, and

[0323] optionally 5 mM to about 200 mM of arginine or a salt thereof,

[0324] the pH of the pharmaceutical composition is about 3.0 to about 7.0; or

[0325] (3) about 0.01 mg / mL to about 200 mg / mL of the FAP / 4-1BB / CD40 binding molecule,

[0326] about 1 mM to about 100 mM of citric acid-sodium citrate or histidine-succinic acid,

[0327] about 0.01 to about 10.0 mg / mL of polysorbate 20 or polysorbate 80,

[0328] about 0.1% (w / v) to about 20% (w / v) sucrose or trehalose, and

[0329] about 5 mM to about 200 mM arginine hydrochloride, optionally present,

[0330] the pH of the pharmaceutical composition is about 3.0 to about 7.0.

[0331] In some specific embodiments, the liquid formulation comprises any of the following:

[0332] (b-1) about 10 mg / mL to about 200 mg / mL of a FAP / 4-1BB / CD40 binding molecule,

[0333] about 10 mM histidine-succinic acid buffer,

[0334] about 8% w / v sucrose, and

[0335] about 0.8 mg / mL polysorbate 80;

[0336] the pH of the pharmaceutical composition is about 4.0 to about 6.0;

[0337] (b-2) about 10 mg / mL to about 200 mg / mL of a FAP / 4-1BB / CD40 binding molecule,

[0338] about 10 mM histidine-succinic acid buffer,

[0339] about 4% w / v sucrose, and

[0340] about 0.4 mg / mL polysorbate 80;

[0341] the pH of the pharmaceutical composition is about 4.0 to about 6.0;

[0342] (b-3) about 10 mg / mL to about 200 mg / mL of a FAP / 4-1BB / CD40 binding molecule,

[0343] about 10 mM histidine-succinic acid buffer,

[0344] about 1% w / v sucrose, 2% w / v mannitol, and

[0345] about 0.4 mg / mL polysorbate 80;

[0346] the pH of the pharmaceutical composition is about 4.0 to about 6.0;

[0347] (b-4) about 10 mg / mL to about 200 mg / mL of a FAP / 4-1BB / CD40 binding molecule,

[0348] about 10 mM histidine-succinate buffer,

[0349] about 0.5% w / v sucrose, about 2.5% w / v mannitol, and

[0350] about 0.4 mg / mL polysorbate 80;

[0351] the pH of the pharmaceutical composition is about 4.0 to about 6.0;

[0352] (b-5) about 10 mg / mL to about 200 mg / mL of the FAP / 4-1BB / CD40 binding molecule,

[0353] about 10 mM histidine-succinate buffer,

[0354] about 3% w / v sucrose,

[0355] about 0.3 mg / mL polysorbate 80,

[0356] the pH of the pharmaceutical composition is about 4.0 to about 6.0.

[0357] The present disclosure also provides a reconstituted solution, which is prepared by reconstituting the aforementioned lyophilized formulation. In some embodiments, the concentration of the FAP / 4-1BB / CD40 binding molecule in the reconstituted solution is greater than the concentration of the FAP / 4-1BB / CD40 binding molecule in the stock solution from which the lyophilized formulation is prepared. In some embodiments, the concentration ratio of the FAP / 4-1BB / CD40 binding molecule in the reconstituted solution to the FAP / 4-1BB / CD40 binding molecule in the stock solution is (1-5): 1. In some specific embodiments, the concentration ratio is (1-2): 1.

[0358] In some embodiments, the reconstituted solution is prepared by reconstituting the aforementioned lyophilized formulation with a solvent to obtain the reconstituted solution. In some specific embodiments, the solvent is preferably water, physiological saline or glucose.

[0359] In some embodiments, the reconstituted solution comprises:

[0360] (1) about 0.01 mg / mL to about 200 mg / mL of the FAP / 4-1BB / CD40 binding molecule,

[0361] about 1 mM to about 100 mM of a buffer,

[0362] about 0.01 to about 10.0 mg / mL of a surfactant,

[0363] about 0.1% (w / v) to about 20% (w / v) of a sugar or alcohol, and

[0364] about 5 mM to about 200 mM of an amino acid or a salt thereof, optionally present,

[0365] the pH of the pharmaceutical composition is about 3.0 to about 7.0; or,

[0366] (2) about 0.01 mg / mL to about 200 mg / mL of a FAP / 4-1BB / CD40 binding molecule,

[0367] about 1 mM to about 100 mM of a histidine salt buffer, a citrate buffer, or a succinate buffer,

[0368] about 0.01 to about 10.0 mg / mL of a polysorbate,

[0369] about 0.1% (w / v) to about 20% (w / v) of sucrose, trehalose, mannitol, or sorbitol, and,

[0370] about 5 mM to about 200 mM of arginine or a salt thereof, optionally present,

[0371] the pH of the pharmaceutical composition is about 3.0 to about 7.0; or,

[0372] (3) about 0.01 mg / mL to about 200 mg / mL of a FAP / 4-1BB / CD40 binding molecule,

[0373] about 1 mM to about 100 mM of citric acid-sodium citrate or histidine-succinic acid,

[0374] about 0.01 to about 10.0 mg / mL of polysorbate 20 or polysorbate 80,

[0375] about 0.1% (w / v) to about 20% (w / v) of sucrose or trehalose, and,

[0376] about 5 mM to about 200 mM of arginine hydrochloride, optionally present,

[0377] the pH of the pharmaceutical composition is about 3.0 to about 7.0.

[0378] In some embodiments, the reconstituted solution comprises any one of the following:

[0379] (c-1) about 10 mg / mL to about 200 mg / mL of a FAP / 4-1BB / CD40 binding molecule,

[0380] about 10 mM histidine-succinic acid buffer,

[0381] about 8% w / v sucrose,

[0382] about 0.8 mg / mL polysorbate 80;

[0383] the pH of the pharmaceutical composition is about 4.0 to about 6.0;

[0384] (c-2) about 10 mg / mL to about 200 mg / mL of a FAP / 4-1BB / CD40 binding molecule,

[0385] about 20 mM histidine-succinate buffer,

[0386] about 8% w / v sucrose,

[0387] about 0.8 mg / mL polysorbate 80;

[0388] the pH of the pharmaceutical composition is about 4.0 to about 6.0;

[0389] (c-3) about 10 mg / mL to about 200 mg / mL of a FAP / 4-1BB / CD40 binding molecule,

[0390] about 20 mM histidine-succinate buffer,

[0391] about 8% w / v sucrose,

[0392] about 0.8 mg / mL polysorbate 80;

[0393] the pH of the pharmaceutical composition is about 4.0 to about 6.0;

[0394] (c-4) about 10 mg / mL to about 200 mg / mL of a FAP / 4-1BB / CD40 binding molecule,

[0395] about 20 mM histidine-succinate buffer,

[0396] about 2% w / v sucrose, about 4% w / v mannitol,

[0397] about 0.8 mg / mL polysorbate 80;

[0398] the pH of the pharmaceutical composition is about 4.0 to about 6.0;

[0399] (c-5) about 10 mg / mL to about 200 mg / mL of a FAP / 4-1BB / CD40 binding molecule,

[0400] about 20 mM histidine-succinate buffer,

[0401] about 1.0% w / v sucrose, about 5% w / v mannitol,

[0402] about 0.8 mg / mL polysorbate 80;

[0403] the pH of the pharmaceutical composition is about 4.0 to about 6.0;

[0404] (c-6) about 10 mg / mL to about 200 mg / mL of the FAP / 4-1BB / CD40 binding molecule,

[0405] about 20 mM histidine-succinate buffer,

[0406] about 8% w / v sucrose,

[0407] about 0.8 mg / mL polysorbate 80;

[0408] the pH of the pharmaceutical composition is about 4.0 to about 6.0;

[0409] (c-7) about 10 mg / mL to about 200 mg / mL of the FAP / 4-1BB / CD40 binding molecule,

[0410] about 30 mM histidine-succinate buffer,

[0411] about 9% w / v sucrose,

[0412] about 0.9 mg / mL polysorbate 80;

[0413] the pH of the pharmaceutical composition is about 4.0 to about 6.0;

[0414] (c-8) about 10 mg / mL to about 200 mg / mL of the FAP / 4-1BB / CD40 binding molecule,

[0415] about 25 mM histidine-succinate buffer,

[0416] about 7.5% w / v sucrose,

[0417] about 0.75 mg / mL polysorbate 80;

[0418] the pH of the pharmaceutical composition is about 4.0 to about 6.0.

[0419] The present disclosure also provides a liquid formulation comprising any one of:

[0420] (a-1) about 10 mg / mL to about 200 mg / mL of the FAP / 4-1BB / CD40 binding molecule,

[0421] about 10 mM histidine-acetate buffer,

[0422] about 8.8% w / v trehalose, and

[0423] about 0.4 mg / mL polysorbate 80,

[0424] the pH of the pharmaceutical composition is about 4 to about 6;

[0425] (a-2) about 10 mg / mL to about 200 mg / mL of the FAP / 4-1BB / CD40 binding molecule,

[0426] about 10 mM histidine-acetate buffer,

[0427] about 8.8% w / v trehalose, and

[0428] about 0.4 mg / mL polysorbate 80,

[0429] the pH of the pharmaceutical composition is about 4 to about 6;

[0430] (a-3) about 10 mg / mL to about 200 mg / mL of the FAP / 4-1BB / CD40 binding molecule,

[0431] about 10 mM histidine-acetate buffer,

[0432] about 8% w / v sucrose, and

[0433] about 0.4 mg / mL polysorbate 80,

[0434] the pH of the pharmaceutical composition is about 4.5 to about 5.5;

[0435] (a-4) about 10 mg / mL to about 200 mg / mL of the FAP / 4-1BB / CD40 binding molecule,

[0436] about 10 mM histidine-acetate buffer,

[0437] about 8% w / v sucrose, and

[0438] about 0.4 mg / mL polysorbate 80,

[0439] the pH of the pharmaceutical composition is about 4 to about 6;

[0440] (a-5) about 10 mg / mL to about 200 mg / mL of the FAP / 4-1BB / CD40 binding molecule,

[0441] about 10 mM histidine-acetate buffer,

[0442] about 8% w / v sucrose, and

[0443] about 0.4 mg / mL polysorbate 80,

[0444] the pH of the pharmaceutical composition is about 4 to about 7;

[0445] (a-6) about 10 mg / mL to about 200 mg / mL of the FAP / 4-1BB / CD40 binding molecule,

[0446] about 10 mM histidine-acetate buffer,

[0447] about 50 mM arginine hydrochloride,

[0448] about 6% w / v sucrose, and

[0449] about 0.8 mg / mL polysorbate 80,

[0450] the pH of the pharmaceutical composition is about 4 to about 6;

[0451] (a-7) about 10 mg / mL to about 200 mg / mL of the FAP / 4-1BB / CD40 binding molecule,

[0452] about 10 mM histidine-acetate buffer,

[0453] about 8% w / v sucrose, and

[0454] about 0.8 mg / mL polysorbate 80,

[0455] the pH of the pharmaceutical composition is about 4 to about 6;

[0456] (a-8) about 10 mg / mL to about 200 mg / mL of the FAP / 4-1BB / CD40 binding molecule,

[0457] about 10 mM citric acid-sodium citrate buffer,

[0458] about 8% w / v sucrose, and

[0459] about 0.8 mg / mL polysorbate 80,

[0460] the pH of the pharmaceutical composition is about 4 to about 6;

[0461] (b-1) about 10 mg / mL to about 200 mg / mL of the FAP / 4-1BB / CD40 binding molecule,

[0462] about 10 mM histidine-succinic acid buffer,

[0463] about 8% w / v sucrose, and

[0464] about 0.8 mg / mL polysorbate 80;

[0465] the pH of the pharmaceutical composition is about 4.0 to about 6.0;

[0466] (b-2) about 10 mg / mL to about 200 mg / mL of the FAP / 4-1BB / CD40 binding molecule,

[0467] about 10 mM histidine-succinate buffer,

[0468] about 4% w / v sucrose, and

[0469] about 0.4 mg / mL polysorbate 80;

[0470] the pH of the pharmaceutical composition is about 4.0 to about 6.0;

[0471] (b-3) about 10 mg / mL to about 200 mg / mL of the FAP / 4-1BB / CD40 binding molecule,

[0472] about 10 mM histidine-succinate buffer,

[0473] about 1% w / v sucrose, 2% w / v mannitol, and

[0474] about 0.4 mg / mL polysorbate 80;

[0475] the pH of the pharmaceutical composition is about 4.0 to about 6.0;

[0476] (b-4) about 10 mg / mL to about 200 mg / mL of the FAP / 4-1BB / CD40 binding molecule,

[0477] about 10 mM histidine-succinate buffer,

[0478] about 0.5% w / v sucrose, about 2.5% w / v mannitol, and

[0479] about 0.4 mg / mL polysorbate 80;

[0480] the pH of the pharmaceutical composition is about 4.0 to about 6.0;

[0481] (b-5) about 10 mg / mL to about 200 mg / mL of the FAP / 4-1BB / CD40 binding molecule,

[0482] about 10 mM histidine-succinate buffer,

[0483] about 3% w / v sucrose,

[0484] about 0.3 mg / mL polysorbate 80,

[0485] the pH of the pharmaceutical composition is about 4.0 to about 6.0;

[0486] (c-1) about 10 mg / mL to about 200 mg / mL of the FAP / 4-1BB / CD40 binding molecule,

[0487] about 10 mM histidine-succinate buffer,

[0488] about 8% w / v sucrose,

[0489] about 0.8 mg / mL polysorbate 80;

[0490] the pH of the pharmaceutical composition is about 4.0 to about 6.0;

[0491] (c-2) about 10 mg / mL to about 200 mg / mL of the FAP / 4-1BB / CD40 binding molecule,

[0492] about 20 mM histidine-succinate buffer,

[0493] about 8% w / v sucrose,

[0494] about 0.8 mg / mL polysorbate 80;

[0495] the pH of the pharmaceutical composition is about 4.0 to about 6.0;

[0496] (c-3) about 10 mg / mL to about 200 mg / mL of the FAP / 4-1BB / CD40 binding molecule,

[0497] about 20 mM histidine-succinate buffer,

[0498] about 8% w / v sucrose,

[0499] about 0.8 mg / mL polysorbate 80;

[0500] the pH of the pharmaceutical composition is about 4.0 to about 6.0;

[0501] (c-4) about 10 mg / mL to about 200 mg / mL of the FAP / 4-1BB / CD40 binding molecule,

[0502] about 20 mM histidine-succinate buffer,

[0503] about 2% w / v sucrose, about 4% w / v mannitol,

[0504] about 0.8 mg / mL polysorbate 80;

[0505] the pH of the pharmaceutical composition is about 4.0 to about 6.0;

[0506] (c-5) about 10 mg / mL to about 200 mg / mL of the FAP / 4-1BB / CD40 binding molecule,

[0507] about 20 mM histidine-succinate buffer,

[0508] about 1.0% w / v sucrose, about 5% w / v mannitol,

[0509] about 0.8 mg / mL polysorbate 80;

[0510] the pH of the pharmaceutical composition is about 4.0 to about 6.0;

[0511] (c-6) about 10 mg / mL to about 200 mg / mL of the FAP / 4-1BB / CD40 binding molecule,

[0512] about 20 mM histidine-succinate buffer,

[0513] about 8% w / v sucrose,

[0514] about 0.8 mg / mL polysorbate 80;

[0515] the pH of the pharmaceutical composition is about 4.0 to about 6.0;

[0516] (c-7) about 10 mg / mL to about 200 mg / mL of the FAP / 4-1BB / CD40 binding molecule,

[0517] about 30 mM histidine-succinate buffer,

[0518] about 9% w / v sucrose,

[0519] about 0.9 mg / mL polysorbate 80;

[0520] the pH of the pharmaceutical composition is about 4.0 to about 6.0;

[0521] (c-8) about 10 mg / mL to about 200 mg / mL of the FAP / 4-1BB / CD40 binding molecule,

[0522] about 25 mM histidine-succinate buffer,

[0523] about 7.5% w / v sucrose,

[0524] about 0.75 mg / mL polysorbate 80;

[0525] the pH of the pharmaceutical composition is about 4.0 to about 6.0.

[0526] In some embodiments, the pharmaceutical composition, reconstituted solution, liquid formulation is an intravenous injection, a subcutaneous injection, an intraperitoneal injection, or a intramuscular injection; in some embodiments, an intravenous or subcutaneous injection.

[0527] The present disclosure also provides an article of manufacture comprising a container filled with the pharmaceutical composition, the lyophilized formulation, the reconstituted solution, or the liquid formulation.

[0528] Therapeutic methods and pharmaceutical uses

[0529] The present disclosure also provides a method of treating or preventing a disease, comprising administering to a subject in need thereof a therapeutically or prophylactically effective amount of the pharmaceutical composition, the lyophilized formulation, the reconstituted solution, the liquid formulation, or the article of manufacture. In some embodiments, the disease is cancer. In some specific embodiments, the cancer is selected from lung cancer, prostate cancer, breast cancer, head and neck cancer, esophageal cancer, gastric cancer, colorectal cancer, bladder cancer, cervical cancer, uterine cancer, ovarian cancer, liver cancer, melanoma, renal cancer, squamous cell carcinoma, and hematological cancer.

[0530] The present disclosure also provides a method of preparing a liquid formulation, comprising the steps of: preparing a stock solution comprising the pharmaceutical composition, subjecting the stock solution to a freeze-drying process to obtain a lyophilized formulation; and reconstituting the lyophilized formulation to obtain the liquid formulation; wherein the concentration of the FAP / 4-1BB / CD40 binding molecule in the liquid formulation is greater than the concentration of the FAP / 4-1BB / CD40 binding molecule in the stock solution. In some embodiments, the concentration ratio of the FAP / 4-1BB / CD40 binding molecule in the liquid formulation to the FAP / 4-1BB / CD40 binding molecule in the stock solution is (1-5):1, preferably (1-3):1.

[0531] The present disclosure also provides a method of preparing a pharmaceutical composition, comprising: preparing a stock solution comprising the pharmaceutical composition, subjecting the stock solution to a freeze-drying process to obtain a lyophilized formulation; and reconstituting the lyophilized formulation with a solvent to obtain a liquid formulation; wherein the concentration of the FAP / 4-1BB / CD40 binding molecule in the liquid formulation is greater than the concentration of the FAP / 4-1BB / CD40 binding molecule in the stock solution, and the solvent is preferably water.

[0532] In some embodiments, the concentration ratio of the FAP / 4-1BB / CD40 binding molecule in the liquid formulation to the FAP / 4-1BB / CD40 binding molecule in the stock solution is (1-5):1, preferably (1-3):1.

[0533] Terminology

[0534] For the purposes of the present disclosure, certain technical and scientific terms are specifically defined below. Unless specifically defined herein, all other technical and scientific terms used in the present disclosure have the meanings that are commonly understood by one of ordinary skill in the art in the field of the present disclosure.

[0535] "Buffer" means a buffering agent that resists changes in pH through the action of its acid-base conjugate components. Examples of buffers that control pH in an appropriate range include acetate, succinate, gluconate, histidine, oxalate, lactate, phosphate, citrate, tartrate, fumarate, glycylglycine, and other organic acid buffers.

[0536] "Histidine buffer" is a buffer that contains histidine ions. Examples of histidine buffers include acetate-histidine, succinate-histidine, histidine-histidine hydrochloride, hydrochloride-histidine, sulfate-histidine, and the like. Illustratively, a histidine-hydrochloride buffer is one formulated with histidine and hydrochloric acid or histidine and histidine hydrochloride.

[0537] "Pharmaceutical composition" means a mixture of one or more of the compounds described herein, or physiologically / pharmaceutically acceptable salts or prodrugs thereof, with other chemical components, such as physiologically / pharmaceutically acceptable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration of the active ingredient to an organism and to facilitate absorption of the active ingredient. As used herein, "pharmaceutical composition" and "formulation" are not mutually exclusive.

[0538] The solvent in the solution form of the pharmaceutical composition described in the present disclosure is water, unless otherwise specified.

[0539] The term "about" as used herein means within an acceptable error range for the particular value as determined by one of ordinary skill in the art to which the claim pertains, which will depend in part on how the value is measured or determined. For example, "about" can mean within 1 or more than 1 standard deviation, per each implementation by one of ordinary skill in the art. Alternatively, "about" or "comprising substantially" can mean a range of up to 20% of a given value. Furthermore, particularly with regard to biological systems or processes, the term can mean up to an order of magnitude or up to 5-fold as compared to a given value. Unless otherwise stated, the meaning of "about" or "comprising substantially" should be assumed to be within the acceptable error range of a particular value or range of values when a particular value appears in the application or claims.

[0540] The pharmaceutical composition described in the present disclosure is capable of achieving a stable effect in which the antibody substantially retains its physical stability and / or chemical stability and / or biological activity after storage, preferably, the pharmaceutical composition substantially retains its physical and chemical stability and its biological activity after storage. The storage period is generally selected based on the intended shelf life of the pharmaceutical composition. There are a variety of analytical techniques for measuring protein stability, which can measure stability after storage at a selected temperature for a selected period of time.

[0541] A stable pharmaceutical antibody formulation is one in which no significant changes are observed: upon 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. In addition, a stable liquid formulation includes one that exhibits the desired characteristics after storage for a period of time including 1 month, 3 months, 6 months at 25°C or 1 month at 40°C. Typical acceptable criteria for stability are that generally no more than about 10%, preferably no more than about 5% of the antibody monomer is degraded as measured by SEC-HPLC. The pharmaceutical antibody formulation is colorless to yellow, clear to slightly opalescent by visual analysis. The concentration, pH, and osmolality of the formulation have no more than a ±10% change. Generally no more than about 10%, preferably no more than about 5% truncation is observed, generally no more than about 10%, preferably no more than about 5% aggregation is formed.

[0542] An antibody "retains its physical stability" in a pharmaceutical formulation if it does not show a significant increase in aggregation, precipitation, and / or denaturation upon visual inspection of color and / or clarity, or as measured 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.

[0543] An antibody "retains its chemical stability" in a pharmaceutical formulation if it does not show a significant chemical change. Chemical stability can be assessed by detecting and quantifying chemically altered forms of the protein. Degradation processes that frequently alter the chemical structure of proteins include hydrolysis or truncation (assessed by methods such as size exclusion chromatography and SDS-PAGE), oxidation (assessed by methods such as peptide mapping in conjunction 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.).

[0544] 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 at the time of manufacture of the pharmaceutical formulation. The biological activity of an antibody can be determined, for example, by an antigen binding assay.

[0545] The three letter and one letter codes for amino acids used herein are as described in J. Biol. Chem., 243, p. 3558 (1968).

