Pharmaceutical compositions containing anti-CD79b antibody-drug conjugates and uses thereof

Abstract

A pharmaceutical composition comprising an anti-CD79b antibody-drug conjugate and use thereof. The pharmaceutical composition comprises a ligand-drug conjugate and a buffer. In the ligand-drug conjugate, the ligand is an anti-CD79b antibody or an antigen-binding fragment thereof, the drug is selected from MMAE or a derivative thereof, exatecan or a derivative thereof, and eribulin or a derivative thereof, and the buffer is selected from acetate buffer, histidine buffer, Tris-hydrochloride buffer, Tris-citrate buffer, phosphate buffer, or succinate buffer. The composition is used for the preparation of a medicament for treating or preventing a proliferative disease.

Description

[Technical field]

[0001] This disclosure claims priority to a Chinese patent application filed on January 26, 2022, bearing application number CN202210093514.0 and entitled "Pharmaceutical Composition Comprising Anti-CD79b Antibody-Drug Conjugate and Use Thereof," the entire contents of which are incorporated herein by reference.

[0002] The present disclosure relates to the field of pharmaceutical formulations, and in particular to pharmaceutical compositions comprising anti-CD79b antibody-drug conjugates and their use as medicines. [Background technology]

[0003] The B cell antigen receptor (BCR) complex is the most important molecule on the surface of B cells. The BCR complex consists of membrane immunoglobulin (mIg), which recognizes and binds antigens, and a heterodimer of Igα (CD79a) and Igβ (CD79b), which transmits antigen-stimulating signals. Igα and Igβ are 47 kDa and 37 kDa glycoproteins, respectively, and belong to the immunoglobulin superfamily. The genes encoding Igα and Igβ are called mb-1 and B29, respectively. Both Igα and Igβ have one Ig-like domain at the amino terminus of the extracellular domain. Both Igα and Igβ can participate in BCR signaling as substrates for protein tyrosine kinases. BCR is widely expressed in B cell lymphomas and normal B cells. In view of the clinical success and proven safety of rituximab, which targets CD20, the development of therapeutic methods targeting BCR should also have good therapeutic efficacy and safety.

[0004] To address the unmet medical needs related to CD79b, currently many international pharmaceutical companies, including Roche Pharmaceuticals, are actively engaged in the development of antibodies against CD79b and related products, such as CD79b antibody-drug conjugates.

[0005] Antibody drug conjugates (ADCs) link monoclonal antibodies or antibody fragments to biologically active cytotoxins via stable chemical linker compounds, taking advantage of the specificity of antibodies to tumor cells or highly expressed antigens and the high efficiency of cytotoxins to avoid toxicity and side effects on normal cells. This means that, compared with traditional chemotherapy drugs, antibody drug conjugates can bind precisely to tumor cells and reduce the impact on normal cells. ADC drugs consist of three parts: antibodies (targeting moieties), linkers and toxins. Among them, a good targeting moiety determines the specificity of ADC drugs, which not only includes specific target binding but also includes effective endocytosis.

[0006] However, antibody drugs, especially ADCs, have larger molecular weights and more complicated structures than other chemical drugs, and are prone to instability due to degradation, polymerization, undesirable chemical modification, etc. In order to enable antibody drug conjugates to be suitable for administration, maintain stability during storage and subsequent use, and achieve better effects, it is particularly important to study stabilized formulations of antibody drugs. The present disclosure provides pharmaceutical compositions comprising CD79b ADCs that are sufficiently stable and more suitable for administration. Summary of the Invention

[0007] The present disclosure provides pharmaceutical compositions comprising the ligand-drug conjugates (e.g., anti-CD79b antibody drug conjugates), methods for preparing the pharmaceutical compositions, and methods for treating or preventing diseases or related pharmaceutical uses thereof.

[0008] The present disclosure provides a pharmaceutical composition comprising a ligand-drug conjugate and a buffer, wherein the ligand is an anti-CD79b antibody or an antigen-binding fragment thereof, and the drug is selected from MMAE or a derivative thereof, exatecan or a derivative thereof, and eribulin or a derivative thereof. In some embodiments, the buffer is selected from acetate buffer, histidine salt buffer, Tris-hydrochloride buffer, Tris-citrate buffer, phosphate buffer, or succinate buffer. In some embodiments, the buffer is a histidine salt buffer or a succinate buffer. In some specific embodiments, the buffer is a histidine-hydrochloride buffer or a succinic acid-sodium succinate buffer. The composition has activity for treating or preventing a disease. In addition, the composition may be characterized by good stability.

[0009] In some embodiments, in the pharmaceutical composition according to any one of the above, the concentration of the ligand-drug conjugate is 0.1 to 50 mg / mL, for example, 0.5 to 50 mg / mL, 1 to 45 mg / mL, 1 to 30 mg / mL, 1 to 25 mg / mL, 5 to 50 mg / mL, 10 to 45 mg / mL, 15 to 40 mg / mL, 20 to 35 mg / mL, 25 to 30 mg / mL, 5 to 30 mg / mL, 10 to 15 mg / mL, 10 to 20 mg / mL, 10 to 25 mg / mL, 10 to 30 mg / mL, 15 to 45 mg / mL, 15 to 35 mg / mL, 15 to 25 mg / mL, 15 to 20 mg / mL, 20 to 25 mg / mL, 20 to 30 mg / mL, or any range between these point values. In some embodiments, the concentration of the ligand-drug conjugate is 10-30 mg / mL.In some embodiments, the concentration of the ligand-drug conjugate is 15-25 mg / mL. In some non-limiting examples, the concentration of the ligand-drug complex is about 0.1 mg / mL, about 0.5 mg / mL, about 1 mg / mL, about 2 mg / mL, about 5 mg / mL, about 8 mg / mL, about 10 mg / mL, about 11 mg / mL, about 12 mg / mL, about 13 mg / mL, about 14 mg / mL, about 15 mg / mL, about 16 mg / mL, about 17 mg / mL, about 18 mg / mL, about 19 mg / mL, about 20 mg / mL, about 21 mg / mL, about 22 mg / mL, about 23 mg / mL, about 24 mg / mL, about 25 mg / mL, about 26 mg / mL, about 27 mg / mL, about 28 mg / mL, about 29 mg / mL, about 30 mg / mL, about 35 mg / mL, about 40 mg / mL, about 45 mg / mL, about 50 mg / mL.

[0010] In some embodiments, the pharmaceutical composition according to any one of the above, wherein the concentration of the buffer is 0.5 to 50 mM, for example, 1 to 40 mM, 1 to 30 mM, 1 to 20 mM, 1 to 15 mM, 1 to 10 mM, 5 to 45 mM, 5 to 35 mM, 5 to 25 mM, 5 to 15 mM, 5 to 10 mM, 10 to 15 mM, 10 to 20 mM, 10 to 30 mM, or any range between these values. In some embodiments, the pharmaceutical composition according to any one of the above, wherein the concentration of the buffer is 1 to 30 mM. In some embodiments, the pharmaceutical composition according to any one of the above, wherein the concentration of the buffer is 5 to 15 mM. In some non-limiting examples, the concentration of the buffer is about 0.5 mM, about 1 mM, about 2 mM, about 3 mM, about 4 mM, about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, about 10 mM, about 11 mM, about 12 mM, about 13 mM, about 14 mM, about 15 mM, about 16 mM, about 17 mM, about 18 mM, about 19 mM, about 20 mM, about 25 mM, about 30 mM, about 35 mM, about 40 mM, about 45 mM, about 50 mM, e.g., about 10 mM.

[0011] In some embodiments, the pH value of the pharmaceutical composition according to any one of the above is 4.0 to 8.5, for example, 4.0 to 8.0, 4.5 to 7.5, 5.0 to 7.0, 5.6 to 6.5, 6.0 to 7.5, 4.5 to 6.5, 5.0 to 6.5, 5.0 to 6.0, 5.0 to 5.5, 5.6 to 6.0, 5.35 to 5.75 (for example, about 5.6), 5.2 to 5.8, 5.5 to 6.5, 4.5 to 6.5, 4.5 to 6.0, 4.5 to 5.5, 4.0 to 6.0, 3.5 to 5.8, or any range between these values. In some embodiments, the pH value of the pharmaceutical composition according to any one of the above is 5.0 to 6.5. In some embodiments, the pH value of the pharmaceutical composition according to any one of the above is 5.5 to 6.0. In some non-limiting examples, the pH value of the pharmaceutical composition is about 4.0, about 4.5, about 4.6, about 4.7, about 4.8, about 4.9, about 4.95, about 5.0, about 5.05, about 5.1, about 5.15, about 5.2, about 5.25, about 5.3, about 5.35, about 5.4, about 5.45, about 5.5, about 5.55, about 5.6, about 5.65, about 5.7, about 5.75, about 5.8, about 5.85, about 5.9, about 5.95, about 6.0, about 6.1, about 6.2, about 6.3, about 6.4, about 6.5, about 7.0, about 7.5, about 8.0, or about 8.5.

[0012] Generally, the pH of the pharmaceutical composition obtained by replacing the buffer is approximately the same as the pH of the buffer.At the same time, as known to those skilled in the art, during the process of pharmaceutical formulation, there may be pH drift, but the pH drift of pharmaceutical formulation is generally very small (within the range of ±0.3).In some embodiments, the pH drift of pharmaceutical formulation is within the range of ±0.1.

[0013] In some embodiments, the pharmaceutical composition according to any one of the above further comprises a surfactant. In some embodiments, the surfactant is a non-ionic surfactant. In some embodiments, the surfactant is a poloxamer (e.g., poloxamer 188), polysorbate (e.g., polysorbate 20 (i.e., PS20), polysorbate 80 (i.e., PS80)), poloxamer, Triton, sodium dodecyl sulfonate, sodium lauryl sulfonate, sodium octyl glucoside, lauryl-sulfobetaine, myristyl-sulfobetaine, linoleic-sulfobetaine, stearic-sulfobetaine, lauryl-sarcosine, myristyl-sarcosine, linoleic-sarcosine, stearic-sarcosine, linoleic-betaine, myristyl -betaine, cetyl-betaine, lauramidopropyl-betaine, cocamidopropyl-betaine, linoleamidopropyl-betaine, myristamidopropyl-betaine, palmitamidopropyl-betaine, isostearamidopropyl-betaine, myristamidopropyl-dimethylamine, palmitamidopropyl-dimethylamine, isostearamidopropyl-dimethylamine, sodium methyl cocoyl, sodium methyl oleyl taurate, polyethylene glycol, polypropylene glycol, copolymers of ethylene and propylene glycol, and the like, or any combination thereof. In some embodiments, the surfactant is a polysorbate or poloxamer. In some embodiments, the surfactant is polysorbate 80, polysorbate 20, or poloxamer 188. In some embodiments, the surfactant is polysorbate 80.

[0014] In some embodiments, the concentration of the surfactant is 0.01 to 1 mg / mL, for example, 0.01 to 0.8 mg / mL, 0.05 to 0.6 mg / mL, 0.08 to 0.5 mg / mL, 0.1 to 0.4 mg / mL, 0.1 to 0.3 mg / mL, 0.15 to 0.25 mg / mL, 0.2 to 0.3 mg / mL, 0.1 to 0.2 mg / mL, or any range between these values. In some embodiments, the concentration of the surfactant is 0.1 to 0.3 mg / mL. In some non-limiting examples, the concentration of the surfactant is about 0.01 mg / mL, about 0.02 mg / mL, about 0.03 mg / mL, about 0.04 mg / mL, about 0.05 mg / mL, about 0.06 mg / mL, about 0.07 mg / mL, about 0.08 mg / mL, about 0.09 mg / mL, about 0.1 mg / mL, about 0.11 mg / mL, about 0.12 mg / mL, about 0.13 mg / mL, about 0.14 mg / mL, about 0.15 mg / mL, about 0.20 mg / mL, about 0.25 mg / mL, about 0.26 mg / mL, about 0.27 mg / mL, about 0.28 mg / mL, about 0.29 mg / mL, about 0.30 mg / mL, about 0.31 mg / mL, about 0.32 mg / mL, about 0.33 mg / mL, about 0.34 mg / mL, about 0.35 mg / mL, about 0.36 mg / mL, about 0.37 mg / mL, about 0.38 mg / mL, about 0.39 mg / mL, about 0.40 mg / mL, about 0.41 mg / mL, about 0.42 mg / mL, about 0.43 mg / mL, about 0.44 mg / mL, about 0.45 mg / mL, about 0.46 mg / mL, about 0.47 mg / mL, about 0.48 mg / mL, about 0.49 mg / mL, about 0.50 mg / mL, about 0.51 mg / mL, about 0.52 mg / mL, about 0.53 mg / mL, about 0.54 mg / mL, about 0.55 mg / mL, about 0.56 mg / mL, about 0.57 mg / mL, about 0.58 mg / L, about 0.16 mg / mL, about 0.17 mg / mL, about 0.18 mg / mL, about 0.19 mg / mL, about 0.2 mg / mL, about 0.21 mg / mL, about 0.22 mg / mL, about 0.23 mg / mL, about 0.24 mg / mL, about 0.25 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, and about 1 mg / mL.

[0015] In some embodiments, the pharmaceutical composition according to any one of the above further comprises an osmolality modifier. In some embodiments, the osmolality modifier is a sugar (including monosaccharides, disaccharides, trisaccharides, polysaccharides, sugar alcohols, reducing sugars, non-reducing sugars, etc.), an amino acid (including arginine, glycine, cysteine, histidine, etc.), or a salt (sodium chloride, potassium chloride, calcium chloride, etc.). In some embodiments, the osmolality modifier is a sugar, and the sugar is selected from glucose, sucrose, trehalose, lactose, fructose, maltose, dextran, glycerin, erythritol, glycerol, arabitol, xylitol, sorbitol (also called sorbitol), mannitol, melibiose, melezitose, raffinose, manninotriose, stachyose, maltose, lactulose, maltulose, maltitol, lactitol, and iso-maltulose. In some embodiments, the osmotic modifier is one or more selected from the group consisting of sucrose, trehalose, sorbitol, arginine, proline, glycine, and sodium chloride. In some embodiments, the osmotic modifier is a non-reducing disaccharide, in some embodiments, the osmotic modifier is trehalose and / or sucrose, and in some embodiments, the osmotic modifier is sucrose.

[0016] In some embodiments, the concentration of the osmotic pressure adjusting agent in any one of the above pharmaceutical compositions is 1 to 300 mg / mL, for example, 5 to 200 mg / mL, 10 to 150 mg / mL, 20 to 140 mg / mL, 30 to 130 mg / mL, 40 to 120 mg / mL, 50 to 110 mg / mL, 60 to 100 mg / mL, 70 to 90 mg / mL (for example, about 80 mg / mL), or any range between these values. In some non-limiting embodiments, the concentration of the osmotic agent is about 10 mg / mL, about 20 mg / mL, about 30 mg / mL, about 40 mg / mL, about 50 mg / mL, about 60 mg / mL, about 70 mg / mL, about 80 mg / mL, about 90 mg / mL, about 100 mg / mL, about 110 mg / mL, about 120 mg / mL, about 130 mg / mL, about 140 mg / mL, about 150 mg / mL, about 160 mg / mL, about 170 mg / mL, about 180 mg / mL, about 190 mg / mL, about 200 mg / mL, about 250 mg / mL, or about 300 mg / mL.

[0017] In some embodiments, the pharmaceutical composition described above comprises any one of a) to o): a) 0.1-50 mg / mL ligand-drug conjugate (e.g., anti-CD79b antibody drug conjugate), 0.5-50 mM, 1-30 mM, 5-15 mM (e.g., about 10 mM) histidine salt buffer or succinate buffer, sucrose or trehalose, and polysorbate.

[0018] b) 10-30 mg / mL of ligand-drug conjugate (e.g., anti-CD79b antibody drug conjugate), 0.5-50 mM, 1-30 mM, 5-15 mM (e.g., about 10 mM) of histidine salt buffer or succinate buffer, sucrose and / or trehalose, and polysorbate.

[0019] c) 15-25 mg / mL (e.g., about 20 mg / mL) ligand-drug conjugate (e.g., anti-CD79b antibody drug conjugate), 0.5-50 mM, 1-30 mM, 5-15 mM (e.g., 10 mM) histidine salt buffer or succinate buffer, sucrose and / or trehalose, and polysorbate.

[0020] l) 10-50 mg / mL of ligand-drug conjugate (e.g., anti-CD79b antibody drug conjugate), 0.5-50 mM, 1-30 mM, 5-15 mM (e.g., about 10 mM) of histidine salt buffer or succinate buffer, sucrose and / or trehalose, and polysorbate.

[0021] wherein the pH of the pharmaceutical compositions in a) to c) is 4.0 to 8.5, 5.0 to 6.5, or 5.5 to 6.0 (e.g., about 5.0, about 5.6); In some embodiments, the concentration of sucrose or trehalose in the pharmaceutical compositions in a) to c) and l) is 1 to 300 mg / mL, 30 to 130 mg / mL, or 70 to 90 mg / mL (e.g., about 70 mg / mL, about 80 mg / mL, about 90 mg / mL, about 100 mg / mL, about 110 mg / mL, or about 120 mg / mL); In some embodiments, the polysorbate in the pharmaceutical compositions in a) to c) and l) is polysorbate 20 or polysorbate 80, and the concentration of the polysorbate is 0.01 to 1 mg / mL, 0.05 to 0.6 mg / mL, or 0.1 to 0.3 mg / mL (e.g., about 0.1 mg / mL, about 0.2 mg / mL, or about 0.3 mg / mL); d) 0.1-50 mg / mL of ligand-drug conjugate (e.g., anti-CD79b antibody drug conjugate), 0.5-50 mM histidine salt buffer or succinate buffer, 1-300 mg / mL of sucrose and / or trehalose, and 0.01-1 mg / mL of polysorbate 80 or polysorbate 20, pH 4.0-8.5.

[0022] m) 10-50 mg / mL of ligand-drug conjugate (e.g., anti-CD79b antibody drug conjugate), 1-30 mM histidine salt buffer or succinate buffer, 30-130 mg / mL of sucrose and / or trehalose, and 0.05-0.6 mg / mL of polysorbate 80 or polysorbate 20, pH 5.0-6.5.

[0023] e) 10-30 mg / mL of ligand-drug conjugate (e.g., anti-CD79b antibody drug conjugate), 1-30 mM histidine salt buffer or succinate buffer, 30-130 mg / mL of sucrose and / or trehalose, and 0.05-0.6 mg / mL of polysorbate 80 or polysorbate 20, pH 5.0-6.5.

[0024] f) 10-30 mg / mL of ligand-drug conjugate (e.g., anti-CD79b antibody drug conjugate), 1-30 mM histidine salt buffer, 30-130 mg / mL of sucrose, and 0.05-0.6 mg / mL of polysorbate 80, pH 5.0-6.5.

[0025] g) 15-25 mg / mL of ligand-drug conjugate (e.g., anti-CD79b antibody drug conjugate), 5-15 mM histidine salt buffer, 70-90 mg / mL of sucrose, and 0.1-0.3 mg / mL of polysorbate 80, pH 5.5-6.0.

[0026] h) 15-25 mg / mL of ligand-drug conjugate (e.g., anti-CD79b antibody drug conjugate), 10 mM histidine salt buffer, about 80 mg / mL sucrose, and 0.1-0.3 mg / mL polysorbate 80, pH 5.5-6.0 (e.g., about 5.6).

[0027] n) 15-25 mg / mL of ligand-drug conjugate (e.g., anti-CD79b antibody-drug conjugate), 5-15 mM histidine-hydrochloride buffer, 70-90 mg / mL of sucrose, and 0.1-0.3 mg / mL of polysorbate 80, pH 5.5-6.0.

[0028] o) 15-25 mg / mL ligand-drug conjugate (e.g., anti-CD79b antibody drug conjugate), 10 mM histidine-hydrochloride buffer, about 80 mg / mL sucrose, and 0.1-0.3 mg / mL polysorbate 80, pH 5.5-6.0 (e.g., about 5.6).

[0029] i) 15-25 mg / mL of ligand-drug conjugate (e.g., anti-CD79b antibody drug conjugate), about 10 mM histidine-hydrochloride buffer, about 80 mg / mL sucrose, and about 0.2 mg / mL polysorbate 80, pH about 5.6.

[0030] p-1) 15 to 25 mg / mL of a ligand-drug conjugate (e.g., an anti-CD79b antibody-drug conjugate), about 10 mM histidine-histidine hydrochloride, about 80 mg / mL of sucrose, and about 0.2 mg / mL of polysorbate 80, pH about 5.6.

