Pharmaceutical composition containing Anti-TF antibody-drug conjugate

Abstract

The present application relates to a pharmaceutical composition containing an anti-TF antibody-drug conjugate. The pharmaceutical composition further contains a buffer. The pharmaceutical composition exhibits good stability.

Description

A pharmaceutical composition comprising an anti-TF antibody drug conjugate Technical Field

[0001] This disclosure pertains to the field of pharmaceutical formulations, specifically relating to a pharmaceutical composition comprising an anti-TF antibody-drug conjugate. Background Technology

[0002] The statements herein are provided only as background information in connection with this disclosure and do not necessarily constitute prior art.

[0003] Tissue Factor, also known as TF / tissue thromboplastin / FIII / CD142, is a transmembrane glycoprotein, 47 kDa in length, consisting of 219 amino acids in the extracellular region, 23 amino acids in the transmembrane region, and 21 amino acids in the intracellular region. The gene name for human Tissue Factor is F3.

[0004] TF is mainly expressed by perivascular cells (such as adventitia fibroblasts and pericytes) and the body surface (such as skin keratinocytes), but it is also expressed by monocytes, brain astrocytes, lung epithelial cells, cardiac fibroblasts, and cardiomyocytes. This widespread expression is beneficial for its formation of a systemic hemostatic barrier.

[0005] Under normal physiological conditions, TF is the promoter of extrinsic coagulation and a cell receptor for coagulation factor VII (FVII) and its activated form FVIIa in the blood. When a blood vessel is damaged, TF forms a complex with FVII in the blood and activates FVII to FVIIa, generating the TF / FVIIa complex. TF / FVIIa can bind to coagulation factor X (FX) in the blood, activating it to FXa and forming the TF / FVIIa / FXa complex, which further triggers the coagulation cascade reaction, activating prothrombin (FII) to thrombin (FIIa). Thrombin converts fibrinogen into insoluble fibrin, ultimately completing coagulation.

[0006] The TF / FVIIa complex can activate the PAR2 (proteinase-activated receptors 2) signaling pathway. The TF / FVIIa complex can also form the TF / FVIIa / Integrinβ1 complex with Integrinβ1. Downstream signaling pathways of PAR2 and Integrinβ1 can promote tumor growth. Downstream products of the TF / FVIIa coagulation cascade, thrombin and fibrin, are associated with tumor metastasis and thrombosis.

[0007] Tissue factor (TF) is highly expressed in various solid tumors under pathological conditions, such as pancreatic cancer, lung cancer, breast cancer, gastric cancer, colorectal cancer, liver cancer, esophageal cancer, ovarian cancer, and bladder cancer. Multiple growth factors and cytokines induce TF expression, while various transcription factors and microRNAs regulate TF gene expression in cancer cells. For example, the hepatocyte growth factor (HGF) / c-Met and epidermal growth factor receptor (EGFR) signaling pathways can upregulate TF expression by activating multiple kinase pathways, including JNK, Src, PI3k / Akt / mTOR, and KRAS / Raf / MEK / ERK. Transforming growth factor β (TGF-β) and vascular endothelial growth factor (VEGF) can also increase TF expression. During inflammation, TF can be upregulated by various cytokines, such as interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α). TF can also be downregulated by some microRNAs such as miR-181b and miR19, or its activity can be inhibited by TFPI (tissue factor pathway inhibitor) on monocytes.

[0008] High expression of TF under pathological conditions can promote tumor growth, metastasis, and thrombus formation, and is associated with poor prognosis. Therefore, developing TF-targeted antibody-drug conjugates (ADCs) can both inhibit tumor growth through antibody signal blocking and achieve specific killing of tumors by TF antibodies.

[0009] Ecinotecan toxoid is a camptothecin derivative that inhibits topoisomerase I, selectively inhibiting DNA replication in proliferating tumor cells. Furthermore, eccinotecan toxoid exhibits excellent membrane permeability, allowing it to penetrate killed cancer cells and continue killing adjacent cancer cells, demonstrating a clear bystander effect in clinical practice.

[0010] Antibody-drug conjugates (ADCs) are obtained by linking antibodies to biologically active drugs via linkers. ADCs fully utilize the specificity of antibodies in binding to antigens on the surface of normal and tumor cells, as well as the high efficiency of drugs (such as cytotoxic agents), while avoiding the drawbacks of low efficacy of antibodies and excessive toxic side effects of drugs. Compared with traditional chemotherapy drugs, antibody-drug conjugates can more precisely kill tumor cells and reduce the impact on normal cells.

[0011] ADCs have a more complex heterogeneous structure than antibodies, thus posing a greater challenge to ADC formulations for therapeutic purposes. Summary of the Invention

[0012] This disclosure provides a pharmaceutical composition comprising an anti-TF antibody-drug conjugate. The pharmaceutical composition exhibits good therapeutic activity, safety, pharmacokinetic properties, and drug-likeness (e.g., stability).

[0013] In some embodiments, this disclosure provides a pharmaceutical composition comprising an anti-TF antibody-drug conjugate and a buffer, wherein:

[0014] The anti-TF antibody-drug conjugate has a structure as shown in Formula I or Formula II:

[0015] in:

[0016] n is between 1 and 10;

[0017] Pc is an anti-TF antibody, which contains a heavy chain variable region and a light chain variable region, wherein:

[0018] The heavy chain variable region HCDR1 contains the amino acid sequence of SEQ ID NO: 40, HCDR2 contains the amino acid sequence of SEQ ID NO: 64, and HCDR3 contains the amino acid sequence of SEQ ID NO: 65; and the light chain variable region LCDR1 contains the amino acid sequence of SEQ ID NO: 66, LCDR2 contains the amino acid sequence of SEQ ID NO: 67, and LCDR3 contains the amino acid sequence of SEQ ID NO: 68 or 73.

[0019] The buffer is a histidine buffer, a citrate buffer, an acetate buffer, or a succinate buffer.

[0020] In some embodiments, the pharmaceutical composition as described above, wherein the buffer is a histidine-histidine hydrochloride buffer, a citrate-sodium citrate buffer, an acetate-sodium acetate buffer, or a succinate-sodium succinate buffer.

[0021] In some embodiments, such as the pharmaceutical composition described in any of the preceding embodiments, the buffer is a histidine-histidine hydrochloride buffer.

[0022] In some embodiments, the pharmaceutical composition as described in any of the preceding embodiments, wherein the anti-TF antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:62, or an amino acid sequence having at least 80% sequence identity with it; and the light chain variable region comprises the amino acid sequence of SEQ ID NO:63 or 72, or an amino acid sequence having at least 80% sequence identity with it.

[0023] In some embodiments, the pharmaceutical composition as described in any of the preceding claims, wherein the anti-TF antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:62, and the light chain variable region comprises the amino acid sequence of SEQ ID NO:63.

[0024] In some embodiments, the pharmaceutical composition as described in any of the preceding claims, wherein the anti-TF antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO: 69, or an amino acid sequence having at least 80% sequence identity therewith; and the light chain comprises the amino acid sequence of SEQ ID NO: 70 or 74, or an amino acid sequence having at least 80% sequence identity therewith.

[0025] In some embodiments, the pharmaceutical composition as described in any of the preceding claims, wherein the anti-TF antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO: 69, and the light chain comprises the amino acid sequence of SEQ ID NO: 70.

[0026] In some embodiments, the pharmaceutical composition as described in any of the preceding embodiments, wherein n is the average number of pharmaceutical modules per anti-TF antibody, and may be an integer or a decimal. In some embodiments, n is 1-10, or 2-10, or 3-10, or 4-10, or 5-10, or 6-10, or 7-10, or 8-10, or 1-9, or 2-9, or 3-9, or 4-9, or 5-9, or 6-9, or 7-9, or 1-8, or 2-8, or 3-8, or 4-8, or 5-8, or 6-8, or 1-7, or 2-7, or 3-7, or 4-7, or 5-7, or 1-6, or 2-6, or 3-6, or 4-6, or 1-5, or 2-5, or 3-5, or 1-4, or 2-4, or 1-3. In some embodiments, n is about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, or about 10.

[0027] In some embodiments, the pharmaceutical composition as described in any of the preceding embodiments, wherein n is 3 to 8. In some embodiments, n is 4 to 8. In some embodiments, n is 5 to 8. In some embodiments, n is 5 to 7. In some embodiments, n is 6 to 8. In some embodiments, n is about 6. In some embodiments, n is 6. In some embodiments, n is about 8. In some embodiments, n is 8.

[0028] In some embodiments, the pharmaceutical composition as described in any of the preceding embodiments, wherein the concentration of the anti-TF antibody drug conjugate is from 1 mg / mL to 50 mg / mL. In some embodiments, the concentration of the anti-TF antibody drug conjugate is from 5 mg / mL to 40 mg / mL. In some embodiments, the concentration of the anti-TF antibody drug conjugate is from 5 mg / mL to 30 mg / mL. In some embodiments, the concentration of the anti-TF antibody drug conjugate is from 15 mg / mL to 25 mg / mL. In some embodiments, the concentration of the anti-TF antibody drug conjugate is from 18 mg / mL to 22 mg / mL. In some embodiments, the concentration of the anti-TF antibody-drug conjugate is about 1 mg / mL, about 5 mg / mL, about 10 mg / mL, about 15 mg / mL, about 16 mg / mL, about 18 mg / mL, about 20 mg / mL, about 22 mg / mL, about 24 mg / mL, about 25 mg / mL, about 30 mg / mL, about 35 mg / mL, about 40 mg / mL, about 45 mg / mL, or about 50 mg / mL. In some embodiments, the concentration of the anti-TF antibody-drug conjugate is about 5 mg / mL. In some embodiments, the concentration of the anti-TF antibody-drug conjugate is about 20 mg / mL. In some embodiments, the concentration of the anti-TF antibody-drug conjugate is about 40 mg / mL. In some embodiments, the concentration of the anti-TF antibody-drug conjugate is 1 mg / mL, 5 mg / mL, 10 mg / mL, 15 mg / mL, 16 mg / mL, 18 mg / mL, 20 mg / mL, 22 mg / mL, 24 mg / mL, 25 mg / mL, 30 mg / mL, 35 mg / mL, 40 mg / mL, 45 mg / mL, or 50 mg / mL, or any range between these values. In some embodiments, the concentration of the anti-TF antibody-drug conjugate is 5 mg / mL. In some embodiments, the concentration of the anti-TF antibody-drug conjugate is 20 mg / mL. In some embodiments, the concentration of the anti-TF antibody-drug conjugate is 40 mg / mL.

[0029] In some embodiments, the pharmaceutical composition as described in any of the preceding embodiments, wherein the concentration of the buffer is from 1 mM to 50 mM. In some embodiments, the concentration of the buffer is from 5 mM to 40 mM. In some embodiments, the concentration of the buffer is from 5 mM to 35 mM. In some embodiments, the concentration of the buffer is from 5 mM to 30 mM. In some embodiments, the concentration of the buffer is from 5 mM to 20 mM. In some embodiments, the concentration of the buffer is from 5 mM to 15 mM. In some embodiments, the concentration of the buffer is from 8 mM to 12 mM. In some embodiments, the concentration of the buffer is from 9 mM to 11 mM. In some embodiments, the concentration of the buffer is from 7 mM to 11 mM. In some embodiments, the concentration of the buffer is about 10 mM. In some embodiments, the concentration of the buffer is from 10 mM to 30 mM. In some embodiments, the concentration of the buffer is from 10 mM to 40 mM. In some embodiments, the concentration of the buffer is 20 mM to 40 mM. In some embodiments, the concentration of the buffer is 25 mM to 35 mM. In some embodiments, the concentration of the buffer is about 30 mM. In some embodiments, the concentration of the buffer is about 1 mM, about 5 mM, about 7 mM, about 8 mM, about 9 mM, about 9.2 mM, about 10 mM, about 11 mM, about 12 mM, about 15 mM, about 20 mM, about 22 mM, about 24 mM, about 25 mM, about 27 mM, about 27.8 mM, about 30 mM, about 33 mM, about 35 mM, about 36 mM, about 40 mM, about 45 mM, or about 50 mM. In some embodiments, the concentration of the buffer is 1 mM, 5 mM, 7 mM, 8 mM, 9 mM, 9.2 mM, 10 mM, 11 mM, 12 mM, 15 mM, 20 mM, 22 mM, 24 mM, 25 mM, 27 mM, 27.8 mM, 30 mM, 33 mM, 35 mM, 36 mM, 40 mM, 45 mM, or 50 mM, or any range between these values. In some embodiments, the concentration of the buffer is 10 mM. In some embodiments, the concentration of the buffer is 30 mM.

[0030] In some embodiments, the pharmaceutical composition as described in any of the preceding embodiments, wherein the concentration of the histidine-histidine hydrochloride buffer is from 1 mM to 50 mM. In some embodiments, the concentration of the histidine-histidine hydrochloride buffer is from 5 mM to 40 mM. In some embodiments, the concentration of the histidine-histidine hydrochloride buffer is from 5 mM to 35 mM. In some embodiments, the concentration of the histidine-histidine hydrochloride buffer is from 5 mM to 30 mM. In some embodiments, the concentration of the histidine-histidine hydrochloride buffer is from 5 mM to 20 mM. In some embodiments, the concentration of the histidine-histidine hydrochloride buffer is from 5 mM to 15 mM. In some embodiments, the concentration of the histidine-histidine hydrochloride buffer is from 8 mM to 12 mM. In some embodiments, the concentration of the histidine-histidine hydrochloride buffer is from 10 mM to 30 mM. In some embodiments, the concentration of the histidine-histidine hydrochloride buffer is from 10 mM to 40 mM. In some embodiments, the concentration of the histidine-histidine hydrochloride buffer is 20 mM to 40 mM. In some embodiments, the concentration of the histidine-histidine hydrochloride buffer is 24 mM to 36 mM. In some embodiments, the concentration of the histidine-histidine hydrochloride buffer is 25 mM to 35 mM. In some embodiments, the concentration of the histidine-histidine hydrochloride buffer is 27 mM to 33 mM. In some embodiments, the concentration of the histidine-histidine hydrochloride buffer is about 1 mM, about 5 mM, about 7 mM, about 8 mM, about 9 mM, about 9.2 mM, about 10 mM, about 11 mM, about 12 mM, about 15 mM, about 20 mM, about 22 mM, about 24 mM, about 25 mM, about 27 mM, about 27.8 mM, about 30 mM, about 33 mM, about 35 mM, about 36 mM, about 40 mM, about 45 mM, or about 50 mM. In some embodiments, the concentration of the histidine-histidine hydrochloride buffer is about 10 mM. In some embodiments, the concentration of the histidine-histidine hydrochloride buffer is about 20 mM. In some embodiments, the concentration of the histidine-histidine hydrochloride buffer is about 30 mM. In some embodiments, the concentration of the histidine-histidine hydrochloride buffer is 1 mM, 5 mM, 7 mM, 8 mM, 9 mM, 9.2 mM, 10 mM, 11 mM, 12 mM, 15 mM, 20 mM, 22 mM, 24 mM, 25 mM, 27 mM, 27.8 mM, 30 mM, 33 mM, 35 mM, 36 mM, 40 mM, 45 mM, or 50 mM, or any range between these values. In some embodiments, the concentration of the histidine-histidine hydrochloride buffer is 10 mM. In some embodiments, the concentration of the histidine-histidine hydrochloride buffer is 20 mM.In some embodiments, the concentration of the histidine-histidine hydrochloride buffer is 30 mM.

[0031] In some embodiments, the pharmaceutical composition as described in any of the preceding embodiments has a pH of 4.5 to 8.0. In some embodiments, the pharmaceutical composition has a pH of 5.0 to 6.5. In some embodiments, the pharmaceutical composition has a pH of 5.0 to 6.0. In some embodiments, the pharmaceutical composition has a pH of 5.2 to 5.8. In some embodiments, the pharmaceutical composition has a pH of 5.3 to 5.5. In some embodiments, the pharmaceutical composition has a pH of 5.5 to 6.0. In some embodiments, the pharmaceutical composition has a pH of 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 7.0, about 7.5, or about 8.0. In some embodiments, the pH of the pharmaceutical composition is about 5.0. In some embodiments, the pH of the pharmaceutical composition is about 5.3. In some embodiments, the pH of the pharmaceutical composition is about 5.5. In some embodiments, the pH of the pharmaceutical composition is about 6.0. In some embodiments, the pH of the pharmaceutical composition is about 6.5. In some embodiments, the pH of the pharmaceutical composition is 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 7.0, 7.5, or 8.0, or any range between these values. In some embodiments, the pH of the pharmaceutical composition is 5.0. In some embodiments, the pH of the pharmaceutical composition is 5.3. In some embodiments, the pH of the pharmaceutical composition is 5.5. In some embodiments, the pH of the pharmaceutical composition is 6.0. In some embodiments, the pH of the pharmaceutical composition is 6.5. When point values ​​are mentioned in this disclosure, it should be understood that these values ​​include a range of error. This range of error is due to factors such as laboratory environment, human operation, instrumentation, methodology, and measurement errors. For example, when measuring pH at approximately 5.5, it should be understood that this includes a range of error. As an example, when measuring a formulation using an industrial pH meter, "approximately 5.5" means 5.5 ± 0.3 (i.e., pH from 5.2 to 5.8).

[0032] In some embodiments, the pharmaceutical composition as described in any of the preceding embodiments comprises a surfactant. In some embodiments, the surfactant is a nonionic surfactant. In some embodiments, the surfactant is selected from polysorbates (e.g., polysorbate 80, polysorbate 20), poloxamer (e.g., poloxamer 188, i.e., PF68 or P188), Triton, sodium lauryl sulfonate, sodium lauryl sulfonate, sodium octyl glycoside, lauryl-sulfobetaine, myristyl-sulfobetaine, linoleyl-sulfobetaine, stearyl-sulfobetaine, lauryl-sarcosine, myristyl-sarcosine, linoleyl-sarcosine, stearyl-sarcosine, linoleyl-saccharide, succinate ... Betaine, myristyl-betaine, cetyl-betaine, lauramidopropyl-betaine, cocarbamate-propyl-betaine, linoleamide-propyl-betaine, myristamidopropyl-betaine, palmitoamide-propyl-betaine, isostearamidopropyl-betaine, myristamidopropyl-dimethylamine, palmitoamide-propyl-dimethylamine, isostearamidopropyl-dimethylamine, sodium methyl cocoyl, sodium methyl oleate, polyethylene glycol, polypropylene glycol, copolymers of ethylene and propylene glycol, etc. In some embodiments, the surfactant is polysorbate. In some embodiments, the surfactant is polysorbate 80 (PS80), polysorbate 20 (PS20), and poloxamer 188. In some embodiments, the surfactant is polysorbate 80 (PS80).

[0033] In some embodiments, the pharmaceutical composition as described in any of the preceding embodiments, wherein the concentration of the surfactant is from 0.01 mg / mL to 2 mg / mL. In some embodiments, the concentration of the surfactant is from 1 mg / mL to 2 mg / mL. In some embodiments, the concentration of the surfactant is from 0.02 mg / mL to 1 mg / mL. In some embodiments, the concentration of the surfactant is from 0.02 mg / mL to 0.8 mg / mL. In some embodiments, the concentration of the surfactant is from 0.02 mg / mL to 0.6 mg / mL. In some embodiments, the concentration of the surfactant is from 0.1 mg / mL to 0.5 mg / mL. In some embodiments, the concentration of the surfactant is from 0.2 mg / mL to 0.4 mg / mL. In some embodiments, the surfactant concentration is about 0.01 mg / mL, about 0.02 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 1.5 mg / mL, or about 2.0 mg / mL. In some embodiments, the surfactant concentration is about 0.02 mg / mL. In some embodiments, the surfactant concentration is about 0.1 mg / mL. In some embodiments, the surfactant concentration is about 0.3 mg / mL. In some embodiments, the surfactant concentration is about 0.5 mg / mL. In some embodiments, the surfactant concentration is about 1 mg / mL. In some embodiments, the surfactant concentration is about 1.5 mg / mL. In some embodiments, the surfactant concentration is 0.01 mg / mL, 0.02 mg / mL, 0.05 mg / mL, 0.1 mg / mL, 0.2 mg / mL, 0.3 mg / mL, 0.4 mg / mL, 0.5 mg / mL, 0.6 mg / mL, 0.7 mg / mL, 0.8 mg / mL, 0.9 mg / mL, 1.0 mg / mL, 1.5 mg / mL, or 2.0 mg / mL, or any range between these values. In some embodiments, the surfactant concentration is 0.02 mg / mL. In some embodiments, the surfactant concentration is 0.1 mg / mL. In some embodiments, the surfactant concentration is 0.3 mg / mL. In some embodiments, the surfactant concentration is 0.5 mg / mL. In some embodiments, the surfactant concentration is 1 mg / mL. In some embodiments, the surfactant concentration is 1.5 mg / mL.