[0546] “CD40” and “CD40 antigen” refer to a -48 kD glycoprotein expressed on the surface of normal and neoplastic B cells that serves as a receptor for signals involved in cell proliferation and differentiation (Ledbetter et al., 1987, J. Immunol. 138:788-785). Cells that endogenously express CD40 are any cells characterized by surface expression of CD40, including but not limited to normal and neoplastic B cells, anaplastic cells, basal epithelial cells, carcinoma cells, macrophages, endothelial cells, follicular dendritic cells, tonsil cells, and bone marrow-derived plasma cells. The disclosure “CD40” refers to any native CD40 from any vertebrate source, including mammals, such as primates (e.g., humans) and rodents (e.g., mice and rats), unless otherwise indicated. A cDNA molecule encoding CD40 has been isolated from a library prepared from the Burkitt’s lymphoma cell line Raji (Stamenkovic et al., 1989, EMBO J. 8:1403). The disclosure “CD40” encompasses full-length, unprocessed CD40 as well as any form of CD40 that results from processing in cells, and also encompasses naturally occurring variants of CD40, such as splice variants or allelic variants. In addition, artificially expressed forms, functional variants, and the like are also included.

[0547] “4-1BB” or “4-1BB antigen”, also known as CD137, tumor necrosis factor receptor superfamily member 9, is a member of the TNF receptor superfamily (TNFRSF) and is a costimulatory molecule expressed on the surface of CD8+ and CD4+ T cells, regulatory T cells (Tregs), NK and NKT cells, B cells, and neutrophils, among other cells. 4-1BB belongs to the costimulatory molecules that are expressed upon activation of immune cells. The human 4-1BB protein is at NCBI Accession No. NP_001552.2. In the disclosure, “4-1BB” can optionally include any such protein or fragment, variant thereof, including but not limited to known or wild-type 4-1BB as described in the disclosure, as well as any naturally occurring splice variant, amino acid variant, or isoform. In addition, artificially expressed forms, functional variants, and the like are also included.

[0548] “Fibroblast activation protein (FAP)” and “FAP antigen”, also known as prolyl endopeptidase FAP or Seprase (EC 3.4.21), refers to any native FAP from any vertebrate source, including mammals, such as primates (e.g., humans), non-human primates (e.g., cynomolgus monkeys), and rodents (e.g., mice and rats), unless otherwise indicated. The disclosure “FAP” encompasses full-length, unprocessed FAP as well as any form of FAP that results from processing in cells, and also encompasses naturally occurring variants of FAP, such as splice variants or allelic variants. In addition, artificially expressed forms, functional variants, and the like are also included.

[0549] The amino acid sequence of human FAP is shown in UniProt (www.uniprot.org) accession no. Q12884, or NCBI (www.ncbi.nlm.nih.gov / ) RefSeq NP_004451.2, GeneBank Accession NO. AAC51668. The extracellular domain (ECD) of human FAP extends from amino acid position 26 to 760.

[0550] The term "functional variant" includes, but is not limited to, homologs, fragments, truncations, mutants, modifications, etc. of wild-type proteins, which have improved, reduced, or maintained protein activity as compared to the wild-type protein.

[0551] "Binding molecule" encompasses any protein, polypeptide, or any molecule comprising the protein or polypeptide, which is capable of specifically binding to an antigen (e.g., CD40, 4-1BB, or FAP), including but not limited to an antibody as defined in the present disclosure.

[0552] "Polypeptide," "peptide," or "protein" are used interchangeably herein in the present disclosure to refer to a polymer of amino acid residues, or a collection of multiple polymer of amino acid residues. These terms apply to amino acid polymers in which one or more amino acid residue is a synthetic chemical mimic of the corresponding naturally occurring amino acid, as well as to naturally occurring and non-naturally occurring amino acid polymers. A polypeptide sequence is typically depicted with its amino-terminal (N-terminal, N-terminus) end on the left and its carboxy-terminal (C-terminal, C-terminus) end on the right.

[0553] "Antibody" encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, monospecific antibodies, multispecific antibodies (e.g., bispecific antibodies, trispecific antibodies), full-length antibodies, and antibody fragments (or antigen-binding fragments, or antigen-binding domains), as long as they exhibit the desired antigen-binding activity.

[0554] An antibody can refer to an immunoglobulin, which is a four polypeptide chain structure connected by interchain disulfide bonds of two heavy chains and two light chains. The amino acid composition and arrangement order of the constant region of the immunoglobulin heavy chain are different, so the antigenicity is also different. Accordingly, the immunoglobulin can be divided into five types, or called isotypes of immunoglobulin, namely IgM, IgD, IgG, IgA and IgE, and the corresponding heavy chains are μ chain, δ chain, γ chain, α chain and ε chain, respectively. The same class of Ig can be divided into different subclasses according to the difference of the amino acid composition of the hinge region and the number and position of the heavy chain disulfide bond, such as IgG can be divided into IgG1, IgG2, IgG3 and IgG4. The light chain is divided into κ chain or λ chain by the constant region. Each of the five types of Ig can have κ chain or λ chain. The sequence of about 110 amino acids near the N-terminus of the heavy chain and light chain of the antibody varies greatly, which is the variable region (V region); the remaining amino acid sequence near the C-terminus is relatively stable, which is the constant region (C region). The variable region includes three hypervariable regions (HVR) and four relatively conserved framework regions (FR). The three hypervariable regions determine the specificity of the antibody, also known as the complementarity determining region (CDR). Each light chain variable region (VL) and heavy chain variable region (VH) is composed of three CDR regions and four FR regions, arranged in the order of FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4 from the amino terminal to the carboxyl terminal. The three CDR regions of the light chain are LCDR1, LCDR2, and LCDR3; the three CDR regions of the heavy chain are HCDR1, HCDR2, and HCDR3.

[0555] The antibodies of the present disclosure can be polyclonal, monoclonal, xenogeneic, allogeneic, syngeneic, or modified versions thereof, with monoclonal antibodies being particularly suitable for use in various embodiments. Generally, the antibodies of the present disclosure are recombinant antibodies. As used herein, “recombinant” refers broadly to products, such as cells or nucleic acids, proteins, or vectors, that indicate the cell, nucleic acid, protein, or vector has been modified by the introduction of a heterologous nucleic acid or protein or alteration of a native nucleic acid or protein, or that the cell is derived from such a modified cell. For example, a recombinant cell expresses a gene that is not present in the native (non-recombinant) form of the cell, or expresses a native gene that is otherwise abnormally expressed, under-expressed, or not expressed at all.

[0556] An "antigen binding domain" encompasses a Fab, a modified Fab, a Fab', a modified Fab', a F(ab')2, a Fv, a Fab-Fv, a Fab-dsFv, a single domain antibody (e.g. a VH or VL or VHH), a scFv, a bivalent or trivalent or tetravalent antibody, a Bis-scFv, a diabody, a triabody, a triabody, a tetrabody, and an epitope binding fragment 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). Methods of generating and making these antigen binding fragments are well known in the art (see e.g. Verma et al., 1998, Journal of Immunological Methods, 216, 165-181).

[0557] A "multispecific antibody" refers to an antibody that binds two or more different target antigens or different epitopes of an antigen. The term "multispecific antibody" includes bispecific, trispecific, tetraspecific, pentaspecific, and hexaspecific. As used herein, the term "trispecific antibody" refers to an antibody that binds three different antigens (e.g., FAP, CD40, or 4-1BB) or three different epitopes.

[0558] For the determination or definition of CDRs, a definitive delineation of CDRs and identification of residues comprising the binding site of an antibody can be accomplished by resolving the structure of the antibody and / or resolving the structure of an antibody-ligand complex. This can be achieved by any of a variety of techniques known to those of skill in the art, e.g., X-ray crystallography. A variety of analytical methods can be used to identify CDRs, including but not limited to the Kabat numbering system, the Chothia numbering system, the AbM numbering system, the IMGT numbering system, contact definition, conformational definition.

[0559] 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 the Chothia numbering system takes into account the location 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 a suite of computer programs produced by Oxford Molecular Group that model antibody structures (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 primary sequences (see Samudrala et al., 1999, "Ab Initio Protein Structure Prediction Using a Combined Hierarchical Approach," in PROTEINS, Structure, Function and Genetics Suppl., 3:194-198). Contact definitions are based on analysis of complex crystal structures available (see, e.g., MacCallum et al., 1996, J. Mol. Biol., 5:732-45). In conformational definitions, the location of CDRs 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). Still other CDR boundary definitions can not strictly follow one of the above methods, but still overlap at least a portion of the Kabat CDRs, although they can be shortened or lengthened depending on the prediction or experimental result that a particular residue or group of residues does not significantly impact antigen binding. As used in the present disclosure, CDR can refer to CDRs defined by any method known in the art, including combinations of methods. Correspondence between the various numbering systems is well known to those of skill in the art, and exemplary is shown in Table 1 below.

[0560] Table 1. Relationship between CDR numbering systems

[0561] 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 known Rabat numbering system.

[0562] A "domain" of a polypeptide or protein refers to a folded protein structure that is capable of maintaining its tertiary structure independently of the remainder of the protein. Generally, a domain is responsible for a single functional property of the protein, and in many cases can be added, removed, or transferred to other proteins without loss of function of the remainder of the protein and / or the domain.

[0563] An "immunoglobulin domain" refers to the globular region of an antibody chain. Immunoglobulin domains are characterized by their ability to maintain the folded character of the antibody molecule.

[0564] An "immunoglobulin variable domain" refers to an immunoglobulin domain consisting essentially of four "framework regions," "framework region 1" or "FR1," "framework region 2" or "FR2," "framework region 3" or "FR3," and "framework region 4" or "FR4," and three "complementarity determining regions" or "CDRs," "complementarity determining region 1" or "CDR1," "complementarity determining region 2" or "CDR2," and "complementarity determining region 3" or "CDR3." Thus, the general structure or sequence of an immunoglobulin variable domain can be represented as: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. An immunoglobulin variable domain confers its specificity for an antigen in that it has an antigen binding site.

[0565] An "antibody framework (FR)" refers to a portion of a variable domain that acts as a scaffold for the antigen binding loops (CDRs) of that variable domain.

[0566] An "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 VH, VHH, or VL domains) 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 a conventional four-chain monoclonal antibody). Examples of "immunoglobulin single variable domains" include nanobodies (including VHH, humanized VHH, and / or camelized VH, e.g., camelized human VH), IgNARs, domains, (single domain) antibodies that are VH domains or derived from VH domains (such as dAbs TM ), and (single domain) antibodies that are VL domains or derived from VL domains (such as dAbs TMImmunoglobulin single variable domains based on and / or derived from heavy chain variable domains (such as VH or VHH domains) are generally preferred. A specific example of an immunoglobulin single variable domain is the “VHH domain” (or simply “VHH”) as defined below.

[0567] The "VHH domain," also known as the heavy chain single-domain antibody, VHH, V HH Domains, VHH antibody fragments, VHH antibodies, and nanobodies are variable domains of antigen-binding immunoglobulins called "heavy chain antibodies" (i.e., "antibodies lacking light chains") (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)). The term "VHH domain" is used to distinguish these variable domains from the VH and VL domains present in conventional tetrapeptide chain antibodies. The VHH domain specifically binds to epitopes without the need for other antigen-binding domains (unlike the VH or VL domains in conventional tetrapeptide chain antibodies, where the epitope is recognized by both the VL and VH domains). The 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", and "V" are used to describe the antibody's structure. HH "structural domain", "VHH antibody fragment", "VHH antibody", as well as"" The terms "Nanobody" and "Nanobody®" (Nanobody® is a trademark of Ablynx N.V., Ghent, Belgium) are used interchangeably. A "VHH domain" includes, but is not limited to, naturally occurring antibodies produced by Camelidae, but can also be humanized after production by Camelidae, or can be obtained by phage display technology. The total number of amino acid residues in a VHH domain will generally range from 110 to 120, often between 112 and 115. It is noted, however, that smaller and longer sequences can also be suitable for the purposes described in the present disclosure. Methods for obtaining VHHs that bind to a specific antigen or epitope have been previously disclosed in R. van der Linden et al., Journal of Immunological Methods, 240 (2000) 185-195; Li et al., J Biol Chem., 287 (2012) 13713-13721; Deffar et al., African Journal of Biotechnology Vol. 8(12), pp. 2645-2652, 17 June, 2009 and WO94 / 04678.

[0568] As is well known in the art for VH and VHH domains, the total number of amino acid residues in each CDR can vary, and can not correspond to the total number of amino acid residues indicated by the numbering of Kabat (i.e. one or more positions according to the numbering of Kabat can not be occupied in the actual sequence, or the actual sequence can contain more amino acid residues than allowed by the numbering of Kabat). This means that in general, the numbering according to Kabat can or can not correspond to the actual numbering of the amino acid residues in the actual sequence. Other numbering systems or coding rules include Chothia, IMGT, AbM.

[0569] A "humanized antibody" (also referred to as a CDR-grafted antibody) is an antibody in which non-human CDR sequences are grafted into a human antibody 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 decrease in immunogenicity while causing a decrease in activity, the human antibody variable region framework sequences can be subjected to minimal back-mutations to maintain activity. Examples of "humanization" include the "humanization" of a VHH domain derived from a Camelid by replacing one or more amino acid residues in the original VHH sequence with one or more amino acid residues present at the corresponding position in a human canonical tetrapeptide chain structure antibody VH domain. The humanized VHH domain can contain one or more fully human framework region sequences, and in some embodiments, can contain a human framework region sequence of IGHV3. Yet another example of "humanization" includes antibodies generated by grafting mouse CDR sequences into a human antibody variable region framework, i.e., different types of human germline antibody framework sequences. This can overcome the strong immune response induced by chimeric antibodies due to the presence of a large number of mouse protein components. Humanization methods such as protein surface amino acid resurfacing and antibody humanization universal framework grafting, i.e., grafting CDRs onto other "scaffolds" including but not limited to human scaffolds or non-immunoglobulin scaffolds. Suitable scaffolds and techniques for CDR grafting are known in the art. Germline DNA sequences of human heavy and light chain variable region genes can be found in the "VBase" human germline sequence database, and in Kabat, E. A. et al., 1991 Sequences of Proteins of Immunological Interest, 5th Ed. Humanized antibodies of the present disclosure also include humanized antibodies that are further affinity matured by phage display of CDRs. In addition, to avoid a decrease in immunogenicity while causing a decrease in activity, the human antibody variable region framework sequences can be subjected to minimal back-mutations or back-mutations to maintain activity.

[0570] A "pre-existing ADA" (i.e., pre-ADA) is an ADA that is already present in a subject or individual who is to be administered or given a drug (e.g., a protein or polypeptide drug, more particularly an antibody drug). The pre-existing ADA can be present in a subject or individual who is being used in an experiment for the first time (i.e., a subject or individual who has never been administered a drug before).

[0571] Generally, "specific binding," "selective binding" refers to the binding of a molecule to an epitope on an antigen. The FAP / 4-1BB / CD40 binding molecules of the present disclosure will bind with a dissociation constant (K -7 of 10 -10 M or less, more preferably 10 -8 M or less, more preferably 10 -10 M or less, even more preferably 10 -9 M or less, and / or an association constant (KA) of at least 10 -10 M, preferably at least 10 D M, more preferably at least 10 -7 M, more preferably at least 10 -8 M, more preferably at least 10 - 9 M, more preferably at least 10 -10 M. Any K -4 value greater than 10 D M is generally considered to indicate non-specific binding. Specific binding of a binding molecule to an antigen or epitope can be determined in any suitable manner known in the art, including, for example, surface plasmon resonance (SPR), enzyme-linked immunosorbent assay (ELISA), and / or flow cytometry sorting (FACS) as described herein.

[0572] An "epitope" is a site on an antigen to which an immunoglobulin or antibody binds. Epitopes can be formed both by contiguous amino acids, or non-contiguous amino acids which are brought into proximity by the tertiary folding of the protein. Epitopes formed from contiguous amino acids are typically retained upon exposure to denaturing solvents, whereas epitopes formed by tertiary folding are typically lost upon treatment with denaturing solvents. Epitopes generally comprise at least 3-15 amino acids in a unique spatial conformation. Methods for determining that an epitope binds to a given antibody are well known in the art, including immunoblot and immunoprecipitation detection assays, etc. Methods for determining the spatial conformation of an epitope include techniques in the art and described herein, such as X-ray crystallography and two-dimensional nuclear magnetic resonance, etc.

[0573] "Binding affinity" or "affinity" is used in the present disclosure as a measure of the strength of the 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 (K D ). K D can be determined by measuring the kinetics of complex formation and dissociation using, for example, surface plasmon resonance (SPR) methods (Biacore).The rate constants for association and dissociation of a biding and unbinding, respectively, corresponding to a monovalent complex are referred to as the association rate constant, ka (or kon), and the dissociation rate constant, kd (or koff). K D The value of K D = kd / ka is related to both ka and kd. The value of the dissociation constant can be determined directly by well-known methods, and even calculated for complex mixtures by methods such as those described in Caceci et al. (1984, Byte 9:340-362). For example, K D can be determined using double-filter nitrocellulose filters in conjunction with assays such as that disclosed in Wong & Lohman (1993, Proc. Natl. Acad. Sci. USA 90:5428-5432). Other standard assays for assessing the binding ability of an antibody to a target antigen are known in the art, including, for example, ELISA, Western blot, RIA and FACS, as well as other assays exemplified elsewhere in the disclosure. The binding kinetics and binding affinities of antibodies can also be evaluated by standard assays known in the art, such as surface plasmon resonance (SPR), for example, by using a Biacore TM system or KinExA. The binding affinities associated with different molecular interactions can be compared by comparing the K D values of the respective antibody / antigen complexes, for example, the comparison of the binding affinities of different antibodies for a given antigen. Similarly, the specificity of an interaction can be evaluated by determining and comparing the K D values of the specific interaction of interest (e.g., between an antibody and an antigen) with the K D values of a non-specific interaction (e.g., a control antibody known not to bind FAP).

[0574] A "conservative substitution" is one in which the amino acid residue is replaced with one having similar properties. For example, lysine, arginine and histidine are similar in that they have basic side chains, and aspartic acid and glutamic acid are similar in that they have acidic side chains. In addition, glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine and tryptophan are similar in that they have uncharged polar side chains, and alanine, valine, leucine, isoleucine, proline, phenylalanine and methionine are similar in that they have nonpolar side chains. Additionally, tyrosine, phenylalanine, tryptophan and histidine have similar properties in that they have aromatic side chains. Thus, it will be apparent to those skilled in the art that even when a substitution is made between amino acid residues that show similar properties as described above, it will not necessarily show a specific change in properties.

[0575] "Identity," "homology," or "sequence identity," refers to sequence similarity between two nucleic acid sequences or between two polypeptides. When a position in both of the compared sequences is occupied by the same nucleotide or amino acid monomer, e.g., if a position in each of two DNA molecules is occupied by adenine, then the molecules are homologous at that position. The percent of homology between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of positions in the comparison times 100%. For example, if 6 of 10 positions in two sequences are matched or homologous, then the two sequences are 60% homologous over die 10 positions. Generally, the comparison is made over the length of the shorter sequence if the lengths of the two sequences are unlike. When comparing two sequences, the comparison is made over the length of the shorter sequence if the lengths of the two sequences are unlike.

[0576] "Nucleic acid" or "polynucleotide" are used interchangeably herein to refer to a molecule of either DNA or RNA of any sequence and any length, either single- or double-stranded, and in the case of single-stranded molecules, its complementary sequence. A nucleic acid is "operably linked" when it is functionally connected to another nucleic acid sequence, such as when a promoter or enhancer affects the transcription of a coding sequence. "Operably linked" means that the nucleic acid sequences being linked are contiguous and, in the case of a control sequence, are in a functional relationship. For example, a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the coding sequence.

[0577] "Host cell" includes an individual cell or cell culture which can be or has been a recipient for a nucleic acid insert. Host cells include progeny of the original host cell that has been subjected to fusion and / or transformation, and which progeny can not necessarily be identical to the original parent cell either at the morphological or the genomic

[0578] The term "pharmaceutically acceptable excipient" or "pharmaceutically acceptable excipient" includes any material that, when combined with an active ingredient, allows the ingredient to retain its biological activity and does not cause an immune response in a subject. Examples include, but are not limited to, any standard pharmaceutical carrier, such as a phosphate buffered saline solution, water, emulsions such as oil / water emulsions, and various types of wetting agents. In some embodiments, the diluent for aerosol or parenteral administration is phosphate buffered saline (PBS) or normal (0.9%) saline. Compositions containing such carriers are formulated by well-known conventional methodology (see, e.g., Remington's Pharmaceutical Sciences, 18th Ed., A. Gennaro, ed., Mack Publishing Co., Easton, PA, 1990; and R Remington, The Science and Practice of Pharmacy 20th Ed. Mack Publishing, 2000).

[0579] "Inhibit" or "block" are used interchangeably and encompass both partial and complete inhibition / blockade. "Inhibit growth" (e.g., in reference to a cell) is intended to include any measurable decrease in cell growth.

[0580] "Prevent growth" or "growth inhibition" refers to the inhibition of growth or proliferation of a cell.

[0581] "Pro liferative disorder" refers to a condition associated with some degree of abnormal cell proliferation. In one embodiment, a proliferative disorder refers to cancer. "Tumor" refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues. "Cancer," "cancerous," "proliferative disorder," and "tumor" are not mutually exclusive when referred to in the present disclosure.

[0582] "Prevent cancer" refers to delaying, inhibiting, or preventing the onset of cancer in a subject in whom the initiation of carcinogenesis or tumorigenesis has not yet been established, but in whom cancer susceptibility has been identified, e.g., by genetic screening or other methods. This also includes treating a subject with a pre-cancerous condition to halt the progression of or cause regression of the pre-cancerous condition to a malignant tumor.

[0583] "Administer," "administering" and "treatment" when applied to an animal, human, experimental subject, cell, tissue, organ or biological fluid, refer to the contact of an exogenous drug, therapeutic agent, diagnostic agent or composition with an animal, human, subject, cell, tissue, organ or biological fluid, for example, therapeutic, pharmacokinetic, diagnostic, research and experimental methods. Treatment of a cell includes contact of the agent with the cell, as well as contact of the agent with a fluid that is in contact with the cell. "Administer," "administering" and "treatment" also mean treatment by an agent, diagnostic, binding composition or by another cell in vitro and ex vivo, for example, a cell. When applied to a human, veterinary or research subject, it refers to therapeutic treatment, prophylaxis or preventative measures, research and diagnostic applications.