[0031] p-2) 15 to 25 mg / mL of a ligand-drug conjugate (e.g., an anti-CD79b antibody-drug conjugate), about 10 mM histidine-histidine hydrochloride, about 80 mg / mL of sucrose, and about 0.2 mg / mL of polysorbate 80, pH about 5.5.

[0032] p-3) 15 to 25 mg / mL of a ligand-drug conjugate (e.g., an anti-CD79b antibody-drug conjugate), about 10 mM histidine-histidine hydrochloride, about 80 mg / mL of sucrose, and about 0.2 mg / mL of polysorbate 80, pH about 5.75.

[0033] p-4) 15 to 25 mg / mL of a ligand-drug conjugate (e.g., an anti-CD79b antibody-drug conjugate), about 10 mM histidine-histidine hydrochloride, about 80 mg / mL of sucrose, and about 0.2 mg / mL of polysorbate 80, pH about 6.0.

[0034] p-5) 15-25 mg / mL of ligand-drug conjugate (e.g., anti-CD79b antibody-drug conjugate), about 10 mM histidine-histidine hydrochloride, about 80 mg / mL of sucrose, and about 0.3 mg / mL of polysorbate 80, pH about 5.5.

[0035] p-6) 15-25 mg / mL of a ligand-drug conjugate (e.g., an anti-CD79b antibody-drug conjugate), about 10 mM histidine-histidine hydrochloride, about 80 mg / mL of sucrose, and about 0.3 mg / mL of polysorbate 80, pH about 5.75.

[0036] p-7) 15-25 mg / mL of a ligand-drug conjugate (e.g., an anti-CD79b antibody-drug conjugate), about 10 mM histidine-histidine hydrochloride, about 80 mg / mL of sucrose, and about 0.3 mg / mL of polysorbate 80, pH about 6.0.

[0037] p-8) 15-25 mg / mL of ligand-drug conjugate (e.g., anti-CD79b antibody-drug conjugate), about 10 mM histidine-histidine hydrochloride, about 80 mg / mL of sucrose, and about 0.1 mg / mL of polysorbate 80, pH about 5.5.

[0038] p-9) 15-25 mg / mL of ligand-drug conjugate (e.g., anti-CD79b antibody-drug conjugate), about 10 mM histidine-histidine hydrochloride, about 80 mg / mL of sucrose, and about 0.1 mg / mL of polysorbate 80, pH about 5.75.

[0039] p-10) 15-25 mg / mL of ligand-drug conjugate (e.g., anti-CD79b antibody-drug conjugate), about 10 mM histidine-histidine hydrochloride, about 80 mg / mL of sucrose, and about 0.1 mg / mL of polysorbate 80, pH about 6.0.

[0040] j) 15-25 mg / mL of ligand-drug conjugate (e.g., anti-CD79b antibody drug conjugate), 5-15 mM succinate buffer, 70-90 mg / mL of sucrose, and 0.1-0.3 mg / mL of polysorbate 80, pH 4.5-6.5 (e.g., 5.0-6.5).

[0041] k) 15-25 mg / mL of a ligand-drug conjugate (e.g., an anti-CD79b antibody drug conjugate), about 10 mM succinic acid-sodium succinate buffer, about 80 mg / mL sucrose, and 0.1-0.3 mg / mL polysorbate 80, pH about 5.0.

[0042] The anti-CD79b antibody-drug conjugate may be ADC-3 of Example 3-1 of the present disclosure, or ADC-5 of Example 3-2, or ADC-6 of Example 3-3, or ADC-7 of Example 3-4, for example, ADC-6 of Example 3-3.

[0043] The pharmaceutical composition of the present disclosure further comprises a solvent. In the pharmaceutical composition, the solvent is selected from physiologically acceptable non-toxic liquid vectors, such as, but not limited to, saline, water for injection, glucose solution (e.g., 5% glucose injection, glucose sodium chloride injection), etc.

[0044] The present disclosure further provides a pharmaceutical composition comprising an anti-CD79b antibody-drug conjugate, a histidine salt, sucrose, and polysorbate 80 in a concentration available for intravenous injection obtained by diluting any one of the above pharmaceutical compositions with 0.9% saline or 5% glucose solution, or after diluting any one of the above pharmaceutical compositions with 0.9% saline or 5% glucose solution.

[0045] Ligand-drug conjugates In some embodiments, the ligand-drug conjugate of any one of the above pharmaceutical compositions, The drug is selected from MMAE or a derivative thereof, exatecan or a derivative thereof, and eribulin or a derivative thereof, and the ligand is an anti-CD79b antibody or an antigen-binding fragment thereof, the anti-CD79b antibody or the antigen-binding fragment thereof comprising a heavy chain variable region (VH) and a light chain variable region (VL), The VH comprises HCDR1, HCDR2, and HCDR3 of the amino acid sequences shown in SEQ ID NOs: 11, 6, and 7, respectively, and the VL comprises LCDR1, LCDR2, and LCDR3 of the amino acid sequences shown in SEQ ID NOs: 8, 9, and 10, respectively; The VH comprises HCDR1, HCDR2 and HCDR3 having the amino acid sequences shown in SEQ ID NOs: 5, 6 and 7, respectively, and the VL comprises LCDR1, LCDR2 and LCDR3 having the amino acid sequences shown in SEQ ID NOs: 8, 9 and 10, respectively.

[0046] In some embodiments, the anti-CD79b antibody, or antigen-binding fragment thereof, in the pharmaceutical composition may be selected from a murine antibody, a chimeric antibody, or a humanized antibody, for example a humanized antibody.

[0047] In an alternative embodiment, the light and heavy chain FR sequences in the light chain and VH of said humanized anti-CD79b antibody in the pharmaceutical composition are derived from human germline light and heavy chain FRs, respectively, or mutated sequences thereof.

[0048] In some embodiments, the anti-CD79b antibody, or antigen-binding fragment thereof, in the pharmaceutical composition comprises an antibody VH and VL, wherein: The VH sequence is SEQ ID NO: 3 or an amino acid sequence having at least 80%, at least 85%, at least 90% identity thereto, and the VL sequence is SEQ ID NO: 4 or an amino acid sequence having at least 80%, at least 85%, at least 90% identity thereto; In some specific embodiments, the anti-CD79b antibody or antigen-binding fragment thereof in the pharmaceutical composition comprises an antibody VH and VL, wherein the VH sequence is set forth in SEQ ID NO:3 and the VL sequence is set forth in SEQ ID NO:4.

[0049] In some embodiments, the anti-CD79b antibody or antigen-binding fragment thereof further comprises a heavy chain constant region and a light chain constant region. In alternative embodiments, the heavy chain constant region is selected from human IgG1, IgG2, IgG3, IgG4 constant regions and common variants thereof, and the light chain constant region is selected from human κ and λ chain constant regions and common variants thereof. In some specific embodiments, the heavy chain constant region is human IgG1, IgG4.

[0050] In some embodiments, the anti-CD79b antibody, or antigen-binding fragment thereof, comprises an IgG Fc region, such as an IgG Fc region, such as an IgG Fc region, or an IgG1, IgG2, or IgG4 Fc region.

[0051] In some embodiments, the anti-CD79b antibody, or antigen-binding fragment thereof, comprises a heavy chain and a light chain, wherein the heavy chain has a sequence set forth in SEQ ID NO: 12, or a sequence having at least 80%, at least 85%, at least 90% identity thereto, and the light chain has a sequence set forth in SEQ ID NO: 13, or a sequence having at least 80%, at least 85%, at least 90% identity thereto.

[0052] In the present disclosure, the above-mentioned "at least 90% identity" covers at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity.

[0053] In some specific embodiments, the anti-CD79b antibody, or antigen-binding fragment thereof, in the pharmaceutical composition comprises an antibody heavy chain and a light chain, wherein the heavy chain sequence is set forth in SEQ ID NO:12 and the light chain sequence is set forth in SEQ ID NO:13.

[0054] In some specific embodiments, the anti-CD79b antibody or fragment thereof may be a variant, wherein the variant has between 1 and 10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) amino acid changes in the VL and / or between 1 and 10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) amino acid changes in the VH.

[0055] In some specific embodiments, the variants have the same or similar biological function or effect as the original anti-CD79b antibody or fragment thereof.

[0056] In some embodiments, the antigen-binding fragment of the anti-CD79b antibody is a Fab, Fv, sFv, Fab', F(ab')2, linear antibody, single chain antibody, scFv, sdAb, sdFv, nanobody, peptibody, domain antibody, and multispecific antibody (diabody, triabody and tetrabody, tandem di-scFv, tandem tri-scFv), such as, in particular, a scFv, Fv, Fab or Fab' fragment.

[0057] Ligand-drug (exatecan or its derivative) conjugate In some embodiments, the ligand-drug conjugate in any one of the above pharmaceutical compositions is a ligand-exatecan or a derivative thereof conjugate, which is represented by the general formula (Pc-LYD) of formula (I): [ka] Among them, Y is -O-(CR a R b ) m -CR 1 R 2 -C(O)-, -O-CR 1 R 2 -(CR a R b ) m -, -O-CR 1 R 2 -, -NH-(CR a R b ) m -CR 1 R 2 -C(O)- and -S-(CR a R b ) m -CR 1 R 2 -C(O)-, R a and R b are the same or different and are each independently selected from a hydrogen atom, a deuterium atom, a halogen, an alkyl group, a haloalkyl group, a deuterated alkyl group, an alkoxy group, a hydroxy group, an amino group, a cyano group, a nitro group, a hydroxyalkyl group, a cycloalkyl group, and a heterocyclyl group; or R a and R b together with the carbon atoms to which they are attached form a cycloalkyl group and a heterocyclyl group, R 1 is selected from a hydrogen atom, a halogen, a haloalkyl group, a deuterated alkyl group, a cycloalkyl group, a cycloalkylalkyl group, an alkoxyalkyl group, a heterocyclyl group, an aryl group, and a heteroaryl group; R 2is selected from a hydrogen atom, a halogen, a haloalkyl group, a deuterated alkyl group, a cycloalkyl group, a cycloalkylalkyl group, an alkoxyalkyl group, a heterocyclyl group, an aryl group, and a heteroaryl group, or R 1 and R 2 together with the carbon atom to which they are attached form a cycloalkyl or heterocyclyl group, Or, R a and R 2 together with the carbon atoms to which they are attached form a cycloalkyl or heterocyclyl group, m is an integer from 0 to 4; n is 1 to 10, n is a decimal number or an integer, and preferably, n is 2 to 8 or 5 to 9; L is a linker unit, Pc is any of the anti-CD79b antibodies or antigen-binding fragments thereof described in this disclosure above.

[0058] In some embodiments, the disclosed ligand-exatecan or derivative thereof conjugate or a pharma- ceutically acceptable salt or solvate thereof, wherein -Y- is -O-(CR a R b )m-CR 1 R 2 -C(O)-, R a and R b are the same or different and each independently selected from a hydrogen atom, a deuterium atom, a halogen, or an alkyl group; R 1 is C 3-6 Cycloalkylalkyl group or C 3-6 a cycloalkyl group, a hydrogen atom, R 2 is a hydrogen atom, a haloalkyl group or C 3-6 cycloalkyl group, preferably a hydrogen atom; Or, R 1 and R 2 are C together with the carbon atoms connected to them. 3-6 Forming a cycloalkyl group, m is 0 or 1.

[0059] In some embodiments, the disclosed ligand-exatecan or derivative thereof conjugate includes a structural unit -Y-, wherein the structural unit is -O-(CH2)m-CR 1 R 2 -C(O)-, R 1 is C 3-6 Cycloalkylalkyl group or C 3-6 a cycloalkyl group, a hydrogen atom, R 2 is a hydrogen atom, a haloalkyl group or C 3-6 cycloalkyl groups, Or, R 1 and R 2 are C together with the carbon atoms connected to them. 3-6 Forming a cycloalkyl group, m is 0 or 1.

[0060] In some embodiments, the disclosed ligand-exatecan or derivative thereof conjugate includes a structural unit -Y-, wherein the structural unit is -O-(CH2)m-CR 1 R 2 -C(O)-, R 1 is C 3-6 Cycloalkylalkyl group or C 3-6 a cycloalkyl group, a hydrogen atom, R 2 is a hydrogen atom, Or, R 1 and R 2 are C together with the carbon atoms connected to them. 3-6 Forming a cycloalkyl group, m is 0 or 1.

[0061] In some embodiments, the disclosed ligand-exatecan or derivative thereof conjugate includes a structural unit -Y-, wherein the structural unit is -O-(CH2)m-CR 1 R 2 -C(O)-, R 1 is C 3-6 Cycloalkylalkyl group or C3-6 a cycloalkyl group, a hydrogen atom, R 2 is a hydrogen atom, Or, R 1 and R 2 are C together with the carbon atoms connected to them. 3-6 Forming a cycloalkyl group, m is 0.

[0062] In some embodiments, the disclosed ligand-exatecan or derivative thereof conjugate includes a structural unit -Y-, wherein the structural unit is -O-(CH2)m-CR 1 R 2 -C(O)-, R 1 is a hydrogen atom, R 2 is a hydrogen atom, Or, R 1 and R 2 are C together with the carbon atoms connected to them. 3-6 Forming a cycloalkyl group, m is 0.

[0063] In some embodiments of the ligand-exatecan or derivative thereof conjugate of the present disclosure, Y is [ka] Selected from Among them, the O-terminus of Y is linked to a linker unit L.

[0064] In another embodiment, the present disclosure provides a ligand-exatecan or a derivative thereof conjugate, which is represented by the general formula (Pc-L-D1) of formula (II): [ka] Among them, R 1 is a hydrogen atom, C 3-6 Cycloalkylalkyl group or C 3-6 is a cycloalkyl group, R2 is a hydrogen atom, a haloalkyl group or C 3-6 cycloalkyl group, preferably a hydrogen atom; Or, R 1 and R 2 are C together with the carbon atoms connected to them. 3-6 Forming a cycloalkyl group, m is 0 or 1; n is 1 to 10 and may be an integer or a decimal point, preferably n is 1 to 8 or 1 to 6 decimal points or an integer, and more preferably n is 1 to 5 or 2 to 4 decimal points or an integer.

[0065] In some specific embodiments, the present disclosure provides a ligand-exatecan or a derivative thereof conjugate, wherein n is 1 to 8, and may be an integer or a decimal, and preferably 1 to 6, and may be an integer or a decimal.

[0066] In some embodiments, the present disclosure provides a ligand-exatecan or derivative thereof conjugate, wherein the linker unit -L- is -L 1 -L 2 -L 3 -L 4 - and L 1 -(succinimid-3-yl-N)-WC(O)-, -CH2-C(O)-NR 3 -WC(O)- or -C(O)-WC(O)-, in which W is selected from C 1-8 Alkyl group, C 1-8 alkyl-cycloalkyl groups or linear heteroalkyl groups of 1 to 8 atoms, said heteroalkyl groups containing 1 to 3 heteroatoms selected from N, O or S, among which the above C 1-8 the alkyl group, the cycloalkyl group, and the straight chain heteroalkyl group are each independently optionally further substituted with one or more substituents selected from halogen, hydroxyl group, cyano group, amino group, alkyl group, chloroalkyl group, deuterated alkyl group, alkoxy group, and cycloalkyl group; L 2 -NR 4(CH2CH2O)p 1 CH2CH2C(O)-, -NR 4 (CH2CH2O)p 1 CH2C(O)-, -S(CH2)p 1 C(O)- or a chemical bond, of which p 1 is an integer from 1 to 20, L 3 is a peptide residue consisting of 2 to 7 amino acids, among which the amino acids are selected from amino acid residues formed by the amino acids phenylalanine, glycine, valine, lysine, citrulline, serine, glutamic acid, and aspartic acid, and are optionally further substituted with one or more substituents selected from halogen, hydroxyl group, cyano group, amino group, alkyl group, chloroalkyl group, deuterated alkyl group, alkoxy group, and cycloalkyl group; L 4 -NR 5 (CR 6 R 7 ) t -, -C(O)NR 5 , -C(O)NR 5 (CH2) t - or a chemical bond, wherein t is an integer from 1 to 6; R 3 , R 4 and R 5 are the same or different and each independently selected from a hydrogen atom, an alkyl group, a haloalkyl group, a deuterated alkyl group, and a hydroxyalkyl group; R 6 and R 7 are the same or different and each independently selected from a hydrogen atom, a halogen, an alkyl group, a haloalkyl group, a deuterated alkyl group, and a hydroxyalkyl group.

[0067] In some embodiments, the present disclosure provides a ligand-exatecan or derivative thereof conjugate, wherein the linker unit L 1 is -(succinimid-3-yl-N)-(CH2)s 1-C(O)-, -(succinimid-3-yl-N)-CH2-cyclohexyl-C(O)-, -(succinimid-3-yl-N)-(CH2CH2O)s 2 -CH2CH2-C(O)-, -CH2-C(O)-NR 3 -(CH2)s 3 -C(O)- or -C(O)-(CH2)s 4 C(O)-, among which s 1 is an integer between 2 and 8, and s 2 is an integer from 1 to 3, and s 3 is an integer from 1 to 8, and s 4 is an integer from 1 to 8, and s 1 is preferably 5.

[0068] In some specific embodiments, the present disclosure provides a ligand-exatecan or a derivative thereof conjugate, wherein the linker unit L 2 -NR 4 (CH2CH2O)p 1 CH2C(O)- or a chemical bond, p 1 is an integer between 6 and 12.

[0069] In some specific embodiments, the present disclosure provides a ligand-exatecan or derivative thereof conjugate, wherein L 4 -NR 5 (CR 6 R 7 )t-, R 5 is selected from a hydrogen atom or an alkyl group, R 6 and R 7 are the same or different and each independently represent a hydrogen atom or an alkyl group; t is 1 or 2, preferably 2; L 4 -NR 5 CR 6 R 7 - is preferred, L 4 is more preferably -NHCH2-.

[0070] In some specific embodiments, the present disclosure provides a ligand-exatecan or derivative thereof conjugate, wherein the linker unit -L- is -L1 -L 2 -L 3 -L 4 - and L 1 teeth [ka] and s 1 is an integer from 2 to 8, L 2 is a chemical bond, L 3 is a tetrapeptide residue, L 4 -NR 5 (CR 6 R 7 )t- and R 5 is selected from a hydrogen atom or an alkyl group, R 6 and R 7 are the same or different and each independently represent a hydrogen atom or an alkyl group; and t is 1 or 2.

[0071] In some specific embodiments, the present disclosure provides a ligand-exatecan or derivative thereof conjugate, wherein the linker unit -L- is -L 1 -L 2 -L 3 -L 4 - and L 1 is -(succinimid-3-yl-N)-CH2-cyclohexyl-C(O)-, L 2 -NR 4 (CH2CH2O)9CH2C(O)-, L 3 is a tetrapeptide residue, L 4 -NR 5 (CR 6 R 7 )t- and R 5 is selected from a hydrogen atom or an alkyl group, R 6 and R 7 are the same or different and each independently represent a hydrogen atom or an alkyl group; and t is 1 or 2.

[0072] In some specific embodiments, the present disclosure provides a ligand-exatecan or derivative thereof conjugate, wherein the L 3 The peptide residue is an amino acid residue formed by one, two or more amino acids selected from phenylalanine (E), glycine (G), valine (V), lysine (K), citrulline, serine (S), glutamic acid (E) and aspartic acid (N), preferably an amino acid residue formed by one, two or more amino acids selected from phenylalanine and glycine, more preferably a tetrapeptide residue, and most preferably a tetrapeptide residue of GGFG (glycine-glycine-phenylalanine-glycine).

[0073] In some embodiments, the present disclosure provides a conjugate of a ligand-exatecan or a derivative thereof, wherein the linker unit -L- is 1 The end is linked to a ligand, L 4 The end is linked to Y.