[0034] In some embodiments, the pharmaceutical composition as described in any of the preceding embodiments, wherein the concentration of polysorbate 80 is from 0.01 mg / mL to 2 mg / mL. In some embodiments, the concentration of polysorbate 80 is from 0.02 mg / mL to 1 mg / mL. In some embodiments, the concentration of polysorbate 80 is from 0.02 mg / mL to 0.8 mg / mL. In some embodiments, the concentration of polysorbate 80 is from 0.02 mg / mL to 0.5 mg / mL. In some embodiments, the concentration of polysorbate 80 is from 0.1 mg / mL to 0.5 mg / mL. In some embodiments, the concentration of polysorbate 80 is from 0.2 mg / mL to 0.4 mg / mL. In some embodiments, the concentration of polysorbate 80 is about 0.01 mg / mL, about 0.02 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 1.5 mg / mL, or 2.0 mg / mL. In some embodiments, the concentration of polysorbate 80 is about 0.02 mg / mL. In some embodiments, the concentration of polysorbate 80 is about 0.1 mg / mL. In some embodiments, the concentration of polysorbate 80 is about 0.3 mg / mL. In some embodiments, the concentration of polysorbate 80 is about 0.5 mg / mL. In some embodiments, the concentration of polysorbate 80 is about 1 mg / mL. In some embodiments, the concentration of polysorbate 80 is 0.01 mg / mL, 0.02 mg / mL, 0.05 mg / mL, 0.1 mg / mL, 0.2 mg / mL, 0.3 mg / mL, 0.4 mg / mL, 0.5 mg / mL, 0.6 mg / mL, 0.7 mg / mL, 0.8 mg / mL, 0.9 mg / mL, 1.0 mg / mL, 1.5 mg / mL, or 2.0 mg / mL, or any range between these values. In some embodiments, the concentration of polysorbate 80 is 0.02 mg / mL. In some embodiments, the concentration of polysorbate 80 is 0.1 mg / mL. In some embodiments, the concentration of polysorbate 80 is 0.3 mg / mL. In some embodiments, the concentration of polysorbate 80 is 0.5 mg / mL. In some embodiments, the concentration of polysorbate 80 is 1 mg / mL.

[0035] In some embodiments, the pharmaceutical composition as described in any of the preceding embodiments, wherein the concentration of polysorbate 20 is from 0.01 mg / mL to 2 mg / mL. In some embodiments, the concentration of polysorbate 20 is from 0.02 mg / mL to 1 mg / mL. In some embodiments, the concentration of polysorbate 20 is from 0.02 mg / mL to 0.8 mg / mL. In some embodiments, the concentration of polysorbate 20 is from 0.02 mg / mL to 0.5 mg / mL. In some embodiments, the concentration of polysorbate 20 is from 0.1 mg / mL to 0.5 mg / mL. In some embodiments, the concentration of polysorbate 20 is from 0.2 mg / mL to 0.4 mg / mL. In some embodiments, the concentration of polysorbate 20 is about 0.01 mg / mL, about 0.02 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 1.5 mg / mL, or about 2.0 mg / mL. In some embodiments, the concentration of polysorbate 20 is about 0.3 mg / mL. In some embodiments, the concentration of polysorbate 20 is 0.01 mg / mL, 0.02 mg / mL, 0.05 mg / mL, 0.1 mg / mL, 0.2 mg / mL, 0.3 mg / mL, 0.4 mg / mL, 0.5 mg / mL, 0.6 mg / mL, 0.7 mg / mL, 0.8 mg / mL, 0.9 mg / mL, 1.0 mg / mL, 1.5 mg / mL, or 2.0 mg / mL, or any range between these values. In some embodiments, the concentration of polysorbate 20 is 0.3 mg / mL.

[0036] In some embodiments, the pharmaceutical composition as described in any of the preceding embodiments, wherein the concentration of poloxamer 188 is from 0.01 mg / mL to 2 mg / mL. In some embodiments, the concentration of poloxamer 188 is from 1 mg / mL to 2 mg / mL. In some embodiments, the concentration of poloxamer 188 is from 1.2 mg / mL to 1.8 mg / mL. In some embodiments, the concentration of poloxamer 188 is about 0.01 mg / mL, about 0.02 mg / mL, about 0.05 mg / mL, about 0.1 mg / mL, about 0.3 mg / mL, about 0.5 mg / mL, about 0.8 mg / mL, about 0.9 mg / mL, about 1.0 mg / mL, about 1.2 mg / mL, about 1.5 mg / mL, about 1.8 mg / mL, about 1.9 mg / mL, or about 2.0 mg / mL. In some embodiments, the concentration of poloxamer 188 is about 1.5 mg / mL. In some embodiments, the concentration of poloxamer 188 is 0.01 mg / mL, 0.02 mg / mL, 0.05 mg / mL, 0.1 mg / mL, 0.3 mg / mL, 0.5 mg / mL, 0.8 mg / mL, 0.9 mg / mL, 1.0 mg / mL, 1.2 mg / mL, 1.5 mg / mL, 1.8 mg / mL, 1.9 mg / mL, or 2.0 mg / mL, or any range between these values. In some embodiments, the concentration of poloxamer 188 is 1.5 mg / mL.

[0037] In some embodiments, the pharmaceutical composition as described in any of the preceding embodiments comprises a sugar. In some embodiments, the sugar is selected from conventional compositions (CH2O). n And its derivatives, including monosaccharides, disaccharides, trisaccharides, polysaccharides, sugar alcohols, reducing sugars, non-reducing sugars, etc. In some embodiments, the sugar is selected from sucrose, trehalose, glucose, lactose, fructose, maltose, dextran, glycerol, erythritol, glycerol, arabinitol, sylitol, sorbitol, mannitol, melitriose, maltotriose, stachyose, maltose, lactulose, maltitol, maltitol, lactitol, isomaltulose, etc. In some embodiments, the sugar is sucrose, trehalose, mannitol, or sorbitol. In some embodiments, the sugar is sucrose.

[0038] In some embodiments, the pharmaceutical composition as described in any of the preceding embodiments, wherein the sugar concentration is from 10 mg / mL to 120 mg / mL. In some embodiments, the sugar concentration is from 40 mg / mL to 100 mg / mL. In some embodiments, the sugar concentration is from 65 mg / mL to 95 mg / mL. In some embodiments, the sugar concentration is from 70 mg / mL to 90 mg / mL. In some embodiments, the sugar concentration is from 10 mg / mL to 80 mg / mL. In some embodiments, the sugar concentration is from 20 mg / mL to 60 mg / mL. In some embodiments, the sugar concentration is from 30 mg / mL to 50 mg / mL. In some embodiments, the sugar concentration is from 32 mg / mL to 48 mg / mL. In some embodiments, the sugar concentration is from 36 mg / mL to 44 mg / mL. In some embodiments, the sugar concentration is about 100 mg / mL. In some embodiments, the sugar concentration is about 10 mg / mL, about 20 mg / mL, about 30 mg / mL, about 33 mg / mL, about 36 mg / mL, about 40 mg / mL, about 44 mg / mL, about 48 mg / mL, about 50 mg / mL, about 60 mg / mL, about 64 mg / mL, about 65 mg / mL, about 70 mg / mL, about 72 mg / mL, about 80 mg / mL, about 88 mg / mL, about 90 mg / mL, about 95 mg / mL, about 96 mg / mL, about 100 mg / mL, about 110 mg / mL, or about 120 mg / mL. In some embodiments, the sugar concentration is about 40 mg / mL. In some embodiments, the sugar concentration is about 80 mg / mL. In some embodiments, the sugar concentration is 10 mg / mL, 20 mg / mL, 30 mg / mL, 33 mg / mL, 36 mg / mL, 40 mg / mL, 44 mg / mL, 48 mg / mL, 50 mg / mL, 60 mg / mL, 64 mg / mL, 65 mg / mL, 70 mg / mL, 72 mg / mL, 80 mg / mL, 88 mg / mL, 90 mg / mL, 95 mg / mL, 96 mg / mL, 100 mg / mL, 110 mg / mL, or 120 mg / mL, or any range between these values. In some embodiments, the sugar concentration is 40 mg / mL. In some embodiments, the sugar concentration is 80 mg / mL. In some embodiments, the sugar concentration is 100 mg / mL.

[0039] In some embodiments, the pharmaceutical composition as described in any of the preceding embodiments, wherein the concentration of sucrose is from 10 mg / mL to 120 mg / mL. In some embodiments, the concentration of sucrose is from 40 mg / mL to 100 mg / mL. In some embodiments, the concentration of sucrose is from 65 mg / mL to 95 mg / mL. In some embodiments, the concentration of sucrose is from 70 mg / mL to 90 mg / mL. In some embodiments, the concentration of sucrose is about 80 mg / mL. In some embodiments, the concentration of sucrose is from 10 mg / mL to 80 mg / mL. In some embodiments, the concentration of sucrose is from 20 mg / mL to 60 mg / mL. In some embodiments, the concentration of sucrose is from 30 mg / mL to 50 mg / mL. In some embodiments, the concentration of sucrose is from 32 mg / mL to 48 mg / mL. In some embodiments, the concentration of sucrose is from 36 mg / mL to 44 mg / mL. In some embodiments, the concentration of sucrose is about 40 mg / mL. In some embodiments, the concentration of the sucrose is about 100 mg / mL. In some embodiments, the concentration of the sucrose is about 10 mg / mL, about 20 mg / mL, about 30 mg / mL, about 33 mg / mL, about 36 mg / mL, about 40 mg / mL, about 44 mg / mL, about 48 mg / mL, about 50 mg / mL, about 60 mg / mL, about 64 mg / mL, about 65 mg / mL, about 70 mg / mL, about 72 mg / mL, about 80 mg / mL, about 88 mg / mL, about 90 mg / mL, about 95 mg / mL, about 96 mg / mL, about 100 mg / mL, about 110 mg / mL, or about 120 mg / mL. In some embodiments, the concentration of sucrose is 10 mg / mL, 20 mg / mL, 30 mg / mL, 33 mg / mL, 36 mg / mL, 40 mg / mL, 44 mg / mL, 48 mg / mL, 50 mg / mL, 60 mg / mL, 64 mg / mL, 65 mg / mL, 70 mg / mL, 72 mg / mL, 80 mg / mL, 88 mg / mL, 90 mg / mL, 95 mg / mL, 96 mg / mL, 100 mg / mL, 110 mg / mL, or 120 mg / mL, or any range between these values. In some embodiments, the concentration of sucrose is 40 mg / mL. In some embodiments, the concentration of sucrose is 80 mg / mL. In some embodiments, the concentration of sucrose is 100 mg / mL.

[0040] In some embodiments, the pharmaceutical composition as described in any of the preceding embodiments further comprises a sodium salt of ethylenediaminetetraacetic acid, ethylenediaminetetraacetic acid hydrate, or a salt thereof.

[0041] In some embodiments, the pharmaceutical composition further comprises ethylenediaminetetraacetic acid hydrate or a salt thereof.

[0042] In some embodiments, the ethylenediaminetetraacetic acid hydrate or its salt is disodium edetate (EDTA-2Na).

[0043] In some embodiments, the pharmaceutical composition further comprises disodium edetate (EDTA-2Na).

[0044] In some embodiments, the concentration of the sodium salt of ethylenediaminetetraacetic acid (EDTA) and EDTA hydrate or a salt thereof, as described in any of the preceding embodiments, is from 0.01 mg / mL to 1 mg / mL. In some embodiments, the concentration of the sodium salt of EDTA and EDTA hydrate or a salt thereof is from 0.01 mg / mL to 0.5 mg / mL. In some embodiments, the concentration of the sodium salt of EDTA and EDTA hydrate or a salt thereof is from 0.01 mg / mL to 0.1 mg / mL. In some embodiments, the concentration of the sodium salt of EDTA and EDTA hydrate or a salt thereof is from 0.02 mg / mL to 0.08 mg / mL. In some embodiments, the concentration of the sodium salt of EDTA and EDTA hydrate or a salt thereof is from 0.04 mg / mL to 0.06 mg / mL. In some embodiments, the concentrations of the sodium salt of ethylenediaminetetraacetic acid (EDTA) and EDTA hydrate or a salt thereof are 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 0.06 mg / mL, about 0.07 mg / mL, about 0.08 mg / mL, about 0.09 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, or about 1 mg / mL. In some embodiments, the concentrations of the sodium salt of EDTA and EDTA hydrate or a salt thereof are about 0.01 mg / mL. In some embodiments, the concentration of the sodium salt of ethylenediaminetetraacetic acid (EDTA) and EDTA hydrate or a salt thereof is about 0.05 mg / mL. In some embodiments, the concentration of the sodium salt of EDTA and EDTA hydrate or a salt thereof is about 0.1 mg / mL. In some embodiments, the concentration of the sodium salt of EDTA and EDTA hydrate or a salt thereof is 0.01 mg / mL, 0.02 mg / mL, 0.03 mg / mL, 0.04 mg / mL, 0.05 mg / mL, 0.06 mg / mL, 0.07 mg / mL, 0.08 mg / mL, 0.09 mg / mL, 0.1 mg / mL, 0.2 mg / mL, 0.3 mg / mL, 0.4 mg / mL, 0.5 mg / mL, 0.6 mg / mL, 0.7 mg / mL, 0.8 mg / mL, 0.9 mg / mL, or 1 mg / mL, or any range between these values. In some embodiments, the concentration of the sodium salt of ethylenediaminetetraacetic acid and ethylenediaminetetraacetic acid hydrate or a salt thereof is 0.01 mg / mL. In some embodiments, the concentration of the sodium salt of ethylenediaminetetraacetic acid and ethylenediaminetetraacetic acid hydrate or a salt thereof is 0.05 mg / mL.In some embodiments, the concentration of the sodium salt of ethylenediaminetetraacetic acid and the hydrate of ethylenediaminetetraacetic acid or its salt is 0.1 mg / mL.

[0045] In some embodiments, the pharmaceutical composition as described in any of the preceding embodiments, wherein the concentration of disodium edetate is from 0.01 mg / mL to 1 mg / mL. In some embodiments, the concentration of disodium edetate is from 0.01 mg / mL to 0.5 mg / mL. In some embodiments, the concentration of disodium edetate is from 0.01 mg / mL to 0.1 mg / mL. In some embodiments, the concentration of disodium edetate is from 0.02 mg / mL to 0.08 mg / mL. In some embodiments, the concentration of disodium edetate is from 0.04 mg / mL to 0.06 mg / mL. In some embodiments, the concentration of the disodium edetate 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 0.06 mg / mL, about 0.07 mg / mL, about 0.08 mg / mL, about 0.09 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, or about 1 mg / mL. In some embodiments, the concentration of the disodium edetate is about 0.01 mg / mL. In some embodiments, the concentration of the disodium edetate is about 0.05 mg / mL. In some embodiments, the concentration of the disodium edetate is about 0.1 mg / mL. In some embodiments, the concentration of disodium edetate is 0.01 mg / mL, 0.02 mg / mL, 0.03 mg / mL, 0.04 mg / mL, 0.05 mg / mL, 0.06 mg / mL, 0.07 mg / mL, 0.08 mg / mL, 0.09 mg / mL, 0.1 mg / mL, 0.2 mg / mL, 0.3 mg / mL, 0.4 mg / mL, 0.5 mg / mL, 0.6 mg / mL, 0.7 mg / mL, 0.8 mg / mL, 0.9 mg / mL, or 1 mg / mL, or any range between these values. In some embodiments, the concentration of disodium edetate is 0.01 mg / mL. In some embodiments, the concentration of disodium edetate is 0.05 mg / mL. In some embodiments, the concentration of disodium edetate is 0.1 mg / mL.

[0046] In some embodiments, the pharmaceutical composition as described in any of the preceding embodiments further comprises an excipient. In some embodiments, the excipient is one or more selected from glycine, DTPA (diethylenetriaminepentaacetic acid), arginine hydrochloride, methionine, proline, histidine, phenylalanine, glutamic acid, aspartic acid, sodium chloride, or calcium chloride. In some embodiments, the excipient is glycine.

[0047] In some embodiments, the pharmaceutical composition as described in any of the preceding embodiments, wherein the concentration of the excipient is from 5 mg / mL to 15 mg / mL. In some embodiments, the concentration of the excipient is from 7 mg / mL to 11 mg / mL. In some embodiments, the concentration of the excipient is from 8 mg / mL to 10 mg / mL. In some embodiments, the concentration of the excipient is 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 11 mg / mL, about 12 mg / mL, about 13 mg / mL, about 14 mg / mL, or about 15 mg / mL. In some embodiments, the concentration of the excipient is about 9 mg / mL. In some embodiments, the concentration of the excipient is 5 mg / mL, 6 mg / mL, 7 mg / mL, 8 mg / mL, 9 mg / mL, 10 mg / mL, 11 mg / mL, 12 mg / mL, 13 mg / mL, 14 mg / mL, or 15 mg / mL, or any range between these values. In some embodiments, the concentration of the excipient is 9 mg / mL.

[0048] In some embodiments, the pharmaceutical composition as described in any of the preceding embodiments, wherein the concentration of glycine is from 5 mg / mL to 15 mg / mL. In some embodiments, the concentration of glycine is from 7 mg / mL to 11 mg / mL. In some embodiments, the concentration of glycine is from 8 mg / mL to 10 mg / mL. In some embodiments, the concentration of glycine is 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 11 mg / mL, about 12 mg / mL, about 13 mg / mL, about 14 mg / mL, or about 15 mg / mL. In some embodiments, the concentration of glycine is about 9 mg / mL. In some embodiments, the concentration of glycine is 5 mg / mL, 6 mg / mL, 7 mg / mL, 8 mg / mL, 9 mg / mL, 10 mg / mL, 11 mg / mL, 12 mg / mL, 13 mg / mL, 14 mg / mL, or 15 mg / mL, or any range between these values. In some embodiments, the concentration of glycine is 9 mg / mL.

[0049] In some embodiments, the pharmaceutical composition as described in any of the preceding embodiments comprises the following components:

[0050] (a) The anti-TF antibody-drug conjugate at concentrations from 1 mg / mL to 50 mg / mL,

[0051] (b) Surfactants ranging from 0.01 mg / mL to 2 mg / mL,

[0052] (c) Sugars ranging from 10 mg / mL to 120 mg / mL,

[0053] (d) Disodium edetate at concentrations ranging from 0.01 mg / mL to 1 mg / mL, and

[0054] (e) A buffer of 1 mM to 50 mM, wherein the pH of the pharmaceutical composition is 5.0 to 6.5.

[0055] In some embodiments, the pharmaceutical composition as described in any of the preceding embodiments comprises the following components:

[0056] (a) The anti-TF antibody-drug conjugate at concentrations ranging from 5 mg / mL to 40 mg / mL,

[0057] (b) Polysorbate 80 at concentrations ranging from 0.02 mg / mL to 1 mg / mL,

[0058] (c) Sucrose at concentrations of 40 mg / mL to 100 mg / mL,

[0059] (d) Disodium edetate at concentrations ranging from 0.01 mg / mL to 0.1 mg / mL, and

[0060] (e) a 5 mM to 30 mM histidine-histidine hydrochloride buffer, citrate-sodium citrate buffer, acetate-sodium acetate buffer, or succinate-sodium succinate buffer, wherein the pH of the pharmaceutical composition is 5.0 to 6.0.

[0061] In some embodiments, the pharmaceutical composition as described in any of the preceding embodiments comprises the following components:

[0062] (a) The anti-TF antibody-drug conjugate at concentrations ranging from 5 mg / mL to 40 mg / mL,

[0063] (b) Polysorbate 80 at concentrations ranging from 0.02 mg / mL to 1 mg / mL,

[0064] (c) Sucrose at concentrations of 40 mg / mL to 100 mg / mL,

[0065] (d) Disodium edetate at concentrations ranging from 0.01 mg / mL to 0.1 mg / mL, and

[0066] (e) A 5 mM to 30 mM histidine-histidine hydrochloride buffer, wherein the pH of the pharmaceutical composition is 5.0 to 6.0.