[0584] "Treatment" means the administration of an internal or external therapeutic agent to a subject, e.g., comprising any of the binding molecules of the disclosure or a pharmaceutical composition thereof as a therapeutic agent, to a subject already suffering from, suspected of suffering from, predisposed to suffering from one or more proliferative diseases or symptoms thereof, where the therapeutic agent is known to have a therapeutic effect on these symptoms. Generally, the therapeutic agent is administered in an amount effective to alleviate one or more symptoms of the disease in the treated subject or population, whether by inducing regression of such symptoms or inhibiting development of such symptoms to any clinically measurable extent. The amount of therapeutic agent effective to alleviate any particular symptom of the disease (also referred to as "therapeutically effective amount") can vary according to factors such as the disease state, age, and weight of the subject, and the ability of the drug to elicit a desired effect in the subject. Whether a disease symptom has been alleviated can be assessed by any clinical detection method generally used by a physician or other professional health care provider to assess the severity or progression of the symptom. Although an embodiment of the disclosure (e.g., a therapeutic method or article of manufacture) can not be effective in alleviating a target disease symptom in a certain subject, it should do so in a statistically significant number of subjects as determined by any statistical test known in the art, such as Student's t-test, the chi-square test, the U test according to Mann and Whitney, the Kruskal-Wallis test (H test), the Jonckheere-Terpstra test, and the Wilcoxon test.

[0585] "Effective amount" includes an amount that is sufficient to ameliorate or prevent the symptoms or conditions of a medical disorder. An effective amount also means an amount that is sufficient to allow or facilitate diagnosis. The effective amount for a subject can vary depending on factors such as the condition to be treated, the overall health status of the subject, the method route and dose of administration, and the severity of side effects. The effective amount can be the maximum dose or administration regimen that avoids significant side effects or toxic effects. A subject of the disclosure can be an animal or a human subject.

[0586] "Optional" or "optionally" means that the subsequently described event or circumstance can or can not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not. "And / or" should be treated as equivalent to the phrase "and / or", meaning that each of the two designated features or components can be present or absent. Thus, as used in phrases such as "A and / or B" in the present disclosure, the term "and / or" includes the instances of "A and B", "A or B", "A" (alone), and "B" (alone). Unless the context clearly requires otherwise, throughout the description and the claims, the words "comprise", "comprising", "include", "including", and the like are to be construed in an inclusive sense as opposed to an exclusive or exhaustive sense; that is to say, in the sense of "including, but not limited to".

[0587] "Subject" or "patient" of the present disclosure means a mammal, especially a primate, and particularly a human. BRIEF DESCRIPTION OF DRAWINGS

[0588] Figure 1 is the FACS binding test results of the tested antibodies to human 4-1BB antigen.

[0589] Figure 2 is the binding of C5_V2, C5_V2-YTI and C5_V2-YTIQ to human 4-1BB on the surface of HEK293 cells.

[0590] Figure 3 is the structural schematic of anti-FAP / 4-1BB / CD40 multispecific antibodies (Fig. 3 a-e) and anti-4-1BB / CD40 (Fig. 3 f).

[0591] Figure 4A is the FACS detection results of the tested antibodies binding to human FAP on the surface of CHOK1 cells. Figure 4B is the FACS detection results of the tested antibodies binding to human 4-1BB on the surface of HEK293 cells. Figure 4C is the FACS detection results of the tested antibodies binding to human CD40 on the surface of HEK293 cells.

[0592] Figure 5A is the FACS detection results of the tested antibodies binding to human FAP on the surface of CHOK1 cells. Figure 5B is the FACS detection results of the tested antibodies binding to human 4-1BB on the surface of HEK293 cells. Figure 5C is the FACS detection results of the tested antibodies binding to human CD40 on the surface of HEK293 cells.

[0593] Figure 6A is the FACS detection results of the tested antibodies binding to human FAP on the surface of CHOK1 cells. Figure 6B is the FACS detection results of the tested antibodies binding to human 4-1BB on the surface of HEK293 cells. Figure 6C is the FACS detection results of the tested antibodies binding to human CD40 on the surface of HEK293 cells.

[0594] FIGS. 7A-7C are FAP / CD40-dependent 4-1BB / NF-κB luciferase reporter assay results of the tested antibodies. FIG. 7A is before cross-linking, FIG. 7B is after FAP cross-linking, and FIG. 7C is after CD40 cross-linking.

[0595] FIGS. 8A-8C are FAP / CD40-dependent 4-1BB / NF-κB luciferase reporter assay results of the tested antibodies. FIG. 8A is before cross-linking, FIG. 8B is after FAP cross-linking, and FIG. 8C is after CD40 cross-linking.

[0596] FIGS. 9A-9C are FAP / CD40-dependent 4-1BB / NF-κB luciferase reporter assay results of the tested antibodies. FIG. 9A is before cross-linking, FIG. 9B is after FAP cross-linking, and FIG. 9C is after CD40 cross-linking.

[0597] FIGS. 10A-10C are FAP / 4-1BB-dependent CD40 / NF-κB luciferase reporter assay results of the tested antibodies. FIG. 10A is before cross-linking, FIG. 10B is after FAP cross-linking, and FIG. 10C is after 4-1BB cross-linking.

[0598] FIGS. 11A-11C are FAP / 4-1BB-dependent CD40 / NF-κB luciferase reporter assay results of the tested antibodies. FIG. 11A is before cross-linking, FIG. 11B is after FAP cross-linking, and FIG. 11C is after 4-1BB cross-linking.

[0599] FIGS. 12A-12C are FAP / 4-1BB-dependent CD40 / NF-κB luciferase reporter assay results of the tested antibodies. FIG. 12A is before cross-linking, FIG. 12B is after FAP cross-linking, and FIG. 12C is after 4-1BB cross-linking.

[0600] FIGS. 13 and 14 are activation of T lymphocytes of donors 1 and 2 by the tested antibodies.

[0601] FIG. 15 is activation of CD3+ T lymphocytes of donors 1 and 2 by the tested antibodies, with RO7122290, MP0310, BNT312, and isotype IgG1 as controls.

[0602] FIGS. 16, 17, and 18 are DC cell maturation promoting effects of the tested antibodies.

[0603] FIG. 19 is cross-linking of 4-1BB / CD40 expressing cells by the tested antibodies, with BNT312 and isotype IgG1 as controls.

[0604] FIG. 20 is MLR of the tested antibodies, with BNT312 and isotype IgG1 as controls.

[0605] Figure 21 is the result of detecting the tumor inhibition effect of the test antibody on MC38-mFAP transplanted tumor, using solvent, RO7122290, Ab10-A12V2-2 as control.

[0606] Figure 22 is the result of the effect of the test antibody on mouse ALT / AST on the 17th day after administration, using solvent, Ab10-A12V2-2, RO7211190 as control.

[0607] Figure 23 is the effect of the test antibody on mouse body weight, using solvent, RO7122290, Ab10-A12V2-2 as control.

[0608] Figure 24 is the effect of the test antibody on the number of neutrophils and platelets in mice, using solvent, RO7122290, Ab10-A12V2-2 as control.

[0609] Figure 25 is the result of the effect of the test antibody on mouse ALT / AST on the 8th day after administration, using solvent, Ab10-A12V2-2, BNT312 as control. DETAILED DESCRIPTION

[0610] The following examples are used to further describe the present disclosure, but these examples do not limit the scope of the present disclosure. The experimental methods in the embodiments of the present disclosure are not specified, and are usually performed according to conventional conditions, such as the Antibody Technology Laboratory Manual of Cold Spring Harbor, Molecular Cloning Manual; or according to the conditions recommended by the manufacturer of the raw materials or products. The reagents not specified by the source are conventional reagents purchased on the market.

[0611] The FAP / 4-1BB / CD40 multispecific antibody in the present disclosure is described in International Application PCT / CN2024 / 079551, and the entire contents of the foregoing patent are incorporated by reference into the present disclosure.

[0612] Example 1. Screening and preparation of anti-FAP monoclonal antibody

[0613] 1.1 Screening of antigen, detection of antigen sequence and preparation

[0614] Human fibroblast activation protein (FAP; GeneBank accession number AAC51668) is a serine oligopeptidase with a molecular weight of about 170 kDa, which is a homodimer. The sequence, source and use of the recombinant FAP protein used in the present disclosure are shown in Table 2.

[0615] Table 2. Source of amino acid sequence of recombinant protein

[0616] 1.2 Screening of h-FAP specific binding antibody fragments from human natural Fab phage display library

[0617] With h-FAP-biotin antigen as the target molecule, magnetic beads with avidin on the surface were used to capture antigen-specific phage, and the phage was enriched with a magnetic stand. Antigen-specific phage was eluted with glycine solution at pH 2.2.

[0618] After two rounds of screening, 284 clones were randomly selected, and positive clones were screened by phage ELISA, including the following specific steps: count the colonies growing on the plate, a total of 284, inoculate 96-well plates, each containing 400 μL of culture medium (2YT+Amp+0.2% glucose), 37°C, 250 rpm shaking culture for 6h. Plate with antigen h-FAP-His, 100 ng / 100 μL / well, 4°C overnight. The next day, after washing and blocking the 96-well plate, add 100 μL of overnight culture to each well, incubate at 37°C for 1h, wash, add secondary antibody (HRP-labeled anti-human IgG-Fab antibody), incubate at 37°C for 40 min, wash, add color developing solution, protect from light for 30 min, and read OD600 values on a microplate reader. Sequence a total of 122 positive clones obtained after two screenings, resulting in 74 unique VHs (constituting a VH enrichment library), 48 unique kappa light chains (constituting a KLC enrichment library), and 10 unique lambda light chains (constituting a LLC enrichment library).

[0619] 1.3 Construction of CHO cell surface full-length antibody display library

[0620] 1) Construction of full-length antibody CHO cell display gene library, including: PCR amplification of VH enrichment library, KLC enrichment library and LLC enrichment library with three sets of primers. After enzyme digestion, the three enrichment libraries are inserted into the corresponding component vectors to construct the corresponding component libraries. Enzyme digestion of KLC, LLC and VH component libraries, electrophoretic analysis of purified enzyme-digested KLC fragment library, LLC fragment library and VH fragment library. Enzyme digestion of full-length antibody cell display vector, purification of vector fragments. Mix and ligate the enzyme-digested and purified vector fragments, KLC fragment library, LLC fragment library and VH fragment library. Purify the ligation product, electrotransform into E. coli, plate, and incubate at 37°C overnight. Count the colonies. After detection, the library capacity reached 3.6 x 10E6, which was more than 800 times the theoretical diversity (74 x 58 = 4292). Collect all colonies, extract vector DNA, and obtain the full-length antibody cell display gene library.

[0621] 2) Construction of h-FAP antigen specific full length antibody cell display library, including: transformation with full length antibody cell display gene library vector DNA, construction of full length antibody CHO cell display library. Screening of cell library with hygromycin pressure. Obtaining stably transformed cell library, double staining cell library with PE labeled mouse anti-human kappa (or lambda) light chain antibody and FITC labeled h-FAP antigen, FACS sorting PE and FITC double positive cells, one cell per well, kappa light chain library sorting 2 pieces of 96 well plate, lambda light chain library sorting 1 piece of 96 well plate, hygromycin pressure culture.

[0622] 3) FACS analysis to identify h-FAP antigen specific antibody single cell clones, including: hygromycin pressure culture for 14 days, obtaining 153 kappa chain single cell clones and 50 lambda chain single cell clones. Digesting cells with 0.5 mM EDTA-PBS buffer, double staining single cell clones with PE labeled mouse anti-human kappa (or lambda) light chain antibody and FITC labeled h-FAP antigen, obtaining 138 PE and FITC fluorescence double positive single cell clones by FACS analysis.

[0623] 4) Cloning of antibody genes, including: determining 45 clones for antibody gene PCR amplification by FACS detection analysis of affinity of positive clones. Centrifuging to collect positive clones, discarding supernatant, extracting cell genomic DNA, PCR amplifying VH and LC, electrophoretically separating amplified fragments, and sequencing analysis. The variable region sequences of anti-human FAP monoclonal antibody Ab10 are as follows:

[0624] > VH

[0625] > VL

[0626] The CDR sequences are as follows:

[0627] Table 3. CDR of anti-FAP antibody Ab10

[0628] 1.4 Expression and purification of anti-FAP antibody

[0629] The heavy and light chain variable regions of the above anti-FAP antibody Ab10 sequence are respectively connected with the constant regions of human HC and LC to form the heavy chain full length sequence (SEQ ID NO: 9) and the light chain full length sequence (SEQ ID NO: 10). Constructing plasmids, transient transfection, expression and purification to obtain the target antibody. The Ab10 full length sequence is as follows, and the underlined part is the constant region sequence:

[0630] > Ab10 heavy chain full length

[0631] > Ab10 light chain full length

[0632] Fab heavy chain

[0633] Fab light chain

[0634] 1.5 ELISA binding test

[0635] Directly coat FAP recombinant protein with His tag, after adding antibody, detect the activity of antibody binding to antigen by adding secondary antibody (HRP conjugated anti-human IgG antibody) and HRP substrate TMB. Including: coat 96-well enzyme plate with human FAP-His protein, 0.5 μg / mL concentration per well 100 μL, 4°C incubate overnight. Wash thoroughly, add 200 μL / well blocking solution, incubate at room temperature for 2 h. Wash thoroughly, add 100 μL per well of anti-FAP antibody to be tested diluted with diluent. Incubate at room temperature for 1 h, wash thoroughly, add 100 μL per well of HRP-labeled goat anti-human IgG secondary antibody diluted 1:20,000 times with diluent. Incubate at room temperature for 1 h, wash thoroughly, add 100 μL per well of TMB, avoid light for 15 min. Add 50 μL / well of 0.16 M sulfuric acid. Thermo MultiSkanFc microplate reader reads OD value at 450 nm, calculates the binding EC 50 value of anti-FAP antibody Ab10 is 0.0920 mg / mL.

[0636] 1.6 Surface plasmon resonance (SPR) binding test

[0637] CM5 sensor chip was chosen, and HBS-EP+ buffer solution (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.05% surfactant P20) was used as the mobile phase. Anti-human IgG (Fc) antibody was prepared into a 30 μg / mL solution with 10 mM sodium acetate buffer (pH 5.0), and was subjected to amino coupling fixation. Each antibody to be tested was prepared with HBS-EP+ buffer solution, and was captured by the anti-human IgG (Fc) antibody on the chip channel. h-FAP-His was prepared as an analyte with HBS-EP+ buffer solution, and was diluted by 2-fold gradient. The diluted antibody was flowed through the experimental channel and the reference channel at a flow rate of 30 μL / min, and was combined for 1 min and dissociated for 15 min. 10 mM glycine pH 1.5 was used as a regeneration buffer, and was operated at a flow rate of 10 μL / min for 30 s. The data were analyzed, and the results in Table 4 show that the affinity of Ab10 to the antigen FAP is 5.3 times higher than that of the control anti-FAP antibody 28H1 (sequences 219 and 233 of patent US9266938B2). The binding affinity of other anti-FAP antibodies obtained by the simultaneous screening of the present disclosure, such as Ab2, Ab4, and Ab5 (sequences not shown), to the antigen is lower than that of 28H1.

[0638] Table 4. SPR affinity Ka, Kd, and K of anti-FAP antibodies D values

[0639] 1.7 FACS binding test

[0640] The binding properties of anti-FAP antibodies on cells were detected by FACS experiments. This included: constructing a CHO cell line overexpressing human FAP, and plating (1E5 / well). The antibody to be tested was added, 100 μL / well, at a maximum concentration of 100 nM, 5-fold dilution, a total of 8 concentrations, and 4°C incubation for 1 h. Anti-hIgG Alexa Fluor-647 was added as a secondary antibody at a ratio of 1:500, and was incubated at 4°C for 30 min, and was detected by FACS. Antibodies that can bind FAP can label cells. The binding EC 50 value of human FAP to antibody Ab10 was 0.0004320 μg / mL.

[0641] Example 2. Screening and preparation of anti-CD40 nanobodies

[0642] 2.1 Immunization antigen, screening of antigen sequences, and preparation

[0643] His-tagged human CD40 recombinant protein (h-CD40-His), C-terminal biotin-tagged human CD40 recombinant protein (h-CD40-biotin), C-terminal His-tagged monkey CD40 recombinant protein (cyno-CD40-His) were selected. The sequences and sources are shown in Table 5. The protein reagents can be used in the following examples.

[0644] Table 5. Recombinant protein amino acid sequence and source

[0645] 2.2 Alpaca immunization process and titer detection

[0646] The alpaca was immunized with h-CD40-his, and immunized once every two weeks for a total of four times. The first immunization was subcutaneous injection of 0.5 mg antigen mixed with Freund's complete adjuvant (CFA) 1 mL, and the last three immunizations were subcutaneous injection of 0.25 mg antigen mixed with Freund's incomplete adjuvant (IFA) 1 mL. Before immunization, blank serum was collected, and 50 mL of peripheral blood lymphocytes were collected one week after the third immunization and one week after the fourth immunization, respectively, to detect the serum titer.

[0647] 2.3 Titer detection and phage library construction

[0648] After four immunizations, the serum titer of the alpaca was detected. After the titer was qualified, PBMCs were isolated, total RNA was extracted, purity was detected, and DNA was reverse transcribed. After two rounds of nested PCR, the purified vector was ligated with the VHH target fragment. Electroporation, clone selection, and phage library A and B were obtained.

[0649] 2.4 Phage library affinity panning and ELISA identification

[0650] Using h-CD40 antigen as the target molecule, Gly-HCL acid elution method was used for screening, and specific phage was eluted. After two rounds of screening, 384 clones were randomly selected from the first and second round of titer determination plates, and positive clones were screened by phage ELISA, and the optical density at 450 nm was detected. According to the sequencing results, sequence alignment and phylogenetic tree analysis were performed. The sequence of A297 is as follows, and the underlined part is the CDR under the Kabat numbering rule: >A297

[0651] The CDR sequence is as follows:

[0652] Table 6. CDR of anti-CD40 antibody A297

[0653] 2.5 Humanization of anti-CD40 nanobody

[0654] Based on the obtained VHH canonical structure of Nanobody A297, the VHH variable region sequence was compared with the antibody germline database to obtain human germline templates with high homology. The preferred human germline templates of the antibody A297 of the present disclosure are all IGHV3-48*03. Then the CDRs were grafted into human FRs, and key amino acids affecting the structure and function of the antibody were subjected to back mutation to restore the binding force and activity. The sequence of A297V3 is as follows, and the underlined part is the CDR under the Kabat numbering rule:

[0655] > A297V3

[0656] 2.6 Detection of the binding ability of anti-CD40 Nanobodies to antigens

[0657] The above anti-CD40 Nanobody sequence was connected with human IgG1-Fc (SEQ ID NO: 16) with C220A, S267E and L328F mutations (according to the Eu numbering system). The plasmid was constructed, transiently transfected, expressed and purified to obtain the target antibody. 9E5-SELFNS was used as a positive control for CD40 agonist (see SEQ ID NO: 58 and SEQ ID NO: 59 of WO2020108611A1).

[0658] > IgG1-Fc

[0659] The binding ability of the humanized antibody to cells highly expressing human antigen protein CD40 was detected by FACS. HEK293 cells were transiently transfected with CD40 plasmid (Sino Biological; HG10774-CF) to obtain HEK293 cells highly expressing CD40, resuspended with flow staining solution (PBS + 2% FBS), and then different concentrations of the antibody to be detected were added after gradient dilution. After incubation on ice for 1 h, PBS was added for washing, and centrifugation was performed at 400g for 5 min. Goat anti-human Fc antibody labeled with fluorescent group Alexa Fluor 647 was added as a secondary antibody, and the cells were stained on ice for 1 h. After washing twice with PBS, the fluorescence signal on the cell surface was detected by FACS. EC 50 The values are shown in Table 7. The humanized antibody (A297V4 sequence not shown) has high affinity to CD40 highly expressing cell lines.

[0660] Table 7. Affinity of each humanized anti-CD40 antibody to human CD40 highly expressing cell lines EC 50 values

[0661] Activation of HEK-Blue™ CD40L cells. The activation of HEK-Blue™ CD40L cells by CD40 antibodies in the presence of FcyRIIb cross-linking was detected by measuring the activity of the reporter gene SEAP.TM The in vitro agonistic activity of CD40 antibodies was evaluated by activating CD40L cells. The S267E / L328F mutation in the Fc region of the humanized CD40 antibody enhances the affinity of IgG1 Fc for FcγRIIb, thus also enhancing the cross-linking of FcγRIIb to the antibody. The activation of CD40 antibodies by FcγRIIb mediated the activity of HEK-Blue antibodies. TM The activation of CD40L cells mimicked the agonist activity of CD40 antibodies under fully cross-linked conditions. HEK293 cells were transiently transfected with the FcγRIIb plasmid (CD32B / Fcgr2b cDNA ORF Clone, Human, N-His tag; Sino Biological; HG10259-NH) to obtain HEK293 cells with high FcγRIIb expression. HEK-Blue... TM CD40L cells were seeded at 5E4 cells / well in a 96-well cell culture plate (medium: DMEM, 10% FBS, 100 μg / mL Normocin). HEK293 cells expressing FcγRIIb were added at 5E4 cells / well. 100 μL of serially diluted test antibody was added to each well, and the plates were incubated overnight at 37°C. Cells were centrifuged, and 20 μL of the cell supernatant was transferred to a new 96-well plate. 180 μL of QUANTI-Blue substrate solution was added, and the plates were incubated in the dark for 30 min. The absorbance at 620 nm was measured using an Envision microplate reader, and the EC50 was calculated. 50 The values ​​and Emax values ​​(relative to the fluorescence intensity of the antibody-free group) are expressed as EC50 values. 50 The value is used to evaluate the in vitro cell agonist activity of CD40 antibodies. EC 50 The values ​​and Emax values ​​(relative fluorescence intensity) are shown in Table 8. The results show that, in the above reporter gene cell system, the humanized CD40 antibody exhibits strong agonist activity when cross-linked with FcγRIIb.

[0662] Table 8. Effects of various humanized anti-CD40 antibodies on HEK-Blue TM Activation of CD40L cells

[0663] Example 3. Preparation and screening of anti-4-1BB nanobodies

[0664] 3.1 Alpaca Immunization, Titer Detection, and Phage Library Affinity Selection

[0665] The llama was immunized with His-tagged human 4-1BB recombinant protein (Acrobiosystems, 41B-H5227), once every two weeks, for a total of four times. At the first immunization, 0.5 mg of antigen was mixed with 1 mL of Freund's complete adjuvant (CFA) for subcutaneous injection, and at the last three immunizations, 0.25 mg of antigen was mixed with 1 mL of Freund's incomplete adjuvant (IFA) for subcutaneous injection. Before immunization, blank serum was collected, and 50 mL of peripheral blood was collected one week after the third immunization and one week after the fourth immunization, respectively, to isolate PBMCs, extract total RNA, detect purity, reverse transcribe into DNA, and after two rounds of nested PCR, the nanobody target fragment was ligated with the phage display vector. Electroporation was performed to obtain the phage library.