[0074] In some embodiments, the disclosure provides a ligand-exatecan or derivative thereof conjugate, wherein -LY- is [ka] and L 1 is -(succinimid-3-yl-N)-(CH2)s 1 -C(O)- or -(succinimid-3-yl-N)-CH2-cyclohexyl-C(O)-; L 2 -NR 4 (CH2CH2O)p 1 CH2C(O)- or a chemical bond, p 1 is an integer between 6 and 12, L 3 is the tetrapeptide residue of GGFG, R 1 is a hydrogen atom, a cycloalkylalkyl group or a cycloalkyl group, preferably C3-6 Cycloalkylalkyl group or C 3-6 is a cycloalkyl group, R 2 is a hydrogen atom, a haloalkyl group or C 3-6 cycloalkyl group, preferably a hydrogen atom; Or, R 1 and R 2 are C together with the carbon atoms connected to them. 3-6 Forming a cycloalkyl group, R 5 is selected from a hydrogen atom or an alkyl group, R 6 and R 7 are the same or different and each independently represent a hydrogen atom or an alkyl group, s 1 is an integer from 2 to 8, preferably 5; m is an integer from 0 to 4.

[0075] In some embodiments, the present disclosure provides a ligand-exatecan or derivative thereof conjugate, Among them, the above -LY- is [ka] and Preferably, [ka] and L 2 -NR 4 (CH2CH2O)9CH2C(O)-, L 3 is the tetrapeptide residue of GGFG, R 1 is a hydrogen atom, a cycloalkylalkyl group or a cycloalkyl group, preferably C 3-6 Cycloalkylalkyl group or C 3-6 is a cycloalkyl group, R 2 is a hydrogen atom, a haloalkyl group or C 3-6 cycloalkyl group, preferably a hydrogen atom; Or, R 1 and R 2 are C together with the carbon atoms connected to them. 3-6 Forming a cycloalkyl group, R 5 is selected from a hydrogen atom or an alkyl group, R 6 and R 7 are the same or different and each independently represent a hydrogen atom or an alkyl group, m is an integer from 0 to 4.

[0076] In some embodiments, the present disclosure provides a ligand-exatecan or a derivative thereof conjugate, wherein the ligand-exatecan or a derivative thereof conjugate comprises a structure represented by formula (III): [ka] Among them, L 2 is a chemical bond, L 3 is the tetrapeptide residue of GGFG, R 1 is a hydrogen atom, C 3-6 Cycloalkylalkyl group or C 3-6 is a cycloalkyl group, R 2 is a hydrogen atom, a haloalkyl group or C 3-6 cycloalkyl groups, Or, R 1 and R 2 are C together with the carbon atoms connected to them. 3-6 Forming a cycloalkyl group, R 5 is selected from a hydrogen atom or an alkyl group, R 6 and R 7 are the same or different and each independently represent a hydrogen atom or an alkyl group, s 1 is an integer from 2 to 8, m is an integer from 0 to 4.

[0077] In some embodiments, the present disclosure provides a ligand-exatecan or a derivative thereof conjugate, wherein the ligand-exatecan or a derivative thereof conjugate comprises a structure represented by the formula (-LY-): [ka] It can be used to obtain a ligand-drug conjugate comprising a drug and a ligand linked via a linking fragment, Among them, L 1 is -(succinimid-3-yl-N)-(CH2)s 1 -C(O)- or -(succinimid-3-yl-N)-CH2-cyclohexyl-C(O)-; L 2 -NR 4 (CH2CH2O)p 1 CH2C(O)- or a chemical bond, p 1 is an integer from 1 to 20, L 3 is the tetrapeptide residue of GGFG, R 1 is a hydrogen atom, a cycloalkylalkyl group or a cycloalkyl group, preferably C 3-6 Cycloalkylalkyl group or C 3-6 is a cycloalkyl group, R 2 is a hydrogen atom, a haloalkyl group or C 3-6 cycloalkyl group, preferably a hydrogen atom; Or, R 1 and R 2 are C together with the carbon atoms connected to them. 3-6 Forming a cycloalkyl group, R 5 , R 6 and R 7 are the same or different and each independently represent a hydrogen atom or an alkyl group, s 1 is an integer from 2 to 8, m is an integer from 0 to 4.

[0078] In some embodiments, the present disclosure provides a ligand-exatecan or a derivative thereof conjugate, wherein the ligand-exatecan or a derivative thereof conjugate comprises a structure represented by the formula (-LY-): [ka] Among them, L 2 is a chemical bond, L 3 is the tetrapeptide residue of GGFG, R 1 is a hydrogen atom, a cycloalkylalkyl group or a cycloalkyl group, preferably C 3-6 Cycloalkylalkyl group or C 3-6 is a cycloalkyl group, R 2 is a hydrogen atom, a haloalkyl group or C 3-6 cycloalkyl group, preferably a hydrogen atom; Or, R 1 and R 2 are C together with the carbon atoms connected to them. 3-6 Forming a cycloalkyl group, R 5 is selected from a hydrogen atom or an alkyl group, R 6 and R 7 are the same or different and each independently represent a hydrogen atom or an alkyl group, s 1 is an integer from 2 to 8, m is an integer from 0 to 4.

[0079] In some embodiments, the present disclosure provides a ligand-exatecan or a derivative thereof conjugate, which has the general formula (Pc-L a -Y-Dr) is a complex of a ligand-exatecan or a derivative thereof, [ka] Among them, W is for C 1-8 Alkyl group, C 1-8alkyl-cycloalkyl groups or linear heteroalkyl groups of 1 to 8 atoms, said heteroalkyl groups containing 1 to 3 heteroatoms selected from N, O or S, among which the above C 1-8 the alkyl group, the cycloalkyl group, and the straight chain heteroalkyl group are each independently optionally further substituted with one or more substituents selected from halogen, hydroxyl group, cyano group, amino group, alkyl group, chloroalkyl group, deuterated alkyl group, alkoxy group, and cycloalkyl group; L 2 -NR 4 (CH2CH2O)p 1 CH2CH2C(O)-, -NR 4 (CH2CH2O)p 1 CH2C(O)-, -S(CH2)p 1 C(O)- or a chemical bond, p 1 is an integer from 1 to 20, L 3 is a peptide residue consisting of 2 to 7 amino acids, wherein the amino acids are optionally further substituted with one or more substituents selected from halogen, hydroxyl group, cyano group, amino group, alkyl group, chloroalkyl group, deuterated alkyl group, alkoxy group, and cycloalkyl group; R 1 is selected from a hydrogen atom, a halogen, a cycloalkylalkyl group, a deuterated alkyl group, a cycloalkyl group, a heterocyclyl group, an aryl group, or a heteroaryl group; R 2 is selected from a hydrogen atom, a halogen, a haloalkyl group, a deuterated alkyl group, a cycloalkyl group, a heterocyclyl group, an aryl group, or a heteroaryl group; Or, R 1 and R 2 together with the carbon atoms to which they are attached form a cycloalkyl or heterocyclyl group, R 4 and R 5 are the same or different and each independently selected from a hydrogen atom, an alkyl group, a haloalkyl group, a deuterated alkyl group, and a hydroxyalkyl group; R 6 and R7 are the same or different and each independently selected from a hydrogen atom, a halogen, an alkyl group, a haloalkyl group, a deuterated alkyl group, and a hydroxyalkyl group; m is an integer from 0 to 4; n is 1 to 10 and may be an integer or a decimal; Pc is an anti-CD79b antibody or antigen-binding fragment thereof provided by this disclosure.

[0080] In some embodiments, the present disclosure provides a ligand-exatecan or derivative thereof conjugate having the general formula (Pc-L b -Y-Dr) is a complex of a ligand-exatecan or a derivative thereof, [ka] Among them, s 1 is an integer from 2 to 8, preferably 5; Pc, R 1 , R 2 , R 5 ~R 7 , m and n are as defined in formula (IV).

[0081] In some embodiments, the linking unit -LY- of the ligand-exatecan or derivative thereof conjugate of the present disclosure includes, but is not limited to, the following: [ka] [ka]

[0082] In some embodiments, the present disclosure provides ligand-exatecan or a derivative thereof conjugates, including, but not limited to, [ka] [ka] [Chem.] [Chem.] n is from 1 to 10 and can be an integer or a decimal, Pc is the anti-CD79b antibody or its antigen-binding fragment of the present disclosure.

[0083] In some embodiments, a method for preparing a complex of a ligand-exatecan or its derivative represented by the general formula (Pc-L a -Y-D), which comprises [Chem.]

[0084] After reducing Pc, performing a coupling reaction with the general formula (L a -Y-D) to obtain a compound represented by the general formula (Pc-L a -Y-D), wherein Pc is the anti-CD79b antibody or its antigen-binding fragment of the present disclosure, and W, L 2 , L 3 , R 1 , R 2 , R 5 ~R 7 , m and n are as defined in formula (IV).

[0085] In the above embodiments, Pc is any anti-CD79b antibody or its antigen-binding fragment according to the present disclosure, preferably the anti-CD79b antibody or its antigen-binding fragment in the examples, and more preferably an antibody comprising the heavy chain represented by SEQ ID NO: 12 and the light chain represented by SEQ ID NO: 13.

[0086] In some specific embodiments, the complex of the ligand-exatecan or its derivative of the present disclosure includes its tautomer, meso form, racemate, enantiomer, diastereomer, deuteride, or a mixture thereof.

[0087] The compounds and preparation methods thereof in WO2020063673 are hereby incorporated by reference in their entirety.

[0088] Ligand-drug (MMAE or its derivative) complex The present disclosure provides new MMAE analogs / derivatives, which are compounds represented by the general formula (D(MMAE)): [ka] or in the form of its tautomers, meso-isomers, racemates, enantiomers, diastereomers or mixtures thereof, or a medicamentous salt thereof, among which: R 8 ~R 13 is selected from a hydrogen atom, a halogen, a hydroxyl group, a cyano group, an alkyl group, an alkoxy group, and a cycloalkyl group, R 14 is selected from a hydrogen atom, an alkyl group, an alkoxy group, and a cycloalkyl group, R 15 ~R 18 any two of the groups form a cycloalkyl group, and the remaining two groups are optionally selected from a hydrogen atom, an alkyl group, and a cycloalkyl group; R 19 is selected from a hydrogen atom or an alkyl group, R 20 ~R 22 is selected from a hydrogen atom, a hydroxyl group, an alkyl group, an alkoxy group, or a halogen atom; R 23 is selected from an aryl group or a heteroaryl group, and the aryl group or the heteroaryl group is optionally further substituted with a substituent selected from a hydrogen atom, a halogen, a hydroxyl group, an alkyl group, an alkoxy group, and a cycloalkyl group.

[0089] In some embodiments of the present disclosure, the compound represented by the above general formula (D(MMAE)) is a compound represented by the general formula (D(MMAE)1): [ka] or in the form of its tautomers, meso-isomers, racemates, enantiomers, diastereomers, or mixtures thereof, or a medicamentable salt thereof; R 16 and R 17 forms a cycloalkyl group, R 9 ~R 15 , R 18 ~R 23 is as defined in general formula (D).

[0090] In some embodiments of the present disclosure, the compound represented by the above general formula (D(MMAE)) is [ka] It is.

[0091] Another aspect of the present disclosure relates to a Ligand-Drug conjugate, or a pharma- ceutically acceptable salt or solvate thereof, wherein said Ligand-Drug conjugate has the structure represented by the formula (-D(MMAE)): [ka] or in the form of its tautomers, meso-isomers, racemates, enantiomers, diastereomers, or mixtures thereof, or a medicamentous salt thereof, among which: R 9 ~R 13 is selected from a hydrogen atom, a halogen, a hydroxyl group, a cyano group, an alkyl group, an alkoxy group, and a cycloalkyl group, R 14 is selected from a hydrogen atom, an alkyl group, an alkoxy group, and a cycloalkyl group, R 15 ~R 18 any two of the groups form a cycloalkyl group, and the remaining two groups are optionally selected from a hydrogen atom, an alkyl group, and a cycloalkyl group; R 19 is selected from a hydrogen atom or an alkyl group, R 20 ~R 22is selected from a hydrogen atom, a hydroxyl group, an alkyl group, an alkoxy group, or a halogen atom; R 23 is selected from an aryl group or a heteroaryl group, the aryl group or the heteroaryl group being optionally further substituted with a substituent selected from a hydrogen atom, a halogen, a hydroxyl group, an alkyl group, an alkoxy group, and a cycloalkyl group; The wavy lines represent hydrogen atoms or alternatively represent covalent bonds to a linker unit or to an antibody that binds to an antigen expressed on a target cell.

[0092] In some embodiments of the present disclosure, the ligand-drug conjugate comprises a compound of formula (-D(MMAE)1), or a pharma- ceutically acceptable salt or solvate thereof, wherein the ligand-drug conjugate comprises a compound of formula (-D(MMAE)1), [ka] Among them, R 16 and R 17 forms a cycloalkyl group, Wavy line, R 9 ~R 15 , R 18 ~R 23 is as defined in the general formula (D(MMAE)).

[0093] In some embodiments of the present disclosure, the ligand-MMAE or derivative thereof conjugate or a pharma- ceutically acceptable salt or solvate thereof, wherein the ligand-MMAE or derivative thereof conjugate comprises the structure represented by the formula: [ka] The wavy lines represent hydrogen atoms or alternatively represent covalent bonds to a linker unit or to an antibody that binds to an antigen expressed on a target cell.

[0094] In some embodiments of the present disclosure, the ligand-MMAE or derivative thereof conjugate, or a pharma- ceutically acceptable salt or solvate thereof, is a ligand-drug conjugate represented by the general formula (Pc-LD(MMAE)), or a pharma- ceutically acceptable salt or solvate thereof: [ka] Among them, R 9 ~R 23 is as defined in the general formula (D(MMAE)), n is 1 to 10 and may be an integer or a decimal; Pc is an anti-CD79B antibody or antigen-binding fragment thereof of the disclosure, and L is a linker unit.

[0095] In some embodiments of the present disclosure, the ligand-MMAE or a derivative thereof conjugate, or a pharma- ceutically acceptable salt or solvate thereof, is a ligand-MMAE or a derivative thereof conjugate represented by the general formula (Pc-L-D1), or a pharma- ceutically acceptable salt or solvate thereof: [ka] Among them, R 9 ~R 23 is as defined in the general formula (-D(MMAE)), Pc, L, and n are as defined in the general formula (Pc-LD(MMAE)).

[0096] In some embodiments of the present disclosure, the ligand-MMAE or a derivative thereof conjugate, or a pharma- ceutically acceptable salt or solvate thereof, is a ligand-MMAE or a derivative thereof conjugate represented by the following general formula, or a pharma- ceutically acceptable salt or solvate thereof: [ka] Pc, L, and n are as defined in the general formula (Pc-LD(MMAE)).

[0097] In some embodiments of the present disclosure, in the ligand-MMAE or derivative thereof conjugate, or a pharma- ceutically acceptable salt or solvate thereof, n is 1 to 8, and may be an integer or a decimal, preferably 1 to 6, and may be an integer or a decimal.

[0098] In some embodiments of the present disclosure, the ligand-MMAE or derivative thereof conjugate or a pharma- ceutically acceptable salt or solvate thereof, wherein the linker unit -L- is -YL 5 -L 6 -L 7 -L 8 and Y is the stretch unit, [ka] or a chemical bond, X1 being selected from a hydrogen atom, an alkyl group, an alkoxy group, an aryl group, or a halogen; and X2 being selected from an alkylene group, the alkylene group being optionally further substituted with one or more substituents selected from a halogen, a hydroxy group, a cyano group, an amino group, an alkyl group, a chloroalkyl group, a deuterated alkyl group, an alkoxy group, and a cycloalkyl group; L 5 is a stretching unit, -(succinimid-3-yl-N)-WC(O)-, -CH2-C(O)-NR 24 -WC(O)- or -C(O)-WC(O)-, in which W is selected from C 1-8 Alkyl group, C 1-8 alkyl-cycloalkyl groups or linear heteroalkyl groups of 1 to 8 atoms, said heteroalkyl groups containing 1 to 3 heteroatoms selected from N, O or S, among which the above C 1-8the alkyl group, the cycloalkyl group, and the straight chain heteroalkyl group are each independently optionally further substituted with one or more substituents selected from halogen, hydroxyl group, cyano group, amino group, alkyl group, chloroalkyl group, deuterated alkyl group, alkoxy group, and cycloalkyl group; L 6 -NR 25 (CH2CH2O)p 1 CH2CH2C(O)-, -NR 25 (CH2CH2O)p 1 CH2C(O)-, -S(CH2)p 1 C(O)- or a chemical bond, of which p 1 is an integer from 1 to 20, preferably a chemical bond; L 7 is a peptide residue consisting of 2 to 7 amino acids, the amino acids being preferably selected from valine, citrulline, and methylvaline, among which the amino acids are optionally further substituted with one or more substituents selected from halogen, hydroxyl group, cyano group, amino group, alkyl group, chloroalkyl group, deuterated alkyl group, alkoxy group, and cycloalkyl group; R 24 and R 25 are the same or different and each independently selected from a hydrogen atom, an alkyl group, a haloalkyl group, a deuterated alkyl group, and a hydroxyalkyl group; L 8 is an extender unit, preferably PAB.

[0099] In some embodiments of the present disclosure, the ligand-MMAE or derivative thereof conjugate or a pharma- ceutically acceptable salt or solvate thereof, wherein Y is [ka] Selected from.

[0100] In some embodiments of the present disclosure, the ligand-MMAE or derivative thereof conjugate or a pharma- ceutically acceptable salt or solvate thereof, wherein L5 is -(succinimid-3-yl-N)-(CH2)s 1 -C(O)-, among which s 1 is an integer of 2 to 8, preferably [ka] It is.

[0101] In some embodiments of the present disclosure, the ligand-MMAE or derivative thereof conjugate or a pharma- ceutically acceptable salt or solvate thereof, wherein L 7 is a dipeptide amino acid unit, preferably selected from valine-citrulline.

[0102] In some embodiments of the present disclosure, the ligand-MMAE or derivative thereof conjugate or a pharma- ceutically acceptable salt or solvate thereof, wherein the linker unit -L- is [ka] Selected from The a-terminus is linked to a ligand and the b-terminus is linked to a drug.

[0103] In some embodiments of the present disclosure, the ligand-MMAE or derivative thereof conjugate or a pharma- ceutically acceptable salt or solvate thereof is selected from the following structural formulas: [ka] Among them, n is 1 to 10 and may be an integer or a decimal; Pc is an anti-CD79B antibody or antigen-binding fragment thereof of the present disclosure, preferably an anti-CD79B antibody or antigen-binding fragment thereof of the Examples, and more preferably an antibody comprising a heavy chain shown in SEQ ID NO:12 and a light chain shown in SEQ ID NO:13.

[0104] Another aspect of the present disclosure relates to a process for preparing a compound of general formula (D(MMAE)) or its tautomeric, meso, racemic, enantiomeric, diastereomeric, or mixture thereof, or a medicamentable salt thereof, comprising: [ka] A step of deprotecting the general formula (DA(MMAE)) to obtain a compound represented by the general formula (DA(MMAE)), Among them, R 9 ~R 23 is as defined in general formula (D).

[0105] Another aspect of the present disclosure is a method for producing a method for manufacturing a semiconductor device comprising the steps of: [ka] or in the form of a tautomer, meso-isomer, racemate, enantiomeric, diastereomeric, or mixture thereof, or a medicamentable salt thereof, which can be used as an intermediate in preparing the ligand-drug conjugate of the present disclosure.

[0106] Another aspect of the present disclosure relates to a method for preparing compound 2 (MMAE) or its tautomeric, meso, racemic, enantiomeric, diastereomeric, or mixture thereof, or a medicamentable salt thereof, comprising: [ka] The method includes a step of subjecting compound 1 (MMAE) to a condensation reaction with compound 2a (MMAE) to obtain compound 2 (MMAE).

[0107] Another aspect of the present disclosure is a method for preparing a ligand-drug conjugate of general formula (Pc-LD(MMAE)) or a pharma- ceutically acceptable salt or solvate thereof, comprising: [ka] The method includes the steps of reducing Pc, and then coupling the Pc with a compound to obtain a compound represented by the general formula (ADC(MMAE)-1), In this regard, Pc and n are as defined in the general formula (Pc-LD(MMAE)).

[0108] In order to achieve the goal of synthesizing MMAE and its derivatives in the present disclosure, the present disclosure adopts the following synthesis technology scheme:

[0109] Technical proposal 1: A method of the present disclosure for the preparation of a compound of general formula (D(MMAE)) or any of its tautomers, meso-isomers, racemates, enantiomers, diastereomers, or mixtures thereof, or a medicamentable salt thereof, comprising: [ka] The compound represented by the general formula (DA(MMAE)) is subjected to a deprotection reaction under basic conditions to obtain a compound represented by the general formula (D(MMAE)), Among them, R 9 ~R 23 is as defined in the general formula (D(MMAE)).