[0067] In some embodiments, the pharmaceutical composition as described in any of the preceding embodiments comprises the following components:

[0068] (a) The anti-TF antibody-drug conjugate at concentrations ranging from 5 mg / mL to 30 mg / mL,

[0069] (b) Polysorbate 80 at concentrations ranging from 0.02 mg / mL to 1 mg / mL,

[0070] (c) Sucrose at concentrations of 40 mg / mL to 100 mg / mL,

[0071] (d) Disodium edetate at concentrations ranging from 0.01 mg / mL to 0.1 mg / mL, and

[0072] (e) A 5 mM to 30 mM histidine-histidine hydrochloride buffer, wherein the pH of the pharmaceutical composition is 5.0 to 6.0.

[0073] In some embodiments, the pharmaceutical composition as described in any of the preceding embodiments comprises the following components:

[0074] (a) The anti-TF antibody-drug conjugate at concentrations of 15 mg / mL to 25 mg / mL,

[0075] (b) Polysorbate 80 at concentrations ranging from 0.1 mg / mL to 0.5 mg / mL.

[0076] (c) Sucrose at concentrations of 65 mg / mL to 95 mg / mL,

[0077] (d) Sodium edetate at concentrations ranging from 0.02 mg / mL to 0.08 mg / mL, and

[0078] (e) A 5 mM to 15 mM histidine-histidine hydrochloride buffer, wherein the pH of the pharmaceutical composition is 5.2 to 5.8.

[0079] In some embodiments, the pharmaceutical composition as described in any of the preceding embodiments comprises the following components:

[0080] (a) The anti-TF antibody-drug conjugate at concentrations of 15 mg / mL to 25 mg / mL,

[0081] (b) Polysorbate 80 at concentrations ranging from 0.1 mg / mL to 0.5 mg / mL.

[0082] (c) Sucrose at concentrations of 65 mg / mL to 95 mg / mL,

[0083] (d) Sodium edetate at concentrations ranging from 0.02 mg / mL to 0.08 mg / mL, and

[0084] (e) an 8 mM to 12 mM histidine-histidine hydrochloride buffer, wherein the pH of the pharmaceutical composition is 5.2 to 5.8.

[0085] In some embodiments, the pharmaceutical composition as described in any of the preceding embodiments comprises the following components:

[0086] (a) The anti-TF antibody-drug conjugate at a concentration of approximately 20 mg / mL,

[0087] (b) Approximately 0.3 mg / mL of polysorbate 80,

[0088] (c) Approximately 80 mg / mL of sucrose,

[0089] (d) Approximately 0.05 mg / mL of disodium edetate, and

[0090] (e) about 10 mM of histidine-histidine hydrochloride buffer, wherein the pH of the pharmaceutical composition is about 5.5.

[0091] In some embodiments, the pharmaceutical composition as described in any of the preceding embodiments comprises the following components:

[0092] (a) The anti-TF antibody-drug conjugate at concentrations from 1 mg / mL to 50 mg / mL,

[0093] (b) Surfactants ranging from 0.01 mg / mL to 2 mg / mL,

[0094] (c) Sugars ranging from 10 mg / mL to 120 mg / mL,

[0095] (d) Disodium edetate at concentrations ranging from 0.01 mg / mL to 1 mg / mL.

[0096] (e) excipients ranging from 0.01 mg / mL to 20 mg / mL, and

[0097] (f) A buffer of 1 mM to 50 mM, wherein the pH of the pharmaceutical composition is 5.0 to 6.5.

[0098] In some embodiments, the pharmaceutical composition as described in any of the preceding embodiments comprises the following components:

[0099] (a) The anti-TF antibody-drug conjugate at concentrations ranging from 5 mg / mL to 40 mg / mL,

[0100] (b) Polysorbate 80 at concentrations ranging from 0.02 mg / mL to 1 mg / mL,

[0101] (c) Sucrose at concentrations ranging from 10 mg / mL to 80 mg / mL,

[0102] (d) Disodium edetate at concentrations ranging from 0.01 mg / mL to 0.1 mg / mL.

[0103] (e) Glycine at concentrations of 5 mg / mL to 15 mg / mL, and

[0104] (f) A 10 mM to 40 mM histidine-histidine hydrochloride buffer, citrate-sodium citrate buffer, acetate-sodium acetate buffer, or succinate-sodium succinate buffer, wherein the pH of the pharmaceutical composition is 5.0 to 6.0.

[0105] In some embodiments, the pharmaceutical composition as described in any of the preceding embodiments comprises the following components:

[0106] (a) The anti-TF antibody-drug conjugate at concentrations ranging from 5 mg / mL to 40 mg / mL,

[0107] (b) Polysorbate 80 at concentrations ranging from 0.02 mg / mL to 1 mg / mL,

[0108] (c) Sucrose at concentrations ranging from 10 mg / mL to 80 mg / mL,

[0109] (d) Disodium edetate at concentrations ranging from 0.01 mg / mL to 0.1 mg / mL.

[0110] (e) Glycine at concentrations of 5 mg / mL to 15 mg / mL, and

[0111] (f) A 10 mM to 40 mM histidine-histidine hydrochloride buffer, wherein the pH of the pharmaceutical composition is 5.0 to 6.0.

[0112] In some embodiments, the pharmaceutical composition as described in any of the preceding embodiments comprises the following components:

[0113] (a) The anti-TF antibody-drug conjugate at concentrations of 15 mg / mL to 25 mg / mL,

[0114] (b) Polysorbate 80 at concentrations ranging from 0.1 mg / mL to 0.5 mg / mL.

[0115] (c) Sucrose at concentrations of 20 mg / mL to 60 mg / mL,

[0116] (d) Disodium edetate at concentrations ranging from 0.02 mg / mL to 0.08 mg / mL.

[0117] (e) Glycine at concentrations of 7 mg / mL to 11 mg / mL, and

[0118] (f) A 25 mM to 35 mM histidine-histidine hydrochloride buffer, wherein the pH of the pharmaceutical composition is 5.2 to 5.8.

[0119] In some embodiments, the pharmaceutical composition as described in any of the preceding embodiments comprises the following components:

[0120] (a) The anti-TF antibody-drug conjugate at a concentration of approximately 20 mg / mL,

[0121] (b) Approximately 0.3 mg / mL of polysorbate 80,

[0122] (c) Approximately 40 mg / mL of sucrose,

[0123] (d) Approximately 0.05 mg / mL of disodium edetate,

[0124] (e) Glycine at approximately 9 mg / mL, and

[0125] (f) about 30 mM of histidine-histidine hydrochloride buffer, wherein the pH of the pharmaceutical composition is about 5.5.

[0126] In some embodiments, the pharmaceutical composition as described in any of the preceding embodiments is a liquid formulation. In some embodiments, the solvent of the liquid formulation is water.

[0127] This disclosure also provides a lyophilized formulation, characterized in that the lyophilized formulation, upon reconstitution, can form a pharmaceutical composition as described in any of the preceding claims.

[0128] This disclosure also provides a lyophilized formulation, which is a lyophilized form of the pharmaceutical composition as described in any of the preceding claims.

[0129] This disclosure also provides a method for preparing a lyophilized formulation, comprising the step of lyophilizing a pharmaceutical composition as described in any of the preceding claims. In some embodiments, the lyophilization as described in any of the preceding claims sequentially comprises the steps of pre-freezing, primary drying, and secondary drying.

[0130] This disclosure also provides a lyophilized formulation obtained by freeze-drying a pharmaceutical composition as described in any of the preceding claims.

[0131] This disclosure also provides a reconstituted solution, characterized in that the reconstituted solution is prepared by reconstituted a lyophilized formulation as described in any of the preceding claims.

[0132] This disclosure also provides a reconstituted solution, which is a reconstituted form of the lyophilized formulation as described in any of the preceding claims.

[0133] In some embodiments, the reconstituted solution as described in any of the preceding embodiments has the same components and contents as the aforementioned pharmaceutical composition.

[0134] In some embodiments, the reconstituted solution as described in any of the preceding embodiments comprises the following components:

[0135] (a) The anti-TF antibody-drug conjugate at concentrations from 1 mg / mL to 50 mg / mL,

[0136] (b) Surfactants ranging from 0.01 mg / mL to 2 mg / mL,

[0137] (c) Sugars ranging from 10 mg / mL to 120 mg / mL,

[0138] (d) Disodium edetate at concentrations ranging from 0.01 mg / mL to 1 mg / mL, and

[0139] (e) A buffer of 1 mM to 50 mM, wherein the pH of the pharmaceutical composition is 5.0 to 6.5.

[0140] In some embodiments, the reconstituted solution as described in any of the preceding embodiments comprises the following components:

[0141] (a) The anti-TF antibody-drug conjugate at concentrations ranging from 5 mg / mL to 40 mg / mL,

[0142] (b) Polysorbate 80 at concentrations ranging from 0.02 mg / mL to 1 mg / mL,

[0143] (c) Sucrose at concentrations of 40 mg / mL to 100 mg / mL,

[0144] (d) Disodium edetate at concentrations ranging from 0.01 mg / mL to 0.1 mg / mL, and

[0145] (e) A 5 mM to 30 mM histidine-histidine hydrochloride buffer, wherein the pH of the pharmaceutical composition is 5.0 to 6.0.

[0146] In some embodiments, the reconstituted solution as described in any of the preceding embodiments comprises the following components:

[0147] (a) The anti-TF antibody-drug conjugate at concentrations ranging from 5 mg / mL to 30 mg / mL,

[0148] (b) Polysorbate 80 at concentrations ranging from 0.02 mg / mL to 1 mg / mL,

[0149] (c) Sucrose at concentrations of 40 mg / mL to 100 mg / mL,

[0150] (d) Disodium edetate at concentrations ranging from 0.01 mg / mL to 0.1 mg / mL, and

[0151] (e) A 5 mM to 30 mM histidine-histidine hydrochloride buffer, wherein the pH of the pharmaceutical composition is 5.0 to 6.0.

[0152] In some embodiments, the reconstituted solution as described in any of the preceding embodiments comprises the following components:

[0153] (a) The anti-TF antibody-drug conjugate at concentrations of 15 mg / mL to 25 mg / mL,

[0154] (b) Polysorbate 80 at concentrations ranging from 0.1 mg / mL to 0.5 mg / mL.

[0155] (c) Sucrose at concentrations of 65 mg / mL to 95 mg / mL,

[0156] (d) Sodium edetate at concentrations ranging from 0.02 mg / mL to 0.08 mg / mL, and

[0157] (e) A 5 mM to 15 mM histidine-histidine hydrochloride buffer, wherein the pH of the pharmaceutical composition is 5.2 to 5.8.

[0158] In some embodiments, the reconstituted solution as described in any of the preceding embodiments comprises the following components:

[0159] (a) The anti-TF antibody-drug conjugate at concentrations of 15 mg / mL to 25 mg / mL,

[0160] (b) Polysorbate 80 at concentrations ranging from 0.1 mg / mL to 0.5 mg / mL.

[0161] (c) Sucrose at concentrations of 65 mg / mL to 95 mg / mL,

[0162] (d) Sodium edetate at concentrations ranging from 0.02 mg / mL to 0.08 mg / mL, and

[0163] (e) an 8 mM to 12 mM histidine-histidine hydrochloride buffer, wherein the pH of the pharmaceutical composition is 5.2 to 5.8.

[0164] In some embodiments, the reconstituted solution as described in any of the preceding embodiments comprises the following components:

[0165] (a) The anti-TF antibody-drug conjugate at a concentration of approximately 20 mg / mL,

[0166] (b) Approximately 0.3 mg / mL of polysorbate 80,

[0167] (c) Approximately 80 mg / mL of sucrose,

[0168] (d) Approximately 0.05 mg / mL of disodium edetate, and

[0169] (e) about 10 mM of histidine-histidine hydrochloride buffer, wherein the pH of the pharmaceutical composition is about 5.5.

[0170] In some embodiments, the reconstituted solution as described in any of the preceding embodiments comprises the following components:

[0171] (a) The anti-TF antibody-drug conjugate at concentrations from 1 mg / mL to 50 mg / mL,

[0172] (b) Surfactants ranging from 0.01 mg / mL to 2 mg / mL,

[0173] (c) Sugars ranging from 10 mg / mL to 120 mg / mL,

[0174] (d) Disodium edetate at concentrations ranging from 0.01 mg / mL to 1 mg / mL.

[0175] (e) excipients ranging from 0.01 mg / mL to 20 mg / mL, and

[0176] (f) A buffer of 1 mM to 50 mM, wherein the pH of the pharmaceutical composition is 5.0 to 6.5.

[0177] In some embodiments, the reconstituted solution as described in any of the preceding embodiments comprises the following components:

[0178] (a) The anti-TF antibody-drug conjugate at concentrations ranging from 5 mg / mL to 40 mg / mL,

[0179] (b) Polysorbate 80 at concentrations ranging from 0.02 mg / mL to 1 mg / mL,

[0180] (c) Sucrose at concentrations ranging from 10 mg / mL to 80 mg / mL,

[0181] (d) Disodium edetate at concentrations ranging from 0.01 mg / mL to 0.1 mg / mL.

[0182] (e) Glycine at concentrations of 5 mg / mL to 15 mg / mL, and

[0183] (f) A 10 mM to 40 mM histidine-histidine hydrochloride buffer, citrate-sodium citrate buffer, acetate-sodium acetate buffer, or succinate-sodium succinate buffer, wherein the pH of the pharmaceutical composition is 5.0 to 6.0.

[0184] In some embodiments, the reconstituted solution as described in any of the preceding embodiments comprises the following components:

[0185] (a) The anti-TF antibody-drug conjugate at concentrations ranging from 5 mg / mL to 40 mg / mL,

[0186] (b) Polysorbate 80 at concentrations ranging from 0.02 mg / mL to 1 mg / mL,

[0187] (c) Sucrose at concentrations ranging from 10 mg / mL to 80 mg / mL,

[0188] (d) Disodium edetate at concentrations ranging from 0.01 mg / mL to 0.1 mg / mL.

[0189] (e) Glycine at concentrations of 5 mg / mL to 15 mg / mL, and

[0190] (f) A 10 mM to 40 mM histidine-histidine hydrochloride buffer, wherein the pH of the pharmaceutical composition is 5.0 to 6.0.

[0191] In some embodiments, the reconstituted solution as described in any of the preceding embodiments comprises the following components:

[0192] (a) The anti-TF antibody-drug conjugate at concentrations of 15 mg / mL to 25 mg / mL,

[0193] (b) Polysorbate 80 at concentrations ranging from 0.1 mg / mL to 0.5 mg / mL.

[0194] (c) Sucrose at concentrations of 20 mg / mL to 60 mg / mL,

[0195] (d) Disodium edetate at concentrations ranging from 0.02 mg / mL to 0.08 mg / mL.

[0196] (e) Glycine at concentrations of 7 mg / mL to 11 mg / mL, and

[0197] (f) A 25 mM to 35 mM histidine-histidine hydrochloride buffer, wherein the pH of the pharmaceutical composition is 5.2 to 5.8.

[0198] In some embodiments, the reconstituted solution as described in any of the preceding embodiments comprises the following components:

[0199] (a) The anti-TF antibody-drug conjugate at a concentration of approximately 20 mg / mL,

[0200] (b) Approximately 0.3 mg / mL of polysorbate 80,

[0201] (c) Approximately 40 mg / mL of sucrose,

[0202] (d) Approximately 0.05 mg / mL of disodium edetate,

[0203] (e) Glycine at approximately 9 mg / mL, and

[0204] (f) about 30 mM of histidine-histidine hydrochloride buffer, wherein the pH of the pharmaceutical composition is about 5.5.

[0205] In some embodiments, the pharmaceutical composition or reconstituted solution as described in any of the preceding embodiments is an intravenous injection formulation, a subcutaneous injection formulation, an intraperitoneal injection formulation, or an intramuscular injection formulation. In some embodiments, the pharmaceutical composition or reconstituted solution as described in any of the preceding embodiments is an intravenous injection formulation.

[0206] In some embodiments, the pharmaceutical composition or reconstituted solution as described in any of the preceding embodiments is suitable for intravenous, subcutaneous, intraperitoneal, or intramuscular injection. In some embodiments, the pharmaceutical composition or reconstituted solution as described in any of the preceding embodiments is suitable for intravenous injection.

[0207] In some embodiments, the pharmaceutical composition, reconstituted solution, or lyophilized formulation described in any of the preceding embodiments is used to prepare a drug for intravenous, subcutaneous, intraperitoneal, or intramuscular injection. In some embodiments, the pharmaceutical composition, reconstituted solution, or lyophilized formulation described in any of the preceding embodiments is used to prepare a drug for intravenous injection.

[0208] This disclosure also provides a medicine box comprising at least one container, each container independently containing a pharmaceutical composition as described in any of the preceding claims, a lyophilized formulation as described in any of the preceding claims, or a reconstituted solution as described in any of the preceding claims.

[0209] In some implementations, this disclosure also provides methods for diagnosing, treating, or alleviating symptoms in a subject, comprising administering to the subject an effective amount of the pharmaceutical composition as described in any of the preceding claims, the lyophilized formulation as described in any of the preceding claims, the reconstituted solution as described in any of the preceding claims, or the kit as described in any of the preceding claims.

[0210] In some embodiments, this disclosure also provides a method for treating or preventing a disease, the method comprising administering to a subject a therapeutically effective amount of a pharmaceutical composition as described in any of the preceding claims, a lyophilized formulation as described in any of the preceding claims, a reconstituted solution as described in any of the preceding claims, or a cassette as described in any of the preceding claims.

[0211] In some embodiments, this disclosure also provides pharmaceutical compositions as described in any of the preceding claims, lyophilized formulations as described in any of the preceding claims, reconstituted solutions as described in any of the preceding claims, or kits as described in any of the preceding claims for the treatment or prevention of disease.

[0212] In one aspect, this disclosure also provides the use of the pharmaceutical compositions as described in any of the preceding claims, the lyophilized formulations as described in any of the preceding claims, the reconstituted solutions as described in any of the preceding claims, or the cassettes as described in any of the preceding claims in the preparation of medicaments for the prevention or treatment of diseases.

[0213] This disclosure also provides pharmaceutical compositions as described in any of the preceding claims, lyophilized formulations as described in any of the preceding claims, reconstituted solutions as described in any of the preceding claims, or kits as described in any of the preceding claims for use as pharmaceuticals. In some embodiments, the pharmaceuticals are used to treat or prevent disease.

[0214] In some implementations, the disease is a tumor or cancer.

[0215] In some implementations, the disease is a disease or condition related to TF.

[0216] In some implementations, the disease is selected from breast cancer, pancreatic cancer, lung cancer (including non-small cell lung cancer and small cell lung cancer), esophageal cancer, ovarian cancer, cervical cancer, endometrial cancer, prostate cancer, bladder cancer, fallopian tube cancer, peritoneal cancer, colorectal cancer (including colon cancer and rectal cancer), head and neck cancer, and squamous cell carcinoma. Attached Figure Description

[0217] Figure 1 shows the results of PT detection of the ADC-1 disclosed herein in the pharmacokinetics experiment of cynomolgus monkeys.

[0218] Figure 2 shows the APTT results of the ADC-1 disclosed herein in the pharmacokinetics experiment in cynomolgus monkeys. Detailed Implementation

[0219] the term

[0220] To facilitate understanding of this disclosure, certain technical and scientific terms are specifically defined below. Unless otherwise expressly defined herein, all other technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this disclosure pertains.

[0221] The singular forms “a,” “an,” and “the” used in this disclosure include plural references unless the context clearly indicates otherwise.

[0222] Unless the context clearly requires otherwise, the words “comprising,” “having,” “including,” etc., in the patent specification and claims should be understood as “including but not limited to,” rather than as exclusive or exhaustive.

[0223] "Optional" or "optionally" means that the event or circumstances described below may, but do not have to, occur, including the circumstances in which the event or circumstances may or may not occur.

[0224] Those skilled in the art will understand that when used as a reference range, cutoff value, or specific value, "about" can mean within one or more standard deviations. Alternatively, "about" can mean a range with a difference of up to 20% (i.e., ±20%). Since many of the values ​​used herein were determined experimentally, those skilled in the art will understand that such determinations can vary between different experiments and are generally true across experiments. Due to this inherent variability, the values ​​used herein should not be unduly restricted. Therefore, the term "about" is used to cover variations of ±20%, ±10%, ±5%, ±1%, ±0.5%, or ±0.1% or less from a specified value.

[0225] Although this disclosure provides content ranges or content values, those skilled in the art will understand that the content ranges or content values ​​cover the acceptable range of error for the specific values ​​measured.

[0226] The three-letter and single-letter codes for amino acids used in this disclosure are as described in J. Biol. Chem., 243, p3558 (1968).