[0666] >Human 4-1BB protein sequence

[0667] To obtain anti-4-1BB nanobodies that recognize both human and monkey, a strategy of cross-screening with human and monkey antigens in two rounds was used. Human 4-1BB and monkey 4-1BB, or monkey 4-1BB and human 4-1BB, were used as screening antigens in the first and second rounds, respectively. Gly-HCl acid elution method was used in each round of screening to elute phages that specifically bind to 4-1BB. From the first and second round titer determination plates, 96 clones were randomly picked (a total of 192 clones), positive clones were screened by phage ELISA, and the optical density at 450 nm was detected. The positive clones were sequenced. According to the sequencing results, sequence alignment and phylogenetic tree analysis were performed, and 14 unique sequences were selected, including H27, C5, C145, etc., among which the C5 sequence is as follows.

[0668] >C5

[0669] Among them, the CDR sequences are as follows:

[0670] Table 9. CDR of anti-4-1BB nanobody C5 (Kabat numbering rule)

[0671] The sequence of C5 was connected with human IgG1-Fc with C220A, S267E and L328F mutations (according to Eu system numbering). The sequence of the VHH-Fc fusion protein after connection is as follows. In addition, L234A, L235A and N297A (according to Eu system numbering) mutations were introduced into the Fc of human IgG1 to completely remove the effector function mediated by FcγR; S228P (according to Eu system numbering) was introduced into the Fc of human IgG4 to stabilize the antibody molecule and prevent half molecule formation, which are all selectable IgG Fc.

[0672] An exemplary antibody sequence of C5 linked to IgGl-Fc (containing C220A, S267E, L328F mutations) is shown in SEQ ID NO: 22, with the IgGl-Fc sequence underlined:

[0673] >C5-Fc

[0674] The plasmid was constructed, the cells were transiently transfected, the antibody was expressed, and purified. The target antibody was obtained after detection.

[0675] 3.2 Detection of antigen binding activity and agonist activity of anti-4-1BB antibodies

[0676] (1) Detection of binding ability to 4-1BB antigen

[0677] The binding activity of anti-4-1BB single domain antibodies to human 4-1BB protein was detected by flow cytometry. HEK293-Hu4-1BB cells were obtained by transient expression of human 4-1BB protein (CD137 cDNA ORF Clone, Human, C-OFPSpark tag; purchased from Sino Biological, Cat# HG10041-ACR) from HEK293 cells (ATCC CRL-1573), and the cell culture medium was DMEM (Gibco, Cat# 11995065) containing 10% fetal bovine serum. The experimental medium was sterile PBS (phosphate buffered saline, pH 7.40) containing 2% fetal bovine serum. 1 x 10 5 HEK293-Hu4-1BB cells were seeded in a 96-well U-bottom plate, different concentrations of anti-4-1BB antibody VHH-Fc samples to be tested were added, and the cells were incubated at 4°C for 1 hour, then washed twice with experimental medium; then goat anti-human IgG (H+L) Alexa Fluor 488 antibody (Thermo, Cat# A11013) was added, washed twice, and the fluorescence signal value was read by flow cytometry. Urelumab monoclonal antibody was used as a positive control, and the MFI values of each antibody are shown in Figure 1. The results show that the anti-4-1BB antibodies involved have different degrees of binding ability to 4-1BB on the surface of HEK293-Hu4-1BB cells, among which C5 has good cell membrane surface antigen binding activity.

[0678] (2) Detection of agonist activity on 4-1BB signaling pathway

[0679] Agonist activity of anti-4-1BB antibodies was evaluated using 4-1BB / NF-κB reporter gene. HEK293 cells (ATCC CRL-1573) were transiently transfected with human 4-1BB gene (CD137 cDNA ORF Clone, Human, C-OFPSpark tag; purchased from Sino Biological, Cat# HG10041-ACR) and NF-κB reporter gene (pGL4.32[luc2P / NF-κB-RE / Hygro] Vector, purchased from Promega, Cat# E849A) to obtain HEK293-Hu4-1BB / NF-κB double-transfected cells, which can be characterized by the level of activation of NF-κB signaling pathway. HEK293 cells were transiently transfected with FcγRIIb plasmid (CD32B / Fcgr2b cDNA ORF Clone, Human, N-His tag; purchased from Sino Biological, Cat# HG10259-NH) to obtain HEK293 cells with high expression of FcγRIIb. The cell culture medium was DMEM (Gibco, Cat# 11995065) containing 10% fetal bovine serum. HEK293-Hu4-1BB / NF-κB cells (2 x 105 / mL) were plated in 96-well cell culture plates at 50 μL per well, and 40 μL of culture medium or HEK293 cells expressing FcγRIIb (2.5 x 105 / mL) was added. 10 x 10 μL of gradient-diluted anti-4-1BB antibodies to be tested was added to each well, and incubated at 37°C for 6 hours. The cells were removed, and an equal volume of Bio-Glo Luciferase Assay System reagent (Promega, Cat# G7940) was added to each well. The fluorescence signal was measured using an Envision microplate reader (PerkinElmer, 2150) after 5 minutes of incubation in the dark. The EC 6 value and Emax value (relative fluorescence intensity of the group without antibody) were calculated. 6 50 50 ​​To evaluate the in vitro cell agonist activity of anti-4-1BB antibodies, the results are shown in Table 10. The results show that the anti-4-1BB antibodies involved show much weaker activation of the 4-1BB / NF-κB luciferase reporter signal pathway than the Urelumab control without the addition of FcγRIIb (i.e., CD32b) positive cross-linking, indicating that the anti-4-1BB antibodies of the present disclosure are safer; the anti-4-1BB antibodies involved show significant activation of the 4-1BB / NF-κB luciferase reporter signal pathway after the addition of FcγRIIb cross-linking, and the activation ability of C5 is the strongest and comparable to the Urelumab control. Based on the activity results, sequence C5 with low background activation before FcγRIIb cross-linking and stronger activation activity after FcγRIIb cross-linking is selected and humanized.

[0680] Table 10. EC of agonist activity of anti-4-1BB antibodies on 4-1BB / NF-κB luciferase reporter 50 Value

[0681] (Note: "-" is the test value beyond the detection limit)

[0682] 3.3 Humanization modification

[0683] According to the amino acid numbering of the CDR and FR regions (human framework region) of the C5 sequence identified according to the Kabat numbering system, the FR1+CDR1 sequence, the FR2+CDR2 sequence, and the FR3+CDR3 sequence are matched with the antibody germline database, respectively, to obtain higher homologous FR human germline templates. FR1 is from IGHV3-64*04, FR2 is from IGHV3-23*03, and FR3 is from IGHV3-74*01. Each of the above human germline FR regions is replaced and inserted into the original sequence to reduce immunogenicity in the human body. Key amino acids affecting antibody structure and function are back-mutated to restore binding force and activity. Each humanized sequence is shown below.

[0684] >C5_V1

[0685] >C5_V2

[0686] >C5_V3

[0687] (Note: underlined is the CDR region)

[0688] The above 3 humanized sequences are respectively connected with human IgG1-Fc (SEQ ID NO: 16). The plasmid is constructed, transiently transfected, expressed and purified.

[0689] 3.4 Detection of antigen-binding activity and agonist activity of humanized anti-4-1BB antibodies

[0690] (1) Detection of binding ability to 4-1BB antigen

[0691] The humanized antibodies were detected for antigen-binding activity using the FACS detection method as described in 3.2. The results are shown in Table 11. The results showed that the humanized anti-4-1BB antibodies involved had different degrees of binding ability to 4-1BB on the surface of HEK293-Hu4-1BB cells, and C5_V1, C5_V2, C5_V3 all maintained good binding activity.

[0692] Table 11. Affinity EC of anti-4-1BB antibodies before / after humanization to human 4-1BB high expression cell strain 50 value

[0693] (2) Detection of agonist activity to 4-1BB signaling pathway

[0694] The humanized antibodies were detected for 4-1BB / NF-κB signal activation dependence using the NF-κB luciferase reporter gene experiment as described in 3.2, and the results are shown in Table 12. The results showed that after adding FcγRIIb cross-linking, the humanized anti-4-1BB antibodies involved showed activation of the 4-1BB / NF-κB luciferase reporter gene signaling pathway, and the activation ability of C5, C5_V1, C5_V2, C5_V3 was comparable to that of Urelumab control.

[0695] Table 12. Activation of NF-κB signaling pathway by anti-4-1BB antibodies before / after humanization

[0696] (Note: Emax change multiple <1.5, defined as "−"; 1.5-2, defined as "+"; 2-2.5, defined as "++"; 2.5-3, defined as "+++"; >3, defined as "++++".)

[0697] 3.5 Removal of TCE sites

[0698] Subsequently, we performed T-cell epitope prediction (T-cell Epitope, TCE) on the C5_V2 sequence, and according to the prediction results, the CDR3 sequence of C5_V2 was modified to reduce the number of TCE. The F in the CDR3 region was mutated to Y (F99Y, according to the Kabat numbering system) to obtain the TCE optimized version of C5_V2 sequence, named C5_V2-YTI, and the sequence is as follows:

[0699] >C5_V2-YTI

[0700] That is, according to the Kabat numbering system, the amino acid sequence of CDR1 in C5_V2-YTI is shown as SEQ ID NO: 19, the amino acid sequence of CDR2 is shown as SEQ ID NO: 20, and the amino acid sequence of CDR3 is shown as HPLTYTIATMNDYDY (SEQ ID NO: 27).

[0701] The above C5_V2-YTI sequence is connected with human IgG1-Fc (SEQ ID NO: 16). The plasmid is constructed, transiently transfected, expressed and purified to obtain the target antibody.

[0702] 3.6 Detection of the binding ability of humanized 4-1BB antibody after removing TCE to antigen

[0703] The humanized antibody C5_V2 is optimized by removing TCE, and the obtained antibody is C5_V2-YTI. The FACS detection method as described in 3.2 is used to compare the activity of C5_V2 and C5_V2-YTI to the antigen binding. The results are shown in Table 13. The results show that the C5_V2-YTI involved maintains good binding activity to 4-1BB on the surface of HEK293-Hu4-1BB cells, which is equivalent to C5_V2.

[0704] Table 13. EC of affinity of C5_V2, C5_V2-YTI to human 4-1BB high expression cell strain 50 Value

[0705] 3.7 Reducing the risk of antibody aggregation

[0706] We found that the C-terminal surface of C5_V2-YTI antibody has a larger hydrophobic region, which can cause the risk of antibody aggregation. By mutating the hydrophobic amino acid Leu (L) in the TLVTVSS sequence to the hydrophilic amino acid Gln (Q), the area of the C-terminal hydrophobic surface region of the antibody can be effectively reduced, and the risk of antibody aggregation can be reduced. Further, to optimize the pi value of the sequence, two acidic amino acids Glu (E) in the sequence are mutated to basic amino acid Gln (Q) and neutral amino acid Ala (A), respectively. The modified antibody is named C5_V2-YTIQ, and its sequence is as follows:

[0707] >C5_V2-YTIQ

[0708] That is, according to the Kabat numbering system, the amino acid sequence of CDR1 in C5_V2-YTIQ is shown as SEQ ID NO: 19, the amino acid sequence of CDR2 is shown as DINSGGQSTFYADSVKG (SEQ ID NO: 29), and the amino acid sequence of CDR3 is shown as SEQ ID NO: 27.

[0709] That is, the anti-4-1BB nanobody has the following general structure:

[0710] CDR1 is SYAMS (SEQ ID NO: 19);

[0711] CDR2 is DINSGGX1STFYX2DSVKG (SEQ ID NO: 30), wherein X1 is E or Q, and wherein X2 is E or A;

[0712] CDR3 is HPLTX3TIATMNDYDY (SEQ ID NO: 31), wherein X3 is F or Y.

[0713] 3.8 Detection of the binding ability of the engineered anti-4-1BB nanobody to antigens

[0714] C5_V2-YTI and C5_V2-YTIQ were connected with human IgG1-Fc (SEQ ID NO: 16). The plasmid was constructed, transiently transfected, expressed and purified to obtain the target antibody.

[0715] Using the FACS detection method, the activities of C5_V2, C5_V2-YTI and C5_V2-YTIQ in binding to antigens were compared, and the results are shown in Figure 2, Table 14, and Urelumab was used as a positive control. The results showed that C5_V2, C5_V2-YTI and C5_V2-YTIQ all maintained good binding activity to 4-1BB on the surface of HEK293-Hu4-1BB cells.

[0716] Table 14. Affinity EC of anti-4-1BB nanobodies to human 4-1BB high expression cell lines 50 Values

[0717] Example 4. Design and preparation of anti-FAP / 4-1BB / CD40 multispecific antibodies

[0718] 4.1 Design and expression and purification of anti-FAP / 4-1BB / CD40 multispecific antibodies

[0719] Fusion proteins of FAP, 4-1BB and CD40 were constructed, and the schematic diagram of the molecular form is shown in Figure 3.

[0720] In the molecule B977703 shown in Fig. 3a, the C-termini of the two heavy chains of the anti-FAP monoclonal antibody Ab10 are connected to the N-termini of the anti-4-1BB nanobody C5V2-YTI, respectively, through a (G4S)2linker, and the C-termini of the two anti-4-1BB nanobodies are connected to the N-termini of the anti-CD40 nanobody A297V3, respectively, through a (G4S)2linker.

[0721] In the molecule B977704 shown in Fig. 3b, the C-termini of the two heavy chains of the anti-FAP monoclonal antibody Ab10 are connected to the N-termini of the anti-4-1BB nanobody C5V2-YTI, respectively, through a (G4S)2linker, and the C-termini of the two light chains of the anti-FAP monoclonal antibody Ab10 are connected to the N-termini of the anti-CD40 nanobody A297V3, respectively, through a (G4S)2linker.

[0722] In the molecule B977707 shown in Fig. 3c, two anti-4-1BB nanobodies C5V2-YTI are connected in sequence through a (G4S)2linker, and the N-termini of the two anti-4-1BB nanobodies are connected to the C-termini of the two heavy chains of the anti-FAP monoclonal antibody Ab10, respectively, through a (G4S)2linker; and the C-termini of the two light chains of the anti-FAP monoclonal antibody Ab10 are connected to the N-termini of the anti-CD40 nanobody A297V3, respectively, through a (G4S)2linker.

[0723] In the molecule B785502 shown in Fig. 3d, the C-termini of the two heavy chains of the anti-FAP monoclonal antibody Ab10 are connected to the N-termini of the anti-CD40 nanobody A297V3, respectively, through a (G4S)2linker, and the C-termini of the two anti-CD40 nanobodies A297V3 are connected to the N-termini of the anti-4-1BB nanobody C5V2-YTI, respectively, through a (G4S)2linker.

[0724] In the molecule B785503 shown in Fig. 3e, the C-termini of the two heavy chains of the anti-FAP monoclonal antibody Ab10 are connected to the N-termini of the anti-CD40 nanobody A297V3, respectively, through a (G4S)2linker, and the C-termini of the two light chains of the anti-FAP monoclonal antibody Ab10 are connected to the N-termini of the anti-4-1BB nanobody C5V2-YTI, respectively, through a (G4S)2linker.

[0725] In the molecule B785511 shown in Fig. 3f, which is a specific antibody 4-1BB / CD40 as a control, two anti-4-1BB nanobodies C5V2-YTI are connected to the N-termini of the Fc region through the hinge region of the heavy chain of IgG1, and the C-termini of the Fc region are connected to the N-termini of the anti-CD40 nanobody A297V3, respectively, through a (G4S)2linker.

[0726] The Fc in the molecules shown in Fig. 3 a-f is human IgGl Fc with mutations of L234A and L235A to remove FcyR-mediated effector functions.

[0727] 4.2 Engineering design and expression purification of anti-FAP / 4-1BB / CD40 multispecific antibodies

[0728] To reduce the binding to pre-existing anti-drug antibodies (pre-ADA) or anti-drug antibodies (ADA), the nanobody at the C-terminus of anti-FAP / 4-1BB / CD40 multispecific antibodies was engineered by mutating the TVSS at the C-terminus of the nanobody to TVSAA. The sequence of A297V3 optimization is A297V3_AA, and the sequence of C5_V2-YTI optimization is C5_V2-YTI_AA.

[0729] A297V3_AA

[0730] C5_V2-YTI_AA

[0731] The anti-CD40 nanobody A297V3 at the C-terminus of the heavy chain in B977703 was engineered to A297V3_AA to obtain B21326101, and the anti-CD40 nanobody A297V3 at the C-terminus of the light chain in B977704 was engineered to A297V3_AA, and the anti-4-1BB nanobody C5_V2-YTI at the C-terminus of the heavy chain in B977704 was engineered to C5_V2-YTI_AA to obtain B21326102. The specific molecular sequences are shown in Table 32.

[0732] 4.3 Engineering design and expression purification of anti-FAP / 4-1BB / CD40 multispecific antibodies to reduce antibody aggregation

[0733] To reduce aggregation, we further engineered B21326101 by replacing C5_V2-YTI with C5_V2-YTIQ (SEQ ID NO: 28), and the optimized molecule was named B21546901.

[0734] 4.4 Control molecules

[0735] BNT312 is a CD40 / 4-1BB bispecific antibody molecule, Ab10-A12V2-2 is a FAP / CD40 bispecific antibody molecule, RO7300490 is a FAP / CD40 bispecific antibody molecule, RO7122290 is a FAP / 4-1BBL bispecific antibody molecule, and MP0310 is a FAP / 4-1BB bispecific antibody molecule, all of which are used as control molecules.

[0736] The above multispecific antibody sequences are shown in Table 32. The sequences of the control molecules are shown in Table 33.

[0737] 4.5 Expression and purification

[0738] Transient transfection and expression: Take the 30 mL expression system as an example, dilute the culture solution with 60 μL transfection reagent, mix with 15 μg plasmid, incubate at 37°C for 15 minutes, then add the mixed transfection solution dropwise into the cell solution, shake while adding, and place on a shaker to culture for expression for one week. Collect the supernatant and centrifuge at 8000 rpm for 5 minutes.

[0739] Antibody purification: First, equilibrate the Protein A affinity chromatography column with 1xPBS at a flow rate of 1 mL / min for 20 mL. The loading flow rate is 1 mL / min. Wash with 1xPBS at a flow rate of 1 mL / min for 20 mL. Use citric acid buffer (pH 3.4) at a flow rate of 1 mL / min for elution, and collect in separate tubes. Use a NanoDrop instrument to read the absorbance value at 280 nm. Finally, transfer the high-concentration protein to a dialysis bag and dialyze in a beaker containing 1xPBS. After detection, the target antibody is obtained.

[0740] Example 5. Detection of antigen binding activity of anti-FAP / 4-1BB / CD40 multispecific antibodies

[0741] 5.1 Detection of antigen binding activity of anti-FAP / 4-1BB / CD40 multispecific antibodies

[0742] The binding activity of anti-FAP / 4-1BB / CD40 multispecific antibodies to human FAP / 4-1BB / CD40 proteins was detected by FACS experiments. CHO-K1-HuFAP cells were obtained by stably expressing human FAP protein in CHO-K1 cells, and the cell culture medium was Ham’s F12 + GlutaMAXTM-I (Gibco, Cat#31765035) containing 10% fetal bovine serum and 800 μg / mL hygromycin B. HEK293-Hu4-1BB cells and HEK293-HuCD40 cells were obtained by overexpressing human 4-1BB and human CD40 proteins in HEK293 cells, respectively, and the cell culture medium was DMEM (Gibco, Cat#11965092) containing 10% fetal bovine serum and 100 μg / mL hygromycin B. The experimental culture medium was sterile PBS (phosphate buffer, pH 7.40) containing 2% fetal bovine serum. Wash the CHO-K1-HuFAP, HEK293-Hu4-1BB, or HEK293-HuCD40 cells twice with the experimental culture medium at 1x10 5Cells were seeded in 96-well U-bottom plates, and different concentrations of the sample to be tested were added. After incubation of the cells at 4°C for 1 hour, the cells were washed twice with experimental culture medium; then Alexa Fluor 647-mouse anti-human (IgG, Fc gamma fragment specific) antibody (Jackson, Cat# 209-605-098) was added, and after washing twice, the fluorescence signal value was read by flow cytometry.

[0743] Table 15, the results of FACS detection in Figure 4A show that the FAP / 4-1BB / CD40 multispecific antibody has strong binding capacity to FAP on the surface of CHO-K1-HuFAP cells, and the binding capacity is comparable to that of the control antibody RO7122290, indicating that the preparation of the multispecific antibody does not affect the binding of the antibody to FAP. Table 16, the results of Figure 4B show that the FAP / 4-1BB / CD40 multispecific antibody has good binding capacity to 4-1BB on the surface of HEK293-Hu4-1BB cells. Table 17, the results of Figure 4C show that the FAP / 4-1BB / CD40 multispecific antibody has good binding to CD40 on the surface of HEK293-HuCD40 cells, indicating that the preparation of the multispecific antibody does not affect the binding of the antibody to CD40.

[0744] Table 15. Results of FAP / 4-1BB / CD40 multispecific antibody binding to CHO-K1-HuFAP cells

[0745] Table 16. Results of FAP / 4-1BB / CD40 multispecific antibody binding to HEK293-Hu4-1BB cells

[0746] Table 17. Results of FAP / 4-1BB / CD40 multispecific antibody binding to HEK293-HuCD40 cells

[0747] 5.2 Detection of antigen binding activity of anti-FAP / 4-1BB / CD40 multispecific antibody

[0748] The antigen binding activity of FAP / 4-1BB / CD40 multispecific antibodies B977703, B977704, B21326101, and B21326102 was detected using the detection method provided in Example 5.1, with RO7122290, Ab10-A12V2-2, BNT312, and IgG1 as controls.

[0749] The FACS detection results (Tables 18-20, FIGS. 5A-5C) show that the FAP / 4-1BB / CD40 multispecific antibody has strong binding capacity to FAP on the surface of CHO-K1-HuFAP cells (Table 18, FIG. 5A). Table 19, FIG. 5B shows that the FAP / 4-1BB / CD40 multispecific antibody has good binding capacity to 4-1BB on the surface of HEK293-Hu4-1BB cells, and the multispecific antibody with modified 4-1BB sequence has no difference in 4-1BB binding, indicating that the modification of 4-1BB sequence has no effect on target binding. Table 20, FIG. 5C shows that the FAP / 4-1BB / CD40 multispecific antibody has binding capacity to CD40 on the surface of HEK293-HuCD40 cells, indicating that the removal of pre-ADA modification in the CD40 sequence has no obvious effect on target binding.