[0110] Reagents that provide basic conditions include organic and inorganic bases, the organic bases including, but not limited to, triethylamine, diethylamine, N-methylmorpholine, pyridine, hexahydropyridine, N,N-diisopropylethylamine, n-butyllithium, lithium diisopropylamide, potassium acetate, sodium tert-butoxide or potassium tert-butoxide, and the inorganic bases including, but not limited to, sodium hydride, potassium phosphate, sodium carbonate, potassium carbonate, cesium carbonate, sodium hydroxide and lithium hydroxide, preferably diethylamine.

[0111] Technical proposal 2: A method for preparing compound 2 (MMAE) of the present disclosure or a medicamentable salt or solvate thereof, the method comprising: [ka] The method includes adding a condensing agent to compound (1(MMAE)) and compound (2a(MMAE)) under basic conditions to carry out a condensation reaction to obtain compound 2.

[0112] Reagents that provide basic conditions include organic and inorganic bases, the organic bases including, but not limited to, triethylamine, diethylamine, N-methylmorpholine, pyridine, hexahydropyridine, N,N-diisopropylethylamine, n-butyllithium, lithium diisopropylamide, potassium acetate, sodium tert-butoxide or potassium tert-butoxide, and the inorganic bases including, but not limited to, sodium hydride, potassium phosphate, sodium carbonate, potassium carbonate, cesium carbonate, sodium hydroxide and lithium hydroxide, preferably N,N-diisopropylethylamine.

[0113] Condensing agents include 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride, 1-hydroxybenzotriazole and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, N,N'-dicyclohexylcarbodiimide, N,N'-diisopropylcarbodiimide, O-benzotriazole-N,N,N',N'-tetramethyluronium tetrafluoroborate, 1-hydroxybenzotriazole, 1-hydroxy-7-azabenzotriazole, O-benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate, 2-(7- azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate, benzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate or benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate, preferably 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride or 1-hydroxybenzotriazole and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, preferably 1-hydroxybenzotriazole.

[0114] Technical proposal 3: A method for the preparation of a ligand-drug conjugate of the general formula (Pc-LD) or a pharma- ceutically acceptable salt or solvate thereof according to the present disclosure, comprising: [ka] The method includes a step of reducing Pc, followed by coupling reaction with compound 2 (MMAE) to obtain a compound represented by the general formula (ADC(MMAE)-1), wherein the reducing agent is preferably TCEP, and particularly preferably a disulfide bond on a reduced antibody; In this regard, Pc and n are as defined in the general formula (Pc-LD(MMAE)).

[0115] Compounds of the Examples The present disclosure provides a ligand-drug conjugate selected from: [ka] wherein Pc is any anti-CD79b antibody or antigen-binding fragment thereof according to the present disclosure, and n is 1 to 10, and may be an integer or a decimal.

[0116] In some specific embodiments, Pc is an anti-CD79b antibody or antigen-binding fragment thereof according to the embodiments of the present disclosure, for example an antibody comprising a heavy chain set forth in SEQ ID NO: 12 and a light chain set forth in SEQ ID NO: 13, and n is an integer or decimal number between 1 and 6.

[0117] In some specific embodiments, the antibody drug conjugates of the disclosure may have an average DAR value of 1-10, e.g., any value between 2-8, or 2-6, or 1-6, or 4-6. In some embodiments, the DAR is between about 1 and about 6, e.g., about 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7.0, 7.5, 8.0. In some embodiments, at least 50% by weight of the sample are compounds having an average DAR ±2, and in some specific embodiments, at least 50% of the sample are conjugates comprising an average DAR ±1. For example, if the DAR is about 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9 ... In some embodiments, a DAR of "about x" means that the measured DAR is within 20% of x.

[0118] In some embodiments, any one of the pharmaceutical compositions may be administered as an intravenous injection, a subcutaneous injection, an intraperitoneal injection, or an intramuscular injection.

[0119] In some embodiments, the disclosure provides a method of preparing any one of the above pharmaceutical compositions, the method comprising replacing a stock solution of ligand-drug conjugate (e.g., anti-CD79b antibody-drug conjugate) with a buffer.

[0120] The present disclosure further provides a liquid formulation comprising any one of the above described pharmaceutical compositions.

[0121] The pharmaceutical compositions described in the present disclosure have sufficient drug stability and can be left stably for a long period of time.

[0122] The present disclosure further provides a method of preparing a lyophilized formulation of a pharmaceutical composition comprising a ligand-drug conjugate (e.g., an anti-CD79b antibody drug conjugate), the method comprising the step of lyophilizing the pharmaceutical composition.

[0123] The present disclosure further provides a lyophilized formulation of a pharmaceutical composition comprising a ligand-drug conjugate (e.g., an anti-CD79b antibody-drug conjugate), the lyophilized formulation being obtainable by lyophilizing any one of the pharmaceutical compositions described above.

[0124] In some embodiments, the lyophilized formulation is stable for at least 1 month, at least 3 months, at least 6 months, at least 12 months, at least 18 months, at least 24 months, or at least 30 months when stored in the dark at 2-8° C.

[0125] In some embodiments, the lyophilized formulation is stable at 25° C. for at least 1 month, at least 3 months, at least 6 months, or at least 12 months.

[0126] In some embodiments, the lyophilized formulation is stable at 40° C. for at least 7 days, at least 14 days, or at least 30 days.

[0127] The present disclosure further provides a reconstituted solution of a pharmaceutical composition comprising a ligand-drug conjugate (e.g., an anti-CD79b antibody-drug conjugate of the present disclosure), the reconstituted solution being prepared by reconstituting any one of the lyophilized formulations described above.

[0128] The present disclosure further provides an article of manufacture comprising a container containing any one of the pharmaceutical compositions, liquid formulations, lyophilized formulations, or reconstituted solutions described above. In some embodiments, the container may be, but is not limited to, a neutral borosilicate glass injection vial.

[0129] The present disclosure further provides the use of any one of the above pharmaceutical compositions, any one of the above liquid formulations, any one of the above lyophilized formulations, any one of the above reconstituted solutions, or any one of the above products in the preparation of a medicament for treating or preventing a proliferative disease or delaying the progression of a proliferative disease.

[0130] The present disclosure further provides any one of the above pharmaceutical compositions, any one of the above liquid formulations, any one of the above lyophilized formulations, any one of the above reconstituted solutions, or any one of the above products for use in treating or preventing a proliferative disease or delaying the progression of a proliferative disease.

[0131] In some embodiments, the proliferative disorder described in any one of the above may be a cancer or tumor, wherein the cancer or tumor is selected from lymphoma, diffuse large B-cell lymphoma, non-Hodgkin's lymphoma (NHL), aggressive NHL, relapsed aggressive NHL, relapsed indolent NHL, refractory NHL, refractory indolent NHL, chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma, leukemia, hairy cell leukemia (HCL), acute lymphocytic leukemia (ALL), and / or mantle cell lymphoma.

[0132] The present disclosure provides a method for treating or preventing a proliferative disease or slowing the progression of a proliferative disease, comprising administering to a subject in need thereof a therapeutically or prophylactically effective amount of any one of the pharmaceutical compositions described above, any one of the liquid formulations described above, any one of the lyophilized formulations described above, any one of the reconstituted solutions described above, or any one of the products described above, wherein the proliferative disease may be a cancer or tumor, and the cancer or tumor is selected from lymphoma, diffuse large B-cell lymphoma, non-Hodgkin's lymphoma (NHL), aggressive NHL, relapsed aggressive NHL, relapsed indolent NHL, refractory NHL, refractory indolent NHL, chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma, leukemia, hairy cell leukemia (HCL), acute lymphocytic leukemia (ALL), and / or mantle cell lymphoma.

[0133] The present disclosure provides a method of enhancing immune function in a subject having a B cell proliferative condition or an autoimmune condition, comprising administering to a subject in need thereof an effective amount of any one of the pharmaceutical compositions described above, any one of the liquid formulations described above, any one of the lyophilized formulations described above, any one of the reconstituted solutions described above, or any one of the products described above, to a subject in need thereof, wherein the B cell proliferative condition is a cancer or tumor, and the B cell proliferative condition is lymphoma, non-Hodgkin's lymphoma (NHL), aggressive NHL, relapsed aggressive NHL, relapsed indolent NHL, refractory NHL, refractory indolent NHL, chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma, leukemia, hairy cell leukemia (HCL), acute lymphocytic leukemia (ALL), and / or mantle cell lymphoma. [Brief description of the drawings]

[0134] [Figure 1] A to C show the therapeutic effects of different ADCs on human diffuse large B-cell lymphoma WSU-DLCL2 tumors subcutaneously transplanted into nude mice. [Diagram 2] FIG. 1 shows the effect of different ADCs on the body weight of tumor-bearing nude mice. [Diagram 3]The therapeutic effect of different ADCs on human diffuse large B-cell lymphoma WSU-DLCL2 tumors subcutaneously transplanted into nude mice. [Figure 4] Effect of different ADCs on the body weight of WSU-DLCL2 tumor-bearing nude mice. [Diagram 5] 1 shows tumor photographs showing the therapeutic effects of different ADCs on human diffuse large B-cell lymphoma WSU-DLCL2 tumors subcutaneously transplanted into nude mice. [Figure 6] Therapeutic effects of different ADCs on human follicular lymphoma DOHH-2 tumors subcutaneously transplanted into nude mice. [Figure 7] Effect of different ADCs on the body weight of DOHH-2 tumor-bearing nude mice. [Figure 8] Screening of the pH value of the buffer system of the ADC composition formulation. Figures 8A and 8B show the trends of change in SEC and RCE, respectively, under different pH / buffer conditions. [Figure 9] Screening of additives and surfactants for ADC compositions. Figures 9A and 9B show the trends of SEC and RCE changes under different pH / buffer conditions, respectively. [Figure 10] 10A and 10B show the change trends of SEC and RCE under different pH / buffer conditions, respectively. Among them, ADC-1, ADC-2, and ADC-4 are other anti-CD79b antibody-drug conjugates obtained by screening in the present disclosure, and their specific structures are not shown. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0135] term In order that the present disclosure may be more readily understood, 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 those of ordinary skill in the art.

[0136] An "antibody drug conjugate (ADC)" is a compound that links an antibody or an antibody fragment to a biologically active cytotoxin or a small molecule drug with cell killing activity via a stable chemical linker compound, taking advantage of the specificity of the antibody to tumor cells or the specificity of the binding to highly expressed antigens and the high efficiency of the cytotoxin to avoid toxicity and side effects on normal cells. Compared with conventional chemotherapy drugs, the antibody drug conjugate can precisely bind to tumor cells and reduce the impact on normal cells.

[0137] "Buffer" refers to a buffer that resists changes in pH by the action of its acid-base conjugate components. Examples of buffers that control pH in the appropriate range include acetate, succinate, gluconate, histidine, oxalate, lactate, phosphate, citrate, tartrate, fumarate, glycylglycine and other organic acid buffers.

[0138] A "histidine salt buffer" is a buffer containing histidine ions. Examples of histidine salt buffers include histidine-hydrochloride, histidine-acetate, histidine-phosphate, histidine-sulfate, etc., and preferably histidine-hydrochloride buffers. Histidine-hydrochloride buffers are prepared with histidine and hydrochloric acid or histidine and histidine hydrochloride.

[0139] "Tris-citrate buffer" is a buffer containing citrate ions. Examples of Tris-citrate buffers include Tris-hydrochloride, Tris-acetate, Tris-phosphate, Tris-sulfate, Tris-citrate, and the like buffers, and preferably Tris-citrate.

[0140] A "Tris-hydrochloride buffer" is a buffer that contains hydrochloride ions. Examples of Tris-hydrochloride buffers include Tris-hydrochloride, Tris-acetate, Tris-phosphate, Tris-sulfate, Tris-citrate, and the like buffers. A preferred citrate buffer is Tris-hydrochloride buffer.

[0141] A "phosphate buffer" is a buffer that contains phosphate ions. Examples of phosphate buffers include disodium hydrogen phosphate-sodium dihydrogen phosphate, disodium hydrogen phosphate-potassium dihydrogen phosphate, etc. A preferred phosphate buffer is disodium hydrogen phosphate-sodium dihydrogen phosphate buffer.

[0142] An "acetate buffer" is a buffer that contains acetate ions. Examples of acetate buffers include acetic acid-sodium acetate, acetic acid-histidine salt, acetic acid-potassium acetate, acetic acid-calcium acetate, acetic acid-magnesium acetate, etc. A preferred acetate buffer is acetic acid-sodium acetate buffer.

[0143] A "succinate buffer" is a buffer containing succinate ions. Examples of succinate buffers include succinic acid-sodium succinate, succinic acid-potassium succinate, succinic acid-calcium succinate, etc. A preferred succinate buffer is succinic acid-sodium succinate. Exemplarily, the succinic acid-sodium succinate may be prepared with succinic acid and sodium hydroxide, or succinic acid and sodium succinate.

[0144] A "pharmaceutical composition" refers to a mixture containing one or more compounds described herein or their physiologically / pharmaceutical acceptable salts or prodrugs, and other chemical components such as physiologically / pharmaceutical acceptable vectors and excipients. The pharmaceutical composition is intended to facilitate administration to a living body and contribute to the absorption of the active ingredient to further exert biological activity. In this specification, the terms "pharmaceutical composition" and "formulation" are not mutually exclusive.

[0145] In the solution forms of the pharmaceutical compositions described in this disclosure, unless otherwise specified, the solvent therein is water.

[0146] The term "about" as used herein means that a numerical value is within an acceptable error range of a specific value as determined by one of ordinary skill in the art, the numerical portion being determined by how it is measured or determined (i.e., the limitations of the measurement system). For example, in the practice of the art, "about" may mean within or more than one standard difference. Alternatively, "about" or "essentially including" may mean a range of at most 20%. Moreover, particularly for biological systems or processes, the term may mean at most an order of magnitude or at most 5 times the numerical value. Unless otherwise indicated, when a specific value appears in the present application and claims, the meaning of "about" or "essentially including" should be assumed to be within an acceptable error range of the specific value.

[0147] The pharmaceutical compositions described in the present disclosure can achieve a stable effect, i.e., the antibody therein essentially retains its physical and / or chemical stability and / or biological activity after storage, and preferably, the pharmaceutical composition essentially retains its physical and chemical stability and its biological activity after storage. The storage period is generally selected based on the desired shelf life of the pharmaceutical composition. Currently, there are several analytical techniques for measuring protein stability, which can measure stability after storage at a given temperature for a given period of time.

[0148] A stable drug antibody formulation is one that does not show significant changes when stored at refrigerated temperatures (2-8°C) for at least 3 months, preferably 6 months, more preferably 1 year, and even more preferably up to 2 years. Stable liquid formulations also include liquid formulations that exhibit the desired characteristics after storage at temperatures including 25°C for periods including 1 month, 3 months, 6 months, or 40°C for periods including 1 month. A typical acceptable criterion for stability is that typically no more than about 10% and preferably no more than about 5% of the antibody monomer is degraded as measured by SEC-HPLC. Upon visual analysis, the drug antibody formulation is colorless or yellow, clear to slightly opalescent. The concentration, pH, and osmolality of the formulation have a variation of no more than ±10%. Typically no more than about 10% and preferably no more than about 5% cleavage is observed, and typically no more than about 10% and preferably no more than about 5% aggregates are formed.

[0149] An antibody "retains its physical stability" in a pharmaceutical formulation if it does not exhibit significant increased aggregation, precipitation, and / or denaturation as determined by visual inspection of color and / or clarity or by UV light scattering, size exclusion chromatography (SEC), and dynamic light scattering (DLS). Changes in protein conformation can be assessed by fluorescence spectroscopy (which determines the tertiary structure of proteins) and by FTIR spectroscopy (which determines the secondary structure of proteins).

[0150] An antibody "retains its chemical stability" in a pharmaceutical formulation if the antibody does not show significant chemical changes. Chemical stability can be assessed by detecting and quantifying chemically altered forms of the protein. Degradation processes that constantly change the chemical structure of a protein include hydrolysis or cleavage (assessed by methods such as size exclusion chromatography and SDS-PAGE), oxidation (assessed by methods such as peptide mapping combined with mass spectrometry or MALDI / TOF / MS), deamidation (assessed by methods such as ion exchange chromatography, capillary isoelectric focusing, peptide mapping, measurement of isoaspartic acid), and isomerization (assessed by measurement of isoaspartic acid content, peptide mapping, etc.).

[0151] An antibody "retains its biological activity" in a pharmaceutical formulation if the biological activity of the antibody over a given time period is within a given range of the biological activity exhibited when the drug formulation was prepared. Antibody biological activity can be determined, for example, by antigen binding assays.

[0152] The engineered antibodies or antigen-binding fragments according to the present disclosure can be prepared and purified by conventional methods. For example, the cDNA sequences encoding the heavy and light chains can be cloned and recombined into a GS expression vector. The recombinant immunoglobulin expression vector can be stably transfected into CHO cells. In one of the more preferred prior art, the mammalian expression system leads to glycosylation of the antibody, especially at the highly conserved N-terminal site of the Fc region. Stable clones are obtained by expressing an antibody that specifically binds to human FXI. Positive clones are expanded in culture in serum-free medium in a bioreactor to produce the antibody. The culture medium in which the antibody is secreted can be purified by conventional techniques. For example, purification is performed on an A or G Sepharose FF column with a regulated buffer. Non-specifically bound components are washed off. Furthermore, the bound antibody is eluted by a pH gradient method, and the antibody fragments are detected and collected by SDS-PAGE. The antibody can be filtered and concentrated by conventional methods. Soluble impurities and multimers may be removed by conventional methods such as molecular sieves, ion exchange, etc. The resulting product should be immediately frozen, such as at -70°C, or lyophilized.

[0153] The three-letter and one-letter codes for amino acids used in this application are as described in J. Biol. Chem, 243, p. 3558 (1968).

[0154] The term "antibody" described in this application is used in the broadest sense and covers various antibody structures. Exemplarily, an antibody refers to an immunoglobulin, which is a tetrapeptide chain structure consisting of two identical heavy chains and two identical light chains linked by interchain disulfide bonds. Immunoglobulins differ in amino acid composition and sequence order of the heavy chain constant region, and therefore their antigenicity also differs. Thus, immunoglobulins can be divided into five types, or immunoglobulin isotypes, IgM, IgD, IgG, IgA, and IgE, whose corresponding heavy chains are μ chain, δ chain, γ chain, α chain, and ε chain, respectively. Ig of the same type can be further divided into different subclasses depending on the amino acid composition of the hinge region and the number and position of the heavy chain disulfide bonds, for example, IgG can be divided into IgG1, IgG2, IgG3, and IgG4. Light chains are divided into κ chains or λ chains depending on the difference in the constant region. Each of the five types of Ig may have a κ chain or a λ chain.

[0155] In the present application, the antibody light chain variable region described herein may further comprise a light chain constant region, said light chain constant region comprising a human or mouse kappa, lambda chain or a variant thereof.

[0156] In the present application, the antibody heavy chain variable region described in the present application may further comprise a heavy chain constant region, said heavy chain constant region comprising human or mouse IgG1, 2, 3, 4 or variants thereof.

[0157] In the antibody heavy and light chains, the sequence of about 110 amino acids near the N-terminus is largely changed and becomes the variable region (V region), and the remaining amino acid sequence near the C-terminus is relatively stable and becomes the constant region (C region). The variable region includes three hypervariable regions (HVR) and four framework regions (FR) whose sequences are relatively conservative. The three hypervariable regions determine the specificity of the antibody and are also called complementarity determining regions (CDR). Each light chain variable region (VL) and heavy chain variable region (VH) consists of three CDR regions and four FR regions, which are arranged in the following order from the amino terminus to the carboxy terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The three CDR regions of the light chain are referred to as LCDR1, LCDR2, and LCDR3, and the three CDR regions of the heavy chain are referred to as HCDR1, HCDR2, and HCDR3. The CDR amino acid residues of the VL and VH regions of the above antibodies or antigen-binding fragments conform in number and position to the known Chothia (ABM) numbering convention.

[0158] The term "recombinant human antibody" includes human antibodies prepared, expressed, engineered or isolated by recombinant techniques and methods well known in the art, such as, for example: (1) Antibodies isolated from transgenic or transchromosomal animals (e.g., mice) containing human immunoglobulin genes, or from hybridomas prepared thereby; (2) an antibody isolated from a host cell transformed to express the antibody, e.g., a transfectoma; (3) Antibodies isolated from a recombinant combinatorial human antibody library, and (4) Antibodies prepared, expressed, created or isolated by any method, such as by splicing human immunoglobulin gene sequences into other DNA sequences.