[0227] The term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimics that function in a manner similar to naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those that are subsequently modified, such as hydroxyproline, γ-carboxyglutamic acid, and O-phosphoserine. Amino acid analogs are compounds that have the same basic chemical structure as naturally occurring amino acids (i.e., the α-carbon bound to hydrogen, carboxyl, amino, and R groups), such as homoserine, ortholeucine, methionine sulfoxide, and methionine methylsulfonium. These analogs have modified R groups (e.g., ortholeucine) or modified peptide backbones but retain the same basic chemical structure as naturally occurring amino acids. Amino acid mimics are chemical compounds that have a structure different from the general chemical structure of amino acids but function in a manner similar to naturally occurring amino acids.

[0228] The term "antibody" is used in the broadest sense and encompasses a wide range of antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies; monospecific antibodies, multispecific antibodies (e.g., bispecific antibodies); full-length antibodies and antigen-binding fragments (or antigen-binding portions), as long as they exhibit the desired antigen-binding activity. "Natural antibody" refers to a naturally occurring immunoglobulin molecule. For example, a natural IgG antibody is a heterotetraglycoprotein of approximately 150,000 Daltons, composed of two identical light chains and two identical heavy chains bound by disulfide bonds. From the N to C terminus, each heavy chain has a variable region (VH), also called a variable heavy domain or heavy chain variable region, followed by a heavy chain constant region. The IgG heavy chain constant region (CH) typically contains three constant domains (CH1, CH2, and CH3); similarly, from the N to C terminus, each light chain has a variable region (VL), also called a variable light domain or light chain variable domain, followed by a constant light domain (light chain constant region, CL).

[0229] The term "variable region" or "variable domain" refers to the domain in the antibody heavy or light chain involved in antibody-antigen binding. In this paper, the antibody heavy chain variable region (VH) and light chain variable region (VL) each contain four conserved frame regions (FRs) and three complementarity-determining regions (CDRs). The term "complementarity-determining region" or "CDR" refers to the region within the variable domain that primarily facilitates antigen binding; "frame" or "FR" refers to the variable domain residues other than the CDR residues. The VH contains three CDR regions: HCDR1, HCDR2, and HCDR3; the VL contains three CDR regions: LCDR1, LCDR2, and LCDR3. Each VH and VL consists of three CDRs and four FRs arranged in the following order from the amino terminus (also called the N-terminus) to the carboxyl terminus (also called the C-terminus): FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. A single VH or VL may be sufficient to confer antigen-binding specificity.

[0230] The amino acid sequence boundaries of CDRs can be determined using various well-known schemes, such as the "Kabat" numbering rule (see Kabat et al. (1991), "Sequences of Proteins of Immunological Interest", 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD), the "Chothia" numbering rule, the "ABM" numbering rule, the "contact" numbering rule (see Martin, ACR. Protein Sequence and Structure Analysis of Antibody Variable Domains[J]. 2001), and the ImMunoGenTics (IMGT) numbering rule (Lefranc, MP et al., Dev. Comp. Immunol., 27, 55-77(2003); Front Immunol. 2018 Oct 16; 9: 2278), etc.; the correspondence between various numbering systems is well known to those skilled in the art and is exemplified as shown in Table 1 below.

[0231] Table 1. Relationship between CDR numbering systems

[0232] Unless otherwise stated, the variable regions and CDR sequences in this disclosure embodiment are subject to the "Kabat" numbering rule.

[0233] The term "monoclonal antibody" refers to a group of substantially homogeneous antibodies, meaning that the antibody molecules contained in this group have the same amino acid sequence, except for the possible small number of naturally occurring mutations. In contrast, polyclonal antibody formulations typically contain multiple different antibodies with different amino acid sequences in their variable structural domains, and they generally specifically target different epitopes. "Monoclonal" indicates the characteristic of an antibody obtained from a substantially homogeneous group of antibodies and should not be construed as requiring the antibody to be produced by any particular method. In some embodiments, the antibodies provided in this disclosure are monoclonal antibodies.

[0234] The antibodies disclosed herein may be derived from animals (such as antibodies from mice, birds, rabbits, camels, monkeys, etc.), chimeric antibodies, or humanized antibodies.

[0235] The term "chimeric" antibody refers to an antibody in which a portion of the heavy and / or light chain is derived from a specific source or species, while the remaining portion of the heavy and / or light chain is derived from another different source or species.

[0236] The term "humanized" antibody refers to an antibody that retains the reactivity of a non-human antibody while exhibiting lower immunogenicity in humans. For example, this can be achieved by retaining the non-human CDR region and replacing the rest of the antibody with its human counterpart (i.e., the frame region portion of the constant region and the variable region).

[0237] The term "affinity" refers to the overall strength of the non-covalent interaction between a single binding site of a molecule (e.g., an antibody) and its binding ligand (e.g., an antigen). Unless otherwise specified, as used herein, binding "affinity" refers to internal binding affinity, which reflects a 1:1 interaction between members of a binding pair (e.g., antibody and antigen). The affinity of molecule X for its ligand Y can typically be represented by the dissociation constant (KD). Affinity can be measured using conventional methods known in the art.

[0238] The terms "anti-TF antibody" and "TF-binding antibody" refer to antibodies capable of binding to TF or its epitopes with sufficient affinity. In one embodiment, the anti-TF antibody binds to unrelated proteins to a degree less than at least about 10% of the antibody's binding to TF, and this binding can be measured by BIACORE surface plasmon resonance assay.

[0239] The term "antigen" refers to a molecule or molecular moiety that can be bound by a selective binder of an antigen-binding protein (such as an antibody). An antigen may have one or more epitopes that can interact with different antigen-binding proteins (such as antibodies).

[0240] The term "epitope" refers to a region on an antigen that can specifically bind to an antibody or its antigen-binding fragment. Epitopes can be formed from consecutive amino acids (linear epitopes) or contain non-consecutive amino acids (conformal epitopes), for example, due to the folding of the antigen (i.e., tertiary folding of the antigen as a protein), which allows non-consecutive amino acids to be spatially close. The difference between conformational and linear epitopes is that antibody binding to a conformational epitope is lost in the presence of a denaturing solvent. An epitope contains at least 3, at least 4, at least 5, at least 6, at least 7, or 8-10 amino acids in a unique spatial conformation. Screening for antibodies that bind to a specific epitope (i.e., those that bind the same epitope) can be performed using methods routine in the art, such as, but not limited to, alanine scanning, Western blotting, peptide cleavage analysis, epitope excision, epitope extraction, chemical modification of the antigen, and cross-blocking.

[0241] The terms "capable of specific binding," "specific binding," or "binding" refer to the ability of an antibody to bind to a specific antigen or epitope with a higher affinity than other antigens or epitopes. Typically, antibodies bind at an affinity of approximately 1 × 10⁻⁶. -7 An equilibrium dissociation constant (KD) of M or less binds to an antigen or epitope. In some embodiments, the KD of antibody binding to an antigen is 10% or less (e.g., 1%) of the KD of the antibody binding to a nonspecific antigen (e.g., BSA, casein). KD can be measured using known methods, such as by FACS or surface plasmon resonance assays. However, antibodies that specifically bind to an antigen or an epitope within an antigen may be cross-reactive to other related antigens, for example, to corresponding antigens from other species (homologous), such as humans or monkeys, such as the cynomolgus (cyno), the chimpanzee (chimp), or the common marmoset (marmoset).

[0242] Antibody drug conjugates (ADCs) are conjugates obtained by linking an antibody (or its antigen-binding fragment) directly or through a linker to a drug.

[0243] A "drug" (abbreviated as D) is any substance that has biological or detectable activity (e.g., therapeutic agents, detectable markers, binders, etc.) and its prodrugs, which are metabolized in the body to become active agents. Examples of therapeutic agents include cytotoxic agents, chemotherapeutic agents, cell growth inhibitors, and immunomodulators. Chemotherapeutic agents are chemical compounds that can be used to treat cancer. Representative therapeutic agents include cytotoxins, cytotoxic agents, and cell growth inhibitors.

[0244] Cytotoxicity refers to the loss, elimination, and / or killing of target cells. Cytotoxic agents are drugs that have cytotoxic and / or cell growth-inhibiting effects on cells. Cell growth inhibition refers to the inhibition of cell proliferation. Cell growth inhibitors are drugs that have a cell growth-inhibiting effect on cells, thereby inhibiting the growth and / or expansion of specific subgroups of cells.

[0245] Additional representative therapeutic agents include radioisotopes, chemotherapeutic agents, immunomodulators, anti-angiogenic agents, antiproliferative agents, apoptosis-promoting agents, and cell-lysing enzymes (e.g., RNase). These drug descriptive terms are not mutually exclusive, and therefore, one or more of the aforementioned terms may be used to describe a therapeutic agent. For example, the selected radioisotope may also be a cytotoxic agent. Therapeutic agents can be prepared as pharmaceutically acceptable salts, acids, or derivatives of any of the above. Generally, conjugates containing a radioisotope as a drug are called radioimmunoconjugates, and those containing a chemotherapeutic agent as a drug are called chemoimmunoconjugates.

[0246] Examples of cytotoxic agents include, but are not limited to, anthracycline, orrisstatin, CC-1065, dolastatin, docalmicin, enediyne, geldanamycin, maytansine, puromycin, taxane, vinca alkaloids, SN-38, tubulolysin, hemiasterlin, eribulin, trabectedin, lurbinectedin, and their stereoisomers, isosteres, analogues, or derivatives. Chemotherapy agents, phytotoxicants, other bioactive proteins, enzymes (i.e., ADEPT), radioactive isotopes, and photosensitizers (i.e., for photodynamic therapy) may also be used.

[0247] The terms "connector unit" and "connector" refer to a chemical structural fragment or bond that is linked to an antibody at one end and a drug at the other. Connectors can also be attached to other connectors before being linked to an antibody or drug. Connector attachment to antibodies can be accomplished in various ways, such as via surface lysine residues, reductive coupling to oxidized carbohydrates, release of cysteine ​​residues via reducing interchain disulfide bonds, modification of reactive cysteine ​​residues at specific sites, and tags containing acyl donor glutamine, or modification of peptides to make them reactive endogenous glutamine in the presence of transglutaminase and amines. Various ADC linker systems are known in the art, including hydrazone-, disulfide-, and peptide-based links.

[0248] The connector may comprise one or more connector elements. Exemplary connector elements include 6-maleiminohexanoyl (“MC”), maleiminopropionyl (“MP”), valine-citrulline (“val-cit” or “vc”), alanine-phenylalanine (“ala-phe”), p-aminobenzyloxycarbonyl (“PAB”), N-succinimino-4-(2-pyridylthio)valerate (“SPP”), N-succinimino-4-(N-maleiminomethyl)cyclohexane-1-carboxylate (“SMCC”, also referred to herein as “MCC”), and N-succinimino-(4-iodo-acetyl)aminobenzoate (“SIAB”).

[0249] The linker may be selected from the following elements or combinations thereof: extensions, spacers, and amino acid units. The linker may be synthesized by methods known in the art, such as those described in US20050238649A1. The linker may be a “cleavable linker” that facilitates drug release into cells. For example, acid-labile linkers (e.g., hydrazones), protease-sensitive linkers (e.g., peptidase-sensitive linkers), photostable linkers, dimethyl linkers, or disulfide-containing linkers may be used (Chari et al., Cancer Research 52:127-131 (1992); US Patent No. 5,208,020).

[0250] “LD” is the connector-drug section formed when the drug (D) is connected to the connector (L).

[0251] "Drug loading," also known as drug-to-antibody ratio (DAR), refers to the average number of drugs conjugated to each antibody in an ADC. It can range from about 1 to about 10 drugs per antibody, and in some embodiments, from about 1 to about 8 drugs per antibody, preferably from the ranges of 2-8, 2-7, 2-6, 2-5, 2-4, 1-3, 3-4, 3-5, 5-6, 5-7, 5-8, and 6-8. The general formula of the ADC disclosed herein includes a set of antibody-drug conjugates within the aforementioned range. In embodiments disclosed herein, drug loading may be expressed as n, which can be a decimal or an integer. Drug loading can be determined using conventional methods such as UV / visible spectroscopy, mass spectrometry, ELISA assays, HIC, and RP-HPLC.

[0252] "Surfactant" refers to a surface-active agent, preferably a nonionic surfactant. Surfactants can reduce protein aggregation and / or particle formation in formulations. The amount of surfactant added is such that it reduces protein aggregation and minimizes particle formation in the formulation.

[0253] Poloxamer is an α-hydro-ω-hydroxy poly(ethylene oxide)a-poly(propylene oxide)b-poly(ethylene oxide)a block copolymer. It is formed by reacting propylene oxide and propylene glycol to form polypropylene glycol, followed by the addition of ethylene oxide to form the block copolymer. Here, 'a' represents the number of ethylene oxide units, and 'b' represents the number of propylene oxide units. Examples of poloxamer include, but are not limited to, poloxamer 188 (P188 or PF68). Specifically, in poloxamer 188, the copolymer contains 75–85 ethylene oxide units (a), 25–30 propylene oxide units (b), an ethylene oxide (EO) content of 79.9%–83.7%, and an average molecular weight of 7680–9510.

[0254] "Disodium edetate" (CAS No.: 6381-92-6, the disodium edetate described in this disclosure was purchased from SIGMA). The company's website lists its alternative names as disodium edetate dihydrate, EDTA disodium salt, EDTA-Na2, EDTA Na2, disodium edetate dihydrate, disodium edetate dihydrate, and calcium sodium edetate. Therefore, in this disclosure, "disodium edetate," "disodium edetate dihydrate," and "EDTA-2Na" are interchangeable, as they all refer to the substance with CAS No.: 6381-92-6.

[0255] "Viscosity modifier" is a conventional pharmaceutical excipient added to adjust the viscosity of a formulation. Viscosity modifiers can be inorganic salts and amino acid salts. Preferably, the inorganic salt is selected from sodium chloride, calcium chloride, and magnesium chloride. More preferably, the amino acid salt is selected from arginine hydrochloride, histidine hydrochloride, lysine hydrochloride, histidine acetate, etc.

[0256] "Buffer" refers to a buffering agent that tolerates pH changes through the action of its acid-base conjugate components. Examples of buffers that maintain pH within an appropriate range include acetate, succinate, gluconate, histidine, oxalate, lactate, phosphate, citrate (also known as tartrate), tartrate, fumarate, glycylglycine, and other organic acid buffers.

[0257] "Histidine buffer" is a buffer containing histidine. Examples of histidine buffers include histidine-histidine acetate, histidine-histidine hydrochloride, histidine-histidine phosphate, and histidine-histidine sulfate buffers, with histidine-histidine acetate buffers being preferred. Histidine-histidine acetate buffers can be prepared by reacting histidine with acetic acid, or by reacting histidine with histidine acetate.

[0258] "Displacement" refers to the replacement of the solvent system in which antibody proteins are dissolved. For example, a buffer system containing antibody proteins may be physically replaced by a high-salt or hypertonic solvent system containing antibody proteins, thereby ensuring the presence of antibody proteins within the stable formulation. Such physical operations include, but are not limited to, ultrafiltration, dialysis, or centrifugation.

[0259] "Pharmaceutical composition" means a mixture containing one or more antibodies described herein or their physiologically / pharmacologically acceptable salts or prodrugs, along with other chemical components, such as physiologically / pharmacologically acceptable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration to a living organism, thereby promoting the absorption of the active ingredient and the exertment of its biological activity.

[0260] The term "pharmaceutically acceptable salt" or "medicinal salt" refers to the salts of the antibody-drug conjugates disclosed herein, which are safe and effective when used in subjects and possess the intended biological activity. As an example, the antibody-drug conjugates disclosed herein contain at least one amino group and can therefore form salts with acids. Non-limiting examples of pharmaceutically acceptable salts include: hydrochloride, hydrobromide, hydroiodide, sulfate, hydrogen sulfate, citrate, acetate, succinate, ascorbate, oxalate, nitrate, sorbate, hydrogen phosphate, dihydrogen phosphate, salicylate, hydrogen citrate, tartrate, maleate, fumarate, formate, benzoate, methanesulfonate, ethanesulfonate, benzenesulfonate, and p-toluenesulfonate.

[0261] "Pharmaceutically acceptable carriers" or "pharmaceutically acceptable excipients" include any material that, when combined with an active ingredient, allows the ingredient to retain its biological activity and does not react with the subject's immune system. Examples include, but are not limited to, any standard pharmaceutical carrier, such as phosphate-buffered saline solutions, 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 physiological (0.9%) saline. Compositions containing such carriers are formulated using well-known conventional methods (see, for example, Remington's Pharmaceutical Sciences, 18th edition, A. Gennaro, editor, Mack Publishing Co., Easton, PA, 1990; and R. Remington, The Science and Practice of Pharmacy, 20th edition, Mack Publishing, 2000).

[0262] "Lyophilized formulation" refers to a pharmaceutical composition or formulation obtained by vacuum freeze-drying a liquid or solution preparation. Typically, freeze-drying includes pre-freezing, primary drying, and secondary drying. Pre-freezing aims to freeze the product to obtain a crystalline solid; in some embodiments, the pre-freezing temperature is set to -45°C, and the pre-freezing rate is set to 1°C / min. Primary drying, also known as main drying, is the main stage of sample freeze-drying and aims to remove ice from the product while maintaining its shape and minimizing damage. Improper selection of the temperature and vacuum level during primary drying can lead to product collapse; higher temperatures and vacuum levels increase freeze-drying efficiency but also increase the risk of product collapse. In some embodiments, the primary drying temperature can be a temperature conventional in the art, such as -30°C to 0°C. Secondary drying, also known as desorption drying, is the main step of removing bound water from the product by applying an ultimate vacuum (0.01 mbar) and increasing the temperature (20°C to 40°C). Because most biological products are temperature-sensitive, the secondary drying temperature is often chosen at the lower end of the temperature range, such as 25°C. The freeze-drying time depends on the freezer, the dosage of the freeze-dried formulation, and the container of the freeze-dried drug. Such adjustments are well known to those skilled in the art.

[0263] Unless otherwise specified, the solvent in the solution form of the pharmaceutical compositions described in this disclosure is water.

[0264] In this disclosure, "pharmaceutical composition" and "formulation" are not mutually exclusive.

[0265] The pharmaceutical compositions disclosed herein achieve a stable effect: the antibodies contained therein substantially retain their physical and / or chemical stability and / or biological activity after storage. Preferably, the pharmaceutical compositions substantially retain their physical and chemical stability and their biological activity after storage. The storage period is generally selected based on the intended shelf life of the pharmaceutical composition. Currently, there are various analytical techniques available for measuring protein stability, which can measure stability after storage at a selected temperature for a selected period of time.

[0266] Stable formulations include those in which no significant changes are observed when stored at refrigerated temperatures (2°C–8°C) for at least 1 month, at least 3 months, at least 6 months, preferably 1 year, and even more preferably up to 2 years. Stable liquid formulations also include those that exhibit the desired characteristics after storage at temperatures including 25°C for periods of 1 month, 3 months, or 6 months. Furthermore, stable liquid formulations also include those that exhibit the desired characteristics after storage at 40°C for periods of 4 weeks, 1 month, 3 months, or 6 months. Typical examples of stability include antibody aggregation or degradation typically not exceeding about 10%, preferably not exceeding about 5%, as determined by SEC-HPLC. Visually, the formulation is a pale yellow, nearly colorless, clear liquid or a colorless, clear liquid, or clear to slightly milky white. The concentration, pH, weight, and molecular osmotic pressure of the formulation exhibit aggregation of not more than about 10%, preferably not more than about 5%.

[0267] If, after visual inspection of color and / or clarity, or by means of UV light scattering, size exclusion chromatography (SEC), and dynamic light scattering (DLS), the antibody does not show significant increase in aggregation, precipitation, and / or denaturation, then the antibody “retains its physical stability” in the pharmaceutical formulation. Changes in protein conformation can be evaluated by fluorescence spectroscopy (which determines the tertiary structure of the protein) and by FTIR spectroscopy (which determines the secondary structure of the protein).

[0268] If an antibody does not exhibit significant chemical changes, then the antibody "retains its chemical stability" in the pharmaceutical formulation. Chemical stability can be assessed by detecting and quantifying the chemically altered form of the protein. Degradation processes that frequently alter the chemical structure of a protein include hydrolysis or truncation (evaluated by methods such as size exclusion chromatography and CE-SDS), oxidation (evaluated by methods such as peptide mapping combined with mass spectrometry or MALDI / TOF / MS), deamidation (evaluated by methods such as ion exchange chromatography, capillary isoelectric focusing, peptide mapping, and isofpartate measurement), and isomerization (evaluated by measuring isofpartate content, peptide mapping, etc.).