[0750] Table 18. Results of FAP / 4-1BB / CD40 multispecific antibody binding to CHO-K1-HuFAP cells

[0751] Table 19. Results of FAP / 4-1BB / CD40 multispecific antibody binding to HEK293-Hu4-1BB cells

[0752] Table 20. Results of FAP / 4-1BB / CD40 multispecific antibody binding to HEK293-HuCD40 cells

[0753] 5.3 Detection of antigen binding activity of anti-FAP / 4-1BB / CD40 multispecific antibody

[0754] The antigen binding activity of FAP / 4-1BB / CD40 multispecific antibodies B21546901 and B21326102 was detected using the detection method provided in Example 5.1, with RO7122290, RO7300490, BNT312, and IgG1 as controls.

[0755] The FACS detection results (Tables 21-23, FIGS. 6A-6C) showed that the FAP / 4-1BB / CD40 multispecific antibody had strong binding ability to FAP on the surface of CHO-K1-HuFAP cells, stronger than RO7300490, and comparable to the binding ability of the control antibody RO7122290. Table 22 showed that the FAP / 4-1BB / CD40 multispecific antibody had good binding ability to 4-1BB on the surface of HEK293-Hu4-1BB cells, indicating that the modification to reduce the risk of antibody aggregation in the 4-1BB sequence had no obvious effect on the binding to 4-1BB. Table 23 showed that the FAP / 4-1BB / CD40 multispecific antibody had good binding ability to CD40 on the surface of HEK293-HuCD40 cells.

[0756] Table 21. Results of FAP / 4-1BB / CD40 multi-functional antibody binding to CHO-K1-HuFAP cells

[0757] Table 22. Results of FAP / 4-1BB / CD40 multi-functional antibody binding to HEK293-Hu4-1BB cells

[0758] Table 23. Results of FAP / 4-1BB / CD40 multi-functional antibody binding to HEK293-HuCD40 cells

[0759] The above results showed that the anti-FAP / 4-1BB / CD40 multispecific antibody had strong binding ability to FAP on the surface of CHO-K1-HuFAP cells, and the binding ability was comparable to that of the control antibody RO7122290, indicating that the preparation of the trispecific antibody did not affect the binding of the antibody to FAP. The anti-FAP / 4-1BB / CD40 multispecific antibody had good binding ability to 4-1BB on the surface of HEK293-Hu4-1BB cells, and there was no difference in 4-1BB binding between B977704 and the modified B21326102, indicating that the modification of the 4-1BB sequence had no effect on the target binding. The anti-FAP / 4-1BB / CD40 multispecific antibody had binding ability to CD40 on the surface of HEK293-HuCD40 cells, indicating that the modification of the CD40 sequence had no obvious effect on the target binding.

[0760] Example 6. Luciferase reporter gene detection of FAP / 4-1BB / CD40 multispecific antibody

[0761] 6.1 4-1BB / NF-κB luciferase reporter gene detection experiment of FAP / 4-1BB / CD40 multispecific antibody

[0762] The 4-1BB / NF-κB reporter assay was used to evaluate the agonist activity of the antibodies. HEK293 cells (ATCC CRL-1573) were transiently transfected with human 4-1BB gene (CD137 cDNA ORF Clone, Human, C-OFPSpark tag; Sino Biological, Cat# HG10041-ACR) and NF-κB genome (pGL4.32[luc2P / NF-κB-RE / Hygro] Vector, Promega, Cat# E849A) to obtain HEK293-Hu4-1BB / NF-κB double-transfected cells, which can be characterized by the activation level of NF-κB signaling pathway. CHO-K1-HuFAP cells were obtained by CHO-K1 cells stably expressing human FAP protein, and the cell culture medium was Ham’s F12 + GlutaMAXTM-I (Gibco, Cat# 31765035) containing 10% fetal bovine serum and 800 μg / mL hygromycin B. HEK293-HuCD40 cells were obtained by HEK293 cells overexpressing human CD40 protein, and the cell culture medium was DMEM (Gibco, Cat# 11965092) containing 10% fetal bovine serum and 100 μg / mL hygromycin B. HEK293-Hu4-1BB / NF-κB cells (2 x 10 6 6 The cells were removed, and an equal volume of Bio-Glo Luciferase Assay System reagent (Promega, Cat# G7940) was added to each well, and the cells were incubated for 5 minutes in the dark. The fluorescence signal was measured using an Envision microplate reader, and the EC50 value and Emax value (relative fluorescence intensity without antibody group) were calculated to evaluate the in vitro cell agonist activity of the anti-4-1BB antibodies.

[0763] The results show (Table 24, FIGS. 7A-7C) that the non-crosslinked FAP / 4-1BB / CD40 multispecific antibodies B977703 and B977704 have no background activation of 4-1BB, and the other antibodies have a certain background activation. The FAP / 4-1BB / CD40 multispecific antibodies all have 4-1BB activity activated by FAP or CD40 crosslinking.

[0764] ​Table 24. FAP / 4-1BB / CD40 multispecific antibody FAP / CD40-dependent 4-1BB / NF-κB luciferase reporter assay results (no crosslinking, FAP crosslinking, CD40 crosslinking)

[0765] 6.2 4-1BB / NF-κB luciferase reporter assay experiment of FAP / 4-1BB / CD40 multispecific antibodies

[0766] The 4-1BB agonist activity of FAP / 4-1BB / CD40 multispecific antibodies was detected by the method provided in Example 6.1. The results are shown in FIGS. 8A-8C and Table 25.

[0767] Table 25 and FIG. 8A results show that the FAP / 4-1BB / CD40 multispecific antibodies without crosslinking have no background activation of 4-1BB. Table 25 and FIG. 8B results show that the agonist activity of 4-1BB of the antibodies B977703, B977704 and B21326101, B21326102 after crosslinking by FAP is comparable, indicating that the 4-1BB sequence modification has no effect on the 4-1BB activation activity. Table 25 and FIG. 8C results show that the FAP / 4-1BB / CD40 multispecific antibodies all have 4-1BB activity activated by CD40 crosslinking, and the agonist activity of 4-1BB of the antibodies after crosslinking by CD40 is stronger than that of BNT312.

[0768] Table 25. FAP / 4-1BB / CD40 multispecific antibody FAP / CD40-dependent 4-1BB / NF-κB luciferase reporter assay results

[0769] 6.3 4-1BB / NF-κB luciferase reporter assay experiment of FAP / 4-1BB / CD40 multispecific antibodies

[0770] The 4-1BB agonist activity of FAP / 4-1BB / CD40 multispecific antibodies was detected by the method provided in Example 6.1. The results are shown in FIGS. 9A-9C and Table 26.

[0771] Table 26 and Figure 9A show that the uncrosslinked B21546901 and B21326102 antibodies have no background activation activity. Table 26 and Figure 9B show that the agonistic activity of the antibodies 4-1BB after crosslinking by FAP is comparable to the activation ability of RO7122290. Table 26 and Figure 9C show that the FAP / 4-1BB / CD40 multispecific antibodies all have 4-1BB activity activated by CD40 crosslinking of the antibodies 4-1BB after crosslinking by CD40 has stronger agonistic activity than BNT312. Table 26. FAP / CD40-dependent 4-1BB / NF-κΒ luciferase reporter assay results of FAP / 4-1BB / CD40 multispecific antibodies (uncrosslinked, FAP crosslinking, CD40 crosslinking)

[0772] The above results show that the uncrosslinked anti-FAP / 4-1BB / CD40 multispecific antibodies B977703, B977704, B21326101, B21326102, B21546901 antibodies have no background activation of 4-1BB; the agonistic activity of the 4-1BB of the multiple anti-FAP / 4-1BB / CD40 multispecific antibodies after crosslinking by FAP has no significant difference, indicating that the 4-1BB sequence modification has no effect on the 4-1BB activation activity; the agonistic activity of the 4-1BB of the multiple anti-FAP / 4-1BB / CD40 multispecific antibodies after crosslinking by CD40 is comparable, and the agonistic activity of the antibodies 4-1BB after crosslinking by CD40 is stronger than BNT312.

[0773] Example 7. Luciferase reporter assay of FAP / 4-1BB / CD40 multispecific antibodies

[0774] 7.1 CD40 / NF-κΒ luciferase reporter assay experiment of FAP / 4-1BB / CD40 multispecific antibodies

[0775] The agonist activity of the antibodies was evaluated using the CD40 / NF-κΒ reporter. HEK-Blue TM CD40L (InvivoGen, hkb-cd40) cells, cell culture medium is DMEM (Gibco, Cat#11965092) containing 10% heat-inactivated fetal bovine serum 100 μg / ml Normocin TM(InvivoGen, ant-nr-1) can characterize HuCD40 activation by measuring the activation level of the NF-κB signaling pathway. CHO-K1-HuFAP cells were obtained by stably transexpressing human FAP protein in CHO-K1 cells. The cell culture medium was Ham's F12+GlutaMAX™-I (Gibco, Cat#31765035) containing 10% fetal bovine serum and 800 μg / mL hygromycin B. HEK293-Hu4-1BB cells were obtained by overexpressing human 4-1BB protein in HEK293 cells. The cell culture medium was DMEM (Gibco, Cat#11965092) containing 10% fetal bovine serum and 100 μg / mL hygromycin B. HEK-Blue... TM CD40L(1×10 6 100 μL of cells / mL were seeded into a 96-well cell culture plate, followed by 80 μL of culture medium and 1.25 × 10⁻⁶ cells of either CHO-K1-HuFAP cells expressing FAP or HEK293-Hu4-1BB cells expressing 4-1BB. 6 Add 20 × 10 μL of serially diluted test antibody to each well and incubate overnight at 37°C. Remove the cells and add 20 μL of each well to 180 μL of QUANTI-Blue. TM (InvivoGen, re-qbs3), incubated in the dark at 37°C for 30 minutes, OD655 was measured using an Envision microplate reader, EC50 and Emax values ​​(relative to the antibody-free group) were calculated, and the in vitro cell agonist activity of the anti-CD40 antibody was evaluated. Results are shown in Figures 10A-10C and Table 27.

[0776] The results in Table 27 and Figure 10B show that the CD40 activating activity of the FAP / 4-1BB / CD40 multispecific antibody after FAP cross-linking is comparable to that of RO7300490 and Ab10-A12V2-2 molecules. The results in Table 27 and Figure 10C show that both the FAP / 4-1BB / CD40 multispecific antibodies exhibit CD40 activity activated by 4-1BB cross-linking.

[0777] Table 27. Results of FAP / 4-1BB-dependent CD40 / NF-κB luciferase reporter gene assay for FAP / 4-1BB / CD40 multispecific antibodies (uncrosslinked, FAP-crosslinked, 4-1BB-crosslinked).

[0778] 7.2 CD40 / NF-κB luciferase reporter gene assay using FAP / 4-1BB / CD40 multispecific antibody

[0779] The CD40 agonist activity of the FAP / 4-1BB / CD40 multispecific antibodies was detected by the method provided in Example 7.1. The results are shown in FIGS. 11A-11C, Table 28.

[0780] Table 28 and FIG. 11B show that the agonist activity of CD40 of the FAP / 4-1BB / CD40 multispecific antibodies after cross-linking by FAP is comparable to that of the Ab10-A12V2-2 molecule, and the activation ability of B977703, B977704 and B21326101, B21326102 is comparable, indicating that the CD40 sequence modification has no effect on the CD40 activation activity. Table 28 and FIG. 11C show that the FAP / 4-1BB / CD40 multispecific antibodies all have 4-1BB cross-linking activation activity, which is comparable to the activation activity of BNT312.

[0781] Table 28. FAP / 4-1BB / CD40 multispecific antibody FAP / 4-1BB-dependent CD40 / NF-κB luciferase reporter gene detection results

[0782] 7.3 CD40 / NF-κB luciferase reporter gene detection experiment of FAP / 4-1BB / CD40 multispecific antibodies

[0783] The CD40 agonist activity of the FAP / 4-1BB / CD40 multispecific antibodies was detected by the method provided in Example 7.1. The results are shown in FIGS. 12A-12C, Table 29.

[0784] Table 29 and FIG. 12B show that the agonist activity of CD40 of the FAP / 4-1BB / CD40 multispecific antibodies involved in the experiment after cross-linking by FAP is comparable to that of the RO7300490 and Ab10-A12V2-2 molecule. Table 29 and FIG. 12C show that the FAP / 4-1BB / CD40 multispecific antibodies all have 4-1BB cross-linking activation activity, and B21546901 is slightly weaker than BNT312, while B21326102 is comparable to BNT312. Table 29. FAP / 4-1BB / CD40 multispecific antibody FAP / 4-1BB-dependent CD40 / NF-κB luciferase reporter gene detection results (no cross-linking, FAP cross-linking, 4-1BB cross-linking)

[0785] The above results show that the agonistic activity of the anti-FAP / 4-1BB / CD40 multispecific antibodies CD40 after cross-linking by FAP has no significant difference, and is equivalent to the activation ability of Ab10-A12V2-2 molecule, indicating that the CD40 sequence modification has no effect on the activation activity of CD40; the anti-FAP / 4-1BB / CD40 multispecific antibodies cross-linked by 4-1BB all have CD40 activation activity.

[0786] Example 8. T lymphocyte activation detection of FAP / 4-1BB / CD40 multispecific antibodies

[0787] 8.1 T lymphocyte activation experiment of FAP / 4-1BB / CD40 multispecific antibodies

[0788] The T lymphocyte activation experiment was used to detect the stimulation ability of the antibody to T cells in the presence or absence of FAP. Freshly isolated and purified PBMC was resuspended in RPMI1640 (Gibco, Cat#10491A-01) containing 10% fetal bovine serum, and the density was adjusted to 1×10 6 CHO-K1-HuFAP cells were obtained by stably expressing human FAP protein in CHO-K1 cells, and the cell culture medium was Ham’s F12+GlutaMAXTM-I (Gibco, Cat#31765035) containing 10% fetal bovine serum and 800 μg / mL hygromycin B. After recovery, the cell state was adjusted by subculture, and the density was adjusted to 1.25×10 6 The 96-well plate was coated with 0.25 μg / mL anti-CD3 antibody OKT3 (Invitrogen, Cat#16-0037-85) per well 100 μL, and the residual antibody was washed away with PBS after incubation at 37°C for 2 hours. Subsequently, 100 μL PBMC cells (1×105 per well) were inoculated in the OKT3 coated 96-well plate with 80 μL of culture medium or CHO-K1-HuFAP cells (1×10 5 The cells were removed from the culture plate, centrifuged (400g, 5 minutes) to collect the cell culture supernatant, and the level of IL-2 was detected by using human IL2 detection kit (Cisbio, Cat#62HIL02PEG). For specific operation, refer to the reagent instruction manual, and the results are shown in Figure 13.

[0789] Figure 13 shows that the FAP / 4-1BB / CD40 multispecific antibodies B977707 have some background activation activity in two donors. After FAP crosslinking, the FAP / 4-1BB / CD40 multispecific antibodies can activate IL-2 secretion, and the activation activity of T lymphocytes is significantly improved.

[0790] 8.2 T lymphocyte activation experiment of FAP / 4-1BB / CD40 multispecific antibodies

[0791] The stimulation ability of T cells of the FAP / 4-1BB / CD40 multispecific antibodies was detected by the experimental method provided in Example 8.1. The results are shown in Figure 14.

[0792] Figure 14 shows that the FAP / 4-1BB / CD40 multispecific antibodies B21326102 and the control antibody BNT312 have some background T cell activation activity in two donors. After FAP crosslinking, the FAP / 4-1BB / CD40 multispecific antibodies can better activate T cells to secrete IL-2, and the activation ability of T cells is significantly improved.

[0793] 8.3 T lymphocyte activation experiment of FAP / 4-1BB / CD40 multispecific antibodies

[0794] Freshly isolated and purified PBMCs were resuspended in MACS Buffer (Miltenyi Biotec, Cat# 130-091-221), and CD3 T cells were isolated using EasySep™ Human T Cell Isolation Kit (Stemcell, Cat# 17951) according to the kit instructions. The isolated and purified CD3 T cells were resuspended in RPMI1640 (Gibco, Cat# 10491A-01) containing 10% fetal bovine serum, and the density was adjusted to 1x10 6 CHO-K1-HuFAP cells were obtained by stably expressing human FAP protein in CHO-K1 cells, and the cell culture medium was Ham’s F12+GlutaMAX™-I (Gibco, Cat# 31765035) containing 10% fetal bovine serum and 800 μg / mL hygromycin B. HEK293-HuCD40 cells were obtained by overexpressing human CD40 protein in HEK293 cells, and the cell culture medium was DMEM (Gibco, Cat# 11965092) containing 10% fetal bovine serum and 100 μg / mL hygromycin B. After recovery, the cell state was adjusted by subculture, and the density was adjusted to 1.25x10 6Cells / mL. 96-well plates were coated with 0.25 μg / mL anti-CD3 antibody OKT3 (Invitrogen, Cat#16-0037-85), 100 μL per well, and incubated at 37°C for 2 hours. Residual antibody was washed away with PBS. Subsequently, 100 μL of PBMC cells (1×10⁻⁶) were added to each well. 5 (cells / well) with 80 μL of culture medium, CHO-K1-FAP cells or HEK293-HuCD40 (1×10⁻⁶ cells / well) and 80 μL of CHO-K1-FAP cells or HEK293-HuCD40 (1×10⁻⁶ cells / well) 5 Cells (number of cells / well) were seeded into OKT3-coated 96-well plates. 20 μL of different concentrations of the test sample were added to each well, and the plates were incubated at 37°C for 3 days in a 5% CO2 incubator. The cell culture plates were then removed, and the cell culture supernatant was collected by centrifugation (400g, 5 minutes). IL-2 levels were measured using a human IL-2 assay kit (Cisbio, Cat#62HIL02PEG). Refer to the kit's instructions for specific procedures. Results are shown in Figure 15.

[0795] Figure 15 shows that, in both donors, after FAP cross-linking, the FAP / 4-1BB / CD40 multispecific antibody could activate IL-2 secretion, stronger than MP0310. After CD40 cross-linking, the FAP / 4-1BB / CD40 multispecific antibody could activate IL-2 secretion, stronger than the control antibody BNT312.

[0796] Example 9. Detection of DC maturation promoted by FAP / 4-1BB / CD40 multispecific antibody

[0797] 9.1 FAP / 4-1BB / CD40 multispecific antibody-induced DC maturation assay

[0798] The ability of the antibody to induce DC maturation was verified using a DC maturation-inducing assay in the presence or absence of FAP. Freshly isolated and purified PBMCs were resuspended in MACS Buffer (Miltenyi Biotec, Cat#130-091-221) and processed using EasySep. TM Human Monocyte Isolation Kit (Stemcell, Cat#19359) was used to isolate monocytes. Specific procedures were followed according to the kit instructions. The isolated monocytes were resuspended in RPMI 1640 (Gibco, Cat#10491A-01) containing 10% fetal bovine serum, 50 ng / mL GM-CSF (Peprotech, 200-04), and 50 ng / mL IL-4 (Peprotech, 300-03), and the density was adjusted to 1×10⁻⁶. 6cells were collected by centrifugation at 400 x g for 10 min, resuspended in RPMI1640 (Gibco, Cat#10491A-01) containing 10% fetal bovine serum, and adjusted to a density of 1 x 10 6 cells were collected by centrifugation at 400 x g for 10 min, resuspended in RPMI1640 (Gibco, Cat#10491A-01) containing 10% fetal bovine serum, and adjusted to a density of 1 x 10 6 cells were collected by centrifugation at 400 x g for 10 min, resuspended in RPMI1640 (Gibco, Cat#10491A-01) containing 10% fetal bovine serum, and adjusted to a density of 1 x 10 6 cells were collected by centrifugation at 400 x g for 10 min, resuspended in RPMI1640 (Gibco, Cat#10491A-01) containing 10% fetal bovine serum, and adjusted to a density of 1 x 10 6 cells were collected by centrifugation at 400 x g for 10 min, resuspended in RPMI1640 (Gibco, Cat#10491A-01) containing 10% fetal bovine serum, and adjusted to a density of 1 x 10

[0799] 48 hours incubation, DCs were transferred to new 96-well U-bottom plates. Centrifuged twice at 300xg for 5 minutes, 100 μL FACS Buffer (sterile PBS (Phosphate Buffered Saline, pH 7.40) containing 2% fetal calf serum) containing 0.1 μL live / dead dye APC-efluor780 (BD Pharmingen, Cat# 565388) was added to each well, incubated at room temperature in the dark for 15 minutes, centrifuged twice at 300xg for 5 minutes. 50 μL FACS Buffer (sterile PBS (Phosphate Buffered Saline, pH 7.40) containing 2% fetal calf serum) containing 1 μL Human Trustain FXTM (Biolegend, Cat# 422302) was added to each well, incubated at room temperature in the dark for 10 minutes, 50 μL FACS Buffer (sterile PBS (Phosphate Buffered Saline, pH 7.40) containing 2% fetal calf serum) containing 2.5 μL PE Mouse Anti-Human CD86 (BD Pharmingen, Cat# 555658) was added to each well, incubated at room temperature in the dark for 30 minutes, centrifuged twice with FACS Buffer after incubation, resuspended in FACS Buffer, and read fluorescence signal values by flow cytometry. The results are shown in Figure 16.

[0800] The results show that the FAP / 4-1BB / CD40 multispecific antibodies all have no background activity to promote DC cell maturation. After FAP cross-linking, the FAP / 4-1BB / CD40 multispecific antibodies can all promote DC cell maturation, and the activity is comparable to that of Ab10-A12V2-2 molecule and RO7300490.

[0801] 9.2 FAP / 4-1BB / CD40 multispecific antibody-promoted DC maturation experiment

[0802] The FAP / 4-1BB / CD40 multispecific antibody-promoted DC maturation activity was detected by the experimental method provided in Example 8.1. The results are shown in Figure 17.

[0803] The results show that the FAP / 4-1BB / CD40 multispecific antibodies all have no background activity to promote DC cell maturation. After FAP cross-linking, the FAP / 4-1BB / CD40 multispecific antibodies can all promote DC cell maturation, and the activity is comparable to that of Ab10-A12V2-2 molecule and RO7300490.

[0804] 9.3 FAP / 4-1BB / CD40 multispecific antibody-promoted DC maturation experiment

[0805] The activity of FAP / 4-1BB / CD40 multispecific antibodies in promoting DC maturation was detected by the experimental method provided in Example 8.1. The results are shown in Figure 18.

[0806] The results show that the FAP / 4-1BB / CD40 multispecific antibodies all have no background activity in promoting DC maturation. After FAP cross-linking, the FAP / 4-1BB / CD40 multispecific antibodies all promote DC cell maturation, which is comparable to the Ab10-A12V2-2 molecule and RO7300490. After 4-1BB cross-linking, the FAP / 4-1BB / CD40 multispecific antibodies all promote DC cell maturation, B21326102 is stronger than the control antibody BNT312, and B21546901 is weaker than the control antibody BNT312.