[0159] Such recombinant human antibodies contain variable and constant regions that utilize specific human germline immunoglobulin sequences encoded by germline genes, but also contain subsequent rearrangements and mutations that occur, for example, during antibody maturation.

[0160] The term "mouse antibody" as used herein refers to a monoclonal antibody against human CD79b or an epitope thereof, prepared by the knowledge and techniques in the art. During preparation, CD79b antigen is injected into a test subject, and hybridomas expressing antibodies with desired sequence or functional properties are isolated. In a specific embodiment of the present application, the mouse CD79b antibody or antigen-binding fragment thereof may further comprise a light chain constant region of a mouse kappa, lambda chain or a variant thereof, or may further comprise a heavy chain constant region of a mouse IgG1, IgG2, IgG3, or IgG4 or a variant thereof.

[0161] The term "human antibody" includes antibodies having variable and constant regions derived from human germline immunoglobulin sequences. Human antibodies according to the present application may contain amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, the term "human antibody" does not include antibodies in which CDR sequences derived from the germline of another mammalian species (e.g., mouse) have been grafted onto human framework sequences (i.e., "humanized antibodies").

[0162] The term "humanized antibody", also known as CDR-grafted antibody, refers to an antibody produced by grafting mouse CDR sequences onto a human antibody variable region framework. This overcomes the strong immune response induced by chimeric antibodies having a large amount of mouse protein components. To avoid the loss of activity associated with reduced immunogenicity, the human antibody variable region can be subjected to minimal back mutations so that activity is maintained.

[0163] The term "chimeric antibody" refers to an antibody in which the variable region of a mouse antibody is fused with the constant region of a human antibody, and can reduce the immune response reaction induced by mouse antibodies. To create a chimeric antibody, first, a hybridoma secreting a mouse-specific monoclonal antibody is created, and then the variable region gene is cloned from the mouse hybridoma cell, and if necessary, the constant region gene of a human antibody is cloned, and the mouse variable region gene and the human constant region gene are linked to form a chimeric gene, which is then inserted into a human vector, and finally, the chimeric antibody molecule is expressed in a eukaryotic or prokaryotic cell system. The constant region of the human antibody may be selected from the heavy chain constant region of human IgG1, IgG2, IgG3, or IgG4 or a variant thereof, and preferably contains a human IgG2 or IgG4 heavy chain constant region, or an IgG1 that has no ADCC (antibody-dependent cell-mediated cytotoxicity) toxicity after amino acid mutation is used.

[0164] The term "antigen-binding fragment" as used herein refers to a Fab fragment, a Fab' fragment, a F(ab')2 fragment having antigen-binding activity, and an Fv fragment or an sFv fragment that binds to human CD79b. An Fv fragment contains an antibody heavy chain variable region and a light chain variable region, but lacks a constant region and has a minimal antibody fragment containing all antigen-binding sites. In general, an Fv antibody further contains a polypeptide linker between the VH and VL domains and can form a structure required for antigen binding. Two antibody variable regions may be linked by different linkers to form a single polypeptide chain called a single chain antibody or single chain Fv (sFv).

[0165] The term "single chain antibody", "single chain Fv" or "scFv" is meant to include molecules of an antibody heavy chain variable domain (or region, VH) and an antibody light chain variable domain (or region, VL) linked by a linker. Such scFv molecules can have the general structures NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH. A suitable prior art linker consists of a repeat of the GGGGS amino acid sequence or a variant thereof, for example a 1-4 repeat variant (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90:6444-6448). Other linkers that can be used in the present disclosure are described in Alfthan et al. (1995), Protein Eng. 8:725-731, Choi et al. (2001), Eur. J. Immunol. 31:94-106, Hu et al. (1996), Cancer Res. 56:3055-3061, Kipriyanov et al. (1999), J. Mol. Biol. 293:41-56, and Roovers et al. (2001), Cancer Immunol.

[0166] The term "CDR" refers to one of the six main hypervariable regions in the variable domain of an antibody that mediate antigen binding. One of the most commonly used definitions of the six CDRs is provided by Kabat EA et al., (1991) Sequences of proteins of immunological interest. NIH Publication 91-3242). As used herein, the Kabat definition of CDR applies only to CDR1, CDR2 and CDR3 (CDR L1, CDR L2, CDR L3 or L1, L2, L3) of the light chain variable domain and CDR2 and CDR3 (CDR H2, CDR H3 or H2, H3) of the heavy chain variable domain. Typically, each heavy chain variable region has three CDRs (HCDR1, HCDR2, HCDR3) and each light chain variable region has three CDRs (LCDR1, LCDR2, LCDR3). The amino acid sequence boundaries of the CDRs can be determined by any one of a variety of known methods, including the "Kabat" numbering convention (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 convention (see Al-Lazikani et al., (1997) JMB 273:927-948), and the ImMunoGenTics (IMGT) numbering convention (Lefranc MP, Immunologist, 7, 132-136 (1999); Lefranc, MP et al., Dev. Comp. Immunol., 27, 55-77 (2003)). For example, in a typical format, according to the Kabat rules, the CDR amino acid residues in the heavy chain variable region (VH) are numbered 31 to 35 (HCDR1), 50 to 65 (HCDR2), and 95 to 102 (HCDR3), and the CDR amino acid residues in the light chain variable region (VL) are numbered 24 to 34 (LCDR1), 50 to 56 (LCDR2), and 89 to 97 (LCDR3).According to the Chothia rules, the CDR amino acid numbers in VH are 26-32 (HCDR1), 52-56 (HCDR2), and 95-102 (HCDR3), and the amino acid residue numbers in VL are 26-32 (LCDR1), 50-52 (LCDR2), and 91-96 (LCDR3). According to the combined CDR definition of Kabat and Chothia, the CDRs are composed of amino acid residues 26-35 (HCDR1), 50-65 (HCDR2), and 95-102 (HCDR3) in human VH, and amino acid residues 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3) in human VL. According to the IMGT rules, the numbers of the CDR amino acid residues in VH are approximately 26 to 35 (CDR1), 51 to 57 (CDR2) and 93 to 102 (CDR3), and the numbers of the CDR amino acid residues in VL are approximately 27 to 32 (CDR1), 50 to 52 (CDR2) and 89 to 97 (CDR3). According to the IMGT rules, the CDR regions of an antibody can be determined by the program IMGT / DomainGap Align.

[0167] The term "antibody framework" refers to a part of a variable domain VL or VH that serves as a support for the antigen binding loops (CDRs) of said variable domain. In effect, it is a variable domain without the CDRs.

[0168] The term "binds to CD79b" as used herein means capable of interacting with CD79b or an epitope thereof, which may be of human origin. The term "antigen-binding site" as used herein refers to a non-contiguous site in an antigen and in three dimensional space that is recognized by an antibody or antigen-binding fragment of the present application.

[0169] The term "epitope" or "antigenic determinant" refers to a site on an antigen that is specifically bound by an immunoglobulin or antibody. An epitope usually includes at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 consecutive or non-consecutive amino acids in a unique spatial conformation. See, e.g., Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, GE Morris, Ed. (1996).

[0170] The terms "specific binding," "selective binding," "selectively binds," and "specifically binds" refer to the binding of an antibody to a predetermined epitope on an antigen. Typically, an antibody binds to an antigen at a concentration of about 10 -7 M is smaller than that, for example, about 10 -8 M, 10 -9 M or 10 -10 It binds with an affinity (KD) lower than or equal to M.

[0171] The "identity" of an amino acid sequence is the percentage of amino acid residues in a first sequence that are identical to those in a second sequence, with alignment of the amino acid sequences and gaps, if necessary, so that the percentage of sequence identity is maximized, and without any conservative substitutions being considered as part of the sequence identity. To measure the percentage of amino acid sequence identity, alignment can be achieved by various methods within the skill of the art, for example, publicly available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR) software. Those skilled in the art can determine the parameters to be applied to measure alignment, including any algorithms required to achieve maximum alignment over the entire length of the sequences to be compared.

[0172] The term "cross-reactivity" refers to the ability of an antibody of the present application to bind to CD79b from a different species. For example, an antibody of the present application that binds to human CD79b may also bind to CD79b of another species. Cross-reactivity is measured by detecting specific reactivity with purified antigen in binding assays (e.g., SPR and ELISA) or binding or functional interaction with cells that physiologically express CD79b. Methods for determining cross-reactivity include standard binding assays as described herein, such as surface plasmon resonance analysis and flow cytometry.

[0173] The terms "suppression" or "inhibition" may be used interchangeably and cover both partial and complete inhibition / inhibition. Inhibition / inhibition of CD79b preferably reduces or alters the normal level or type of activity that occurs when binding of CD79b occurs in the absence of inhibition or inhibition. Inhibition and inhibition are also intended to include any measurable reduction in CD79b binding affinity when contacted with an anti-CD79b antibody, as compared to CD79b not contacted with an anti-CD79b antibody.

[0174] The term "inhibition of growth" (eg, of a cell) is intended to include any measurable decrease in cell growth.

[0175] The terms "induction of an immune response" and "enhancement of an immune response" may be used interchangeably and refer to the stimulation of an immune response (i.e., passive or adaptive) to a particular antigen. With respect to induction of CDC or ADCC, the term "induction" refers to the stimulation of a specific direct cell killing mechanism.

[0176] As used herein, "ADCC", i.e. antibody-dependent cell-mediated cytotoxicity, refers to the direct killing of antibody-coated target cells by cells expressing Fc receptors by recognizing the Fc segment of the antibody. Modifications to the Fc segment of IgG can reduce or eliminate the ADCC effector function of the antibody. The above modifications refer to mutations in the heavy chain constant region of the antibody, and are selected from, for example, N297A, L234A, L235A of IgG1, IgG2 / 4 chimera, F235E of IgG4, or L234A / E235A mutations.

[0177] The term "connexon, linker unit, linker or linking fragment" refers to a chemical structure fragment or bond that is linked at one end to a ligand and at the other end to an agent, and may be linked to another linker and then to an agent.

[0178] The linker may include one or more linker elements. Exemplary linker elements include those derived from coupling with linker reagents such as 6-maleimidocaproyl ("MC"), maleimidopropionyl ("MP"), valine-citrulline ("val-cit" or "vc"), alanine-phenylalanine ("ala-phe"), p-aminobenzyloxycarbonyl ("PAB"), and N-succinimidyl 4-(2-pyridylthio)pentanoate ("SPP"), N-succinimidyl 4-(N-maleimidomethyl)cyclohexane-1 carboxylate ("SMCC", also referred to herein as "MCC"), and N-succinimidyl (4-iodo-acetyl)aminobenzoate ("SIAB"). The linker may include a stretch unit, a spacer unit, an amino acid unit, and an extension unit, and may be synthesized by methods known in the art, such as those described in US2005-0238649A1. The linker may be a "cleavable linker" that facilitates the release of the drug in cells. For example, an acid-labile linker (e.g., hydrazone), a protease-sensitive (e.g., peptidase-sensitive) linker, a photolabile linker, a dimethyl linker, or a disulfide-containing linker (Chari et al., Cancer Research 52:127-131 (1992), U.S. Patent No. 5,208,020) may be used.

[0179] The term “amino acid unit” refers to a unit having the following structural formula Y R The carbonyl group in Y can be linked to an extender unit, and in the absence of an extender unit, R refers to an amino acid that can be directly linked to a cytotoxic drug, and in an embodiment of the disclosure, the amino acid unit is -K k - Denoted by: [ka] -K k- is a dipeptide, tripeptide, tetrapeptide, pentapeptide, hexapeptide, heptapeptide, octapeptide, nonapeptide, or decapeptide, and each -K- unit independently has the following structural formula K a Or K b where k is an integer between 0 and 10: [ka] Among them, R in the above amino acid unit 23 is -H or a methyl group, R 24 stands for H, methyl, isopropyl, isobutyl, sec-butyl, benzyl, p-hydroxybenzyl, -CH2OH, -CH(OH)CH3, -CH2CH2SCH3, -CH2CONH2, -CH2COOH, -CH2CH2CONH2, -CH2CH2COOH, -(CH2)3NHC(=NH)NH2, -(CH2)3NH2, -(CH2)3NHCOCH3, -(CH2)3NHCHO, -(CH2)4NHC(=NH)NH2, -(CH2)4NH2, -(CH2)4NHCOCH3, -(CH2)4NHCHO, -(CH2)3NHCONH2, -(CH2)4NHCONH2, -CH2CH2CH(OH)CH2NH2, 2-pyridylmethyl-, 3-pyridylmethyl-, 4-pyridylmethyl-, phenyl, cyclohexyl, [ka] and R 25 is -aryl-, -alkyl-aryl-, -cycloalkyl-, -alkyl-cycloalkyl-, -cycloalkyl-alkyl-, -alkyl-cycloalkyl-alkyl-, -heterocyclyl-, -alkyl-heterocyclyl-, -heterocyclyl-alkyl-, -alkyl-heterocyclyl-alkyl-, -aryl-, -alkyl-aryl-, -aryl-alkyl-, -alkyl-aryl-alkyl-, -heteroaryl-, -alkyl-heteroaryl-, -heteroaryl-alkyl-, -alkyl-heteroaryl-alkyl-.

[0180] In one embodiment, -K k - is a dipeptide, preferably -valine-citrulline-, -phenylalanine-lysine- or -N-methylvaline-citrulline-, more preferably -valine-citrulline-.

[0181] The term "stretch unit" refers to a fragment of a chemical structure that is covalently attached at one end to a ligand via a carbon atom and linked at the other end to a cytotoxic drug via a sulfur atom.

[0182] The term "spacer unit" refers to a fragment of a bifunctional compound structure that can be used to couple a linking unit with a cytotoxic drug to ultimately form a ligand-cytotoxic drug conjugate, and such a coupling scheme can selectively bind the cytotoxic drug to the linking unit.

[0183] The term "extender unit" refers to a chemical structure that, in the presence of the amino acid unit, can couple the amino acid unit to a cytotoxic drug, or, in the absence of the amino acid unit, can couple to a cytotoxic drug via a carbonyl group in YR. In an embodiment of the disclosure, the extender unit is -Q q -, where q is selected from 0, 1, or 2.

[0184] In the present disclosure, the extender unit is PAB, which has the structure of a 4-iminobenzylcarbamoyl fragment, the structure of which is shown in the following formula and is linked to D: [ka] Abbreviation Linker elements include, but are not limited to, the following: MC=6-maleimidocaproyl, with the following structure: [ka] Val-Cit or "vc" = valine-citrulline (an exemplary dipeptide in a protease-cleavable linker) Citrulline = 2-amino-5-ureidopentanoic acid PAB = p-aminobenzyloxycarbonyl (an example of a "self-immolative" linker element) Me-Val-Cit = N-methyl-valine-citrulline (in which the linker peptide bond has been modified to prevent cleavage by cathepsin B) MC(PEG)6-OH = Maleimidocaproyl-polyethylene glycol (can be attached to antibody cysteines) SPP = N-Succinimidyl 4-(2-pyridylthio)pentanoate SPDP = N-Succinimidyl 3-(2-pyridyldithio)propionate SMCC = succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate IT=iminothiolane PBS = phosphate buffered saline

[0185] The term "drug loading" refers to the average amount of cytotoxic drugs loaded on each ligand in an ADC and may be expressed as the ratio of the amount of drug to the amount of antibody, and the range of drug loading is such that each antibody (Pc) may be linked to 1 to 20, preferably 1 to 10, cytotoxic drugs (D). In an embodiment of the present disclosure, the drug loading is represented by n or k, and may be illustratively 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or an average value between any two of these values. Preferably, it is 1 to 10, and more preferably, it is an average value of 1 to 8, or 1 to 8, or 1 to 7, or 2 to 8, or 2 to 7, or 2 to 6, or 2 to 5, or 2 to 3, or 1 to 2, or 2 to 4, or 1 to 4, or 1 to 5, or 1 to 6, or 3 to 8, or 3 to 7, or 3 to 6, or 4 to 7, or 4 to 6, or 4 to 5. The average quantity of drug in each ADC molecule after the coupling reaction can be specifically identified by conventional methods such as UV / visible spectroscopy, mass spectrometry, ELISA test, monoclonal antibody molecular size variant assay (CE-SDS) and HPLC.

[0186] The monoclonal antibody molecular size variant assay (CE-SDS) disclosed herein can quantitatively measure the purity of recombinant monoclonal antibody products according to the capillary electrophoresis method (2015 edition of Chinese Pharmacopoeia 0542) according to the molecular weight size under reducing and non-reducing conditions by sodium dodecyl sulfate capillary electrophoresis (CE-SDS) ultraviolet detection method.

[0187] In one embodiment of the disclosure, the cytotoxic drug is coupled to the N-terminal amino group and / or the ε-amino group of a lysine residue of the ligand via a linking unit, and typically, in a coupling reaction, the number of drug molecules that can be coupled to the antibody is less than the theoretical maximum.

[0188] The loading of the ligand-cytotoxic drug conjugate is (1) controlling the molar ratio of the linking reagent to the monoclonal antibody; (2) controlling reaction time and temperature; (3) selecting different reaction reagents; The amount of oxygen can be controlled by a number of methods, including but not limited to:

[0189] Although for a particular conjugate molecule, the drug to antibody ratio has a precise value (e.g., n in formula (I)), it should be understood that when used to describe a sample containing many molecules, the value is always an average value, due to some degree of heterogeneity typically associated with the conjugation step. The average loading of an immunoconjugate sample is referred to herein as the drug to antibody ratio or "DAR." In some embodiments, the DAR is between about 1 and about 6, and typically is about 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7.0, 7.5, 8.0. In some embodiments, at least 50% of the sample by weight is a compound having an average DAR ±2, and preferably at least 50% of the sample is a conjugate containing an average DAR ±1. Examples include those having a DAR of about 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.4, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0 In some embodiments, a DAR of "about x" means that the measured DAR is within 20% of x.

[0190] A method for detecting DAR is, for example, to estimate the DAR value from the LC-MS data of reduced and deglycosylated samples. LC / MS allows quantification of the average number of payload (drug moiety) molecules linked to the antibody in the ADC. HPLC separates the antibody into light and heavy chains, and separates the heavy chain (HC) and light chain (LC) according to the number of linker-payload groups in each chain. Mass spectrometry data allows identification of the types of components in the mixture, e.g., LC, LC+1, LC+2, HC, HC+1, HC+2, etc. The average loading of LC and HC chains allows calculation of the average DAR of the ADC. The DAR of a given immunoconjugate sample indicates the average number of drug (payload) molecules linked to a tetrameric antibody containing two light chains and two heavy chains. For example, the method for detecting DAR in WO2018142322.

[0191] The term "alkyl group" refers to a saturated aliphatic hydrocarbon group that is a straight or branched chain group containing from 1 to 20 carbon atoms, preferably an alkyl group containing from 1 to 12 carbon atoms, more preferably an alkyl group containing from 1 to 10 carbon atoms, and most preferably an alkyl group containing from 1 to 6 carbon atoms. Non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 2,2-dimethylbutyl, 1,3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2,3-dimethylbutyl, n-heptyl, 2-methylhexyl, 3-methylhexyl, 4-methylhexyl, 5-methylhexyl, 6-methylhexyl, 7-methylhexyl, 8-methylhexyl, 9-methylhexyl, 10-methylhexyl, 11-methylhexyl, 12-methylhexyl, 13-methylhexyl, 14-methylhexyl, 15-methylhexyl, 16-methylhexyl, 17-methylhexyl, 18-methylhexyl, 19-methylhexyl, 20-methylhexyl, 21-methylhexyl, 22-methylhexyl, 23-methylhexyl, 24-methylhexyl, 25-methylhexyl, 26-methylhexyl, 27-methylhexyl, 28-methylhexyl, 29-methylhexyl, 30-methylhexyl, 31-methylhexyl, 32-methylhexyl, 33-methylhexyl, 34-methylhexyl, 35-methylhexyl, 36-methylhexyl, 37-methylhexyl, 38-methylhexyl, 39-methylhexyl, 40-methylhexyl, 41-methylhexyl, 42-methylhexyl, 43-methylhex Examples of such groups include 2,3-dimethylpentyl, 2,4-dimethylpentyl, 2,2-dimethylpentyl, 3,3-dimethylpentyl, 2-ethylpentyl, 3-ethylpentyl, n-octyl, 2,3-dimethylhexyl, 2,4-dimethylhexyl, 2,5-dimethylhexyl, 2,2-dimethylhexyl, 3,3-dimethylhexyl, 4,4-dimethylhexyl, 2-ethylhexyl, 3-ethylhexyl, 4-ethylhexyl, 2-methyl-2-ethylpentyl, 2-methyl-3-ethylpentyl, n-nonyl, 2-methyl-2-ethylhexyl, 2-methyl-3-ethylhexyl, 2,2-diethylpentyl, n-decyl, 3,3-diethylhexyl, 2,2-diethylhexyl, and various branched chain isomers thereof.More preferred are lower alkyl groups containing 1 to 6 carbon atoms, non-limiting examples of which include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 2,2-dimethylbutyl, 1,3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2,3-dimethylbutyl, and the like. The alkyl group may be substituted or unsubstituted, and when substituted, the substituents may be substituted at any available attachment point, and preferably the substituents are independently one or more groups selected from alkyl groups, alkenyl groups, alkynyl groups, alkoxy groups, alkylthio groups, alkylamino groups, halogens, mercapto groups, hydroxy groups, nitro groups, cyano groups, cycloalkyl groups, heterocycloalkyl groups, aryl groups, heteroaryl groups, cycloalkoxy groups, heterocycloalkoxy groups, cycloalkylthio groups, heterocycloalkylthio groups, and oxo groups.