[0269] If the antibody's biological activity at a given time is within a predetermined range of the biological activity exhibited when the pharmaceutical formulation is prepared, then the antibody "retains its biological activity" in the pharmaceutical formulation.

[0270] "Administration," "giving," and "treatment," when applied to animals, humans, experimental subjects, cells, tissues, organs, or biological fluids, refer to the contact of an exogenous drug, therapeutic agent, diagnostic agent, or composition with the animal, human, subject, cell, tissue, organ, or biological fluid. "Administration," "giving," and "treatment" can refer to, for example, therapeutic, pharmacokinetic, diagnostic, research, and experimental methods. Cellular treatment includes contact between a reagent and cells, as well as contact between a reagent and a fluid, wherein the fluid is in contact with the cells. "Administration," "giving," and "treatment" also mean, by means of a reagent, diagnostic agent, conjugate composition, or by means of another cell in vitro and ex vivo, such as cells. "Treatment," when applied to humans, veterinary, or research subjects, refers to therapeutic treatment, preventative or prophylactic measures, research, and diagnostic applications.

[0271] "Treatment" means administering an oral or topical therapeutic agent, such as a pharmaceutical composition comprising any of the substances disclosed herein, to a patient who has symptoms of one or more diseases, and the therapeutic agent is known to have a therapeutic effect on these symptoms. Typically, a therapeutic agent is administered in a treated patient or population in an amount that effectively relieves symptoms of one or more diseases, in order to induce the regression of such symptoms or inhibit their progression to any clinically measurable degree. The amount of a therapeutic agent that effectively relieves any specific disease symptom (also referred to as a "therapeuticly effective amount") can vary depending on a variety of factors, such as the patient's disease state, age, and weight, and the drug's ability to produce the desired therapeutic effect in the patient. Whether the disease symptoms have been relieved can be evaluated using any clinical test method commonly used by a physician or other healthcare professional to assess the severity or progression of the symptoms. Although the embodiments disclosed herein (e.g., treatment methods or products) may be ineffective in alleviating symptoms of each target disease, they should reduce symptoms of the target disease in a statistically significant number of patients, as determined by any statistical test known in the art, such as the Student t-test, chi-square test, U-test according to Mann and Whitney, Kruskal-Wallis test (H-test), Jonckheere-Terpstra test, and Wilcoxon test.

[0272] "Effective amount" includes an amount sufficient to improve or prevent the symptoms or condition of a medical condition. Effective amount also means an amount sufficient to allow or facilitate a diagnosis. The effective amount used on a subject may vary depending on factors such as the condition to be treated, the subject's overall health, the route and dosage of administration, and the severity of side effects. Effective amount may be the maximum dose or administration regimen that avoids significant side effects or toxicity. Subjects disclosed herein may be animal or human subjects.

[0273] The pharmaceutical compositions disclosed herein may be administered by any suitable means, including parenteral, intrapulmonary, and intranasal administration, and, if local treatment is required, intralesional administration. Parenteral infusion includes intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. Administration may be carried out via any suitable route, such as by injection, such as intravenous or subcutaneous injection. Various dosing schedules are considered herein, including, but not limited to, single or multiple administrations at multiple time points, bolus administration, and pulsatile infusion. In some embodiments, the pharmaceutical compositions disclosed herein are administered by intravenous injection.

[0274] The pharmaceutical compositions disclosed herein will be formulated, administered, and applied in accordance with good medical practice. Factors considered in this context include the specific condition being treated, the specific mammal being treated, the individual patient's clinical condition, the cause of the condition, the site of delivery of the agent, the method of administration, the timing of administration, and other factors known to a medical practitioner. Optionally, the pharmaceutical composition may also be formulated with one or more other agents for the prevention or treatment of said condition. The effective amount of such other agents depends on the amount of antigen-binding molecules present in the pharmaceutical composition, the type of condition or treatment, and other factors. It may be used at the same dosage and route of administration as described herein, or at about 1% to 99% of the dosage described herein, or at any dosage and any route of administration determined empirically / clinically to be appropriate.

[0275] Details of one or more embodiments of this disclosure are set forth in the foregoing description. While any methods and materials similar to or the same as those described herein may be used to implement or test this disclosure, preferred methods and materials are described below. Other features, objects, and advantages of this disclosure will be apparent from the description and claims. In the description and claims, the singular form includes plural references unless the context clearly indicates otherwise. Unless otherwise defined, all technical and scientific terms used herein have their general meaning as understood by one of ordinary skill in the art to which this disclosure pertains. All patents and publications referenced in the description are incorporated herein by reference. The following embodiments are presented to illustrate preferred embodiments of this disclosure more fully. These embodiments should not be construed in any way as limiting the scope of this disclosure, which is defined by the claims.

[0276] Example

[0277] The present disclosure is further described below with reference to embodiments, but these embodiments are not intended to limit the scope of the invention.

[0278] Experimental methods not specifying specific conditions in the examples or test cases disclosed herein are generally performed under standard conditions or as recommended by the raw material or product manufacturer. Reagents not specifying their source are commercially available, standard reagents.

[0279] I. Antibody Preparation

[0280] PCT / CN2024 / 090058 (Application date: 2024-04-26; Priority patent application numbers: CN 202310476753.9, CN 202311512744.7) is included herein by reference in its entirety.

[0281] Example 1: Cloning and Construction of Antigens and Antibodies

[0282] Molecular clones of antibodies (containing both light and heavy chains) and antigens were constructed using overlap extension PCR or gene synthesis methods known in the art. The antibody and protein antigen sequences were separately constructed into the pTT5 vector and analyzed using Expi293F. TM Cells (A14528, Gibco) were transfected to obtain the corresponding antibodies and antigen proteins.

[0283] antigen protein sequence

[0284] The human TF (hTF) protein sequence was obtained from NCBI (AAA61151.1), and its extracellular region (ECD) contains 219 amino acids (Ser33-Glu251). The monkey TF (cyno TF, cTF) protein sequence was obtained from NCBI (XP_005542723.1), and its extracellular region (ECD) contains 220 amino acids (Ser33-Glu252). The rat TF (rTF) protein sequence was obtained from NCBI (AAA16966.1), and its extracellular region (ECD) contains 224 amino acids (Ala29-Glu252). The mouse TF (mTF) protein sequence was obtained from UniProtKB (P20352), and its extracellular region (ECD) contains 223 amino acids (Ala29-Glu251). The extracellular region sequence of TF may be tagged with His or human Fc region (hFc) or mouse Fc region (mFc).

[0285] For TF immunization and detection, the plasmids used to prepare the proteins involved were prepared using the pTT5 vector, or using ExpiFectamine. TM The 293 transfection kit (A14525, Gibco) was used to transfect the plasmid into Expi293F. TM Cells acquire target proteins.

[0286] The antigen protein sequence is as follows:

[0287] Human TF complete sequence:

[0288] Cyno TF full sequence:

[0289] Rat TF full sequence:

[0290] Mouse TF complete sequence:

[0291] Human TF ECD-His sequence (hTF-his):

[0292] Human TF ECD-hFc sequence (hTF-hFc):

[0293] cyno TF ECD-hFc sequence (cTF-hFc):

[0294] Cyno TF ECD-His sequence (cTF-his):

[0295] cyno TF ECD-mFc sequence (cTF-mFc):

[0296] Rat TF ECD-His sequence:

[0297] Mouse TF ECD-His sequence:

[0298] The human-rat hybrid antigen HuRatTF was constructed by combining amino acids 1-130 (Met1-Ala130) of rat TF with amino acids 130-245 (Pro130-Glu245) of human TF and adding a His tag. Some amino acids affecting coagulation were replaced with amino acid Ala, and certain sites in the rat TF sequence were mutated to their corresponding positions in the human TF sequence. The protein sequence (including the signal peptide) is as follows: Note: Italics indicate human TF sequences, dashed lines indicate signal peptides, double underlines indicate rat TF amino acid mutations to corresponding human TF amino acids, underlines indicate site mutations affecting coagulation to alanine (Ala), and wavy lines indicate His tags.

[0299] Sequences used to construct antigen cell lines

[0300] The plasmids used in TF immunization and detection were prepared according to the vector in Cell, 01 Aug 2005, 122(3):473-48. The piggyBac transposon plasmid containing the antigen sequence was transfected into NIH / 3T3 (ATCC, CRL-1658) and / or CHO-K1 (ATCC, CCL-61) cells using Lipofectamine 3000 transfection reagent (Invitrogen, L3000015) and the transposase hyPBase plasmid, and then sorted by flow cytometry (BD FACSAria). TM The target cell line was obtained using a Fusion Cell Sorter. The antigen sequence used to construct the cell line consisted of the full-length TF antigen, the autoclavicle protein (2A), and the green fluorescent protein (EGFP) sequence. Details are as follows:

[0301] piggyBac transposon plasmid constituent elements (PB 3'LTR and PB 5'LTR, NCBI-KF658273.1):

[0302] PB 3'LTR-CMV promoter-antigen sequence-beta-globin poly(A)-PB 5'LTR-Ampicillin resisitance gene-bla promoter

[0303] Transposase hyPBase plasmid constituent elements (hyPBase, NCBI-OL519599.1):

[0304] EF1a promoter-hyPBase-BGH polyA-f1 origin-SV40 early promoter and origin-Neomycin resistance gene-SV40 polyA-pUC origin-Ampicillin resisitance gene-bla promoter

[0305] Human TF-2A-EGFP sequence (hTF):

[0306] Cyno TF-2A-EGFP sequence (cTF): Note: Italics represent TF sequences, double underlines represent 2A sequences, and wavy lines represent EGFP sequences.

[0307] Referring to patent WO2010066803A2, a segment of the human TF sequence was replaced with the corresponding sequence of mouse TF, resulting in the preparation of seven human-mouse chimeric antigens: hTF1 (1-41 mm), hTF2 (42-84 mm), hTF3 (85-122 mm), hTF4 (123-137 mm), hTF7 (138-184 mm), hTF5 (185-225 mm), and hTF6 (226-250 mm). These seven antigens are abbreviated as hTF1, hTF2, hTF3, hTF4, hTF7, hTF5, and hTF6. Note that hTF7 is not mentioned in patent WO2010066803A2 and is a new addition in this disclosure. The human TF antigenic epitopes corresponding to these seven antigens in the test examples are shown in Table 2.

[0308] Table 2

[0309] The seven chimeric antigen sequences are as follows:

[0310] hTF1 (1-41mm) sequence:

[0311] hTF2 (42-84mm) sequence:

[0312] hTF3 (85-122mm):

[0313] hTF4 (123-137 mm):

[0314] hTF7 (138-184 mm):

[0315] hTF5 (185-225 mm):

[0316] hTF6 (226-250mm):

[0317] Note: Italics represent human TF sequences, underlines represent mouse TF sequences, double underlines represent 2A sequences, and wavy lines represent EGFP sequences.

[0318] Epitope analysis was performed using FACS binding assays, revealing that the disclosed antibody binds to specific epitopes of TF.

[0319] Positive control antibody sequence

[0320] The positive control TF011 antibody sequence was derived from the monoclonal antibody sequence of tisotumab vedotin, and the variable region sequence of the H39 antibody was derived from WO2018036117A1.

[0321] TF011 antibody heavy chain sequence (monoclonal antibody sequence of tisotumab vedotin):

[0322] TF011 antibody light chain sequence (monoclonal antibody sequence of tisotumab vedotin):

[0323] H39 antibody heavy chain sequence (variable region + human IgG1 constant region):

[0324] H39 antibody light chain sequence (variable region + human κ constant region):

[0325] Negative control antibody hIgG1 (where VH / VL is derived from the C25 antibody of patent US6114143A):

[0326] The amino acid sequence of the hIgG1 antibody heavy chain (C25 heavy chain variable region + human IgG1 constant region):

[0327] The amino acid sequence of the hIgG1 antibody light chain (C25 light chain variable region + human κ constant region):

[0328] Note: The underlined part is the variable region sequence.

[0329] Example 2: Purification of antigen and antibody proteins

[0330] His-tagged antigens were affinity purified using a Ni Sepharose excel 25 mL column (GE Healthcare, 17-3712-01), while Fc-tagged antigens or antibodies were affinity purified using a Protein A-MabSelect SuRe 25 mL column (GE Healthcare, 17-5438-01). The affinity-purified proteins were then subjected to size exclusion chromatography to remove aggregates or fragments. The specific methods are as follows:

[0331] Nickel column purification: The nickel column was equilibrated by rinsing with PBS solution. The supernatant from cell transfection was centrifuged at high speed to remove impurities before loading the cells. When the nickel column was rinsed with PBS solution to A... 280The reading drops to the baseline. The nickel column is rinsed with PBS solution containing 10 mM imidazole to remove non-specifically bound miscellaneous proteins, and then the target protein is eluted with PBS solution containing 300 mM imidazole. The elution peak liquid is collected and changed to PBS for storage.

[0332] Size exclusion purification: The SEC column (GE, superdex75) is rinsed and equilibrated with PBS solution. The protein obtained by nickel column purification or Protein A purification is loaded (loading volume ≤ 3% of the column volume), and eluted with PBS solution as the mobile phase. Each elution peak is collected, and the fraction where the target protein is located is identified by SDS-PAGE.

[0333] Affinity chromatography of antibody or Fc fusion protein: The supernatant after cell transfection is centrifuged at high speed to remove impurities and then loaded onto the Protein A column. When the Protein A column is rinsed with PBS solution to A 280 The reading drops to the baseline. The target protein is eluted with 100 mM acetic acid at pH 3.5, and the eluate is neutralized with 1 M Tris-HCl at pH 8.0 and changed to PBS. If necessary, gel filtration (Superdex200, GE) can be used subsequently to further remove aggregates.

[0334] Example 3: Preparation of anti-human TF monoclonal antibody

[0335] 3.1 Immunization

[0336] The anti-human TF monoclonal antibody is obtained by hybridoma technology. SJL white mice, female, 6 - 8 weeks old (Shanghai Slake Experimental Animal Co., Ltd., animal production license number: SCXK(Shanghai)2017 - 0005) are used in the experiment. Feeding environment: SPF level. After the mice are purchased, they are raised in the laboratory environment for 1 week, with a 12 / 12 hour light / dark cycle adjustment, temperature 20 - 25 °C; humidity 40 - 60%. The mice that have adapted to the environment are immunized according to the following protocol.

[0337] 3.2 Immunization protocol

[0338] The immunizing antigens of anti-human TF antibody 67 are hTF-NIH / 3T3 cells (SEQ ID NO:12), cTF-NIH / 3T3 cells (SEQ ID NO:13), hTF-his protein (SEQ ID NO:5) and cTF-mFc protein (SEQ ID NO:75), and the immunization method is cell and protein immunization. For the first immunization, hTF-NIH / 3T3 cells are used. Before immunizing the cells, Gold Adjuvant (Sigma, T2684) 0.1 mL / mouse is injected intraperitoneally into the mouse, and half an hour later, 0.1 mL of normal saline diluted to 10 8Cell suspension of 10 cells / mL. A second immunization was administered on day 14 using cTF-NIH / 3T3 cells, with 0.1 mL of physiological saline diluted to 10 cells / mL injected intraperitoneally into each mouse. 8 Cell suspension of cells / mL. A third immunization was administered on day 35, using hTF-his protein, 50 μg / animal / dose, antigen and adjuvant (…). The ratio of Gold Adjuvant (1:1) was used, and the antigen and adjuvant were fully emulsified before vaccination. A fourth immunization was administered on day 49, using hTF-his protein, 50 μg / animal / dose, with a ratio of antigen to adjuvant (…). The ratio of Gold Adjuvant was 1:1, and the antigen and adjuvant were fully emulsified before inoculation. Blood samples were collected on days 29, 47, and 61, and the antibody titers in mouse serum were determined using ELISA and FACS. After the fourth immunization, mice with high antibody titers in their serum that were trending towards a plateau were selected for spleen cell fusion. Three days before spleen cell fusion, a booster immunization was performed by intraperitoneal (ip) injection of 50 μg / mouse of a 1:1 mass mixture of hTF-his and cTF-mFc antigen-protein solution prepared with physiological saline.

[0339] 3.3 Spleen cell fusion

[0340] Splenic lymphocytes were electrofusion-mediated to fused with myeloma cells Sp2 / O-Ag14 (ATCC, CRL-8287) TM Hybridoma cells were obtained by fusing the cells together. The hybridoma cells were then subjected to a process of 3-4 × 10⁻⁶. 5 The cells were resuspended at a density of 1 / mL in complete medium (IMDM medium containing 20% ​​FBS, 1×HAT, and 1×OPI) and seeded at 150 μL / well in 96-well cell culture plates. After incubation at 37°C and 5% CO2 for 3–4 days, the supernatant was removed, and 200 μL / well of HT complete medium (IMDM medium containing 20% ​​FBS, 1×HT, and 1×OPI) was added. The cells were then incubated at 37°C and 5% CO2 for 3–5 days before detection.

[0341] 3.4 Hybridoma cell screening

[0342] Once the hybridoma cell density reaches 90%, the cell supernatant is aspirated, and initial screening is performed in 96-well plates using a cell-based ELISA method. The selected positive wells are then transferred to 24-well plates. After 2-3 days, when the cell density reaches 90%, the cell supernatant is aspirated, and secondary screening is performed in 24-well plates using FACS and / or competition and / or coagulation assays. Single-well cells are selected for subcloning to obtain single-cell clones. The supernatant from the subcloned cells is collected for retesting. Hybridoma clones obtained through the above experiments are used for subsequent experiments.

[0343] Example 4: Determination of the amino acid sequence of the variable region of a mouse monoclonal antibody

[0344] The hybridoma monoclonal cell line obtained in Example 3 was subjected to variable region amino acid sequencing. The mouse hybridoma variable region sequence was combined with the human constant region sequence to form a human-mouse chimeric antibody (CHAb) sequence. The chimeric antibody was then expressed and identified. The specific method is as follows:

[0345] First, total RNA was obtained from hybridoma monoclonal cell lines using conventional and well-known methods, and then the reverse transcription kit PrimeScript was used. TM IV. cDNA was prepared using the first-strand cDNA Synthesis Mix (TAKARA, 6215A). PCR was performed using the cDNA as a template to amplify PCR fragments containing variable regions of the light and heavy chains. The corresponding variable region sequences were obtained by sequencing the PCR fragments. Using standard methods, the obtained light and heavy chain sequences were cloned into the pTT5 vector to express recombinant monoclonal antibodies, and their activity was verified.

[0346] The amino acid residues of the VH / VL CDR of the anti-human TF antibody were determined and annotated using the Kabat numbering system.

[0347] Sequence of mouse hybridoma cell monoclonal antibody 67#:

[0348] 67# Heavy chain variable region:

[0349] 67# Light Chain Variable Zone:

[0350] Table 3. CDR of 67# murine antibody

[0351] Human IgG1 heavy chain constant region:

[0352] Human κ light chain constant region:

[0353] The heavy chain and light chain sequences of the chimeric antibody CH67 are as follows:

[0354] CH67 heavy chain:

[0355] CH67 Light Chain:

[0356] Example 5: Humanization of anti-human TF antibody 67#

[0357] The light and heavy chain sequences of the murine anti-TF monoclonal antibody 67# obtained in Example 4 were compared for homology with those in the IMGT human antibody heavy and light chain variable region germline gene database using MOE software. Germline genes of the heavy and light chain variable regions with high homology to 67# were selected as templates, and the CDRs of this murine antibody were transplanted into the corresponding human templates, forming variable region sequences in the order FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Exemplarily, in the following specific examples, the CDR amino acid residues were determined and annotated using the Kabat numbering system.

[0358] For antibody #67, the human lineage light chain template is selected from FR1, FR2, and FR3 of IGKV4-1*01 or IGKV1-33*01, and FR4 of IGKJ4*01; the human lineage heavy chain template is selected from FR1, FR2, and FR3 of IGHV2-26*01 or IGHV4-30-4*01, and FR4 of IGHJ6*01. Optionally, amino acid residues at positions 8, 9, 10, 43, and / or 73 on the variable region of the light chain of the humanized antibody are substituted; and / or amino acid residues at positions 1, 6, 16, 27, 29, 30, 42, 44, 49, 71, 73, 76, 78, and / or 81 on the variable region of the heavy chain of the humanized antibody are substituted.

[0359] Table 4. Human-centered design of #67 Note: For example, G42R means that the 42-bit G was mutated back to R according to the Kabat numbering system.