[0807] Example 10. Detection of 4-1BB / CD40 expression cell cross-linking ability of FAP / 4-1BB / CD40 multispecific antibodies

[0808] From the clinical results of BNT312, cross-linking activation of CD40-4-1BB in the periphery can cause severe liver toxicity. Therefore, our tri-specific molecules need to achieve tumor targeting through strong FAP binding ability, while reducing the cross-linking ability between CD40-4-1BB to reduce peripheral activation and reduce the production of liver toxicity. The ability of FAP / 4-1BB / CD40 multispecific antibodies to cross-link 4-1BB / CD40 expression cells was detected by FACS experiment. HEK293-Hu4-1BB cells and HEK293-HuCD40 cells were obtained by overexpressing human 4-1BB and human CD40 proteins in HEK293 cells, respectively, and the cell culture medium was DMEM (Gibco, Cat#11965092) containing 10% fetal bovine serum and 100 μg / mL hygromycin B. The experimental medium was sterile PBS (phosphate buffer, pH 7.40) containing 2% fetal bovine serum. The HEK293-Hu4-1BB and HEK293-HuCD40 cells were collected by centrifugation at 300xg for 5 minutes, washed twice with sterile PBS (phosphate buffer, pH 7.40), and resuspended in sterile PBS (phosphate buffer, pH 7.40) to a density of 2x10 6cells / mL, 5 mM Cell Trace Violet (ThermoFisher, Cat#C345557A) and 5 mM Cell Trace Far red (ThermoFisher, Cat#C34564 A) were added, and incubated at 37 °C for 10 min in the dark. After 10 min, the same volume of RPMI1640 (Gibco, Cat#10491A-01) containing 40% fetal bovine serum was added to terminate the reaction in the dark for 10 min, and then washed twice with sterile PBS, resuspended in FACS Buffer (sterile PBS (Phosphate Buffered Salts, pH 7.40) containing 2% fetal bovine serum), and adjusted to a density of 2 x 10 6 cells / mL, 5 mM Cell Trace Violet (ThermoFisher, Cat#C345557A) and 5 mM Cell Trace Far red (ThermoFisher, Cat#C34564 A) were added, and incubated at 37 °C for 10 min in the dark. After 10 min, the same volume of RPMI1640 (Gibco, Cat#10491A-01) containing 40% fetal bovine serum was added to terminate the reaction in the dark for 10 min, and then washed twice with sterile PBS, resuspended in FACS Buffer (sterile PBS (Phosphate Buffered Salts, pH 7.40) containing 2% fetal bovine serum), and adjusted to a density of 2 x 10

[0809] The results showed that the FAP / 4-1BB / CD40 multispecific antibodies could all crosslink 4-1BB / CD40 expressing cells. In terms of crosslinking ability, the FAP / 4-1BB / CD40 multispecific antibodies were all weaker than the control antibody BNT312.

[0810] Example 11. Mixed lymphocyte culture detection of FAP / 4-1BB / CD40 multispecific antibodies

[0811] The CD40 / 4-1BB crosslinking ability of the triabody and the control molecule BNT312 was detected using mixed lymphocyte culture. The frozen PBMC was resuspended in MACS Buffer (Miltenyi Biotec, Cat#130-091-221), and the CD14+ monocytes were isolated using EasySep TM Human Monocyte Isolation Kit (Stemcell, Cat#19359) to isolate monocytes, according to the instructions of the kit. The isolated monocytes were resuspended in RPMI1640 (Gibco, Cat#10491A-01) containing 10% fetal bovine serum, 50 ng / mL GM-CSF (Peprotech, 200-04) and 50 ng / mL IL4 (Peprotech, 300-03), and adjusted to a density of 1 x 10 6cells were collected by centrifugation at 400 g for 10 min, resuspended in RPMI1640 (Gibco, Cat#10491A-01) containing 10% fetal bovine serum, 1 ng / mL Staphylococcus aureus enterotoxin B (SEB, Toxin Technology, Cat#BT202), and adjusted to a density of 2 x 10 5 6 cells were collected by centrifugation at 400 g for 10 min, resuspended in RPMI1640 (Gibco, Cat#10491A-01) containing 10% fetal bovine serum, 1 ng / mL Staphylococcus aureus enterotoxin B (SEB, Toxin Technology, Cat#BT202), and adjusted to a density of 2 x 10 6 cells were collected by centrifugation at 400 g for 10 min, resuspended in RPMI1640 (Gibco, Cat#10491A-01) containing 10% fetal bovine serum, 1 ng / mL Staphylococcus aureus enterotoxin B (SEB, Toxin Technology, Cat#BT202), and adjusted to a density of 2 x 10 6 cells were collected by centrifugation at 400 g for 10 min, resuspended in RPMI1640 (Gibco, Cat#10491A-01) containing 10% fetal bovine serum, 1 ng / mL Staphylococcus aureus enterotoxin B (SEB, Toxin Technology, Cat#BT202), and adjusted to a density of 2 x 10

[0812] The results show that the IL-2 secreted by the control antibody BNT312 is higher than the FAP / 4-1BB / CD40 multispecific antibodies involved at two concentrations.

[0813] In summary, the FAP / 4-1BB / CD40 multispecific antibodies are characterized as non-FAP dependent molecules in the cross-linking of 4-1BB / CD40 expressing cells and stimulating IL-2 secretion experiments. The results of the FAP / 4-1BB / CD40 multispecific antibodies being weaker than the control antibody BNT312 show that the background activation of the FAP / 4-1BB / CD40 multispecific antibodies in the non-FAP dependent case is low when the 4-1BB and CD40 ends are simultaneously involved. The FAP / 4-1BB / CD40 multispecific antibodies have the ability of FAP-mediated tumor localization while reducing peripheral non-FAP dependent activation, which can reduce toxicity.

[0814] Example 12 In vivo efficacy and toxicity study of anti-FAP / 4-1BB / CD40 multispecific antibodies

[0815] 12. In vivo efficacy of FAP / 4-1BB / CD40 multispecific antibodies in mouse colon cancer model MC38-mFAP

[0816] MC38-mFAP cells (constructed in-house) were inoculated subcutaneously at 2 x 10 5 cells / 100 μL / mouse in B-h4-1BB / CD40 humanized mice (provided by BioSensory) and when the tumors grew to about 110 mm 3 in diameter, 48 mice were selected by tumor volume and randomly divided into 8 groups of 6 mice each, namely: vehicle, B977703 (6 mg / kg), B977703 (2 mg / kg), B977704 (6 mg / kg), B977704 (2 mg / kg), B977707 (2.28 mg / kg), RO7122290 (1.76 mg / kg), and Ab10-A12V2-2 (1.72 mg / kg). Drug administration was twice a week, and tumor volume was measured twice a week during the drug administration and observation period, and the measurement values were recorded. Among them, B977703 (2 mg / kg), B977704 (2 mg / kg), B977707 (2.28 mg / kg), RO7122290 (1.76 mg / kg), and Ab10-A12V2-2 (1.72 mg / kg) were administered at equimolar doses.

[0817] Tumor volume (TV) was calculated using the formula TV = 1 / 2 x a x b2, where a and b represent the long and short diameters of the measured tumor, respectively.

[0818] Relative tumor growth rate T / C% = (T-T0) / (C-C0) x 100%; tumor inhibition rate TGI% = 1-T / C%.

[0819] CR% (proportion of complete tumor regression) = number of mice with complete tumor regression (<110 mm 3 ) / number of mice enrolled.

[0820] The results are shown in Figure 21 and Table 30. The results show that the FAP / 4-1BB / CD40 multispecific antibodies B977703, B977704, and B977707 of different doses all have good antitumor activity, and their antitumor activity is better than that of the control antibodies RO7122290 and Ab10-A12V2-2.

[0821] Table 30. Tumor inhibition effect of different FAP / 4-1BB / CD40 multispecific antibodies on mouse transplanted tumors

[0822] (P value: * p < 0.05, ** P < 0.01, *** P < 0.001)

[0823] 12.2 Hepatotoxicity detection of FAP / 4-1BB / CD40 multispecific antibody in mouse colon cancer model MC38-mFAP

[0824] In the 12.1 experiment, blood was drawn on day 17 to test aspartate aminotransferase (AST) and alanine aminotransferase (ALT), and the results are shown in Figure 22; and body weight was measured twice a week, and the results are shown in Figure 23.

[0825] The results showed that the mice maintained a stable body weight during the administration process, suggesting that the involved FAP / 4-1BB / CD40 multispecific antibodies had no obvious toxic side effects (Figure 23). The ALT / AST test results on day 17 (Figure 22) showed that none of the involved FAP / 4-1BB / CD40 multispecific antibodies showed significant hepatotoxicity.

[0826] Hematologic toxicity assay of 12.3FAP / 4-1BB / CD40 multifunctional antibody in mouse colon cancer model MC38-mFAP

[0827] In the 12.1 trial, blood samples were taken 24 hours after the first administration to perform a complete blood count.

[0828] The results (Figure 24) showed that none of the FAP / 4-1BB / CD40 multispecific antibodies involved caused a decrease in neutrophils (NEUT#) and platelets (PLT), and there was no blood toxicity.

[0829] In vivo efficacy of 12.4FAP / 4-1BB / CD40 multispecific antibody in mouse colon cancer model MC38-mFAP

[0830] MC38-mFAP cells (in-house constructed) were used at a rate of 2 × 10⁻⁶. 5 One 100 μL / mouse was subcutaneously injected into B-h4-1BB / CD40 humanized mice (provided by Biocytogen), and the tumors were allowed to grow to approximately 110 mm. 3The 42 mice were randomly divided into 7 groups according to the tumor volume, 6 mice in each group, namely: solvent, B21326101 (6 mg / kg), B21326101 (2 mg / kg), B21326101 (0.5 mg / kg), Ab10-A12V2-2 (0.43 mg / kg), BNT312 (4.47 mg / kg) and BNT312 (1.49 mg / kg). B21326101 (6 mg / kg), B21326101 (2 mg / kg), BNT312 (4.47 mg / kg) and BNT312 (1.49 mg / kg) were administered once every 3 days for 3 times. Among them, B21326101 (6 mg / kg) and BNT312 (4.47 mg / kg) were administered in equimolar amount, B21326101 (2 mg / kg) and NT312 (1.49 mg / kg) were administered in equimolar amount, and B21326101 (0.5 mg / kg) and Ab10-A12V2-2 (0.43 mg / kg) were administered in equimolar amount. B21326101 (0.5 mg / kg) and Ab10-A12V2-2 (0.43 mg / kg) were administered once every 3 days for 4 times. The tumor volume was measured twice a week during the administration and observation period, and the measurement values were recorded. On the 46th day after administration, the CR mice were subcutaneously inoculated with MC38-mFAP cells (2×10 B-h4-1BB / CD40 humanized mice were subcutaneously inoculated with MC38-mFAP cells (2×10 5 The tumor volume (TV), relative tumor growth rate T / C%, tumor inhibition rate TGI%, and CR% (tumor complete regression rate) were calculated according to 12.1.

[0831] The results are shown in Table 31. The results show that the different doses of FAP / 4-1BB / CD40 multispecific antibody B21326101 involved have good antitumor activity, which is better than the control antibodies BNT312 and Ab10-A12V2-2.

[0832] Table 31. Tumor inhibition effect of different FAP / 4-1BB / CD40 multispecific antibodies on mouse transplanted tumors

[0833] 12.5 FAP / 4-1BB / CD40 multispecific antibody in a mouse colon cancer model MC38-mFAP

[0834] In the 12.4 test, blood was taken on the 8th day to detect AST and ALT.

[0835] The results are shown in Figure 25. The ALT / AST test results on day 8 showed that the FAP / 4-1BB / CD40 multi-functional antibodies involved did not have liver toxicity, and had good safety. BNT312 had obvious liver toxicity at high and low doses.

[0836] Table 32. Amino acid sequences of anti-FAP / 4-1BB / CD40 trispecific antibodies

[0837] Table 33. Amino acid sequences of control antibodies

[0838] The following is for the antibody B21546901 (heavy chain as shown in SEQ ID NO: 49, light chain as shown in SEQ ID NO: 50) to develop a preparation.

[0839] Example 13. Screening of FAP / 4-1BB / CD40 multispecific antibody preparation pH value and buffer system

[0840] The following buffers were prepared, with the addition of corresponding excipients and surfactants, to prepare an antibody preparation of FAP / 4-1BB / CD40 multispecific antibody at a concentration of 40 mg / mL, for stability studies.

[0841] 1) F1 10 mM histidine-acetic acid buffer pH 4.0, with the addition of 8.8% w / v trehalose, 0.4 mg / mL polysorbate 80;

[0842] 2) F2 10 mM histidine-acetic acid buffer pH 4.5, with the addition of 8.8% w / v trehalose, 0.4 mg / mL polysorbate 80;

[0843] 3) F3 10 mM histidine-acetic acid buffer pH 5.0, with the addition of 8% w / v sucrose, 0.4 mg / mL polysorbate 80;

[0844] 4) F4 10 mM histidine-acetic acid buffer pH 5.5, with the addition of 8% w / v sucrose, 0.4 mg / mL polysorbate 80;

[0845] 5) F5 10 mM histidine-acetic acid buffer pH 6.0, with the addition of 8% w / v sucrose, 0.4 mg / mL polysorbate 80.

[0846] The equipment and methods used during the detection process are as follows:

[0847] Appearance: The sample bottle was cleaned by visual method, under the light intensity of 1000-1500 lx, the sample color, clarity and visible impurities were observed on the white background and black background of the clarity detector. Appearance detection instrument: Jingzuo instrument YB-2A clarity detector.

[0848] SEC molecular exclusion chromatography: an analytical method for separating solutes according to the relative relationship between the pore size of the gel and the size of the polymer sample molecules. SEC monomer content percentage = Amonomer / Atotal*100% (Amonomer is the peak area of the main monomer peak in the sample, and Atotal is the sum of all peak areas). SEC determination instrument: Waters Arc HPLC; chromatographic column: Waters, XBrige BEH SEC (300x7.8mm 3.5μm)

[0849] NR-CE capillary gel electrophoresis: a method of electrophoresis in which the gel is moved into a capillary as a support medium and separated under a certain voltage according to the size of the sample molecular weight. Non-reduced CE purity percentage = Apeak / Atotal*100% (Apeak is the peak area of the main peak in the sample, and Atotal is the sum of all peak areas. NR-CE determination instrument: Sciex model PA800 plus

[0850] icIEF imaging capillary isoelectric focusing electrophoresis: a technique for separating proteins according to their isoelectric points pI.

[0851] icIEF main peak content percentage = main peak peak area / total area*100% (total area is the sum of acidic peak, main peak and basic peak areas).

[0852] icIEF acidic peak content percentage = acidic peak peak area / total area*100% (total area is the sum of acidic peak, main peak and basic peak areas).

[0853] icIEF basic peak content percentage = basic peak peak area / total area*100% (total area is the sum of acidic peak, main peak and basic peak areas).

[0854] icIEF determination instrument manufacturer: Protein Simple, model Muarice.

[0855] Particle size: determination of protein particle size based on dynamic light scattering technology. Particle size determination instrument manufacturer: Wyatt, model NanoStarII.

[0856] The experimental results are shown in Tables 34-35.

[0857] Table 34. pH and buffer system screening-pH, protein concentration, particle size T0 detection results

[0858] Table 35. pH and buffer system screening - appearance, SEC stability results

[0859] Both pH and protein concentration results for T0(Table 34) were within the target range.

[0860] Appearance results(Table 35) showed that the color was light yellow for each formulation T0and during the stability process, and there was no significant difference between formulations. After shaking for 3 days, there was no obvious change in opalescence for each formulation compared with T0. After repeated freeze-thawing for 5 times, the opalescence of each formulation was slightly enhanced. After 5W at 40°C, there was no obvious change in opalescence for each formulation compared with T0, and there was no difference between formulations. After 1W, 2W, 5W at 40°C, freeze-thawing for 5 times, and shaking for 3 days, no particles appeared in each formulation.

[0861] SEC(Table 35) results showed that there was no obvious decrease in SEC monomer purity for each formulation after shaking for 3 days and repeated freeze-thawing for 5 times.

[0862] Example 14. Screening of buffer system and excipients for FAP / 4-1BB / CD40 multispecific antibody formulation

[0863] The following buffers were prepared, with the addition of corresponding excipients and surfactants, to prepare FAP / 4-1BB / CD40 multispecific antibody formulations for stability studies. The experimental results are shown in Tables 36-37.

[0864] (1) F6 10 mM histidine-acetic acid buffer pH 5.0, with the addition of 50 mM arginine hydrochloride, 6% w / v sucrose, 0.8 mg / mL polysorbate 80; antibody concentration: 40 mg / mL;

[0865] (2) F7 10 mM histidine-acetic acid buffer pH 5.0, with the addition of 8% w / v sucrose, 0.8 mg / mL polysorbate 80; antibody concentration: 40 mg / mL;

[0866] (3) F9 10 mM citric acid-sodium citrate buffer pH 5.0, with the addition of 8% w / v sucrose, 0.8 mg / mL polysorbate 80; antibody concentration: 40 mg / mL.

[0867] Table 36. Screening of buffer system and excipients - pH, protein concentration T0detection results

[0868] Table 37. Screening of buffer system and excipients - stability results

[0869] The T0 (Table 36) data showed that the pH and protein concentration results of all samples were within the target range.

[0870] The appearance results (Table 37) showed that the opalescence of formulations F6 and F9 (10 mM citrate system, pH 5.0) with arginine hydrochloride added at T0 increased. After 1 W, 3 W, 4 W at 40 °C, 5 freeze-thaw cycles and 3 days of shaking, the appearance of each formulation did not change significantly.

[0871] The SEC (Table 37) results showed that after 3 days of shaking and 5 repeated freeze-thaw cycles, the SEC monomer purity of each formulation did not change significantly. After 1 W at 40 °C, each formulation showed a trend of decrease in SEC monomer content. After 4 W at 40 °C, the SEC monomer purity of F6 (containing arginine hydrochloride excipient) decreased by 15.04%, F9 (10 mM citrate system, pH 5.0) decreased by 21.54%, and F7 formulation decreased the least, by 3.89%.

[0872] Example 15. FAP / 4-1BB / CD40 multispecific antibody formulation prescription confirmation

[0873] The following buffers were prepared, with the addition of corresponding excipients and surfactants, to prepare FAP / 4-1BB / CD40 multispecific antibody formulations for stability studies. The experimental results are shown in Tables 38-39.

[0874] (1) F10 10 mM histidine-acetic acid buffer pH 5.0, 8% w / v sucrose, 0.8 mg / mL polysorbate 80 added; antibody concentration: 40 mg / mL.

[0875] Table 38. Low concentration prescription confirmation - pH, protein concentration T0 detection results

[0876] Table 39. Low concentration prescription confirmation - stability results

[0877] The T0 (Table 38) data showed that the pH and protein concentration results of the samples were within the target range.

[0878] The appearance results (Table 39) showed that after 3 days of shaking, 5 repeated freeze-thaw cycles, and 4 W at 40 °C, the appearance of the formulations did not change significantly.

[0879] The SEC (Table 39) results showed that after 3 days of shaking and 5 repeated freeze-thaw cycles, the SEC monomer purity of the formulations increased slightly. After 1 W at 40 °C, each formulation showed a trend of decrease in SEC monomer content, and after 4 W, the SEC monomer purity decreased by 3.26%, which was within the acceptable quality range.

[0880] NR-CE (Table 39) results showed that the main peak content of the prescription NR-CE did not change significantly after shaking for 3 days and repeated freezing and thawing for 5 times. After being placed at 40°C for 4W, the main peak content of the prescription NR-CE decreased by 3.88%, which was within the quality standard range.

[0881] icIEF (Table 39) results showed that the main peak purity of the prescription icIEF did not change significantly after shaking for 3 days and repeated freezing and thawing for 5 times. After being placed at 40°C for 1W, the main peak of the prescription began to decrease, and after 4W, the main peak purity decreased by 28.75%.

[0882] Example 16. High-concentration formulation protein concentration and pH screening

[0883] The following buffers were prepared, with the addition of corresponding excipients and surfactants, to prepare FAP / 4-1BB / CD40 multispecific antibody formulations for stability studies. The experimental results are shown in Tables 40-41.

[0884] (1) F11 10mM histidine-acetic acid buffer pH 5.0, with the addition of 8% w / v sucrose, 0.8mg / mL polysorbate 80; antibody concentration: 150mg / mL;

[0885] (2) F12 10mM histidine-acetic acid buffer pH 5.0, with the addition of 8% w / v sucrose, 0.8mg / mL polysorbate 80; antibody concentration: 100mg / mL;

[0886] (3) F13 10mM histidine-acetic acid buffer pH 4.5, with the addition of 8% w / v sucrose, 0.8mg / mL polysorbate 80; antibody concentration: 100mg / mL;

[0887] (4) F14 10mM histidine-acetic acid buffer pH 5.5, with the addition of 8% w / v sucrose, 0.8mg / mL polysorbate 80; antibody concentration: 100mg / mL;

[0888] Table 40. High-concentration formulation protein concentration and pH screening - pH, protein concentration T0 detection results

[0889] Table 41. High-concentration formulation protein concentration and pH screening - stability results

[0890] T0 (Table 40) data showed that the pH and protein concentration results of all samples were within the target range.

[0891] Appearance results (Table 41) showed that after shaking for 3 days, repeated freezing and thawing for 5 times, and being placed at 40°C for 4W, the appearance of each prescription did not change significantly.

[0892] SEC (Table 41) results showed that after shaking for 3 days and repeated freeze-thaw for 5 times, the SEC monomer purity of each formulation did not show significant changes. After being placed at 40°C for 1W, each formulation showed a trend of decrease in SEC monomer content. With the increase of concentration, the decrease of monomer content was more obvious. Compared at the same pH and buffer system, the SEC monomer purity of F11 (protein concentration 150 mg / ml) decreased by 22.56% after being placed at 40°C for 4W, and the SEC monomer purity of F12 (protein concentration 100 mg / ml) decreased by 11.36% after being placed at 40°C for 4W, indicating that under the same pH and buffer system, the higher the protein concentration, the more obvious the decrease of SEC monomer content. Under the same protein concentration (100 mg / ml), the SEC monomer purity of F12-14 (pH 4.5-5.5) after being placed at 40°C for 4W decreased by 11.36%, 14.2%, and 12.4%, respectively.

[0893] NR-CE (Table 41) results showed that after shaking for 3 days and repeated freeze-thaw for 5 times, the main peak content of NR-CE of each formulation did not show significant changes. After being placed at 40°C for 4W, the main peak content of NR-CE of each formulation slightly decreased.

[0894] icIEF (Table 41) results showed that after shaking for 3 days and repeated freeze-thaw for 5 times, the main peak of icIEF of each formulation did not show significant changes. After being placed at 40°C for 1W, each formulation showed a trend of obvious decrease in main peak.