[0192] The term "heteroalkyl group" refers to an alkyl group containing one or more heteroatoms selected from N, O or S, wherein the alkyl group is as defined above.

[0193] The term "alkylene group" refers to a saturated straight or branched chain aliphatic hydrocarbon group having two residues derived by removing two hydrogen atoms from the same carbon atom or from two different carbon atoms of a parent alkane, and is a straight or branched chain alkylene group containing 1 to 20 carbon atoms, preferably 1 to 12 carbon atoms, and more preferably 1 to 6 carbon atoms. Non-limiting examples of alkylene groups include, but are not limited to, methylene (-CH2-), 1,1-ethylidene (-CH(CH3)-), 1,2-ethylidene (-CH2CH2)-, 1,1-propylidene (-CH(CH2CH3)-), 1,2-propylidene (-CH2CH(CH3)-), 1,3-propylidene (-CH2CH2CH2-), 1,4-butylidene (-CH2CH2CH2CH2-), and 1,5-butylidene (-CH2CH2CH2CH2CH2-). An alkylene group may be substituted or unsubstituted, and when substituted, the substituents may be substituted at any available point of attachment, and said substituents are preferably independently and optionally substituted with one or more substituents selected from alkyl groups, alkenyl groups, alkynyl groups, alkoxy groups, alkylthio groups, alkylamino groups, halogens, mercapto groups, hydroxy groups, nitro groups, cyano groups, cycloalkyl groups, heterocyclyl groups, aryl groups, heteroaryl groups, cycloalkoxy groups, heterocycloalkoxy groups, cycloalkylthio groups, heterocycloalkylthio groups, and oxo groups.

[0194] The term "alkoxy group" refers to -O-(alkyl group) and -O-(unsubstituted cycloalkyl group), where alkyl group or cycloalkyl group is defined above. Non-limiting examples of alkoxy groups include methoxy group, ethoxy group, propoxy group, butoxy group, cyclopropoxy group, cyclobutoxy group, cyclopentyloxy group, and cyclohexyloxy group. An alkoxy group may be optionally substituted or unsubstituted, and when substituted, the substituents are preferably one or more groups independently selected from alkyl group, alkenyl group, alkynyl group, alkoxy group, alkylthio group, alkylamino group, halogen, mercapto group, hydroxy group, nitro group, cyano group, cycloalkyl group, heterocycloalkyl group, aryl group, heteroaryl group, cycloalkoxy group, heterocycloalkoxy group, cycloalkylthio group, and heterocycloalkylthio group.

[0195] The term "cycloalkyl group" refers to a saturated or partially unsaturated monocyclic or polycyclic cyclic hydrocarbon substituent, where the cycloalkyl ring contains from 3 to 20 carbon atoms, preferably from 3 to 12 carbon atoms, more preferably from 3 to 10 carbon atoms, and most preferably from 3 to 8 carbon atoms. Non-limiting examples of monocyclic cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, cycloheptyl, cycloheptatrienyl, cyclooctyl, and the like, and polycyclic cycloalkyl groups include spiro, fused, and bridged ring cycloalkyl groups.

[0196] The term "heterocyclyl group" refers to a saturated or partially unsaturated monocyclic or polycyclic cyclic hydrocarbon substituent containing 3 to 20 ring atoms, of which one or more of the ring atoms is nitrogen, oxygen or S(O). m(wherein m is an integer of 0-2), but does not include the ring moieties -OO-, -OS-, or -SS-, with the remaining ring atoms being carbon. Preferably, the ring contains 3-12 ring atoms, of which 1-4 are heteroatoms, and more preferably the cycloalkyl ring contains 3-10 ring atoms. Non-limiting examples of monocyclic heterocyclyl groups include pyrrolidinyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, homopiperazinyl, and the like. Polycyclic heterocyclyl groups include spiro, fused, and bridged heterocyclyl groups.

[0197] The term "spiroheterocyclyl group" refers to a 5- to 20-membered polycyclic heterocyclyl group in which the monocyclic rings share one atom (called a spiro atom) in which one or more of the ring atoms is nitrogen, oxygen, or S(O). m (wherein m is an integer from 0 to 2), and the remaining ring atoms are carbon. It may contain one or more double bonds, but none of the rings has a completely conjugated π-electron system. For example, it may have 6 to 14 members, or for example, 7 to 10 members. Depending on the number of spiro atoms shared between the rings, spiroheterocyclyl groups are divided into monospiroheterocyclyl groups, bisspiroheterocyclyl groups, and polyspiroheterocyclyl groups, and are preferably monospiroheterocyclyl groups and bisspiroheterocyclyl groups. For example, 4-membered / 4-membered, 4-membered / 5-membered, 4-membered / 6-membered, 5-membered / 5-membered, or 5-membered / 6-membered monospiroheterocyclyl groups. Non-limiting examples of spiroheterocyclyl groups are: [ka] Includes.

[0198] The term "fused heterocyclyl group" refers to a polycyclic heterocyclyl group having 5 to 20 members in which each ring in the system shares an adjacent pair of atoms with another ring in the system, one or more of the rings may contain one or more double bonds, but none of the rings has a completely conjugated pi-electron system, and in which one or more of the ring atoms is nitrogen, oxygen, or S(O) m(wherein m is an integer of 0 to 2), and the remaining ring atoms are carbon. For example, 6 to 14 members, or for example, 7 to 10 members. Depending on the number of rings, the heterocyclyl group can be divided into bicyclic, tricyclic, tetracyclic or polycyclic fused heterocyclyl groups, for example, bicyclic or tricyclic, or for example, 5-membered / 5-membered or 5-membered / 6-membered bicyclic fused heterocyclyl groups. Non-limiting examples of fused heterocyclyl groups are: [ka] Includes.

[0199] The term "bridged heterocyclyl group" refers to a 5- to 14-membered polycyclic heterocyclyl group in which any two rings share two atoms that are not directly connected, and which may contain one or more double bonds, but in which no ring has a completely conjugated pi-electron system, and in which one or more ring atoms are not nitrogen, oxygen, or S(O) m (wherein m is an integer of 0 to 2), and the remaining ring atoms are carbon. For example, it is 6 to 14-membered, or, for example, 7 to 10-membered. Depending on the number of rings, it can be divided into bicyclic, tricyclic, tetracyclic or polycyclic bridged heterocyclyl groups, for example, bicyclic, tricyclic or tetracyclic, more preferably bicyclic or tricyclic. Non-limiting examples of bridged heterocyclyl groups are: [ka] Includes.

[0200] The heterocyclyl ring may be fused to an aryl group, a heteroaryl group, or a cycloalkyl ring, where the ring connected to the parent structure is a heterocyclyl group, non-limiting examples of which are: [ka] etc.

[0201] The heterocyclyl group may be optionally substituted or unsubstituted, and when substituted, the substituents are preferably one or more groups independently selected from alkyl groups, alkenyl groups, alkynyl groups, alkoxy groups, alkylthio groups, alkylamino groups, halogens, mercapto groups, hydroxy groups, nitro groups, cyano groups, cycloalkyl groups, heterocycloalkyl groups, aryl groups, heteroaryl groups, cycloalkoxy groups, heterocycloalkoxy groups, cycloalkylthio groups, heterocycloalkylthio groups, and oxo groups.

[0202] The term "aryl group" refers to a 6-14 membered all carbon monocyclic or fused polycyclic (i.e. rings sharing adjacent pairs of carbon atoms) group having a conjugated pi electron system, e.g., 6-10 membered, e.g., phenyl and naphthyl groups, specifically e.g., phenyl. The aryl ring may be fused to a heteroaryl, heterocyclyl or cycloalkyl ring, of which the ring connected to the parent structure is an aryl ring, non-limiting examples of which are: [ka] Includes.

[0203] The aryl group may be substituted or unsubstituted. When it is substituted, the substituents are preferably one or more groups independently selected from an alkyl group, an alkenyl group, an alkynyl group, an alkoxy group, an alkylthio group, an alkylamino group, a halogen, a mercapto group, a hydroxy group, a nitro group, a cyano group, a cycloalkyl group, a heterocycloalkyl group, an aryl group, a heteroaryl group, a cycloalkoxy group, a heterocycloalkoxy group, a cycloalkylthio group, and a heterocycloalkylthio group.

[0204] The term "heteroaryl group" refers to a heteroaromatic system containing 1-4 heteroatoms and 5-14 ring atoms, where the heteroatoms are selected from oxygen, sulfur and nitrogen. The heteroaryl group is preferably 5-10 membered, more preferably 5 or 6 membered, such as furanyl, thienyl, pyridyl, pyrrolyl, N-alkylpyrrolyl, pyrimidinyl, pyrazinyl, imidazolyl, tetrazolyl, etc. The heteroaryl ring may be fused to an aryl, heterocyclyl or cycloalkyl ring, where the ring connected to the parent structure is a heteroaryl ring, non-limiting examples of which are: [ka] Includes.

[0205] The heteroaryl group may be optionally substituted or unsubstituted, and when substituted, the substituents are preferably one or more groups independently selected from alkyl groups, alkenyl groups, alkynyl groups, alkoxy groups, alkylthio groups, alkylamino groups, halogens, mercapto groups, hydroxy groups, nitro groups, cyano groups, cycloalkyl groups, heterocycloalkyl groups, aryl groups, heteroaryl groups, cycloalkoxy groups, heterocycloalkoxy groups, cycloalkylthio groups, and heterocycloalkylthio groups.

[0206] The term "amino protecting group" refers to a group that protects an amino group with an easily removable group so that the amino group is not changed when other parts of the molecule react. Non-limiting examples include 9-fluorenylmethyloxycarbonyl group, tert-butoxycarbonyl group, acetyl group, benzyl group, allyl group, and p-methoxybenzyl group. These groups can be optionally substituted with 1 to 3 substituents selected from halogen, alkoxy group, or nitro group. The amino protecting group is preferably 9-fluorenylmethyloxycarbonyl group.

[0207] The term "aminoheterocyclyl group" refers to a heterocyclyl group substituted with one or more amino groups, preferably one amino group, wherein the heterocyclyl group is as defined above, wherein "amino group" refers to -NH2. Representative examples of the present disclosure are as follows: [ka]

[0208] The term "heterocyclylamino group" refers to an amino group substituted with one or more heterocyclyl groups, preferably one heterocyclyl group, wherein the amino group is as defined above and the heterocyclyl group is as defined above. Representative examples of the present disclosure are as follows: [ka]

[0209] The term "cycloalkylamino group" refers to an amino group substituted with one or more cycloalkyl groups, preferably one cycloalkyl group, wherein the amino group is as defined above, and wherein the cycloalkyl group is as defined above. Representative examples of the present disclosure are as follows: [ka]

[0210] The term "cycloalkylalkyl group" refers to an alkyl group substituted with one or more cycloalkyl groups, preferably one cycloalkyl group, wherein the alkyl group is as defined above and wherein the cycloalkyl group is as defined above.

[0211] The term "haloalkyl group" refers to an alkyl group substituted with one or more halogens, wherein the alkyl group is as defined above.

[0212] The term "deuterated alkyl group" refers to an alkyl group substituted with one or more deuterium atoms, wherein the alkyl group is as defined above.

[0213] The term "hydroxy" refers to an -OH group.

[0214] The term "halogen" refers to fluorine, chlorine, bromine or iodine.

[0215] The term "amino group" refers to -NH2.

[0216] The term "nitro group" refers to --NO.sub.2.

[0217] The abbreviation "Me" in the chemical formula is the methyl group.

[0218] The present disclosure further includes various deuterated forms of the compound. Each available hydrogen atom connected to a carbon atom may be independently replaced with a deuterium atom. Those skilled in the art can synthesize deuterated forms of the compound by referring to related literature. When preparing deuterated forms of the compound, they may use commercially available deuterated starting materials or may be synthesized by conventional techniques with deuterated reagents, including but not limited to deuterated borane, tritium borane tetrahydrofuran solution, lithium aluminum deuteride, deuterated iodoethane and deuterated iodomethane.

[0219] In another embodiment, hydrogen in the functional groups of the compounds described herein can be deuterated to obtain the corresponding deuterated compounds, which retain the same selectivity and potency as the hydrogen analogs, while the more stable deuterium bonds provide clinically beneficial effects through different "ADME" or "toxicokinetics", which is the process of absorption, distribution, metabolism, and excretion of exogenous chemicals by the living body.

[0220] "Optionally" or "optionally" means that the subsequently described event or circumstance may, but may not, occur, and the description includes cases where the event or circumstance occurs and cases where it does not. For example, "a heterocyclyl group optionally substituted with an alkyl group" means that the alkyl group may, but may not, be present, and the description includes cases where the heterocyclyl group is substituted with an alkyl group and cases where the heterocyclyl group is not substituted with an alkyl group.

[0221] "Substituted" refers to one or more hydrogen atoms in a group, preferably 5 or less, more preferably 1 to 3 hydrogen atoms, being substituted with a corresponding number of substituents independently of each other. Of course, the substituents are only located at their chemically possible sites, and a person skilled in the art can determine (by experiment or theory) possible or impossible substitutions without much effort. For example, an amino group or a hydroxy group having a free hydrogen may be unstable when bonded to a carbon atom having an unsaturated (e.g., olefinic) bond.

[0222] "Administration" and "treatment", when applied to an animal, human, experimental subject, cell, tissue, organ, or biological fluid, refers to contact of an exogenous agent, therapeutic agent, diagnostic agent, or composition with an animal, human, subject, cell, tissue, organ, or biological fluid. "Administration" and "treatment" may refer, for example, to therapeutic, pharmacokinetic, diagnostic, research, and experimental methods. Treatment of cells includes contact of a reagent with a cell, and contact of a reagent with a fluid, where the fluid is in contact with the cell. "Administration" and "treatment" also refer to treating, for example, cells ex vivo and in vitro with a reagent, diagnostic, binding composition, or through another cell. "Treatment", when applied to a human, veterinary, or research subject, refers to therapeutic treatment, preventative or prophylactic measures, research and diagnostic applications.

[0223] "Treatment" refers to, for example, administering an oral or topical therapeutic agent comprising a composition of any one of the binding compounds of the present disclosure to a patient, the patient having one or more disease symptoms, the therapeutic agent being known to have a therapeutic effect on those symptoms. Typically, the patient or population being treated is administered an amount of therapeutic agent that effectively relieves one or more disease symptoms, thereby inducing the resolution of those symptoms or inhibiting those symptoms from progressing to any clinically measurable extent. The amount of therapeutic agent that effectively relieves any particular disease symptom (also referred to as a "therapeutically effective amount") can vary depending on a variety of factors, including the disease state, age and weight of the patient, and the ability of the drug to produce the required therapeutic effect in the patient. Reduction of disease symptoms can be assessed by any clinical detection method commonly used by physicians or other professional health care providers to assess the severity or progression of the condition. Although embodiments of the present disclosure (e.g., methods of treatment or products) may be ineffective in alleviating the respective target disease symptoms, any statistical testing method known in the art, such as Student's t-test, chi-square test, Mann and Whitney U test, Kruskal-Wallis test (H test), Jonckheere-Terpstra test, and Wilcoxon test, will determine that they should reduce the target disease symptoms in a statistically significant number of patients.

[0224] An "effective amount" includes an amount sufficient to ameliorate or prevent a symptom or condition of a medical disease. Effective amount also refers to an amount sufficient to enable or facilitate diagnosis. The effective amount used in a particular patient or veterinary subject can vary depending on factors such as, for example, the condition being treated, the overall health of the patient, the route and dose of administration, and the severity of side effects. An effective amount may be the maximum dose or dosing regimen in which significant side effects or toxic effects are avoided. EXAMPLES

[0225] The present disclosure will be further described below in conjunction with examples, but these examples are not intended to limit the scope of the present disclosure. Experimental methods for which specific conditions are not specified in the examples of the present disclosure generally follow standard conditions, such as those in the Cold Spring Harbor Antibody Technology Laboratory Manual, the Molecular Cloning Manual, etc., or follow conditions suggested by manufacturers of raw materials or products. Reagents for which a specific source is not specified are standard commercially available reagents.

[0226] Example 1. Preparation of anti-CD79b antibodies The present disclosure provides an anti-CD79b antibody obtained by screening using human CD79b extracellular domain (ECD) immunized mice, and the screening and functional identification process is specifically described in WO2020156439A. The amino acid residues of the VH / VL CDRs of the antibody are determined and annotated according to the Chothia numbering system. The sequences of the monoclonal antibody mAb015 from mouse hybridoma cells and its humanized antibody hAb015-10 are as follows:

[0227] TIFF2025504419000056.tif37160

[0228] TIFF2025504419000057.tif32160

[0229] TIFF2025504419000058.tif37160

[0230] TIFF2025504419000059.tif32160

[0231] [Table 1]

[0232] TIFF2025504419000061.tif80160

[0233] TIFF2025504419000062.tif48160

[0234] Example 2. Preparation of Compounds Experimental methods for which specific conditions are not specified in the examples of this disclosure generally follow conventional conditions or conditions suggested by the manufacturers of materials or products. Reagents for which a specific source is not specified are conventional reagents that are commercially available.

[0235] For the drug moieties of the antibody-drug conjugates of the present disclosure, see WO2020063676A, CN202010073671.6, US7098308, US6884869, CN201911390425.7, and the synthesis and testing of related compounds are all incorporated by reference into this disclosure. [ka] The preparation method of compound D is incorporated by reference in WO2020063676A, for example in Example 9 thereof.

[0236] Example 3. Preparation of anti-CD79b antibody-drug conjugate Experimental objectives and principles of ADC bulk drug loading analysis: ADC stock solution is one of the antibody crosslinker class drugs, and its mechanism of treating diseases is to rely on the targeting of antibodies to deliver toxin molecules to cells and then kill the cells. The amount of drug loaded plays a crucial role in the efficacy of the drug. The amount of drug loaded in the ADC stock solution was measured by ultraviolet light method.

[0237] Experimental Method The cuvettes containing the sodium succinate buffer were placed in the reference absorption cell and the sample measurement absorption cell, respectively, and then after subtracting the solvent blank, the cuvette containing the sample solution was placed in the sample measurement absorption cell and the absorbance at 280 nm and 370 nm was measured.

[0238] Calculation of results: The loading amount of ADC stock solution was measured by ultraviolet spectrophotometry (instrument: Thermo nanodrop2000 ultraviolet spectrophotometer), the principle being that the total absorbance value of ADC stock solution at a certain wavelength is equal to the sum of the absorbance values ​​of the cytotoxic drug and the monoclonal antibody at the same wavelength, i.e. (1)A 280nm =ε mab-280 bC mab +ε Drug-280 bC Drug ε Drug-280 The drug has an average molar extinction coefficient of 5100 at 280 nm. C Drug : Drug concentration, ε mab-280 : The average molar extinction coefficient at 280 nm of the monoclonal antibody stock solution is 214600, C mab : Concentration of monoclonal antibody stock solution, B: The optical path length is 1 cm.