[0360] The heavy chain variable region and light chain variable region sequences of humanized antibody #67 are as follows:

[0361] >hu67-VH1:

[0362] >hu67-VH2:

[0363] >hu67-VH3:

[0364] >hu67-VH4:

[0365] >67VL-VL1:

[0366] >67VL-VL2:

[0367] >67VL-VL3:

[0368] >67VL-VL4:

[0369] Note: In the above sequences, the order is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4; the underlined part is the CDR sequence determined according to the Kabat numbering system; the non-underlined part is the FR sequence; the double-underlined bold part is the amino acid point mutation.

[0370] The heavy chain variable region and light chain variable region of the 67# humanized antibody were recombined with the human heavy chain IgG1 constant region (SEQ ID NO: 54) and the human light chain Kappa constant region (SEQ ID NO: 55), respectively, to obtain the humanized antibodies shown in Table 5 below.

[0371] Table 5. Humanized Antibody #67

[0372] The heavy chain and light chain sequences of the humanized antibody are as follows:

[0373] Heavy chain sequences hu67-1 / hu67-5 / hu67-9 / hu67-13:

[0374] Heavy chain sequences hu67-2 / hu67-6 / hu67-10 / hu67-14:

[0375] hu67-3 / hu67-7 / hu67-11 / hu67-15 heavy chain sequences:

[0376] hu67-4 / hu67-8 / hu67-12 / hu67-16 heavy chain sequences:

[0377] The light chain sequence hu67-1 / hu67-2 / hu67-3 / hu67-4:

[0378] Light chain sequence hu67-5 / hu67-6 / hu67-7 / hu67-8:

[0379] Light chain sequences hu67-9 / hu67-10 / hu67-11 / hu67-12:

[0380] Light chain sequences hu67-13 / hu67-14 / hu67-15 / hu67-16:

[0381] Example 6: Affinity maturation of hu67-11

[0382] The human anti-TF monoclonal antibody hu67-11 obtained in Example 5 was used as an antigen to construct an antibody yeast library for affinity maturation, thereby improving the cell-binding affinity of the target antibody. The above antibodies were cloned, expressed, purified, and then tested to select the optimal humanized antibody. Details are as follows.

[0383] To obtain better anti-TF hu67-11 related antibodies, hu67-11 was maturated for affinity using yeast display platform technology. Based on hu67-11-scFv (SEQ ID NO:71), affinity-matured yeast libraries targeting human TF and monkey TF binding to CDR were designed and prepared, and new mutants were screened from them.

[0384] Construction of yeast libraries: Degenerate primers were designed, and the designed mutant amino acids were introduced into the hu67-11-scFv mutant library by PCR. The size of each library was 10. 9 The constructed yeast library was then validated for diversity using next-generation sequencing.

[0385] In the first round of screening, yeast from the hu67-11-scFv mutant library was incubated with biotinylated hTF-hFc protein in 50 mL of 0.1% bovine serum albumin (BSA)-phosphate buffer (PBSA) at room temperature for 1 hour. The mixture was then washed three times with PBSA to remove unbound antibody fragments. Streptomycin beads (Milenvi Biotec, Auburn, CA) were then added to the mutant library bound to biotinylated hTF-hFc protein and loaded onto an AutoMACS system for sorting. Antibody libraries with high affinity for human TF were collected and amplified. The cultures were then induced to express the antibodies. The resulting enriched libraries were subjected to a second round of screening targeting binding to biotinylated cTF-hFc (SEQ ID NO:7).

[0386] For the third and fourth rounds of screening, the library cells from the previous round were incubated with biotinylated hTF-hFc and Mouse Anti-cMyc (9E10, Sigma) antibodies in PBSA at room temperature for 1 hour. The mixture was washed three times with PBSA to remove unbound antibody fragments. Goat anti-mouse-Alexa488 (Life Technologies, A-11001) and Strepavidin-PE (Life Technologies, S-866) were added and incubated at 4°C for 1 hour. The mixture was washed three times with PBSA to remove unbound antibody fragments. Finally, antibodies with high affinity were screened using FACS (BD FACSAria™ FUSION).

[0387] The mutant library was constructed using biotinylated TF-hFc antigen and underwent 2-3 rounds of MACS screening (streptomycin beads, Invitrogen) and 2-3 rounds of FACS screening (BD FACSAria™ FUSION). Approximately 600 yeast clones were then selected, cultured, and induced to express the mutant. FACS (BD FACSCanto II) was used to detect the binding of yeast clones to the aforementioned hTF-hFc (SEQ ID NO:6) and cTF-hFc (SEQ ID NO:7). Yeast clones with better affinity than the wild-type (hu67-11-scfv) were selected for sequencing verification. The sequenced clones were compared and analyzed; redundant sequences were removed, and the non-redundant sequences were converted into full-length human IgG for expression in mammalian cells.

[0388] hu67-11-scFv sequence:

[0389] Example 7: Modification of the hu67-11 affinity maturation molecule

[0390] The humanized antibody hu67-11 obtained in Example 6 was subjected to CDR point mutation (single point and / or combination), and a P40S point mutation was further introduced into the FR region to further improve cell binding affinity. The antibodies were cloned, expressed, purified, and tested. The variable region sequences of antibodies hu67Y116 and hu67Y118, with further enhanced cell binding affinity, were obtained through screening.

[0391] Antibody hu67Y116 / hu67Y118 heavy chain variable region sequence:

[0392] Antibody hu67Y116 light chain variable region sequence:

[0393] Antibody hu67Y118 light chain variable region sequence:

[0394] Table 6. CDR of antibody hu67Y116

[0395] Table 7. CDR of antibody hu67Y118

[0396] hu67Y116 / hu67Y118 heavy chain sequence:

[0397] hu67Y116 light chain sequence:

[0398] hu67Y118 light chain sequence:

[0399] II. Preparation of ADC

[0400] DAR value determination of ADC

[0401] The method for calculating the DAR value of the ADC disclosed herein uses RP-HPLC (reversed-phase high-performance liquid chromatography).

[0402] The pharmaceutical portion of the conjugate disclosed herein can be any suitable pharmaceutical product. Particularly suitable pharmaceutical products are described, for example, in WO2020063676A1 (included herein in its entirety by reference). Compound 9A of this disclosure (i.e., compound 9-A of Example 9 of WO2020063676A1) has the following structure:

[0403] This disclosure describes the preparation of antibody-drug conjugates, as shown in the general formula (TF-9A) of ADCs, by adjusting reaction parameters using the following method.

[0404] ADC Example 1 ADC-1

[0405] At 37°C, a prepared aqueous solution of tris(2-carboxyethyl)phosphonic acid hydrochloride (TCEP.HCl) (10 mM, 131.8 μL, 1.318 μmol) was added to a PBS buffer solution of antibody hu67Y116 (pH = 6.5, 0.05 M) (10.0 mg / mL, 7.4 mL, 511 nmol). The solution was placed in a constant temperature shaker and the reaction was carried out at 37°C for 3 hours with shaking. The reaction was then stopped. The reaction solution was cooled to 25°C in a water bath.

[0406] Compound 9A (5.49 mg, 5.11 μmol) was dissolved in 370 mL of DMSO and added dropwise to the above reaction solution. The mixture was placed in a constant temperature shaker and shaken at 25 °C for 3 hours, after which the reaction was stopped. The reaction solution was purified by desalting using a Sephadex G25 gel column (elution phase: His buffer aqueous solution at pH 5.0) to obtain the exemplary product ADC-1 (hu67Y116-9A) of conjugate TF-9A in His buffer (3.8 mg / mL, 17.6 mL), which was stored at 4 °C. RP-HPLC calculated average: DAR = 4.14.

[0407] ADC Example 2 ADC-2

[0408] At 37°C, a prepared aqueous solution of tris(2-carboxyethyl)phosphonic acid hydrochloride (TCEP.HCl) (10 mM, 178.9 μL, 1.789 μmol) was added to a PBS buffer solution of antibody hu67Y116 (pH = 6.5, 0.05 M) (10.0 mg / mL, 7.4 mL, 511 nmol). The solution was placed in a water bath and shaken at 37°C for 3 hours, after which the reaction was stopped. The reaction solution was then cooled to 25°C in a water bath.

[0409] Compound 9A (6.586 mg, 6.132 μmol) was dissolved in 370 μL DMSO and added to the above reaction solution. The mixture was placed in a water bath and shaken at 25 °C for 3 hours, after which the reaction was stopped. The reaction solution was purified by desalting using a Sephadex G25 gel column (elution phase: His buffer aqueous solution at pH 5.0) to obtain the exemplary product ADC-2 (hu67Y116-9A) of conjugate TF-9A in PBS buffer (3.52 mg / mL, 17.9 mL), which was stored at 4 °C. RP-HPLC calculated average: DAR = 5.91.

[0410] ADC Example 3 ADC-3

[0411] At 37°C, a prepared aqueous solution of tris(2-carboxyethyl)phosphonic acid hydrochloride (TCEP.HCl) (10 mM, 48.3 μL, 483 nmol) was added to a PBS buffer solution of antibody hu67Y116 (pH = 6.5, 0.05 M) (10.0 mg / mL, 1 mL, 69 nmol). The solution was placed in a water bath and shaken at 37°C for 3 hours, after which the reaction was stopped. The reaction solution was then cooled to 25°C in a water bath.

[0412] Compound 9A (1.1 mg, 1.035 μmol) was dissolved in 50 μL DMSO and added to the above reaction solution. The mixture was placed in a water bath and shaken at 25 °C for 3 hours, after which the reaction was stopped. The reaction solution was purified by desalting using a Sephadex G25 gel column (elution phase: His buffer aqueous solution at pH 5.0) to obtain the exemplary product of conjugate TF-9A, ADC-3 (hu67Y116-9A), in PBS buffer (0.8 mg / mL, 11.5 mL), which was stored at 4 °C. RP-HPLC calculated average: DAR = 7.23.

[0413] ADC Example 4 ADC-4

[0414] At 37°C, a prepared aqueous solution of tris(2-carboxyethyl)phosphonic acid hydrochloride (TCEP.HCl) (10 mM, 16.6 μL, 166 nmol) was added to the PBS buffered aqueous solution of antibody hIgG1 (pH = 6.5, 0.05 M PBS buffered aqueous solution; 10.0 mg / mL, 1 mL, 67.6 nmol). The solution was placed in a water bath and shaken at 37°C for 3 hours, after which the reaction was stopped. The reaction solution was then cooled to 25°C in a water bath.

[0415] Compound 9A (0.73 mg, 676 nmol) was dissolved in 60 μL of dimethyl sulfoxide and added dropwise to the above reaction solution. The mixture was placed in a water bath and shaken at 25 °C for 3 hours, after which the reaction was stopped. The reaction solution was then purified by desalting using a Sephadex G25 gel column (elution phase: 0.05 M PBS buffer solution at pH 6.5 containing 0.001 M EDTA) to obtain PBS buffer (0.91 mg / mL, 9.8 mL) of the title product ADC-4, which was stored at 4 °C.

[0416] RP-HPLC calculated average: n = 4.73.

[0417] III. Examples of Antibody and ADC Tests

[0418] Test Example 1: Binding Experiment of Antibody with Free Human and Monkey TF Protein

[0419] AffiniPure Goat Anti-Human IgG (Jackon, 109-005-008) protein was diluted to 2 μg / mL with PBS (Shanghai Yuanpei Biotechnology Co., Ltd., B320KJ) buffer at pH 7.4 and added to a 96-well microplate at a volume of 100 μL / well. The plate was incubated overnight at 4°C. After discarding the liquid, 300 μL of 5% skim milk (BD, 232100) diluted in PBS was added to each well for blocking, and the plate was incubated at 37°C for 2 hours. After blocking, the blocking solution was discarded, and the plate was washed three times with PBST buffer (pH 7.4 PBS containing 0.1% Tween-20). Then, 50 μL of the antibody solution diluted to 2 μg / mL was added to each well, and the plate was incubated at 37°C for 1 hour. After incubation, wash the plate three times with PBST. Add 50 μL of serially diluted (10 μg / mL, 5-fold serial dilution) biotinylated human or monkey TF-his protein (SEQ ID No. 5 / 8) (Biotin-labeled kit: DO JINDO, LK03-10) to each well and incubate at 37°C for 1 hour. After incubation, wash the plate three times with PBST. Add Streptavidin-Peroxidase Polymer (sigma, S2438-250UG) diluted 1:8000 and incubate at 37°C for 1 hour. After washing the plate three times with PBST, add 50 μL of TMB chromogenic substrate (KPL, 5120-0077) to each well and incubate at room temperature for 5 minutes. Stop the reaction by adding 50 μL of 1M H2SO4 to each well. Read the absorbance at 450 nm using a microplate reader. Fit the antibody-antigen binding curve using GraphPad Prism and calculate the EC50. 50 value.

[0420] Table 8: Binding of antibodies to free TF

[0421] The antibody disclosed herein exhibits good binding activity to the TF antigen.

[0422] Test Example 2: Antibody-cell binding experiment

[0423] MDA-MB-231 cells, NCI-H358 cells (H358 cells for short), and HCT116 cells were prepared into 1×10⁻⁶ cells using FACS buffer (1% BSA, pH 7.4 PBS). 6Cell suspension of 100 μL / well was added to 96-well round-bottom plates containing 100 cells / mL. After centrifugation to remove the supernatant, 100 μL / well of the target antibody diluted with FACS buffer at different concentrations (60 μg / mL, 3-fold serial dilution) was added, and the plates were incubated at 4°C in the dark for 1 hour. After washing three times with FACS buffer at 300g, the working concentration of APC Rat Anti-human IgG Fc (BioLegend, 410712) was added, and the plates were incubated at 4°C in the dark for 40 minutes. After washing three times with FACS buffer at 300g, the geometric mean fluorescence intensity was measured using a Thermo Attune Nxt flow cytometer to calculate EC50. 50 value.

[0424] Table 9. Antibody-cell binding

[0425] The antibody disclosed herein exhibits good binding activity with cells expressing TF antigen.

[0426] Test Example 3: Affinity Binding Experiment

[0427] Antibody samples were prepared into a 4 μg / mL solution using 1×HBS-EP buffer (pH 7.4) (Cytiva, BR-1006-69). Antibodies were affinity-captured using a Protein A biosensor chip (Cytiva, 29127556). A specific concentration of the antigen molecule, human TF-His protein (SEQ ID NO:5) or monkey TF-His protein (SEQ ID NO:8), was then passed through the chip surface. The reaction signal was monitored in real-time using a Biacore T200 (Cytiva) instrument to obtain binding and dissociation curves. After dissociation in each experimental cycle, the biosensor chip was washed and regenerated using 10 mM glycine-HCl (pH 1.5) (Cytiva, BR-1003-54). A 1:1 binding model was used for data fitting. The detection results are shown in Table 10.

[0428] Table 10. Antibody Affinity

[0429] The disclosed antibody has a high affinity for the TF antigen.

[0430] Test Example 4: Antibody Binding Experiment with Mouse and Rat TF Protein

[0431] Mouse TF-His protein (SEQ ID NO:10) or rat TF-His protein (SEQ ID NO:9) was diluted to 4 μg / mL with PBS buffer at pH 7.4 and added to a 96-well microplate at a volume of 100 μL / well. The plate was incubated overnight at 4°C. After discarding the supernatant, 300 μL of 5% skim milk (BD, 232100) diluted in PBS was added to each well for blocking, and the plate was incubated at 37°C for 2 hours. After blocking, the blocking solution was discarded, and the plate was washed three times with PBST buffer (pH 7.4 PBS containing 0.1% Tween-20). Then, 100 μL of serially diluted antibody solution was added to each well, and the plate was incubated at 37°C for 1 hour. After incubation, the plate was washed three times with PBST. 100 μL of 1:4000 diluted Peroxidase Affini Pure Goat Anti-Human IgG (H+L) (Jackson, 109-035-003) was added to each well, and the plate was incubated at 37°C for 1 hour. After washing three times with PBST, 50 μL of TMB chromogenic substrate (KPL, 5120-0077) was added to each well, and the plate was incubated at room temperature for 5 minutes. The reaction was terminated by adding 50 μL of 1M H2SO4 to each well. The absorbance at 450 nm was read using a microplate reader, and the antibody-antigen binding curve was plotted using GraphPad Prism. The results showed that hu67Y116 did not bind to mouse or rat TF-His protein.

[0432] Test Example 5: Factor-VII-Human Chromogenic-Activity Coagulation Pathway Experiment

[0433] In the TF-FVII-FX pathway, TF affects coagulation by influencing the formation of FX substrates. This assay is used to detect the effects of different antibodies on the coagulation pathway.

[0434] Following the instructions in the Factor-VII-Human Chromogenic-Activity kit (Abcam, ab 108830), 96-well plates were incubated at room temperature for approximately 10 minutes. Then, 100 μL / well of 0.5 μg / mL FVIIa (recombinant human coagulation factor VIIa for injection, i.e., NovoSeven) was added, and the plates were incubated at 37°C for 2 hours. After incubation, the plates were washed 5 times with wash buffer (provided in the kit). 40 μL / well of diluted antibody was added, followed by 40 μL / well of a mixture of MDA-MB-231 cells and FX (provided in the kit). The plates were incubated at 37°C for half an hour. 20 μL / well of substrate (provided in the kit) was added to initiate the reaction. Absorbance values ​​at 405 nm were read at 3-minute intervals for 30 minutes. The values ​​in wells without antibody were taken as the maximum (MAX), and the values ​​in wells without FVIIa were taken as the minimum (Min). Data within the linear range were used to calculate the ratio of the effects on the coagulation pathway. The coagulation ratios are shown in Table 11.

[0435] Table 11. Coagulation Tests

[0436] The results showed that none of the three antibodies disclosed in this study affected the coagulation pathway, while the positive controls TF011 and H39 did affect the coagulation pathway.

[0437] Test Example 6: DT3C Internalization Experiment

[0438] After the DT3C protein enters the cell, activated diphtheria toxin (DT) kills the cell, indirectly reflecting the antibody endocytosis. (Based on IC50) 50 Imax was used to evaluate the in vitro endocytic activity of the antibody.

[0439] DT3C is a recombinantly expressed fusion protein composed of fragment A (toxin only) of diphtheria toxin and fragment 3C (IgG binding portion) of group G streptococcus. This protein exhibits high affinity for the Fc structure of antibodies and enters the cell along with the antibody during endocytosis. Under the action of intracellular furin protease, it releases the toxic DT. DT inhibits EF2-ADP ribosylation activity, blocking protein translation and ultimately leading to cell death. DT3C that does not enter the cell does not possess cytotoxic activity. The endocytic activity of the antibody is evaluated based on its cytotoxic effect.

[0440] MDA-MB-231 cell suspension was prepared using fresh cell culture medium containing 20% ​​low IgG FBS (Gibco, 1921005PJ) at a cell density of 4 × 10⁻⁶ cells / mL. 41000 cells / mL, add 50 μL / well to the cell culture plate, i.e., 2000 cells per well, and incubate at 37°C with 5% carbon dioxide for 16 hours.

[0441] DT3C (120 μg / mL) at a 4× concentration was prepared using serum-free RPMI 1640 medium and sterilized by filtration through a 0.22 μm filter. Antibody (40 μg / mL) at a 4× concentration was prepared using serum-free RPMI 1640 medium. 80 μL of DT3C (approximately 70 KD) and 80 μL of antibody (approximately 145 KD) were mixed in a 1:1 volume ratio and incubated at room temperature for 30 minutes. Then, 40 μL of the mixture was added to 120 μL of serum-free RPMI 1640 medium, leaving only 160 μL of serum-free RPMI 1640 medium in each well. The final antibody concentration was 10 μg / mL. The wells with only medium were used as the zero point for calculating endocytosis. 50 μL of the diluted antibody was added to 50 μL of cells and incubated at 37°C with 5% CO2 for three days. 50 μL of CellTiter-Glo was added to each well. TM (CTG)(Promega, G7573) was incubated at room temperature in the dark for 10 minutes, and the chemiluminescence was read on ENVISION. The results are shown in Table 12.

[0442] Table 12. Antibody endocytic activity

[0443] The results showed that the disclosed antibody has endocytic activity.