[0895] Example 17. First round of screening of FAP / 4-1BB / CD40 multispecific antibody formulation lyophilization concentration and excipients

[0896] The following buffers were prepared, and the corresponding excipients and surfactants were added to prepare 1.7 mL of FAP / 4-1BB / CD40 multispecific antibody formulations for lyophilization.

[0897] After lyophilization, the product of F15 was reconstituted with 1.5 mL of water, and the products of F16-F17 were reconstituted with 0.6 mL of water. The results of the appearance of the lyophilized products, the reconstitution time, and the appearance after reconstitution are shown in Table 42-2. The lyophilization process was pre-freezing, primary drying, and secondary drying.

[0898] Table 42-1

[0899] Table 42-2. Results of reconstitution time and appearance of the first round of screening of lyophilization concentration and excipients

[0900] As shown in the reconstitution time results in Table 42-2, the reconstitution time of F15 and F16 high concentration formulations is relatively long, and the reconstitution time of F17 formulation using dilute freeze-concentrated solution (50 mg / mL concentration freeze-dried, protein concentration after reconstitution is 100 mg / mL) is relatively short. Therefore, it is considered to reduce the protein concentration of the formulation and develop the freeze-dried formulation of the formulation using dilute freeze-concentrated solution.

[0901] Example 18. Second round of screening of FAP / 4-1BB / CD40 multispecific antibody formulation freeze-dried formulation concentration and excipients

[0902] The following buffers were prepared, and the corresponding excipients and surfactants were added to prepare FAP / 4-1BB / CD40 multispecific antibody formulations. The filling volume of F18-F20 was 1.2 mL, and the filling volume of F21 was 5.4 mL. After freeze-drying, 0.5 mL of water was added to reconstitute F18-F20, and 1.5 mL of filtered water was added to reconstitute F21, and stability studies were performed. The experimental results are shown in Tables 44-1 and 44-2.

[0903] Table 43

[0904] Table 44-1. Second round of screening of freeze-dried formulation concentration and excipients reconstitution time and appearance results

[0905] Table 44-2. Summary of stability results of second round of screening of freeze-dried formulation concentration and excipients

[0906] As shown in the results in Tables 44-1 and 44-2, the reconstitution time of F18-F19 (mannitol added formulation group) is slightly shorter than that of F20 (pure sucrose group), indicating that the addition of mannitol reduces the reconstitution time of the product to some extent. After scaling up the westlin bottle specification of formulation F21 compared to F20, the reconstitution time increases slightly. The purity data shows that after 1W at 40°C, the SEC monomer purity of F18-F19 (mannitol added formulation group) decreases by 2.07% and 3.2%, respectively; the SEC monomer purity of F20 (pure sucrose group) decreases by 0.34% after 1W at 40°C. The NR-CE main peak purity of each group does not change significantly before and after freeze-drying and after 1W at high temperature 40°C. The icIEF main peak purity of each formulation does not decrease significantly before and after freeze-drying, and decreases slightly after 1W at high temperature 40°C, and there is no significant difference between the formulations.

[0907] Example 19. Confirmation of FAP / 4-1BB / CD40 multispecific antibody freeze-dried formulation

[0908] The following buffers were prepared, with the addition of the corresponding excipients and surfactants, to prepare 6.10 mL FAP / 4-1BB / CD40 multispecific antibody formulations for stability studies. The experimental results are shown in Tables 46-1 and 46-2.

[0909] Table 45

[0910] Table 46-1. Lyophilized formulation confirmation reconstitution time and appearance after reconstitution results

[0911] Table 46-2. Lyophilized formulation concentration and excipient second round of screening stability results summary

[0912] As shown in the results of Table 46-1, the appearance of the lyophilizate, the reconstitution time, and the appearance after reconstitution results were all good, in line with expectations. As shown in the results of Table 46-2, the lyophilizate was stable after being placed at 40°C for 4W, and the SEC monomer purity, NR-CE main peak purity, and icIEF main peak purity all did not change significantly.

[0913] Example 20 FAP / 4-1BB / CD40 multispecific antibody formulation

[0914] This example provides the following formulations:

[0915] (1) 40 mg / mL FAP / 4-1BB / CD40, 10 mM histidine-succinate buffer pH 5.0, with the addition of 3% w / v sucrose, 0.3 mg / mL polysorbate 80;

[0916] (2) 30 mg / mL FAP / 4-1BB / CD40, 10 mM histidine-succinate buffer pH 5.0, with the addition of 3% w / v sucrose, 0.3 mg / mL polysorbate 80;

[0917] (3) 50 mg / mL FAP / 4-1BB / CD40, 10 mM histidine-succinate buffer pH 5.0, with the addition of 3% w / v sucrose, 0.3 mg / mL polysorbate 80;

[0918] (4) 40 mg / mL FAP / 4-1BB / CD40, 8 mM histidine-succinate buffer pH 5.0, with the addition of 3% w / v sucrose, 0.3 mg / mL polysorbate 80;

[0919] (5) 40 mg / mL FAP / 4-1BB / CD40, 12 mM histidine-succinate buffer pH 5.0, with the addition of 3% w / v sucrose, 0.3 mg / mL polysorbate 80;

[0920] (6) 40 mg / mL FAP / 4-1BB / CD40, 10 mM Histidine-Succinate buffer pH 4.5, 3% w / v sucrose, 0.3 mg / mL polysorbate 80 added;

[0921] (7) 40 mg / mL FAP / 4-1BB / CD40, 10 mM Histidine-Succinate buffer pH 5.5, 3% w / v sucrose, 0.3 mg / mL polysorbate 80 added;

[0922] (8) 40 mg / mL FAP / 4-1BB / CD40, 10 mM Histidine-Succinate buffer pH 5.0, 2% w / v sucrose, 0.3 mg / mL polysorbate 80 added;

[0923] (9) 40 mg / mL FAP / 4-1BB / CD40, 10 mM Histidine-Succinate buffer pH 5.0, 4% w / v sucrose, 0.3 mg / mL polysorbate 80 added;

[0924] (10) 40 mg / mL FAP / 4-1BB / CD40, 10 mM Histidine-Succinate buffer pH 5.0, 3% w / v sucrose, 0.2 mg / mL polysorbate 80 added;

[0925] (11) 40 mg / mL FAP / 4-1BB / CD40, 10 mM Histidine-Succinate buffer pH 5.0, 3% w / v sucrose, 0.4 mg / mL polysorbate 80 added.

[0926] The above samples were reconstituted after lyophilization to obtain the following formulations:

[0927] (1) 100 mg / mL FAP / 4-1BB / CD40, 25 mM Histidine-Succinate buffer pH 5.0, 7.5% w / v sucrose, 0.75 mg / mL polysorbate 80 added;

[0928] (2) 75 mg / mL FAP / 4-1BB / CD40, 25 mM Histidine-Succinate buffer pH 5.0, 7.5% w / v sucrose, 0.75 mg / mL polysorbate 80 added;

[0929] (3) 125 mg / mL FAP / 4-1BB / CD40, 25 mM Histidine-Succinate buffer pH 5.0, 7.5% w / v sucrose, 0.75 mg / mL polysorbate 80 added;

[0930] (4) 100 mg / mL FAP / 4-1BB / CD40, 20 mM Histidine-Succinate Buffer pH 5.0, with 7.5% w / v sucrose, 0.75 mg / mL polysorbate 80;

[0931] (5) 100 mg / mL FAP / 4-1BB / CD40, 30 mM Histidine-Succinate Buffer pH 5.0, with 7.5% w / v sucrose, 0.75 mg / mL polysorbate 80;

[0932] (6) 100 mg / mL FAP / 4-1BB / CD40, 25 mM Histidine-Succinate Buffer pH 4.5, with 7.5% w / v sucrose, 0.75 mg / mL polysorbate 80;

[0933] (7) 100 mg / mL FAP / 4-1BB / CD40, 25 mM Histidine-Succinate Buffer pH 5.5, with 7.5% w / v sucrose, 0.75 mg / mL polysorbate 80;

[0934] (8) 100 mg / mL FAP / 4-1BB / CD40, 25 mM Histidine-Succinate Buffer pH 5.0, with 5% w / v sucrose, 0.75 mg / mL polysorbate 80;

[0935] (9) 100 mg / mL FAP / 4-1BB / CD40, 25 mM Histidine-Succinate Buffer pH 5.0, with 10% w / v sucrose, 0.75 mg / mL polysorbate 80;

[0936] (10) 100 mg / mL FAP / 4-1BB / CD40, 25 mM Histidine-Succinate Buffer pH 5.0, with 7.5% w / v sucrose, 0.5 mg / mL polysorbate 80;

[0937] (11) 100 mg / mL FAP / 4-1BB / CD40, 25 mM Histidine-Succinate Buffer pH 5.0, with 7.5% w / v sucrose, 1.0 mg / mL polysorbate 80.

Claims

1. A pharmaceutical composition comprising a FAP / 4-1BB / CD40 binding molecule, and a buffer; wherein the FAP / 4-1BB / CD40 binding molecule comprises a first antigen binding domain that specifically binds FAP, a second antigen binding domain that specifically binds CD40, and a third antigen binding domain that specifically binds 4-1BB; wherein the third antigen binding domain comprises at least one immunoglobulin single variable domain; the immunoglobulin single variable domain in the third antigen binding domain comprises CDR1, CDR2 and CDR3 in an amino acid sequence as set forth in any one of SEQ ID NOs: 28, 18, 23-26 and 33, as defined according to the Kabat, IMGT, Chothia, AbM or Contact numbering system; wherein the buffer is selected from one or more of histidine salt buffer, acetate buffer, citrate buffer and succinate buffer; more preferably one or more of histidine-acetate, citrate-sodium citrate and histidine-succinate.

2. The pharmaceutical composition according to claim 1, wherein, the immunoglobulin single variable domain in the third antigen binding domain comprises CDR1, CDR2 and CDR3 as set forth in SEQ ID NOs: 19, 30 and 31, respectively; Preferably, the immunoglobulin single variable domain in the third antigen binding domain comprises CDR1, CDR2 and CDR3 as set forth in any one of the following: a-1) CDR1, CDR2 and CDR3 as set forth in SEQ ID NOs: 19, 29 and 27, respectively; a-2) CDR1, CDR2 and CDR3 as set forth in SEQ ID NOs: 19, 20 and 27, respectively; or a-3) CDR1, CDR2 and CDR3 as set forth in SEQ ID NOs: 19, 20 and 21, respectively; Preferably, the immunoglobulin single variable domain in the third antigen binding domain comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 28, 18, 23-26 and 33, or an amino acid sequence having at least 90% sequence identity thereto; Preferably, the immunoglobulin single variable domain in the third antigen binding domain is humanized, affinity matured, T-cell epitope depleted, deamidation-reduced, aggregation-reduced, isomerization-reduced, pre-existing anti-drug antibody (pre-ADA) binding-reduced, and / or anti-drug antibody (ADA) binding-reduced engineered.

3. The pharmaceutical composition according to any one of claims 1-2, wherein, the second antigen binding domain comprises at least one immunoglobulin single variable domain, the immunoglobulin single variable domain in the second antigen binding domain comprises CDR1, CDR2 and CDR3 in an amino acid sequence as set forth in SEQ ID NO: 32, 11 or 15, as defined according to the Kabat, IMGT, Chothia, AbM or Contact numbering system; Preferably, the immunoglobulin single variable domain in the second antigen binding domain comprises CDR1, CDR2 and CDR3 as set forth in SEQ ID NOs: 12, 13 and 14, respectively; more preferably, the immunoglobulin single variable domain in the second antigen binding domain comprises an amino acid sequence as set forth in SEQ ID NO: 32, 11 or 15, or an amino acid sequence having at least 90% sequence identity thereto; Preferably, the immunoglobulin single variable domain in the second antigen binding domain is humanized, affinity matured, T-cell epitope removed, antibody deamidation reduced, antibody aggregation reduced, antibody isomerization reduced, binding to pre-existing anti-drug antibodies (pre-ADAs) reduced, and / or binding to anti-drug antibodies (ADAs) reduced engineered; more preferably, the humanized engineered heavy chain framework region module of the human germline gene is derived from IGHV3-48*03.

4. The pharmaceutical composition according to any one of claims 1-3, wherein, The first antigen binding domain comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein: The VH comprises HCDR1, HCDR2 and HCDR3 as set forth in SEQ ID NOs: 3, 4 and 5, respectively; and the VL comprises LCDR1, LCDR2 and LCDR3 as set forth in SEQ ID NOs: 6, 7 and 8, respectively; Preferably, the heavy chain variable region comprises an amino acid sequence as set forth in SEQ ID NO: 1, or an amino acid sequence having at least 90% sequence identity thereto; and, the light chain variable region comprises an amino acid sequence as set forth in SEQ ID NO: 2, or an amino acid sequence having at least 90% sequence identity thereto; Preferably, the first antigen binding domain is selected from a Fab, a Fab’, a Fv or a ScFv, more preferably a Fab; More preferably, the first antigen binding domain comprises a Fab heavy chain and a Fab light chain, wherein: The Fab heavy chain comprises an amino acid sequence as set forth in SEQ ID NO: 66, or an amino acid sequence having at least 90% sequence identity thereto; and, the Fab light chain comprises an amino acid sequence as set forth in SEQ ID NO: 67, or an amino acid sequence having at least 90% sequence identity thereto.

5. The pharmaceutical composition of any one of claims 1-4, the FAP / 4-1BB / CD40 binding molecule further comprises a Fc region and / or a linker; Preferably, the Fc region is derived from a Fc region of human IgG1, IgG2 or IgG4; more preferably, the Fc region of human IgG1 has at least one of the following mutations: 234A, 235A, 220A, 297A or 297Q, 267E, 328F; more preferably, the Fc region of human IgG4 has at least one of the following mutations: 228P, 234A, 235A, 447A; the above mutations are numbered according to the EU numbering rule; Preferably, the linker is located between any two of the first, second and third antigen binding domains; preferably the linker is (G x S) y linker, wherein, x is selected from an integer from 1-5, and y is selected from an integer from 1-6; more preferably, x is 4, and y is 1, 2 or 3.

6. The pharmaceutical composition according to any one of claims 1-5, wherein, The valence ratio of the first, second and third antigen binding domains is (1-2):(1-5):(1-5); preferably 1:2:2, 1:2:4, 1:1:1 or 1:1:

2.

7. The pharmaceutical composition according to any one of claims 1-6, wherein, The FAP / 4-1BB / CD40 binding molecule comprises a first polypeptide chain and a second polypeptide chain as shown in any one of the following: The first polypeptide chain and the second polypeptide chain respectively comprise the amino acid sequences as shown in SEQ ID NOs: 49 and 50; The first polypeptide chain and the second polypeptide chain respectively comprise the amino acid sequences as shown in SEQ ID NOs: 34 and 35; The first polypeptide chain and the second polypeptide chain respectively comprise the amino acid sequences as shown in SEQ ID NOs: 36 and 37; The first polypeptide chain and the second polypeptide chain respectively comprise the amino acid sequences as shown in SEQ ID NOs: 38 and 39; The first polypeptide chain and the second polypeptide chain respectively comprise the amino acid sequences as shown in SEQ ID NOs: 40 and 41; The first polypeptide chain and the second polypeptide chain respectively comprise the amino acid sequences as shown in SEQ ID NOs: 42 and 43; The first polypeptide chain and the second polypeptide chain respectively comprise the amino acid sequences as shown in SEQ ID NOs: 45 and 46; The first polypeptide chain and the second polypeptide chain respectively comprise the amino acid sequences as shown in SEQ ID NOs: 47 and 48; or, combinations of amino acid sequences having at least 90% sequence identity to the first polypeptide chain and the second polypeptide chain of any one of the preceding groups, respectively.

8. A pharmaceutical composition comprising a FAP / 4-1BB / CD40 binding molecule, and a buffer; wherein, The FAP / 4-1BB / CD40 binding molecule comprises a first antigen binding domain specifically binding to FAP, a second antigen binding domain specifically binding to CD40, and a third antigen binding domain specifically binding to 4-1BB; The buffer is selected from one or more of histidine salt buffer, acetate buffer, citrate buffer and succinate buffer; more preferably one or more of histidine-acetate, citrate-sodium citrate and histidine-succinate.

9. The pharmaceutical composition of any one of claims 1-8, wherein the buffer or the pharmaceutical composition has a pH of about 3.0 to about 7.0, preferably about 4.0 to about 6.0, more preferably about 4.5 to about 5.

5.

10. The pharmaceutical composition of any one of claims 1-9, wherein the buffer has a concentration of about 1 mM to about 100 mM, preferably about 2 mM to about 50 mM, more preferably about 5 mM to about 30 mM.

11. The pharmaceutical composition of any one of claims 1-10, wherein the FAP / 4-1BB / CD40 binding molecule has a concentration of about 0.01 mg / mL to about 200 mg / mL, preferably about 10 mg / mL to about 180 mg / mL, more preferably about 20 mg / mL to about 160 mg / mL.

12. The pharmaceutical composition of any one of claims 1-11, further comprising a surfactant. Preferably, the surfactant is polysorbate, preferably polysorbate 20 or polysorbate 80, more preferably polysorbate 80. Preferably, the concentration of the surfactant is about 0.01 to about 10.0 mg / mL, preferably about 0.1 to about 5.0 mg / mL, more preferably about 0.2 to about 2.0 mg / mL.

13. The pharmaceutical composition of any one of claims 1-12, further comprising a sugar or alcohol selected from the group consisting of one or more of sucrose, trehalose, mannitol, sorbitol, preferably sucrose or trehalose, more preferably sucrose. Optionally, the pharmaceutical composition further comprises an amino acid or a salt thereof, preferably arginine or a salt thereof, more preferably arginine hydrochloride.

14. The pharmaceutical composition of claim 13, wherein the concentration of the sugar or alcohol is about 0.1% (w / v) to about 20% (w / v), preferably about 0.2% (w / v) to about 15% (w / v), more preferably about 0.5% (w / v) to about 12% (w / v). The concentration of the arginine or a salt thereof is about 5 mM to about 200 mM, preferably about 10 mM to about 160 mM, more preferably about 20 mM to about 120 mM.

15. The pharmaceutical composition of any one of claims 1-14, comprising: (1) about 0.01 mg / mL to about 200 mg / mL of a FAP / 4-1BB / CD40 binding molecule, about 1 mM to about 100 mM of a buffer, about 0.01 to about 10.0 mg / mL of a surfactant, about 0.1% (w / v) to about 20% (w / v) of a sugar or alcohol, and, optionally, about 5 mM to about 200 mM of an amino acid or a salt thereof, the pH of the pharmaceutical composition is about 3.0 to about 7.0; or, (2) about 0.01 mg / mL to about 200 mg / mL of a FAP / 4-1BB / CD40 binding molecule, about 1 mM to about 100 mM of a histidine salt buffer, acetate buffer, citrate buffer, or succinate buffer, about 0.01 to about 10.0 mg / mL of polysorbate, about 0.1% (w / v) to about 20% (w / v) of sucrose, trehalose, mannitol, or sorbitol, and, optionally, 5 mM to about 200 mM of arginine or a salt thereof, the pH of the pharmaceutical composition is about 3.0 to about 7.0; or, (3) about 0.01 mg / mL to about 200 mg / mL of a FAP / 4-1BB / CD40 binding molecule, about 1 mM to about 100 mM of histidine-acetate, citrate-sodium citrate, or histidine-succinate, about 0.01 to about 10.0 mg / mL of polysorbate 20 or polysorbate 80, about 0.1% (w / v) to about 20% (w / v) of sucrose or trehalose, and, optionally, about 5 mM to about 200 mM of arginine hydrochloride, the pH of the pharmaceutical composition is about 3.0 to about 7.

0. about 3.0 to about 7.0; or, (4) about 0.01 mg / mL to about 200 mg / mL of the FAP / 4-1BB / CD40 binding molecule, about 1 mM to about 100 mM histidine-acetate, about 0.01 to about 10.0 mg / mL polysorbate 80, about 0.1% (w / v) to about 20% (w / v) sucrose, and, optionally about 5 mM to about 200 mM arginine hydrochloride, about 3.0 to about 7.0; or, (5) about 10 mg / mL to about 180 mg / mL of the FAP / 4-1BB / CD40 binding molecule, about 2 mM to about 50 mM histidine-acetate, about 0.1 to about 5.0 mg / mL polysorbate 80, about 0.2% (w / v) to about 15% (w / v) sucrose, and, optionally about 10 mM to about 160 mM arginine hydrochloride, about 4.0 to about 6.0; or, (6) about 20 mg / mL to about 160 mg / mL of the FAP / 4-1BB / CD40 binding molecule, about 5 mM to about 30 mM histidine-acetate, about 0.2 to about 2.0 mg / mL polysorbate 80, about 0.5% (w / v) to about 12% (w / v) sucrose, and, optionally about 20 mM to about 120 mM arginine hydrochloride, about 4.5 to about 5.5; (7) about 10 mg / mL to about 200 mg / mL of the FAP / 4-1BB / CD40 binding molecule, about 5 mM to about 50 mM histidine-succinate buffer, about 1% w / v to about 10% w / v sucrose, and about 0.1 mg / mL to about 1.0 mg / mL polysorbate 80; about 4.0 to about 6.0; (8) about 10 mg / mL to about 200 mg / mL of the FAP / 4-1BB / CD40 binding molecule, about 10 mM to about 25 mM histidine-succinate buffer, about 3% w / v to about 7.5% w / v sucrose, and about 0.3 mg / mL to about 0.75 mg / mL polysorbate 80; about 4.5 to about 5.

5.

16. A pharmaceutical composition comprising any one of: (a-1) about 10 mg / mL to about 200 mg / mL of the FAP / 4-1BB / CD40 binding molecule, about 10 mM histidine-acetate buffer, about 8.8% w / v trehalose, and, about 0.4 mg / mL polysorbate 80, about 3 to about 7; (a-2) about 10 mg / mL to about 200 mg / mL of the FAP / 4-1BB / CD40 binding molecule, about 10 mM histidine-acetate buffer, about 8.8% w / v trehalose, and, about 0.4 mg / mL polysorbate 80, about 4 to about 6; (a-3) about 10 mg / mL to about 200 mg / mL of the FAP / 4-1BB / CD40 binding molecule, about 10 mM histidine-succinate buffer, about 8.8% w / v trehalose, and, about 0.4 mg / mL polysorbate 80, about 4.5 to about 5.