[0239] Similarly, the equation for the total absorbance value at 370 nm of the sample can be obtained: (2)A 370nm =ε mab-370 bC mab +ε Drug-370 bC Drug ε Drug-370 The drug has an average molar extinction coefficient of 19,000 at 370 nm. C Drug : Drug concentration, ε mab-370 : The extinction coefficient of the monoclonal antibody stock solution at 370 nm is 0, C mab : Concentration of monoclonal antibody stock solution, B: The optical path length is 1 cm.

[0240] The two equations (1) and (2) can be used to calculate the drug loading amount in combination with the data on the extinction coefficients and concentrations of the monoclonal antibody and drug at the two detection wavelengths. Drug loading = C Drug / C mab

[0241] Example 3-1. ADC-3 [ka]

[0242] Under the condition of 37°C, the prepared aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 85.0 μL, 0.850 μmol) was added to an aqueous solution of antibody hAb017-10 in PBS buffer (0.05 M aqueous solution of PBS buffer with pH=6.5, 10.0 mg / mL, 5.0 mL, 0.338 μmol), placed in a water bath shaker, and reacted with shaking at 37°C for 3 hours to stop the reaction. The reaction solution was cooled to 25°C in a water bath.

[0243] The compound MC-vc-PAB-MMAE (4.45 mg, 3.380 μmol) was dissolved in 250 μL of dimethyl sulfoxide, added to the above reaction solution, placed on a water bath shaker, and reacted with shaking at 25 ° C for 3 hours to stop the reaction. The reaction solution was desalted and purified on a Sephadex G25 gel column (elution phase: 0.05 M PBS buffer solution at pH 6.5, containing 0.001 M EDTA) to obtain the title product ADC-3 (i.e., hAb015-10-cys-MC-vc-PAB-MMAE) in this example in PBS buffer (2.79 mg / mL, 17.4 mL), which was stored frozen at 4 ° C. The mean value was calculated by CE-SDS: n = 3.09.

[0244] Example 3-2. ADC-5 [ka]

[0245] Under the condition of 37°C, the prepared aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 16.2 μL, 0.162 μmol) was added to an aqueous solution of antibody hAb015-10 in PBS buffer (0.05 M PBS buffer solution at pH=6.5, 10.0 mg / mL, 1.5 mL, 0.101 μmol), placed in a water bath shaker, and reacted with shaking at 37°C for 3 hours to stop the reaction. The reaction solution was cooled to 25°C in a water bath.

[0246] Compound D (0.87 mg, 0.810 μmol) of this example was dissolved in 37 μL of dimethyl sulfoxide, added to the above reaction solution, placed on a water bath shaker, and reacted with shaking at 25 ° C. for 3 hours to stop the reaction. The reaction solution was desalted and purified on a Sephadex G25 gel column (elution phase: 0.05 M PBS buffer solution at pH 6.5, containing 0.001 M EDTA) to obtain the title product ADC-5 of this example (i.e., HAB015-10-cys-D, DAR value about 2) in PBS buffer solution (0.90 mg / mL, 14.0 mL), which was then frozen and stored at 4 ° C. The mean value was calculated by RP-HPLC: n = 1.81.

[0247] Example 3-3. ADC-6 [ka]

[0248] Under the condition of 37°C, the prepared aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 25.3 μL, 0.253 μmol) was added to an aqueous solution of antibody hAb015-10 in PBS buffer (0.05 M PBS buffer solution at pH=6.5, 10.0 mg / mL, 1.5 mL, 0.101 μmol), placed in a water bath shaker, and reacted with shaking at 37°C for 3 hours to stop the reaction. The reaction solution was cooled to 25°C in a water bath.

[0249] Compound D (1.09 mg, 1.015 μmol) in Example 2 was dissolved in 45 μL of dimethyl sulfoxide, added to the above reaction solution, placed on a water bath shaker, and reacted with shaking at 25 ° C. for 3 hours to stop the reaction. The reaction solution was desalted and purified with a Sephadex G25 gel column (elution phase: 0.05 M PBS buffer solution at pH 6.5, containing 0.001 M EDTA) to obtain the title product ADC-6 of this example (i.e., hAb015-10-cys-D, DAR value about 4) in PBS buffer solution (0.71 mg / mL, 14.0 mL), which was stored frozen at 4 ° C. The mean value was calculated by RP-HPLC: n=3.46.

[0250] Example 3-4. ADC-7 [ka]

[0251] Under the condition of 37°C, the prepared aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 50.7 μL, 0.507 μmol) was added to an aqueous solution of antibody hAb015-10 in PBS buffer (0.05 M PBS buffer solution at pH=6.5, 10.0 mg / mL, 1.5 mL, 0.101 μmol), placed in a water bath shaker, and reacted with shaking at 37°C for 3 hours to stop the reaction. The reaction solution was cooled to 25°C in a water bath.

[0252] Compound D (1.63 mg, 1.518 μmol) in Example 2 was dissolved in 68 μL of dimethyl sulfoxide, added to the above reaction solution, placed on a water bath shaker, and reacted while shaking at 25 ° C. for 3 hours, and the reaction was stopped. The reaction solution was desalted and purified with a Sephadex G25 gel column (elution phase: 0.05 M PBS buffer solution at pH 6.5, containing 0.001 M EDTA) to obtain the title product ADC-7 (i.e., hAb015-10-cys-D, DAR value about 6) of this example in PBS buffer solution (0.81 mg / mL, 13.5 mL), which was stored frozen at 4 ° C. The mean value was calculated by RP-HPLC: n = 5.84.

[0253] Example 3-5. ADC-8 [ka]

[0254] Under the condition of 37°C, the prepared aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 1.388 mL, 13.88 μmol) was added to the PBS buffer solution of antibody SN8 (0.05 M PBS buffer solution with pH=6.5, 10.0 mg / mL, 79 mL, 5.338 μmol), placed in a water bath shaker, and reacted with shaking at 37°C for 3 hours to stop the reaction. The reaction solution was cooled to 25°C in a water bath.

[0255] The compound MC-VC-PAB-MMAE (70.3 mg, 53.40 μmol) was dissolved in 3.5 mL of dimethyl sulfoxide, added to the above reaction solution, placed on a water bath shaker, and reacted with shaking at 25 ° C. for 3 hours to stop the reaction. The reaction solution was desalted and purified on a Sephadex G25 gel column (elution phase: 0.05 M PBS buffer aqueous solution at pH 6.5, containing 0.001 M EDTA) to obtain the title product ADC-8 of this example (i.e., SN8-cys-MC-PAB-MMAE, DAR value about 4) in PBS buffer (5.83 mg / mL, 132 mL), which was stored frozen at 4 ° C. The mean value was calculated by CE-SDS: n = 3.59.

[0256] Example 4, Biological Evaluation Example 4-1. Biacore affinity experiments The affinity of the CD79b antibodies (hAb015-10 and SN8) and ADCs to the CD79b protein was measured by Biacore. The affinity values ​​were obtained by fitting the data with the (1:1) Langmuir model using BIAevaluation version 4.1, GE software.

[0257] The affinity detection results are shown in Table 2. For human CD79b protein, the naked antibody and the different ADCs have similar binding avidity, and both are higher than the positive drug Polivy.

[0258] Of which, the SN8 (i.e., antibody in Polivy) sequence is as follows: TIFF2025504419000069.tif80160

[0259] TIFF2025504419000070.tif48160

[0260] [Table 2]

[0261] Example 4-2. In vitro cell endocytosis experiment Cell endocytosis experiments were performed using DOHH-2 cells (DSMZ, ACC 47), which highly expressed human CD79b protein, to evaluate the endocytosis ability of different ADCs.

[0262] As can be seen from the data in Table 3, the different ADCs and DoHH2 cells have good endocytosis ability, with the endocytosis rate after 4 h incubation all being greater than 65%. The endocytosis rate of each ADC is comparable to that of the positive drug Polivy.

[0263] [Table 3]

[0264] Example 4-3. Cell proliferation experiment This example evaluated the effects of different ADCs on the proliferation of in vitro cultured DoHH2, WSU-DLCL2 and Raji cells. According to literature reports (Leukemia. 2015 Jul, 29(7):1578-86; Blood. 2007 Jul 15, 110(2):616-23), DoHH2 is a high CD79b expressing cell, WSU-DLCL2 is a low CD79b expressing cell, and Raji is a negative CD79b expressing cell.

[0265] Drugs: ADC-3 (DAR=3.59), ADC-5 (DAR=1.81), ADC-6 (DAR=3.46), ADC-7 (DAR=5.84), ADC-8 (DAR=3.59), all stored in the dark in sealed containers at 4°C.

[0266] Cell lines: DOHH2 were purchased from DSMZ, WSU-DLCL-2 cells were purchased from American Type Culture Collection (ATCC), and Raji cells were purchased from American Type Culture Collection (ATCC).

[0267] A certain amount of logarithmic growth phase cells were seeded in a 96-well culture plate, and different concentrations of drugs were added for 72 hours. After the drug action was completed, MTT working solution was added and allowed to act for 4 hours, after which the three solutions were added to dissolve the blue-purple crystal formazan. The OD value was measured at wavelengths of 570 nm and 690 nm using a plate reader, and the cell growth inhibition rate was calculated using the following formula: Inhibition rate = (control wells OD570nm-OD690nm -Dosing well OD570nm-OD690nm ) / Control wells OD570nm-OD690nm ×100% The median inhibitory concentration (IC) was calculated by PrismGraph 8 based on the inhibition rate of each concentration. 50 The results are shown in Table 4.

[0268] [Table 4]

[0269] Example 4-4. Therapeutic effect of ADC on human diffuse large B-cell lymphoma WSU-DLCL2 tumors subcutaneously transplanted into nude mice Drugs: ADC-3, ADC-6, and ADC-8 are the same as in Example 4-3.

[0270] Cells and mice: Human diffuse large B-cell lymphoma WSU-DLCL2 cells were purchased from the American Type Culture Collection. Female BALB / c-nu nude mice at 35 days of age were purchased from Beijing Huafukang Biotechnology Co., Ltd.

[0271] Experimental procedures: 2.1×10 7 WSU-DLCL2 cells were inoculated subcutaneously into each nude mouse. When the tumor grew to 100 - 150 mm 3 , the animals were grouped according to tumor volume (D0). The mice were administered by intravenous injection (IV) with a dosing volume of 10 mL / kg. The specific dosing amounts and dosing schedules are shown in Table 5. The tumor volume was measured twice a week, the body weight of the mice was weighed, and the data were recorded.

[0272] Experimental indicators and statistical analysis: The formula for calculating tumor volume (V) is V = 1 / 2 × a × b 2 [where a and b represent length and width respectively].

[0273] T / C(%) = (T - T0) / (C - C0) × 100 [where T and C are the tumor volumes at the end of the experiment, and T0 and C0 are the tumor volumes at the start of the experiment. Among them, T is the tumor volume administered with ADC, and C is the tumor volume administered with IgG1 as the control group]

[0274] Tumor growth inhibition rate % (TGI%) = 100 - T / C(%)

[0275] When tumor regression appears, the tumor growth inhibition rate % (TGI%) = 100 - (T - T0) / T0 × 100.

[0276] When the tumor becomes smaller than the initial volume, that is, when T < T0 or C < C0, it is defined as partial regression (PR) of the tumor. When the tumor completely disappears, it is defined as complete regression (CR) of the tumor.

[0277] Unless otherwise stated, two-way ANOVA test was used to compare tumor volumes between two groups, and P<0.05 was defined as a statistically significant difference.

[0278] The tumor inhibition rate of WSU-DLCL2 by ADC-3 (3 mg / kg, IV, D0) was 76%, with 1 / 6 tumors partially regressing, the tumor inhibition rate of WSU-DLCL2 by ADC-6 (3 mg / kg, IV, D0) was 86%, with 2 / 6 tumors partially regressing, and the tumor inhibition rates of WSU-DLCL2 by ADC-8 (3 mg / kg, 10 mg / kg, IV, D0) were 39% and 93%, respectively, with 4 / 6 tumors partially regressing in the 10 mg / kg dose group. The tumor-bearing mice tolerated all of the above drugs relatively well, with no significant weight loss or other symptoms. IgG1 was used as a negative control.

[0279] See Table 5 and Figures 1 and 2 for the results.

[0280] [Table 5]

[0281] A single intravenous injection of 3 mg / kg or 10 mg / kg of ADC-3, ADC-6 and ADC-8 all showed remarkable therapeutic effects on human diffuse large B-cell lymphoma WSU-DLCL2 tumors subcutaneously transplanted in nude mice, causing partial tumor regression, and the drugs showed a remarkable dose-dependency, and the efficacy of each ADC coupled with hAb015-10 at the same dose was superior to that of the positive drug ADC-8 (i.e., Polivy). Tumor-bearing mice were able to tolerate all of the above drugs relatively well.

[0282] Example 4-5. Therapeutic effect of ADC on human diffuse large B-cell lymphoma WSU-DLCL2 tumors subcutaneously transplanted into nude mice The concentrations of drugs: ADC-5, ADC-6, ADC-7, and ADC-8 are the same as in Example 4-3.

[0283] The experimental materials, procedures and indicators were the same as those in Example 4-4, and the statistical analysis was as follows: Unless otherwise stated, two-tailed Student's t test was used to compare tumor volumes between two groups, with P<0.05 defined as a statistically significant difference.

[0284] The tumor inhibition rates of human diffuse large B-cell lymphoma WSU-DLCL2 tumors implanted subcutaneously in nude mice by ADC-5 (3 mg / kg, 6 mg / kg, 12 mg / kg, IV, D0) were 69%, 86%, and 88%, respectively. Of these, 1 / 6 and 1 / 6 tumors in the 6 mg / kg and 12 mg / kg dose groups, respectively, partially regressed. The tumor inhibition rates of WSU-DLCL2 tumors implanted subcutaneously in nude mice by ADC-6 (1.5 mg / kg, 3 mg / kg, 6 mg / kg, IV, D0) were 66% and 108%, respectively. and 125%, of which 5 / 6 and 6 / 6 tumors partially regressed in the 3 mg / kg and 6 mg / kg dose groups, respectively, the tumor inhibition rate of the subcutaneously implanted WSU-DLCL2 tumors by ADC-7 (1 mg / kg, IV, DO) was 91%, 1 / 6 tumors partially regressed, and the tumor inhibition rate of the subcutaneously implanted WSU-DLCL2 tumors by ADC-8 (3 mg / kg, IV, DO) was 10%. The tumor-bearing mice tolerated all of the above drugs relatively well and did not experience symptoms such as weight loss.

[0285] Please refer to Table 6 and Figures 3 to 5 for specific results.

[0286] [Table 6]

[0287] Examples 4-6. Therapeutic effect of ADC on human B cell lymphoma DoHH2 tumors subcutaneously transplanted into nude mice The concentrations of drugs: ADC-6 and ADC-8 are the same as in Example 4-3.

[0288] Cells and mice: Human follicular lymphoma DOHH-2 cells were purchased from DSMZ, Germany. 4-5 week-old female BALB / c-nu nude mice were purchased from Shanghai Lingchang Biological Technology Co., Ltd.

[0289] Experimental procedure: Each nude mouse was subcutaneously injected with 3 × 10 7 DOHH-2 cells were inoculated into the tumor, and the tumor size was 100–150 mm 3 When the tumors grew to 100 mg / kg, the animals were divided into groups according to tumor volume (D0). The mice were administered intravenously (IV), with a volume of 10 mL / kg, and the specific dose and administration schedule are shown in Table 7. The tumor volumes were measured twice a week, and the mice were weighed and the data were recorded.

[0290] The experimental parameters and statistical analysis were the same as those in Example 4-3.

[0291] The tumor inhibition rates of ADC-1, ADC-6, and ADC-8 (1 mg / kg, IV, DO) in human follicular lymphoma DOHH-2 nude mice subcutaneously implanted tumors were 82% (1 / 6 PR), 127% (5 / 6 PR), and 41%, respectively. The tumor-bearing mice tolerated all of the above drugs relatively well, with no significant weight loss or other symptoms.

[0292] See Table 7 and Figures 6 and 7 for the results.

[0293] [Table 7]

[0294] A single intravenous injection of 1 mg / kg of ADC-6 had a remarkable therapeutic effect on human follicular lymphoma DoHH2 subcutaneously transplanted tumors in nude mice, causing partial tumor regression, and both drugs were more effective than the positive drug ADC-8 (i.e., Polivy). Tumor-bearing mice tolerated both drugs relatively well.

[0295] The CD79b antibody-drug conjugate used in Examples 5 to 8 was ADC-6 prepared in Example 3-3 above.

[0296] Example 5. Screening of pH values ​​of buffer systems for ADC composition formulations The following buffer solution was prepared, and an antibody formulation with a CD79b antibody-drug conjugate (ADC-6) concentration of 20 mg / mL was prepared. Samples were taken to examine the stability at a high temperature of 40°C and at freezing and thawing.

[0297] 1) 10 mM succinic acid-sodium succinate, pH 5.0 2) 10 mM succinic acid-sodium succinate, pH 5.5 3) 10 mM succinic acid-sodium succinate, pH 6.0 4) 10 mM citric acid-sodium citrate, pH 5.0 5) 10 mM citric acid-sodium citrate, pH 5.5 6) 10 mM citric acid-sodium citrate, pH 6.0 7) 10 mM histidine-histidine hydrochloride, pH 5.5 8) 10 mM histidine-histidine hydrochloride, pH 6.0

[0298] [Table 8-1] [Table 8-2] [Table 8-3]

[0299] As shown by the appearance results, after five freeze-thaw cycles, F1-F8 did not change in appearance from T0, and as the pH increased, the 40°C 1W samples of 10mM succinic acid-sodium succinate (F1-F3) and 10mM citric acid-sodium citrate (F4-F6) systems changed from no particles to particles, indicating that low pH can inhibit particle production. When the pH was increased to 6.0, the formulations of F3 and F6 both produced particles at 40°C 1W, but the 10mM histidine-histidine hydrochloride system (F8) showed no obvious particles, indicating that the 10mM histidine-histidine hydrochloride system was superior to the other two systems.

[0300] As shown by the results of SEC (Figure 8A), after freezing and thawing, there was no significant difference in SEC compared to T0, and all of the different formulations at 40°C were reduced, of which the 10 mM histidine system was reduced to a smaller extent than the succinic acid-sodium succinate and citric acid-sodium citrate systems.

[0301] As shown by the results of RCE (Figure 8B), after freezing and thawing, the RCE was not significantly different from that at T0, and all of the different formulations at 40°C were reduced, of which the 10 mM histidine system was reduced the slowest, followed by the 10 mM succinic acid system.

[0302] As shown by the results of DAR, there was no significant difference in DAR after freeze-thawing and at 40°C compared to T0.

[0303] As a result of examining the appearance and purity of the samples, 10 mM succinic acid-sodium succinate pH 5.0 and 10 mM histidine-histidine hydrochloride pH 5.5-6.0 systems were selected for further consideration.

[0304] Example 6. Screening of additives and surfactants for ADC compositions A 10 mM succinic acid-sodium succinate pH 5.0 system was selected to prepare an ADC composition formulation containing 20 mg / mL antibody-drug conjugate (ADC-6), different types and concentrations of additives, and different concentrations of surfactants. The sample was frozen and thawed five times at -35°C / room temperature, and shaken at 1W (25°C, 300 rpm), 40°C 1W, 40°C 2W, and 40°C 1M to examine its stability: 1) 10 mM succinic acid-sodium succinate pH 5.0, 60 mg / mL sucrose, 2) 10 mM succinic acid-sodium succinate pH 5.0, 90 mg / mL sucrose; 3) 10 mM succinic acid-sodium succinate pH 5.0, 60 mg / mL trehalose; 4) 10 mM succinic acid-sodium succinate pH 5.0, 90 mg / mL trehalose; 5) 10 mM succinic acid-sodium succinate pH 5.0, 0.2 mg / mL polysorbate 80, 60 mg / mL sucrose; 6) 10 mM succinic acid-sodium succinate pH 5.0, 0.4 mg / mL polysorbate 80, 60 mg / mL sucrose, 7) 10 mM succinic acid-sodium succinate pH 5.0, 0.6 mg / mL polysorbate 80, 60 mg / mL sucrose.

[0305] [Table 9-1] [Table 9-2]

[0306] As shown by the appearance results, after 5 freeze-thaw cycles, the appearance was not significantly different compared to T0, and the formulation with polysorbate 80 did not produce particles in any of the preparations after shaking at 1W (25°C, 300 rpm), indicating that the addition of polysorbate 80 had a significant effect on particle production in the formulations after shaking, with particles appearing in all of the formulations at 40°C 2W, but no flocculence was produced in the formulation with polysorbate 80.