[0444] Test Example 7: Prothrombin Time (PT) Test

[0445] The effect of anti-TF antibodies on the in vitro PT (proton pressure) of human plasma was evaluated by measuring the PT value of human plasma containing anti-TF antibodies. Venous blood was drawn from the upper limb veins of volunteers and poured into 15 mL centrifuge tubes pre-added with 3.8% trisodium citrate anticoagulant (Sinopharm Chemical Reagent Co., Ltd., 6132-04-3) (anticoagulant: whole blood = 1:9). The tubes were centrifuged at 2500 rpm for 10 minutes at room temperature, and plasma was collected. The antibody was prepared into working solutions with concentrations of 16000 nM, 8000 nM, 4000 nM, 2000 nM, 1000 nM, and 500 nM (final concentrations of 1600 nM, 800 nM, 400 nM, 200 nM, 100 nM, and 50 nM) using PBS (Meilun Biotechnology, PWL050). PT reagent (Shanghai Dacheng Medical Equipment Co., Ltd., OUHP29) was added to 4 mL of purified water and incubated at 37°C for 30 minutes. Following the operating manual of the CA600 coagulation analyzer (sysmex), human plasma, PT reagent, test antibody, blank control PBS, and Clean I cleaning agent (Shanghai Dacheng Medical Equipment Co., Ltd., GSA-500A) were placed in the designated locations on the instrument. The program was started, and the PT value was measured. The results were obtained by curve fitting using GraphPad Prism, and the calculated values ​​were analyzed (Table 13). The effect of the antibody on PT was mainly determined based on the maximum antibody concentration reading.

[0446] Table 13. PT Test Results

[0447] The results showed that none of the three antibodies disclosed herein affected coagulation, and their impact on prothrombin time (PT) was less than that of TF011 and H39. TF011 showed a relatively significant inhibitory effect on coagulation in the PT experiment.

[0448] Test Example 8: Antibody and Human-Rat Hybrid TF Protein HuRatTF Binding Experiment

[0449] To screen for antibodies that bind to specific epitopes of human TF without affecting human coagulation function, the human-rat hybrid antigen HuRatTF protein (SEQ ID No: 11) from Example 1 was constructed and used for antibody binding experiments. The HuRatTF protein has some coagulation-affecting sites mutated to alanine Ala and contains partial sequences from both humans and rats.

[0450] Dilute HuRatTF protein to 4 μg / mL with PBS buffer (pH 7.4) and add 100 μL / well to each well of a 96-well microplate. Incubate overnight at 4°C. After discarding the supernatant, add 300 μL of 5% skim milk (BD, 232100) diluted in PBS to each well for blocking and incubate at 37°C for 2 hours. After blocking, discard the blocking solution and wash the plate three times with PBST buffer (pH 7.4 PBS containing 0.1% Tween-20). Add 100 μL of serially diluted antibody solution (starting at 20 μg / mL, with 5-fold serial dilutions) to each well and incubate at 37°C for 1 hour. After incubation, wash the plate three times with PBST and add 100 μL of 1:4000 diluted Peroxidase Affini Pure Goat Anti-Human IgG (H+L) (Jackson, 109-035-003) to each well and incubate at 37°C for 1 hour. After washing the plate three times with PBST, add 50 μL of TMB chromogenic substrate (KPL, 5120-0077) to each well and incubate at room temperature for 5 minutes. Then, add 50 μL of 1M H2SO4 to each well to stop the reaction. Read the absorbance at 450 nm using a microplate reader. Fit the antibody-antigen binding curve using GraphPad Prism. The data are shown in Table 14.

[0451] Table 14. Results of antibody-HuRatTF protein binding experiments

[0452] The results showed that the antibody TF011, which affects coagulation function, did not bind to the HuRatTF protein, while the antibody disclosed herein, which does not affect coagulation function, could bind to the HuRatTF protein.

[0453] Test Example 9: IL-8 Cytokine Assay Experiment

[0454] MDA-MB-231 cells were prepared into 3×10⁶ cells using serum-free culture medium. 5 Cell suspension of 100 μL / well was added to a 96-well plate containing 100 cells / mL and incubated at 37°C for 2 hours. The supernatant was discarded, and 100 μL / well of serially diluted antibody (starting at 10 μg / mL, 6-fold serial dilutions) prepared in serum-free medium was added. The plate was incubated at 37°C for 0.5 hours. The supernatant was discarded, and 120 μL / well of FVIIa (recombinant human coagulation factor VIIa for injection, NovoSeven) prepared in serum-free medium was added. The plate was incubated at 37°C for 5 hours. After incubation, the plate was centrifuged at 300g for 5 minutes, and 100 μL of the supernatant was transferred to a 96-well plate in an IL-8 kit (Xinbosheng Biotechnology, EHC008). IL-8 assay was performed according to the manufacturer's instructions. The absorbance at 450 nm was read using a microplate reader, and the antibody-antigen binding curve was plotted using GraphPad Prism. The EC50 was calculated.50 The values ​​are shown in Table 15.

[0455] Table 15. IL8 Experiment

[0456] The results showed that the antibody disclosed herein had a strong inhibitory effect on IL8.

[0457] Test Example 10: ADC Molecular Cellular Activity Experiment

[0458] The purpose of this experiment was to detect the cell-killing effect of ADC samples, based on IC50. 50 Imax was used to evaluate the in vitro activity of the ADC.

[0459] HPAFII cells (human pancreatic cancer cells), MDA-MB-231 cells (human breast cancer cells), H358 cells (human non-small cell lung cancer cells), and CHO-K1 cells (hamster ovary cells) were digested with trypsin, neutralized with fresh culture medium, centrifuged at 1000 rpm, and resuspended in culture medium. After counting, the cell suspension densities were adjusted to: 3700 HPAFII cells / mL, 7400 MDA-MB-231 cells / mL, 7400 H358 cells / mL, and 3700 CHO-K1 cells / mL, respectively. 135 μL of the cell suspension was added to a 96-well cell culture plate, i.e., 500 HPAFII cells per well, 1000 MDA-MB-231 cells per well, 1000 H358 cells per well, and 500 CHO-K1 cells per well. The 11th column was not seeded with cells, but only 135 μL of culture medium was added. The cells were cultured at 37°C for 16 hours with 5% CO2.

[0460] The ADC sample was diluted with PBS to 3.333 μM (10× concentration). This was used as the initial concentration, and the sample was serially diluted five times with PBS to obtain a total of nine concentrations. 15 μL of the 10× serial dilution solution was added to each well, with wells 10 and 11 containing culture medium. Each concentration point was replicated. The sample was incubated at 37°C with 5% carbon dioxide for 6 days.

[0461] After culture, add 50 μL of CTG to each well and incubate at room temperature in the dark for 10 minutes. Place the white substrate membrane on the bottom of the cell culture plate and read the chemiluminescence using an ENVISION scanner. The in vitro cell-killing activity results are shown in Table 16.

[0462] Table 16. In vitro cell-killing activity of ADCs

[0463] The results showed that the above-mentioned ADC had a strong killing effect on cell lines HPAFⅡ, MDA-MB-231, and H358.

[0464] Test Example 11: In vivo pharmacodynamic experiment of HPAFII in a CDX model

[0465] This test case evaluates and compares the efficacy of various ADC drugs on subcutaneous xenografts in NUNU mice.

[0466] Each NUNU mouse (4-6 weeks old, female, SPF grade, Beijing Vital River Laboratory Animal Co., Ltd.) was subcutaneously inoculated with 4 × 10 6 / 200μL (containing 50% Matrigel) HPAF-II cells (human pancreatic cancer), until the tumor grows to 250-300mm 3 After evaluation, the animals were grouped into groups (D0) based on tumor volume. Mice were administered the drug via intraperitoneal injection (IP) once a week for a total of three weeks. The weekly administration volume was 10 mL / kg; specific dosages and administration regimens are shown in Table 17. Tumor volume was measured twice a week, and mouse weight was recorded.

[0467] The experimental indicators were to examine the effect of the drug on tumor growth, specifically the T / C percentage or tumor inhibition rate (TGI%). Tumor diameter was measured twice weekly using calipers. Tumor volume (V) was calculated using the formula: V = 1 / 2 × a × b 2 Where a and b represent length and width, respectively. T / C% = (T-T0) / (C-C0) × 100, where T and C are the tumor volumes at the end of the experiment; T0 and C0 are the tumor volumes at the beginning of the experiment. Where T is the tumor volume of the drug-treated tumor and C is the tumor volume of the control group. Tumor growth inhibition rate % (TGI%) = 100 - T / C%; when tumor regression occurs, tumor growth inhibition rate % (TGI%) = 100 - (T-T0) / T0 × 100; if the tumor shrinks compared to its initial volume, i.e., T < T0 or C < C0, it is defined as partial tumor regression (PR); if the tumor completely disappears, it is defined as complete tumor regression (CR). Unless otherwise specified, a two-way ANOVA test was used to compare the tumor volumes of the two groups, and P < 0.05 was defined as statistically significant. See Table 17 for the results.

[0468] Table 17. Efficacy of ADC in treating subcutaneous xenografts in HPAF-II nude mice

[0469] P-value refers to the difference between the control group and the control group; IP: intraperitoneal injection; partial regression: tumor volume is smaller than D0 at D25.

[0470] After three administrations, the ADC-1 (2 mg / kg) group antibodies showed significant efficacy against subcutaneous xenograft tumors in HPAF-II nude mice, with a significant difference compared to the blank control group; the drugs exhibited a clear dose-dependent effect. Tumor-bearing mice tolerated all of the above drugs well.

[0471] Test Example 12: In vivo pharmacodynamic experiment of the CDX model of H358

[0472] This test case evaluates and compares the efficacy of various ADC drugs on subcutaneous xenografts in nude mice.

[0473] Each nude mouse (4-6 weeks old, female, SPF grade, Shanghai Slack Laboratory Animal Co., Ltd.) was subcutaneously inoculated with 5 × 10 6 / 200μL (containing 50% Matrigel) H358 cells (human non-small cell lung cancer), until the tumor grows to 300mm 3 After evaluation, the animals were grouped (D0) according to tumor volume. Mice were administered the drug via intraperitoneal injection (IP) once a week for a total of 3 weeks. The weekly administration volume was 10 mL / kg; specific dosage and administration regimen are shown in Table 26. Tumor volume was measured twice a week, and mouse weight was recorded.

[0474] The experimental indicators were to examine the effect of the drug on tumor growth, specifically the T / C percentage or tumor inhibition rate (TGI%). Tumor diameter was measured twice weekly using calipers. Tumor volume (V) was calculated using the formula: V = 1 / 2 × a × b 2 Where a and b represent length and width, respectively. T / C% = (T-T0) / (C-C0) × 100, where T and C are the tumor volumes at the end of the experiment; T0 and C0 are the tumor volumes at the beginning of the experiment. Where T is the tumor volume of the drug-treated tumor and C is the tumor volume of the control group. Tumor growth inhibition rate % (TGI%) = 100 - T / C%; when tumor regression occurs, tumor growth inhibition rate % (TGI%) = 100 - (T-T0) / T0 × 100; if the tumor shrinks compared to its initial volume, i.e., T < T0 or C < C0, it is defined as partial tumor regression (PR); if the tumor completely disappears, it is defined as complete tumor regression (CR). Unless otherwise specified, a two-way ANOVA test was used to compare the tumor volumes of the two groups, and P < 0.05 was defined as statistically significant. See Table 18 for the results.

[0475] Table 18. Efficacy of ADC in subcutaneous xenografts in H358 nude mice

[0476] P-value refers to the comparison with the negative antibody group; IP: intraperitoneal injection; partial regression: tumor volume is smaller than D0 at D32.

[0477] ADC-1 (3 mg / kg, 1 mg / kg) showed good tumor-suppressing effects after three administrations, causing partial tumor regression; the drug exhibits a certain dose-dependency. Tumor-bearing mice tolerated all of the above drugs well.

[0478] Test Example 13: In vivo pharmacodynamic experiment of MDA-MB-231 using a CDX model

[0479] This test case evaluates and compares the efficacy of various ADC drugs on subcutaneous xenografts in NOD scid mice.

[0480] Each NOD scid mouse (4-6 weeks old, female, SPF grade, Shanghai Slack Laboratory Animal Co., Ltd.) was subcutaneously inoculated with 3×10 6 / 100μL (containing 50% Mastiff) MDA-MB-231 cells (human breast cancer), until the tumor grows to 250-300mm 3 After evaluation, the animals were grouped into groups (D0) based on tumor volume. Mice were administered the drug via intraperitoneal injection (IP) once a week for a total of three weeks. The weekly administration volume was 10 mL / kg. Tumor volume was measured twice a week, and mouse weight was recorded.

[0481] The experimental indicators were to examine the effect of the drug on tumor growth, specifically the T / C percentage or tumor inhibition rate (TGI%). Tumor diameter was measured twice weekly using calipers. Tumor volume (V) was calculated using the formula: V = 1 / 2 × a × b 2 Where a and b represent length and width, respectively. T / C% = (T-T0) / (C-C0) × 100, where T and C are the tumor volumes at the end of the experiment; T0 and C0 are the tumor volumes at the beginning of the experiment. Where T is the tumor volume of the drug-treated tumor and C is the tumor volume of the control group. Tumor growth inhibition rate % (TGI%) = 100 - T / C%; when tumor regression occurs, tumor growth inhibition rate % (TGI%) = 100 - (T-T0) / T0 × 100; if the tumor shrinks compared to its initial volume, i.e., T < T0 or C < C0, it is defined as partial tumor regression (PR); if the tumor completely disappears, it is defined as complete tumor regression (CR). Unless otherwise specified, a two-way ANOVA test was used to compare the tumor volumes of the two groups, and P < 0.05 was defined as statistically significant. Tumor-bearing mice tolerated the above drugs well, and no significant weight loss or other symptoms occurred. PBS was used as a blank control. See Table 19 for the results.

[0482] Table 19. Efficacy of ADCs on subcutaneous xenografts in MDA-MB-231 mice

[0483] P-value refers to the difference between the control group and the control group; IP: intraperitoneal injection; partial regression: tumor volume is smaller than D0 at D21.

[0484] After three administrations, the antibodies in the ADC-1 (3 mg / kg, 1 mg / kg), ADC-2 (1 mg / kg), ADC-3 (1 mg / kg), and ADC-4 (hIgG-9A) (3 mg / kg) groups all showed significant efficacy against subcutaneous xenografts in MDA-MB-231 mice, with significant differences compared to the blank control group; the drugs exhibited a clear dose-dependent effect. Tumor-bearing mice tolerated all of the above drugs well.

[0485] Test Example 14: Observation Experiment on PK and Toxicity in Cynomolgus Monkeys

[0486] This test case evaluates the pharmacokinetic comparison of ADC-1 and ADC-2 in cynomolgus monkeys and simultaneously observes toxicity.

[0487] Each cynomolgus monkey (Suzhou Xishan Zhongke Experimental Animal Co., Ltd.; SCXK(Su)2018-0001) was administered the drug via intravenous infusion at a dose of 12 mg / kg. Blood samples were collected at 0.083 h (5 min ± 1 min), 1 h (± 5 min), 6 h (± 15 min), 12 h (± 15 min), 24 h (± 30 min), 48 h (± 30 min), 96 h (± 30 min), 168 h (± 30 min), 240 h (± 60 min), 336 h (± 60 min), and 504 h (± 60 min) after administration for drug concentration testing and other toxicological observations.

[0488] The PK assay used the LBA (Delfia) method to quantitatively detect the concentrations of total antibody and intact ADC-1 in cynomolgus monkey serum.

[0489] Table 20. Total ADC-1 Pharmacokinetic Antibody and Intact ADC Blood Concentration in Cynomolgus Monkeys

[0490] Table 21. Total ADC-2 Pharmacokinetic Antibody and Intact ADC Blood Concentration in Cynomolgus Monkeys

[0491] The half-life and AUC of the total antibody and intact ADC of ADC-1 and ADC-2 molecules in cynomolgus monkeys were basically the same, and the stability of ADC-1 and ADC-2 molecules in cynomolgus monkeys was good. Due to the presence of immunogenicity, the half-life and AUC of the second dose were lower than those of the first dose.

[0492] For ADC-1 pharmacokinetic testing, blood samples were collected from all animals before the first administration, on the second day, and on the eighth day for coagulation function testing. Animals were fed overnight before blood collection. PT and APTT were measured using either a Sysmex CS-2000i automated coagulation analyzer or a Sysmex CA-1500 automated coagulation analyzer. The PT and APTT results are shown in Figures 1 and 2. The results showed no significant prolongation of clotting time or increased bleeding risk compared to pre-administration levels.

[0493] Test Example 15, TGA Experiment

[0494] This test case examines whether the antibody hu67Y116 affects the coagulation activity of TF.

[0495] Human plasma contains extrinsic coagulation factors other than TF, such as FVII, FX, FV, FII, and FI. If the antibody does not affect the coagulation activity of TF, it will not affect the coagulation time and peak value of human plasma. Human plasma was prepared according to the instructions of the Technothrombin TGA Kit. Then, 20X antibody was prepared and mixed with plasma at a ratio of 1:19. 40 μL / well was added to a black 96-well plate, followed by 10 μL / well of RCH (TGA reagent C high, included in the kit), and finally 50 μL / well of substrate to initiate the reaction. Fluorescence was read using a microplate reader (~360 nm / ~460 nm, excitation / emission). The results are shown in Table 22. TF011 showed significant coagulation inhibition compared to the negative control antibody, while hu67Y116 had virtually no effect on coagulation.

[0496] Table 22. Experimental Results of TGA

[0497] IV. Preparation Examples - Anti-TF Antibody Drug Conjugate Formulation

[0498] 1) SEC size exclusion chromatography:

[0499] An analytical method for separating solutes based on the relative relationship between the pore size of the gel and the coil size of the polymer sample molecules.

[0500] SEC% polymer (SEC polymer content percentage) = A polymer / A total * 100% (A polymer is the peak area of ​​the polymer peak in the sample, and A total is the sum of the peak areas of all peaks.)

[0501] △SEC aggregate% represents the difference between the measured value of this test item after placement under various conditions and the initial placement value.

[0502] Instrument used for SEC determination: Agilent HPLC 1260;

[0503] Pillars: Waters, BioResolve TM SEC mAb 2.5μm 7.8×300mm Column

[0504] 2) R-CE capillary gel electrophoresis:

[0505] An electrophoresis method in which a gel is transferred into a capillary as a supporting medium and then separated according to the molecular weight of the sample under a certain voltage.

[0506] R-CE% fragments (R-CE fragment content percentage) = A fragments / A total * 100% (A fragments is the peak area of ​​fragments in the sample, and A total is the sum of the peak areas of all peaks).

[0507] Instrument used for R-CE determination: Beckman capillary electrophoresis apparatus, model PA 800plus

[0508] △R-CE fragments% represents the difference between the value of this test item after placement under each condition and the value at the beginning of placement.

[0509] 3) Osmotic pressure measurement:

[0510] The freezing point method for determining osmotic pressure is based on the principle that the freezing point depression is directly proportional to the molar concentration of the solution. It uses a highly sensitive temperature sensing element to measure the freezing point of the solution and converts the electrical charge into osmotic pressure.

[0511] Instrument used for osmotic pressure measurement: Loser, model OM815.

[0512] 4) Protein concentration determination:

[0513] The concentrations of antibody-drug conjugates disclosed herein are expressed as protein concentrations, i.e., the concentrations of the antibody portion within the antibody-drug conjugate.

[0514] Because the toxin in the antibody-drug conjugate absorbs at the characteristic absorption wavelength of proteins at 280 nm, and also absorbs at 370 nm, the protein concentration is calculated using the following formula: A 280nm =(C drug ×E drug-280 +C mAb ×E mAb-280 )×l A 370nm =C drug ×E drug-370 ×l

[0515] Pick

[0516] Right now:

[0517] In the formula, A280nm The average absorbance of a single sample of the test solution at a wavelength of 280 nm when the optical path length is 1 cm;

[0518] A 370nm The average absorbance of a single sample of the test solution at a wavelength of 370 nm when the optical path length is 1 cm;

[0519] E mAb-280 The mass extinction coefficient of the protein at a wavelength of 280 nm is 1.429 g. -1 cm -1 L;

[0520] E drug-280 The mass extinction coefficient of the toxin at a wavelength of 280 nm is 5.17 g. -1 cm -1 L;

[0521] E drug-370 The mass extinction coefficient of the toxin at a wavelength of 370 nm is 17.89 g. -1 cm -1 L;

[0522] R: The ratio of the toxin extinction coefficient at 370nm to 280nm is 3.46;

[0523] C mAb Protein concentration, mg / mL;

[0524] l: Optical path length, cm (the optical path length here is 1 cm).

[0525] If the test solution is diluted, the protein concentration is: C (mg / ml) = C mAb ×N, where N is the dilution factor.

[0526] Protein concentration measurement instrument: UV-Vis spectrophotometer, model: Nano Drop 2000.