5. (a-3) about 10 mg / mL to about 200 mg / mL of the FAP / 4-1BB / CD40 binding molecule, about 10 mM histidine-acetate buffer, about 8% w / v sucrose, and about 0.4 mg / mL polysorbate 80, the pH of the pharmaceutical composition is about 4.5 to about 5.5; (a-4) about 10 mg / mL to about 200 mg / mL of the FAP / 4-1BB / CD40 binding molecule, about 10 mM histidine-acetate buffer, about 8% w / v sucrose, and about 0.4 mg / mL polysorbate 80, the pH of the pharmaceutical composition is about 4 to about 6; (a-5) about 10 mg / mL to about 200 mg / mL of the FAP / 4-1BB / CD40 binding molecule, about 10 mM histidine-acetate buffer, about 8% w / v sucrose, and about 0.4 mg / mL polysorbate 80, the pH of the pharmaceutical composition is about 4 to about 7; (a-6) about 10 mg / mL to about 200 mg / mL of the FAP / 4-1BB / CD40 binding molecule, about 10 mM histidine-acetate buffer, about 50 mM arginine hydrochloride, about 6% w / v sucrose, and about 0.8 mg / mL polysorbate 80, the pH of the pharmaceutical composition is about 4 to about 6; (a-7) about 10 mg / mL to about 200 mg / mL of the FAP / 4-1BB / CD40 binding molecule, about 10 mM histidine-acetate buffer, about 8% w / v sucrose, and about 0.8 mg / mL polysorbate 80, the pH of the pharmaceutical composition is about 4 to about 6; (a-8) about 10 mg / mL to about 200 mg / mL of the FAP / 4-1BB / CD40 binding molecule, about 10 mM citrate-sodium citrate buffer, about 8% w / v sucrose, and about 0.8 mg / mL polysorbate 80, the pH of the pharmaceutical composition is about 4 to about 6; (b-1) about 10 mg / mL to about 200 mg / mL of the FAP / 4-1BB / CD40 binding molecule, about 10 mM histidine-succinate buffer, about 8% w / v sucrose, and about 0.8 mg / mL polysorbate 80; the pH of the pharmaceutical composition is about 4.0 to about 6.0; (b-2) about 10 mg / mL to about 200 mg / mL of the FAP / 4-1BB / CD40 binding molecule, about 10 mM histidine-succinate buffer, about 4% w / v sucrose, and about 0.4 mg / mL polysorbate 80; the pH of the pharmaceutical composition is about 4.0 to about 6.0; (b-3) about 10 mg / mL to about 200 mg / mL of the FAP / 4-1BB / CD40 binding molecule, about 10 mM histidine-succinate buffer, about 1% w / v sucrose, 2% w / v mannitol, and about 0.4 mg / mL polysorbate 80; the pH of the pharmaceutical composition is about 4.0 to about 6.0; (b-4) about 10 mg / mL to about 200 mg / mL of the FAP / 4-1BB / CD40 binding molecule, about 10 mM histidine-succinate buffer, about 0.5% w / v sucrose, about 2.5% w / v mannitol, and about 0.4 mg / mL polysorbate 80; the pH of the pharmaceutical composition is about 4.0 to about 6.0; (b-5) about 10 mg / mL to about 200 mg / mL of the FAP / 4-1BB / CD40 binding molecule, about 10 mM histidine-succinate buffer, about 3% w / v sucrose, about 0.3 mg / mL polysorbate 80, the pH of the pharmaceutical composition is about 4.0 to about 6.0; (c-1) about 10 mg / mL to about 200 mg / mL of the FAP / 4-1BB / CD40 binding molecule, about 10 mM histidine-succinate buffer, about 8% w / v sucrose, about 0.8 mg / mL polysorbate 80; the pH of the pharmaceutical composition is about 4.0 to about 6.0; (c-2) about 10 mg / mL to about 200 mg / mL of the FAP / 4-1BB / CD40 binding molecule, about 20 mM histidine-succinate buffer, about 8% w / v sucrose, about 0.8 mg / mL polysorbate 80; the pH of the pharmaceutical composition is about 4.0 to about 6.0; (c-3) about 10 mg / mL to about 200 mg / mL of the FAP / 4-1BB / CD40 binding molecule, about 20 mM histidine-succinate buffer, about 8% w / v sucrose, about 0.8 mg / mL polysorbate 80; the pH of the pharmaceutical composition is about 4.0 to about 6.0; (c-4) about 10 mg / mL to about 200 mg / mL of the FAP / 4-1BB / CD40 binding molecule, about 20 mM histidine-succinate buffer, about 2% w / v sucrose, about 4% w / v mannitol, about 0.8 mg / mL polysorbate 80; the pH of the pharmaceutical composition is about 4.0 to about 6.0; (c-5) about 10 mg / mL to about 200 mg / mL of the FAP / 4-1BB / CD40 binding molecule, about 20 mM histidine-succinate buffer, about 1.0% w / v sucrose, about 5% w / v mannitol, about 0.8 mg / mL polysorbate 80; the pH of the pharmaceutical composition is about 4.0 to about 6.0; (c-6) about 10 mg / mL to about 200 mg / mL of the FAP / 4-1BB / CD40 binding molecule, about 20 mM histidine-succinate buffer, about 8% w / v sucrose, about 0.8 mg / mL polysorbate 80; the pH of the pharmaceutical composition is about 4.0 to about 6.0; (c-7) about 10 mg / mL to about 200 mg / mL of the FAP / 4-1BB / CD40 binding molecule, about 30 mM histidine-succinate buffer, about 9% w / v sucrose, about 0.9 mg / mL polysorbate 80; the pH of the pharmaceutical composition is about 4.0 to about 6.0; (c-8) FAP / 4-1BB / CD40 binding molecules at approximately 10 mg / mL to approximately 200 mg / mL Approximately 25 mM histidine-succinate buffer. Approximately 7.5% w / v sucrose, Approximately 0.75 mg / mL of polysorbate 80; The pH of the pharmaceutical composition is from about 4.0 to about 6.0; Preferably, the FAP / 4-1BB / CD40 binding molecule is as defined in any one of claims 1-7.

17. A lyophilized formulation, wherein the lyophilized formulation is obtained by freeze-drying the pharmaceutical composition according to any one of claims 1 to 16, or the lyophilized formulation can be reconstituted to form the pharmaceutical composition according to any one of claims 1 to 16.

18. A lyophilized formulation obtained by freeze-drying a liquid formulation comprising a pharmaceutical composition; wherein, The liquid formulation comprises: (1) FAP / 4-1BB / CD40 binding molecules at concentrations of approximately 0.01 mg / mL to approximately 200 mg / mL. Buffers ranging from approximately 1 mM to approximately 100 mM Surfactants ranging from approximately 0.01 to approximately 10.0 mg / mL Sugars or alcohols ranging from about 0.1% (w / v) to about 20% (w / v), and, Choose any amino acid or its salt that is present at a concentration of approximately 5 mM to approximately 200 mM. The pH of the pharmaceutical composition is from about 3.0 to about 7.0; or, (2) FAP / 4-1BB / CD40 binding molecules at concentrations of approximately 0.01 mg / mL to approximately 200 mg / mL. Histidine, citrate, or succinate buffers ranging from approximately 1 mM to approximately 100 mM. Polysorbate at concentrations of approximately 0.01 to approximately 10.0 mg / mL From about 0.1% (w / v) to about 20% (w / v) of sucrose, trehalose, mannitol or sorbitol, and, Arginine or its salt, which may be present at a concentration of 5 mM to approximately 200 mM, may be used. The pH of the pharmaceutical composition is from about 3.0 to about 7.0; or, (3) FAP / 4-1BB / CD40 binding molecules at concentrations of approximately 0.01 mg / mL to approximately 200 mg / mL. Citric acid-sodium citrate or histidine-succinic acid, approximately 1 mM to approximately 100 mM. Polysorbate 20 or polysorbate 80, at concentrations of approximately 0.01 to approximately 10.0 mg / mL. Approximately 0.1% (w / v) to approximately 20% (w / v) of sucrose or trehalose, and, Arginine hydrochloride, which is optionally present at a concentration of approximately 5 mM to approximately 200 mM, The pH of the pharmaceutical composition is from about 3.0 to about 7.0; or, (4) FAP / 4-1BB / CD40 binding molecules at concentrations of approximately 10 mg / mL to approximately 180 mg / mL Histidine-acetic acid at approximately 2 mM to approximately 50 mM Polysorbate 80 at concentrations of approximately 0.1 to approximately 5.0 mg / mL Sucrose from about 0.2% (w / v) to about 15% (w / v), and, Arginine hydrochloride, optionally present at a concentration of approximately 10 mM to approximately 160 mM, The pH of the pharmaceutical composition is from about 4.0 to about 6.0; or, (5) FAP / 4-1BB / CD40 binding molecules at concentrations of approximately 20 mg / mL to approximately 160 mg / mL Histidine-acetic acid at approximately 5 mM to approximately 30 mM about 0.2 to about 2.0 mg / mL polysorbate 80, about 0.5% (w / v) to about 12% (w / v) sucrose, and optionally about 20 mM to about 120 mM arginine hydrochloride, the pH of the pharmaceutical composition is about 4.5 to about 5.5; or (6) about 10 mg / mL to about 200 mg / mL of the FAP / 4-1BB / CD40 binding molecule. about 5 mM to about 50 mM histidine-succinate buffer, about 1% w / v to about 10% w / v sucrose, and about 0.1 mg / mL to about 1.0 mg / mL polysorbate 80; the pH of the pharmaceutical composition is about 4.0 to about 6.0; or (7) about 10 mg / mL to about 200 mg / mL of the FAP / 4-1BB / CD40 binding molecule. about 10 mM to about 25 mM histidine-succinate buffer, about 3% w / v to about 7.5% w / v sucrose, and about 0.3 mg / mL to about 0.75 mg / mL polysorbate 80; the pH of the pharmaceutical composition is about 4.5 to about 5.5; preferably comprising any one of: (b-1) about 10 mg / mL to about 200 mg / mL of the FAP / 4-1BB / CD40 binding molecule, about 10 mM histidine-succinate buffer, about 8% w / v sucrose, and about 0.8 mg / mL polysorbate 80; the pH of the pharmaceutical composition is about 4.0 to about 6.0; (b-2) about 10 mg / mL to about 200 mg / mL of the FAP / 4-1BB / CD40 binding molecule, about 10 mM histidine-succinate buffer, about 4% w / v sucrose, and about 0.4 mg / mL polysorbate 80; the pH of the pharmaceutical composition is about 4.0 to about 6.0; (b-3) about 10 mg / mL to about 200 mg / mL of the FAP / 4-1BB / CD40 binding molecule, about 10 mM histidine-succinate buffer, about 1% w / v sucrose, 2% w / v mannitol, and about 0.4 mg / mL polysorbate 80; the pH of the pharmaceutical composition is about 4.0 to about 6.0; (b-4) about 10 mg / mL to about 200 mg / mL of the FAP / 4-1BB / CD40 binding molecule, about 10 mM histidine-succinate buffer, about 0.5% w / v sucrose, about 2.5% w / v mannitol, and about 0.4 mg / mL polysorbate 80; the pH of the pharmaceutical composition is about 4.0 to about 6.0; (b-5) about 10 mg / mL to about 200 mg / mL of the FAP / 4-1BB / CD40 binding molecule, about 10 mM histidine-succinate buffer, about 3% w / v sucrose, about 0.3 mg / mL polysorbate 80, the pH of the pharmaceutical composition is about 4.0 to about 6.0; Preferably, the FAP / 4-1BB / CD40 binding molecule comprises a first antigen binding domain that specifically binds FAP, a second antigen binding domain that specifically binds CD40, and a third antigen binding domain that specifically binds 4-1BB; wherein the third antigen binding domain comprises at least one immunoglobulin single variable domain; the immunoglobulin single variable domain in the third antigen binding domain comprises CDR1, CDR2 and CDR3 as set forth in any one of SEQ ID NOs: 28, 18, 23-26 and 33, which CDRs are defined according to the Kabat, IMGT, Chothia, AbM or Contact numbering system; Preferably, the immunoglobulin single variable domain in the third antigen binding domain comprises CDR1, CDR2 and CDR3 as set forth in SEQ ID NOs: 19, 30 and 31, respectively; More preferably, the immunoglobulin single variable domain in the third antigen binding domain comprises CDR1, CDR2 and CDR3 as set forth in any one of: a-1) CDR1, CDR2 and CDR3 as set forth in SEQ ID NOs: 19, 29 and 27, respectively; a-2) CDR1, CDR2 and CDR3 as set forth in SEQ ID NOs: 19, 20 and 27, respectively; or a-3) CDR1, CDR2 and CDR3 as set forth in SEQ ID NOs: 19, 20 and 21, respectively.

19. A reconstituted solution, which is prepared by reconstituting the lyophilized formulation of claim 17 or 18; Preferably, the concentration of the FAP / 4-1BB / CD40 binding molecule in the reconstituted solution is greater than the concentration of the FAP / 4-1BB / CD40 binding molecule in the stock solution from which the lyophilized formulation is prepared; Preferably, the concentration ratio of the FAP / 4-1BB / CD40 binding molecule in the reconstituted solution to the FAP / 4-1BB / CD40 binding molecule in the stock solution is (1-5): 1, more preferably 2.5: 1 or 2:

1.

20. The reconstituted solution of claim 19, which is prepared by reconstituting the lyophilized formulation of claim 17 or 18 with a solvent; preferably, the solvent is water, physiological saline or dextrose.

21. The reconstituted solution of claim 19 or 20, which comprises: (1) about 0.01 mg / mL to about 200 mg / mL of the FAP / 4-1BB / CD40 binding molecule, about 1 mM to about 100 mM of a buffering agent, about 0.01 to about 10.0 mg / mL of a surfactant, about 0.1% (w / v) to about 20% (w / v) of a saccharide or an alcohol, and, optionally, about 5 mM to about 200 mM of an amino acid or a salt thereof, the pH of the pharmaceutical composition is about 3.0 to about 7.0; or, (2) about 0.01 mg / mL to about 200 mg / mL of the FAP / 4-1BB / CD40 binding molecule, about 1 mM to about 100 mM histidine, citrate, or succinate, about 0.01 to about 10.0 mg / mL polysorbate, about 0.1% (w / v) to about 20% (w / v) sucrose, trehalose, mannitol, or sorbitol, and, optionally about 5 mM to about 200 mM arginine or a salt thereof, the pH of the pharmaceutical composition is about 3.0 to about 7.0; or, (3) about 0.01 mg / mL to about 200 mg / mL of a FAP / 4-1BB / CD40 binding molecule, about 1 mM to about 100 mM citric acid-sodium citrate or histidine-succinic acid, about 0.01 to about 10.0 mg / mL polysorbate 20 or polysorbate 80, about 0.1% (w / v) to about 20% (w / v) sucrose or trehalose, and, optionally about 5 mM to about 200 mM arginine hydrochloride, the pH of the pharmaceutical composition is about 3.0 to about 7.0; or, (4) about 10 mg / mL to about 180 mg / mL of a FAP / 4-1BB / CD40 binding molecule, about 2 mM to about 50 mM histidine-acetic acid, about 0.1 to about 5.0 mg / mL polysorbate 80, about 0.2% (w / v) to about 15% (w / v) sucrose, and, optionally about 10 mM to about 160 mM arginine hydrochloride, the pH of the pharmaceutical composition is about 4.0 to about 6.0; or, (5) about 20 mg / mL to about 160 mg / mL of a FAP / 4-1BB / CD40 binding molecule, about 5 mM to about 30 mM histidine-acetic acid, about 0.2 to about 2.0 mg / mL polysorbate 80, about 0.5% (w / v) to about 12% (w / v) sucrose, and, optionally about 20 mM to about 120 mM arginine hydrochloride, the pH of the pharmaceutical composition is about 4.5 to about 5.5; or, (6) about 10 mg / mL to about 200 mg / mL of a FAP / 4-1BB / CD40 binding molecule. about 5 mM to about 50 mM histidine-succinic acid buffer, about 1% w / v to about 10% w / v sucrose, and about 0.1 mg / mL to about 1.0 mg / mL polysorbate 80; the pH of the pharmaceutical composition is about 4.0 to about 6.0; or, (7) about 10 mg / mL to about 200 mg / mL of a FAP / 4-1BB / CD40 binding molecule. about 10 mM to about 25 mM histidine-succinic acid buffer, about 3% w / v to about 7.5% w / v sucrose, and about 0.3 mg / mL to about 0.75 mg / mL polysorbate 80; the pH of the pharmaceutical composition is about 4.5 to about 5.5; preferably comprising any one of: (c-1) about 10 mg / mL to about 200 mg / mL of a FAP / 4-1BB / CD40 binding molecule, about 10 mM histidine-succinic acid buffer, about 8% w / v sucrose, about 0.8 mg / mL polysorbate 80; the pH of the pharmaceutical composition is about 4.0 to about 6.0; (c-2) about 10 mg / mL to about 200 mg / mL of a FAP / 4-1BB / CD40 binding molecule, about 20 mM histidine-succinate buffer, about 8% w / v sucrose, about 0.8 mg / mL polysorbate 80; the pH of the pharmaceutical composition is about 4.0 to about 6.0; (c-3) about 10 mg / mL to about 200 mg / mL of a FAP / 4-1BB / CD40 binding molecule, about 20 mM histidine-succinate buffer, about 8% w / v sucrose, about 0.8 mg / mL polysorbate 80; the pH of the pharmaceutical composition is about 4.0 to about 6.0; (c-4) about 10 mg / mL to about 200 mg / mL of a FAP / 4-1BB / CD40 binding molecule, about 20 mM histidine-succinate buffer, about 2% w / v sucrose, about 4% w / v mannitol, about 0.8 mg / mL polysorbate 80; the pH of the pharmaceutical composition is about 4.0 to about 6.0; (c-5) about 10 mg / mL to about 200 mg / mL of a FAP / 4-1BB / CD40 binding molecule, about 20 mM histidine-succinate buffer, about 1.0% w / v sucrose, about 5% w / v mannitol, about 0.8 mg / mL polysorbate 80; the pH of the pharmaceutical composition is about 4.0 to about 6.0; (c-6) about 10 mg / mL to about 200 mg / mL of a FAP / 4-1BB / CD40 binding molecule, about 20 mM histidine-succinate buffer, about 8% w / v sucrose, about 0.8 mg / mL polysorbate 80; the pH of the pharmaceutical composition is about 4.0 to about 6.0; (c-7) about 10 mg / mL to about 200 mg / mL of a FAP / 4-1BB / CD40 binding molecule, about 30 mM histidine-succinate buffer, about 9% w / v sucrose, about 0.9 mg / mL polysorbate 80; the pH of the pharmaceutical composition is about 4.0 to about 6.0; (c-8) about 10 mg / mL to about 200 mg / mL of a FAP / 4-1BB / CD40 binding molecule, about 25 mM histidine-succinate buffer, about 7.5% w / v sucrose, about 0.75 mg / mL polysorbate 80; the pH of the pharmaceutical composition is about 4.0 to about 6.0; Preferably, the FAP / 4-1BB / CD40 binding molecule comprises a first antigen binding domain that specifically binds FAP, a second antigen binding domain that specifically binds CD40, and a third antigen binding domain that specifically binds 4-1BB; wherein, the third antigen binding domain comprises at least one immunoglobulin single variable domain; the immunoglobulin single variable domain in the third antigen binding domain comprises CDR1, CDR2 and CDR3 as set forth in any one of the amino acid sequences of SEQ ID NOs: 28, 18, 23-26 and 33, the CDRs being defined according to the Kabat, IMGT, Chothia, AbM or Contact numbering system; the immunoglobulin single variable domain in the third antigen binding domain comprises CDR1, CDR2 and CDR3 as set forth in any one of the amino acid sequences of SEQ ID NOs: 28, 18, 23-26 and 33, the CDRs being defined according to the Kabat, IMGT, Chothia, AbM or Contact numbering system; Preferably, the immunoglobulin single variable domain in the third antigen binding domain comprises a CDR1, a CDR2 and a CDR3 as set forth in SEQ ID NOs: 19, 30 and 31, respectively; More preferably, the immunoglobulin single variable domain in the third antigen binding domain comprises a CDR1, a CDR2 and a CDR3 as set forth in any one of the following: a-1) a CDR1, a CDR2 and a CDR3 as set forth in SEQ ID NOs: 19, 29 and 27, respectively; a-2) a CDR1, a CDR2 and a CDR3 as set forth in SEQ ID NOs: 19, 20 and 27, respectively; or a-3) a CDR1, a CDR2 and a CDR3 as set forth in SEQ ID NOs: 19, 20 and 21, respectively.

22. The pharmaceutical composition of any one of claims 1 to 16, the lyophilized formulation of any one of claims 17-18, or the reconstituted solution of any one of claims 19-21; which is an intravenous injection, a subcutaneous injection, an intraperitoneal injection or a intramuscular injection; preferably an intravenous or subcutaneous injection.

23. An article of manufacture comprising a container filled with a pharmaceutical composition of any one of claims 1 to 16, a lyophilized formulation of claim 17 or 18, or a reconstituted solution of any one of claims 19-21.

24. A method of treating or preventing a disease, comprising administering to a subject in need thereof a therapeutically or prophylactically effective amount of a pharmaceutical composition of any one of claims 1 to 16, a lyophilized formulation of claim 17 or 18, a reconstituted solution of any one of claims 19-21, a liquid formulation of claim 23, or an article of manufacture of claim 23, wherein: Preferably, the disease is a cancer; More preferably, the cancer is selected from lung cancer, prostate cancer, breast cancer, head and neck cancer, esophageal cancer, gastric cancer, colorectal cancer, bladder cancer, cervical cancer, uterine cancer, ovarian cancer, liver cancer, melanoma, renal cancer, squamous cell carcinoma and hematological cancer.

25. A method of preparing a liquid formulation, comprising the steps of: preparing a stock solution comprising a pharmaceutical composition of any one of claims 1-16, subjecting the stock solution to a freeze-drying process to obtain a lyophilized formulation; and, reconstituting the lyophilized formulation to obtain a liquid formulation; wherein, the concentration of the FAP / 4-1BB / CD40 binding molecule in the liquid formulation is greater than the concentration of the FAP / 4-1BB / CD40 binding molecule in the stock solution; Preferably, the concentration ratio of the FAP / 4-1BB / CD40 binding molecule in the liquid formulation to the FAP / 4-1BB / CD40 binding molecule in the stock solution is (1-5): 1, preferably 2: 1 or 2.5:

1.

26. A method of preparing a pharmaceutical composition, comprising: preparing a stock solution comprising a pharmaceutical composition of any one of claims 1-16, subjecting the stock solution to a freeze-drying process to obtain a lyophilized formulation; and, reconstituting the lyophilized formulation to obtain a liquid formulation; ​ The concentration of the FAP / 4-1BB / CD40 binding molecule in the liquid preparation is greater than the concentration of the FAP / 4-1BB / CD40 binding molecule in the stock solution, and the solvent is preferably water; Preferably, the concentration ratio of the FAP / 4-1BB / CD40 binding molecule in the liquid preparation to the FAP / 4-1BB / CD40 binding molecule in the stock solution is (1-5): 1, preferably 2: 1 or 2.5: 1.

Images