[0527] The concentration of the anti-TF antibody drug conjugate (DAR: approximately 6, which can be prepared with reference to ADC-2 above; hereinafter referred to as "ADC") disclosed herein is calculated based on the protein concentration, that is, based on the concentration of the anti-TF antibody portion in the anti-TF antibody drug conjugate.

[0528] Formulation Example 1, pH and Buffer System Screening

[0529] Formulas containing 20 mg / mL ADC, 0.3 mg / mL polysorbate 80 (PS80), 0.05 mg / mL disodium edetate (hereinafter referred to as "EDTA-2Na"), and 80 mg / mL sucrose were prepared using the buffer systems shown in Table 23. Forced degradation studies (incubation at 40°C for one month) were conducted on the samples, and the effects of different buffer systems on protein stability were investigated using SEC and R-CE as evaluation indicators.

[0530] The results are shown in Table 23. The data show that after being placed at 40℃ for one month, the polymer content of the formulation containing 10mM His-HCl (pH 5.0-6.0) was lower than that of other groups.

[0531] Table 23. Screening Results of pH and Buffer Systems Note: His-HCl represents histidine-histidine hydrochloride; SA represents succinic acid-sodium succinate; 40℃

[0532] M1: Store at 40℃ for one month, the same applies below.

[0533] Formulation Example 2: Supplementary Screening of Different Buffer Systems

[0534] The buffer systems shown in Table 24 were used to prepare formulations containing 20 mg / mL ADC, 0.3 mg / mL PS80, 0.05 mg / mL EDTA-2Na, and 80 mg / mL sucrose. Accelerated experimental studies (incubated at 25°C for one month) were conducted on the samples, using SEC and R-CE as evaluation indicators to investigate the effects of different buffer systems on protein stability.

[0535] The results are shown in Table 24. The data show that after being placed at 25°C for one month, the stability of each buffer system was good, and the content of polymers and fragments in the formulations did not change significantly.

[0536] Table 24. Results of Supplemental Screening for Different Buffer Systems Note: CA represents citric acid-sodium citrate; AA represents acetic acid-sodium acetate, and so on.

[0537] Formulation Example 3: Screening of Ionic Strength of Buffer System

[0538] Formulations containing 20 mg / mL ADC, 0.3 mg / mL PS80, 0.05 mg / mL EDTA-2Na, and 80 mg / mL sucrose were prepared using 10 mM–30 mM pH 5.5 His-HCl buffer. Stability studies were conducted on the samples (incubated at 25°C and 2–8°C for one month), using appearance after displacement, SEC, and R-CE as evaluation indicators to investigate the effect of buffer systems with different ionic strengths on stability.

[0539] The results are shown in Table 25. After being stored at 25℃ and 2–8℃ for one month, the formulations with ionic strengths of 10 mM and 30 mM were both transparent and clear in appearance, with similar aggregate and fragment contents and no significant difference in purity.

[0540] Table 25. Long-term and accelerated data for ion strength screening of buffer systems

[0541] Formulation Example 4: Screening of sucrose concentration

[0542] Formulations containing 40–80 mg / mL sucrose, 20 mg / mL ADC, 0.3 mg / mL PS80, and 0.05 mg / mL EDTA-2Na were prepared using 30 mM pH 5.5 His-HCl buffer; and formulations containing 80–100 mg / mL sucrose, 20 mg / mL ADC, 0.3 mg / mL PS80, and 0.05 mg / mL EDTA-2Na were prepared using 10 mM pH 5.5 His-HCl buffer. Forced degradation studies (incubation at 40°C for one month) were conducted on the samples. The effects of different sucrose concentrations on stability were investigated using appearance, SEC, and R-CE as evaluation indicators. The results are shown in Tables 26 and 27. After one month of incubation at 40°C, all groups showed clear and transparent appearance, with no significant difference in purity.

[0543] Table 26. Screening results of different sucrose concentrations at 30 mM ionic strength.

[0544] Table 27. Screening results of different sucrose concentrations at 10 mM ionic strength.

[0545] Formulation Example 5: Screening of EDTA-2Na Concentration

[0546] Formulations containing 0–0.1 mg / mL EDTA-2Na, 20 mg / mL ADC, 0.3 mg / mL PS80, and 80 mg / mL sucrose were prepared using 10 mM pH 5.5 His-HCl buffer. Forced degradation studies (incubation at 40°C for one month) were conducted on the samples, and the effects of different EDTA-2Na concentrations on stability were investigated using appearance, SEC, and R-CE as evaluation indicators.

[0547] The results are shown in Table 28. After being stored at 40℃ for one month, all groups were clear and transparent in appearance. There was no significant difference in purity among the preparations containing 0.01–0.1 mg / mL EDTA-2Na, and the ΔSEC polymer percentage was better than that of the control group without EDTA-2Na.

[0548] Table 28. EDTA-2Na Concentration Screening Table

[0549] Formulation Example 6: Screening of Surfactant Types

[0550] Formulations containing 10 mM pH 5.5 His-HCl buffer, 20 mg / mL ADC, 0.05 mg / mL EDTA-2Na, and 80 mg / mL sucrose were prepared using different surfactants shown in Table 29. Forced degradation studies (incubation at 40°C for one month) were conducted on the samples, and the effects of different surfactants on protein stability were investigated using appearance, SEC, and R-CE as evaluation indicators.

[0551] The results are shown in Table 29. After being stored at 40℃ for one month, all groups showed clear and transparent appearance, with no significant difference in purity.

[0552] Table 29. Surfactant Selection Table

[0553] Formulation Example 7: Screening of PS80 Concentration

[0554] Formulations containing 0.02–1 mg / mL PS80, 20 mg / mL ADC, 0.05 mg / mL EDTA-2Na, and 80 mg / mL sucrose were prepared using 10 mM pH 5.5 His-HCl buffer. Forced degradation studies (incubation at 40°C for one month) were conducted on the samples, and the effects of different PS80 concentrations on stability were investigated using appearance, SEC, and R-CE as evaluation indicators.

[0555] The results are shown in Table 30. After being stored at 40℃ for one month, all groups showed clear and transparent appearance, with no significant difference in purity.

[0556] Table 30. PS80 Concentration Screening Table

[0557] Formulation Example 8: Screening of Protein Concentration

[0558] Formulations containing 5–40 mg / mL ADC, 0.3 mg / mL PS80, 0.05 mg / mL EDTA-2Na, and 80 mg / mL sucrose were prepared using 10 mM pH 5.5 His-HCl buffer. Forced degradation studies (incubation at 40°C for one month) were conducted on the samples, and the effects of different protein concentrations on stability were investigated using appearance, SEC, and R-CE as evaluation indicators.

[0559] The results are shown in Table 31. After being stored at 40℃ for one month, all groups were clear and transparent in appearance. The SEC polymer content increased slightly at a concentration of 40 mg / mL, while there were no significant differences in other purity items.

[0560] Table 31. Protein Concentration Screening Table

[0561] Formulation Example 9: Investigation of the Lyophilized Formulation

[0562] A formulation containing 20 mg / mL ADC, 0.3 mg / mL PS80, 0.05 mg / mL EDTA-2Na, 40 mg / mL sucrose, and 9 mg / mL glycine was prepared using 30 mM pH 5.5 His-HCl buffer and then lyophilized. The appearance of the lyophilized product and its stability after being stored at 40°C for one month were investigated. The lyophilization procedure is shown in Table 32 below.

[0563] Table 32. Freeze-drying process

[0564] The results are shown in Table 33. After freeze-drying, the powder compact appeared uniform and full, without any collapse.

[0565] Table 33. Results of freeze-drying screening of excipients and osmotic pressure regulators

[0566] The results are shown in Table 34. After being stored at 40℃ for one month, the reconstituted sample of the prescription was clear and transparent, and there was no significant change in purity.

[0567] Table 34. Stability data of lyophilized formulations

[0568] Formulation Example 10, Prescription Confirmation

[0569] A formulation containing 20 mg / mL ADC, 10 mM His-HCl, pH 5.5, 80 mg / mL sucrose, 0.3 mg / mL PS80, and 0.05 mg / mL EDTA-2Na was prepared. Forced degradation stability studies were conducted on the samples, using appearance, SEC, and R-CE as evaluation indicators. Stability was assessed at 2–8℃ for 3 months, 12 months, 25℃ accelerated for 3 months, and 25℃ accelerated for 6 months.

[0570] The results are shown in Table 35. There were no significant changes in appearance, SEC, and R-CE, indicating that the protein exhibited good stability.

[0571] Table 35. Results of Forced Degradation Stability

Claims

1. A pharmaceutical composition comprising an anti-TF antibody-drug conjugate and a buffer, wherein: The anti-TF antibody-drug conjugate has a structure as shown in Formula I or II: in: n is between 1 and 10; Pc is an anti-TF antibody, which contains a heavy chain variable region and a light chain variable region, wherein: The heavy chain variable region HCDR1 contains the amino acid sequence of SEQ ID NO: 40, HCDR2 contains the amino acid sequence of SEQ ID NO: 64, and HCDR3 contains the amino acid sequence of SEQ ID NO: 65; and the light chain variable region LCDR1 contains the amino acid sequence of SEQ ID NO: 66, LCDR2 contains the amino acid sequence of SEQ ID NO: 67, and LCDR3 contains the amino acid sequence of SEQ ID NO: 68 or 73. The buffer is a histidine buffer, a citrate buffer, an acetate buffer, or a succinate buffer. Preferably, the buffer is a histidine-histidine hydrochloride buffer, a citrate-sodium citrate buffer, an acetate-sodium acetate buffer, or a succinate-sodium succinate buffer. More preferably, the buffer is a histidine-histidine hydrochloride buffer.

2. The pharmaceutical composition according to claim 1, wherein the anti-TF antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:62, or an amino acid sequence having at least 80% sequence identity with it; and the light chain variable region comprises the amino acid sequence of SEQ ID NO:63 or 72, or an amino acid sequence having at least 80% sequence identity with it. Preferably, the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:62, and the light chain variable region comprises the amino acid sequence of SEQ ID NO:63; More preferably, the anti-TF antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO: 69, or an amino acid sequence having at least 80% sequence identity with it; and the light chain comprises the amino acid sequence of SEQ ID NO: 70 or 74, or an amino acid sequence having at least 80% sequence identity with it; More preferably, the anti-TF antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO: 69, and the light chain comprises the amino acid sequence of SEQ ID NO:

70.

3. The pharmaceutical composition according to claim 1 or 2, wherein n is 3 to 8; preferably, n is 5 to 8.

4. The pharmaceutical composition according to any one of claims 1 to 3, wherein the concentration of the anti-TF antibody drug conjugate is from 1 mg / mL to 50 mg / mL; Preferably, the concentration of the anti-TF antibody drug conjugate is from 5 mg / mL to 40 mg / mL; More preferably, the concentration of the anti-TF antibody drug conjugate is from 15 mg / mL to 25 mg / mL; Most preferably, the concentration of the anti-TF antibody-drug conjugate is about 20 mg / mL.

5. The pharmaceutical composition according to any one of claims 1 to 4, wherein the concentration of the buffer is from 1 mM to 50 mM; Preferably, the concentration of the buffer is from 5 mM to 30 mM; More preferably, the concentration of the buffer is 5 mM to 15 mM; Most preferably, the concentration of the buffer is about 10 mM or about 30 mM.

6. The pharmaceutical composition according to any one of claims 1 to 5, wherein the pH of the pharmaceutical composition is 5.0 to 6.5; Preferably, the pH of the pharmaceutical composition is 5.0 to 6.0; More preferably, the pH of the pharmaceutical composition is 5.2 to 5.8; More preferably, the pH of the pharmaceutical composition is about 5.

5.

7. The pharmaceutical composition according to any one of claims 1 to 6, wherein the pharmaceutical composition comprises a surfactant; Preferably, the surfactant is polysorbate or poloxamer; More preferably, the surfactant is selected from polysorbate 20, polysorbate 80 and poloxamer 188; Most preferably, the surfactant is polysorbate 80.

8. The pharmaceutical composition according to claim 7, wherein the concentration of the surfactant is from 0.01 mg / mL to 2.0 mg / mL; Preferably, the concentration of the surfactant is from 0.02 mg / mL to 1 mg / mL; More preferably, the concentration of the surfactant is from 0.1 mg / mL to 0.5 mg / mL; Most preferably, the concentration of the surfactant is about 0.3 mg / mL.

9. The pharmaceutical composition according to claim 7, wherein the surfactant is poloxamer 188 at a concentration of 1.0 mg / mL to 2.0 mg / mL; preferably, the concentration of poloxamer 188 is about 1.5 mg / mL.

10. The pharmaceutical composition according to any one of claims 1 to 9, wherein the pharmaceutical composition comprises sugar; Preferably, the sugar is sucrose, trehalose, mannitol, or sorbitol; More preferably, the sugar is sucrose.

11. The pharmaceutical composition according to claim 10, wherein the concentration of the sugar is from 10 mg / mL to 120 mg / mL; Preferably, the concentration of the sugar is from 40 mg / mL to 100 mg / mL; More preferably, the concentration of the sugar is from 65 mg / mL to 95 mg / mL; Most preferably, the concentration of the sugar is about 80 mg / mL.

12. The pharmaceutical composition according to claim 10, wherein the concentration of the sugar is from 10 mg / mL to 80 mg / mL; More preferably, the concentration of the sugar is from 20 mg / mL to 60 mg / mL; Most preferably, the concentration of the sugar is about 40 mg / mL.

13. The pharmaceutical composition according to any one of claims 1 to 12, wherein the pharmaceutical composition further comprises a sodium salt of ethylenediaminetetraacetic acid, ethylenediaminetetraacetic acid hydrate, or a salt thereof; Preferably, the pharmaceutical composition further comprises ethylenediaminetetraacetic acid hydrate or a salt thereof; More preferably, the ethylenediaminetetraacetic acid hydrate or its salt is disodium edetate.

14. The pharmaceutical composition according to claim 13, wherein the concentration of the sodium salt of ethylenediaminetetraacetic acid and the ethylenediaminetetraacetic acid hydrate or a salt thereof is from 0.01 mg / mL to 1 mg / mL; Preferably, its concentration is from 0.01 mg / mL to 0.1 mg / mL; More preferably, its concentration is from 0.02 mg / mL to 0.08 mg / mL; Most preferably, its concentration is about 0.05 mg / mL.

15. The pharmaceutical composition according to any one of claims 1 to 14, wherein the pharmaceutical composition further comprises an excipient selected from one or more of glycine, DTPA, arginine hydrochloride, methionine, proline, histidine, phenylalanine, glutamic acid, aspartic acid, sodium chloride, and calcium chloride; Preferably, the excipient is glycine.

16. The pharmaceutical composition according to claim 15, wherein the concentration of the excipient is from 0.01 mg / mL to 20 mg / mL; Preferably, the concentration of the excipient is from 5 mg / mL to 15 mg / mL; More preferably, the concentration of the excipient is from 7 mg / mL to 11 mg / mL; Most preferably, the concentration of the excipient is about 9 mg / mL.

17. The pharmaceutical composition according to claim 1, comprising the following components: (a) The anti-TF antibody-drug conjugate at concentrations from 1 mg / mL to 50 mg / mL, (b) Surfactants ranging from 0.01 mg / mL to 2 mg / mL, (c) Sugars ranging from 10 mg / mL to 120 mg / mL, (d) Disodium edetate at concentrations ranging from 0.01 mg / mL to 1 mg / mL, and (e) a buffer of 1 mM to 50 mM, wherein the pH of the pharmaceutical composition is 5.0 to 6.5; Preferably, the pharmaceutical composition comprises the following components: (a) The anti-TF antibody-drug conjugate at concentrations ranging from 5 mg / mL to 40 mg / mL, (b) Polysorbate 80 at concentrations ranging from 0.02 mg / mL to 1 mg / mL, (c) Sucrose at concentrations of 40 mg / mL to 100 mg / mL, (d) Disodium edetate at concentrations ranging from 0.01 mg / mL to 0.1 mg / mL, and (e) a 5 mM to 30 mM histidine-histidine hydrochloride buffer, citrate-sodium citrate buffer, acetate-sodium acetate buffer, or succinate-sodium succinate buffer, wherein the pH of the pharmaceutical composition is 5.0 to 6.

0. More preferably, the pharmaceutical composition comprises the following components: (a) The anti-TF antibody-drug conjugate at concentrations of 15 mg / mL to 25 mg / mL, (b) Polysorbate 80 at concentrations ranging from 0.1 mg / mL to 0.5 mg / mL. (c) Sucrose at concentrations of 65 mg / mL to 95 mg / mL, (d) Sodium edetate at concentrations ranging from 0.02 mg / mL to 0.08 mg / mL, and (e) a 5 mM to 15 mM histidine-histidine hydrochloride buffer, wherein the pH of the pharmaceutical composition is 5.2 to 5.8; Most preferably, the pharmaceutical composition comprises the following components: (a) The anti-TF antibody-drug conjugate at a concentration of approximately 20 mg / mL, (b) Approximately 0.3 mg / mL of polysorbate 80, (c) Approximately 80 mg / mL of sucrose, (d) Approximately 0.05 mg / mL of disodium edetate, and (e) about 10 mM of histidine-histidine hydrochloride buffer, wherein the pH of the pharmaceutical composition is about 5.

5.

18. The pharmaceutical composition according to claim 1, comprising the following components: (a) The anti-TF antibody-drug conjugate at concentrations from 1 mg / mL to 50 mg / mL, (b) Surfactants ranging from 0.01 mg / mL to 2 mg / mL, (c) Sugars ranging from 10 mg / mL to 120 mg / mL, (d) Disodium edetate at concentrations ranging from 0.01 mg / mL to 1 mg / mL. (e) excipients ranging from 0.01 mg / mL to 20 mg / mL, and (f) a buffer of 1 mM to 50 mM, wherein the pH of the pharmaceutical composition is 5.0 to 6.5; Preferably, the pharmaceutical composition comprises the following components: (a) The anti-TF antibody-drug conjugate at concentrations ranging from 5 mg / mL to 40 mg / mL, (b) Polysorbate 80 at concentrations ranging from 0.02 mg / mL to 1 mg / mL, (c) Sucrose at concentrations ranging from 10 mg / mL to 80 mg / mL, (d) Disodium edetate at concentrations ranging from 0.01 mg / mL to 0.1 mg / mL. (e) Glycine at concentrations of 5 mg / mL to 15 mg / mL, and (f) a 10 mM to 40 mM histidine-histidine hydrochloride buffer, citrate-sodium citrate buffer, acetate-sodium acetate buffer or succinate-sodium succinate buffer, wherein the pH of the pharmaceutical composition is 5.0 to 6.

0. More preferably, the pharmaceutical composition comprises the following components: (a) The anti-TF antibody-drug conjugate at concentrations of 15 mg / mL to 25 mg / mL, (b) Polysorbate 80 at concentrations ranging from 0.1 mg / mL to 0.5 mg / mL. (c) Sucrose at concentrations of 20 mg / mL to 60 mg / mL, (d) Disodium edetate at concentrations ranging from 0.02 mg / mL to 0.08 mg / mL. (e) Glycine at concentrations of 7 mg / mL to 11 mg / mL, and (f) a 25 mM to 35 mM histidine-histidine hydrochloride buffer, wherein the pH of the pharmaceutical composition is 5.2 to 5.8; Most preferably, the pharmaceutical composition comprises the following components: (a) The anti-TF antibody-drug conjugate at a concentration of approximately 20 mg / mL, (b) Approximately 0.3 mg / mL of polysorbate 80, (c) Approximately 40 mg / mL of sucrose, (d) Approximately 0.05 mg / mL of disodium edetate, (e) Glycine at approximately 9 mg / mL, and (f) about 30 mM of histidine-histidine hydrochloride buffer, wherein the pH of the pharmaceutical composition is about 5.

5.

19. The pharmaceutical composition according to any one of claims 1 to 18, wherein it is an intravenous injection preparation, a subcutaneous injection preparation, an intraperitoneal injection preparation, or an intramuscular injection preparation; preferably an intravenous injection preparation.

20. A lyophilized formulation, wherein the lyophilized formulation, upon reconstitution, can form a pharmaceutical composition according to any one of claims 1 to 18.

21. Use of the pharmaceutical composition according to any one of claims 1 to 18, or the lyophilized formulation according to claim 20, in the preparation of a medicament for treating tumors or cancer; Preferably, the tumor or cancer is selected from breast cancer, pancreatic cancer, lung cancer, esophageal cancer, ovarian cancer, cervical cancer, endometrial cancer, prostate cancer, bladder cancer, fallopian tube cancer, peritoneal cancer, colorectal cancer, head and neck cancer, and squamous cell carcinoma.

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