[0307] As shown by the results of SEC (FIG. 9A), the SEC purity was reduced for all the different formulations at 40°C, and the SEC purity was not significantly different for different sugar types and concentrations (F1-F4), but with increasing concentrations of polysorbate 80 (F5-F7) (0.2 mg / mL, 0.4 mg / mL, and 0.6 mg / mL), the SEC purity was gradually reduced.

[0308] As shown by the results of RCE (Figure 9B), the different formulations at 40°C all had reduced RCE purity, and the different sugar concentrations (F1-F4) had no significant difference in RCE. The formulations (F3, F4) containing 1 M trehalose at 40°C had a slightly higher RCE than the formulations containing sucrose (F1, F2). With increasing concentrations of polysorbate 80 (F5-F7) (0.2 mg / mL, 0.4 mg / mL, and 0.6 mg / mL), the RCE purity gradually decreased.

[0309] As shown by the results of free mAb and DAR value, after high temperature, the free mAb of different formulations was increased, and the DAR value was slightly decreased, and there was no significant difference between the different formulations.

[0310] As mentioned above, in the 10 mM succinic acid-sodium succinate pH 5.0 system, the type and concentration of sugar have no significant effect on the protein appearance, SEC, RCE, free mAb and DAR value. Considering the economic cost and the osmotic pressure of the human body, the target additive is 8% sucrose, and in the 10 mM succinic acid-sodium succinate pH 5.0 system, the concentration of polysorbate 80 is relatively good for the SEC and RCE purity of the antibody-drug conjugate under high temperature conditions of 40°C.

[0311] Example 7. Fine Screening of pH and Polysorbate 80 Concentration of ADC Compositions Considering the particle problem, fine screening of pH and screening of polysorbate 80 concentration can be performed by selecting a 10 mM histidine-histidine hydrochloride system. A 10 mM histidine-histidine hydrochloride pH 5.5-6.0 buffer system was selected to prepare an ADC composition formulation containing 20 mg / mL protein (ADC-6), 0.4-0.6 mg / mL polysorbate 80, and 80 mg / mL sucrose as an additive, and the stability at a high temperature of 40°C and at freezing and thawing was examined: 1) 10 mM histidine-histidine hydrochloride pH 5.5, 0.4 mg / mL polysorbate 80, 80 mg / mL sucrose, 2) 10 mM histidine-histidine hydrochloride pH 5.5, 0.6 mg / mL polysorbate 80, 80 mg / mL sucrose; 3) 10 mM histidine-histidine hydrochloride pH 5.8, 0.4 mg / mL polysorbate 80, 80 mg / mL sucrose; 4) 10 mM histidine-histidine hydrochloride pH 5.8, 0.6 mg / mL polysorbate 80, 80 mg / mL sucrose; 5) 10 mM histidine-histidine hydrochloride pH 6.0, 0.4 mg / mL polysorbate 80, 80 mg / mL sucrose; 6) 10 mM histidine-histidine hydrochloride pH 6.0, 0.6 mg / mL polysorbate 80, 80 mg / mL sucrose.

[0312] [Table 10-1] [Table 10-2]

[0313] As shown by the appearance results, after five freeze-thaw cycles, there was no significant change in appearance, particles appeared in all of the 40°C 2W formulations, and there was no significant difference in the particle counts of different formulations.

[0314] As shown by the results of SEC (FIG. 10A), the SEC purity was reduced for all different formulations at 40°C. In the pH range of 5.5 to 6.0, the SEC purity of 40°C 4W decreased with increasing pH, and the concentration of polysorbate 80 (0.4 mg / mL, 0.6 mg / mL) did not significantly affect the SEC purity.

[0315] As shown by the results of RCE (FIG. 10B) and DAR values, the different formulations at 40° C. both had decreased RCE purity and DAR values, with no significant difference between the different formulations.

[0316] As shown by the results of free mAb, after high temperature, the free mAb of the different formulations was all increased, and there was no significant difference between the different formulations.

[0317] As described above, in the 10 mM histidine-histidine hydrochloride system, in the range of pH 5.5 to 6.0, a relatively low pH is advantageous for the SEC purity of the antibody-drug conjugate. However, considering the robustness of the formulation, the target pH was determined to be 5.6. Also, considering that there is no significant difference in the effect of polysorbate 80 concentration on the SEC and RCE of the protein, and that the concentration of polysorbate 80 in the 10 mM succinic acid-sodium succinate pH 5.0 system is relatively good for the SEC and reduced CE of the antibody-drug conjugate under the condition of 40°C, the target concentration of polysorbate 80 was finally determined to be 0.2 mg / mL. Considering the stability and appearance of the sample comprehensively, the formulation was initially determined to be 10 mM histidine-histidine hydrochloride, 80 mg / mL sucrose, 0.2 mg / mL polysorbate 80, pH 5.6.

[0318] Example 8. Comprehensive screening of ADC composition formulation components and lyophilization stability To further optimize the antibody-drug conjugate concentration, polysorbate 80 concentration, and pH, a DoE design was performed using JMP software to prepare CD79b antibody-drug conjugate formulations with different protein concentrations, different polysorbate 80 concentrations, and 80 mg / mL sucrose in a 10 mM histidine-histidine hydrochloride buffer system, with a filling volume of 3.8 mL / vial, and the formulation design is as follows: 1) 0.3 mg / mL polysorbate 80, pH 5.5, 15 mg / mL ADC-6 2) 0.2 mg / mL polysorbate 80, pH 6.0, 25 mg / mL ADC-6 3) 0.1 mg / mL polysorbate 80, pH 6.0, 18.15 mg / mL ADC-6 4) 0.2 mg / mL polysorbate 80, pH 5.75, 15 mg / mL ADC-6 5) 0.3 mg / mL polysorbate 80, pH 6.0, 15 mg / mL ADC-6 6) 0.1 mg / mL polysorbate 80, pH 5.5, 15 mg / mL ADC-6 7) 0.1 mg / mL polysorbate 80, pH 5.75, 25 mg / mL ADC-6 8) 0.2 mg / mL polysorbate 80, pH 5.5, 20 mg / mL ADC-6 9) 0.3 mg / mL polysorbate 80, pH 5.75, 20 mg / mL ADC-6 10) 0.3 mg / mL polysorbate 80, pH 5.5, 25 mg / mL ADC-6 The formulation was subjected to preliminary freezing, primary drying, and secondary drying to obtain a freeze-dried product. The freeze-dried product had a good cake shape, was transparent after reconstitution, and had no obvious changes in pH and purity, suggesting that the freeze-drying process was satisfactory. The freeze-dried product was left at 40°C at 1M to examine its stability.

[0319] [Table 11]

[0320] As shown by the data, under the forced degradation conditions of 40°C 1M, all formulations had SEC monomer peak areas of 99% or more, RCE purity of 95% or more, neutral peak areas of 57% or more, and stable DAR values ​​of about 4. There was no significant difference between the different formulations compared to T0. That is, the CD79b antibody-drug conjugate (ADC-6) formulations had no significant difference in purity within the ranges of 15-25 mg / mL, pH 5.5-6.0, and 0.1-0.3 mg / mL polysorbate 80, and the lyophilized formulations could exist stably.

Claims

1. 1. A pharmaceutical composition comprising a ligand-drug conjugate and a buffering agent, wherein the ligand is an anti-CD79b antibody or an antigen-binding fragment thereof, and the drug is selected from MMAE or a derivative thereof, exatecan or a derivative thereof, and eribulin or a derivative thereof; the buffer is selected from acetate buffer, histidine buffer, Tris-hydrochloride buffer, Tris-citrate buffer, phosphate buffer or succinate buffer; the anti-CD79b antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1 having the amino acid sequence shown in SEQ ID NO: 11 or 5, and HCDR2 and HCDR3 having the amino acid sequences shown in SEQ ID NO: 6 and SEQ ID NO: 7, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3 having the amino acid sequences shown in SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10, Preferably, the buffer is a histidine salt buffer or a succinate buffer, preferably, the buffer is a histidine-hydrochloride buffer or a succinic acid-sodium succinate buffer. Pharmaceutical compositions.

2. The pharmaceutical composition according to claim 1, wherein the pH value of the buffer or the pharmaceutical composition is 4.0 to 8.5, preferably 5.0 to 6.5, more preferably 5.5 to 6.

0.

3. 2. The pharmaceutical composition of claim 1, wherein the concentration of the buffering agent is 1 to 30 mM, preferably 5 to 15 mM, more preferably about 10 mM.

4. 2. The pharmaceutical composition of claim 1, wherein the concentration of the ligand-drug conjugate is 0.1 to 50 mg / mL, preferably 10 to 30 mg / mL, more preferably 15 to 25 mg / mL.

5. further comprising a surfactant, Preferably, the surfactant is selected from poloxamer 188, polysorbate 80 or polysorbate 20; More preferably, the surfactant is polysorbate 80. The pharmaceutical composition of claim 1.

6. The pharmaceutical composition according to claim 5, wherein the concentration of the surfactant is 0.01 to 1 mg / mL, preferably 0.05 to 0.6 mg / mL, more preferably 0.1 to 0.3 mg / mL.

7. further comprising an osmolality adjuster; Preferably, the osmotic pressure adjusting agent is one or a combination of two or more selected from sucrose, trehalose, sorbitol, arginine, proline, glycine, and sodium chloride; More preferably, the osmolality adjusting agent is sucrose. The pharmaceutical composition of claim 1.

8. 8. The pharmaceutical composition of claim 7, wherein the concentration of the osmotic agent is 1 to 300 mg / mL, preferably 30 to 130 mg / mL, more preferably about 80 mg / mL.

9. 1. A pharmaceutical composition comprising: (a) 0.1 to 50 mg / mL of a ligand-drug conjugate; (b) 0.5 to 50 mM histidine salt buffer or succinate buffer; (c) 1 to 300 mg / mL of sucrose or trehalose; (d) 0.01 to 1 mg / mL of polysorbate; the pH of the pharmaceutical composition is 4.0 to 8.5; Or, (a) 10-30 mg / mL of a ligand-drug conjugate; (b) 1 to 30 mM histidine salt buffer or succinate buffer; (c) 30 to 130 mg / mL of sucrose or trehalose; (d) 0.05 to 0.6 mg / mL of polysorbate 80; the pH of the pharmaceutical composition is 5.0 to 6.5; Or, (a) 10-50 mg / mL of a ligand-drug conjugate; (b) 1 to 30 mM histidine salt buffer or succinate buffer; (c) 30 to 130 mg / mL of sucrose or trehalose; (d) 0.05 to 0.6 mg / mL of polysorbate 80; the pH of the pharmaceutical composition is 5.0 to 6.5; Or, (a) 15-25 mg / mL of a ligand-drug conjugate; (b) 5-15 mM histidine salt buffer or succinate buffer; (c) 70-90 mg / mL sucrose; (d) 0.1 to 0.3 mg / mL of polysorbate 80; the pH of the pharmaceutical composition is 5.0 to 6.5; Or, (a) 15-25 mg / mL of a ligand-drug conjugate; (b) about 10 mM histidine salt buffer; (c) about 80 mg / mL sucrose; and (d) 0.1 to 0.3 mg / mL of polysorbate 80; the pH of the pharmaceutical composition is 5.5 to 6.0; Or, (a) 15-25 mg / mL of a ligand-drug conjugate; (b) about 10 mM succinate buffer; (c) about 80 mg / mL sucrose; and (d) 0.1 to 0.3 mg / mL of polysorbate 80; the pH of the pharmaceutical composition is 5.0 to 6.0; Or, (a) 15-25 mg / mL of a ligand-drug conjugate; (b) about 10 mM histidine-hydrochloride buffer; (c) about 80 mg / mL sucrose; and (d) 0.1 to 0.3 mg / mL of polysorbate 80; the pH of the pharmaceutical composition is 5.5 to 6.0; Or, (a) 15-25 mg / mL of a ligand-drug conjugate; (b) about 10 mM succinic acid-sodium succinate buffer; (c) about 80 mg / mL sucrose; and (d) 0.1 to 0.3 mg / mL of polysorbate 80; the pH of the pharmaceutical composition is 5.0 to 6.0; Preferably, the pharmaceutical composition comprises: (a) 15-25 mg / mL of a ligand-drug conjugate; (b) about 10 mM histidine-histidine hydrochloride; (c) about 80 mg / mL sucrose; and (d) about 0.2 mg / mL polysorbate 80; the pH of the pharmaceutical composition is 5.6; Or, (a) 15-25 mg / mL of a ligand-drug conjugate; (b) about 10 mM histidine-histidine hydrochloride; (c) about 80 mg / mL sucrose; and (d) about 0.2 mg / mL polysorbate 80; the pH of the pharmaceutical composition is about 5.5; Or, (a) 15-25 mg / mL of a ligand-drug conjugate; (b) about 10 mM histidine-histidine hydrochloride; (c) about 80 mg / mL sucrose; and (d) about 0.2 mg / mL polysorbate 80; the pH of the pharmaceutical composition is about 5.75; Or, (a) 15-25 mg / mL of a ligand-drug conjugate; (b) about 10 mM histidine-histidine hydrochloride; (c) about 80 mg / mL sucrose; and (d) about 0.2 mg / mL polysorbate 80; the pharmaceutical composition has a pH of about 6.0; Or, (a) 15-25 mg / mL of a ligand-drug conjugate; (b) about 10 mM histidine-histidine hydrochloride; (c) about 80 mg / mL sucrose; and (d) about 0.3 mg / mL polysorbate 80; the pH of the pharmaceutical composition is about 5.5; Or, (a) 15-25 mg / mL of a ligand-drug conjugate; (b) about 10 mM histidine-histidine hydrochloride; (c) about 80 mg / mL sucrose; and (d) about 0.3 mg / mL polysorbate 80; the pH of the pharmaceutical composition is about 5.75; Or, (a) 15-25 mg / mL of a ligand-drug conjugate; (b) about 10 mM histidine-histidine hydrochloride; (c) about 80 mg / mL sucrose; and (d) about 0.3 mg / mL polysorbate 80; the pharmaceutical composition has a pH of about 6.0; Or, (a) 15-25 mg / mL of a ligand-drug conjugate; (b) about 10 mM histidine-histidine hydrochloride; (c) about 80 mg / mL sucrose; and (d) about 0.1 mg / mL polysorbate 80; the pH of the pharmaceutical composition is about 5.5; Or, (a) 15-25 mg / mL of a ligand-drug conjugate; (b) about 10 mM histidine-histidine hydrochloride; (c) about 80 mg / mL sucrose; and (d) about 0.1 mg / mL polysorbate 80; the pH of the pharmaceutical composition is about 5.75; Or, (a) 15-25 mg / mL of a ligand-drug conjugate; (b) about 10 mM histidine-histidine hydrochloride; (c) about 80 mg / mL sucrose; and (d) about 0.1 mg / mL polysorbate 80; the pharmaceutical composition has a pH of about 6.0; the ligand is an anti-CD79b antibody or an antigen-binding fragment thereof, the anti-CD79b antibody or antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising HCDR1 having the amino acid sequence shown in SEQ ID NO: 11 or 5, and HCDR2 and HCDR3 having the amino acid sequences shown in SEQ ID NO: 6 and SEQ ID NO: 7, and the light chain variable region comprising LCDR1, LCDR2 and LCDR3 having the amino acid sequences shown in SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 10, The drug is selected from MMAE or a derivative thereof, exatecan or a derivative thereof, and eribulin or a derivative thereof, and is preferably exatecan or a derivative thereof. Pharmaceutical compositions.

10. 2. The pharmaceutical composition of claim 1, wherein the anti-CD79b antibody or antigen-binding fragment thereof comprises an antibody heavy chain variable region and a light chain variable region, wherein the heavy chain variable region sequence comprises SEQ ID NO: 3 or an amino acid sequence having at least 90% identity thereto, and the light chain variable region sequence comprises SEQ ID NO: 4 or an amino acid sequence having at least 90% identity thereto.

11. 2. The pharmaceutical composition of claim 1, wherein the anti-CD79b antibody or antigen-binding fragment thereof comprises a heavy chain and a light chain, wherein the heavy chain sequence comprises SEQ ID NO: 12, or an amino acid sequence having at least 80% identity thereto, and the light chain sequence comprises SEQ ID NO: 13, or an amino acid sequence having at least 80% identity thereto.

12. The ligand-drug conjugate is represented by the general formula (Pc-LY-D) of formula (I): 【Chemistry 1】 During the ceremony, Y is —O—(CR ａ R ｂ ) ｍ -CR １ R ２ -C(O)-, -O-CR １ R ２ - (CR ａ R ｂ ) ｍ -, -O-CR １ R ２ -, -NH-(CR ａ R ｂ ) ｍ -CR １ R ２ -C(O)- and -S-(CR ａ R ｂ ) ｍ -CR １ R ２ —C(O)—, R ａ and R ｂ are the same or different and are each independently selected from a hydrogen atom, a deuterium atom, a halogen, an alkyl group, a haloalkyl group, a deuterated alkyl group, an alkoxy group, a hydroxy group, an amino group, a cyano group, a nitro group, a hydroxyalkyl group, a cycloalkyl group, and a heterocyclyl group, or R ａ and R ｂ together with the carbon atoms to which they are attached form a cycloalkyl group and a heterocyclyl group, R １ is selected from a hydrogen atom, a halogen, a haloalkyl group, a deuterated alkyl group, a cycloalkyl group, a cycloalkylalkyl group, an alkoxyalkyl group, a heterocyclyl group, an aryl group, and a heteroaryl group; R ２ is selected from a hydrogen atom, a halogen, a haloalkyl group, a deuterated alkyl group, a cycloalkyl group, a cycloalkylalkyl group, an alkoxyalkyl group, a heterocyclyl group, an aryl group, and a heteroaryl group, or R １ and R ２ together with the carbon atoms to which they are attached form a cycloalkyl or heterocyclyl group, Or, R ａ and R ２ together with the carbon atoms to which they are attached form a cycloalkyl or heterocyclyl group, m is an integer from 0 to 4; n is 1 to 10, n is a decimal or an integer, preferably n is 2 to 8 or 5 to 9; L is a linker unit, Pc is an anti-CD79b antibody or an antigen-binding fragment thereof; Preferably, the ligand-drug conjugate has the structure shown in the formula: 【Chemistry 2】 n is 1 to 10, and n is a decimal or an integer; More preferably, n is 1 to 6, and n is a decimal or an integer. The pharmaceutical composition of claim 1.

13. 13. A lyophilized formulation obtainable by lyophilizing the pharmaceutical composition of any one of claims 1 to 12, or which, when reconstituted, can form the pharmaceutical composition of any one of claims 1 to 12.

14. A method for preparing a lyophilized formulation, comprising the step of lyophilizing the pharmaceutical composition according to any one of claims 1 to 12.

15. A reconstituted solution prepared by reconstituting the freeze-dried formulation according to claim 13, which is preferably an intravenous injection, a subcutaneous injection, an intraperitoneal injection, or an intramuscular injection, more preferably an intravenous injection.

16. The pharmaceutical composition according to any one of claims 1 to 12, which is an intravenous injection, a subcutaneous injection, an intraperitoneal injection or an intramuscular injection, preferably an intravenous injection.

17. A product comprising: the pharmaceutical composition according to any one of claims 1 to 12; a freeze-dried preparation obtained by freeze-drying the pharmaceutical composition according to any one of claims 1 to 12; a freeze-dried preparation that can form the pharmaceutical composition according to any one of claims 1 to 12 when reconstituted; or a container containing a reconstituted solution prepared by reconstituting the freeze-dried preparation.

18. 13. Use of the pharmaceutical composition according to any one of claims 1 to 12, a lyophilized formulation obtained by lyophilizing the pharmaceutical composition according to any one of claims 1 to 12, a lyophilized formulation which can form the pharmaceutical composition according to any one of claims 1 to 12 upon reconstitution, or a reconstituted solution prepared by reconstituting the lyophilized formulation, in the preparation of a medicament for treating or preventing a proliferative disease or delaying the progression of a proliferative disease, Preferably, the proliferative condition is a cancer or tumor; More preferably, the cancer or tumor is selected from lymphoma, diffuse large B-cell lymphoma, non-Hodgkin's lymphoma (NHL), aggressive NHL, relapsed aggressive NHL, relapsed indolent NHL, refractory NHL, refractory indolent NHL, chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma, leukemia, hairy cell leukemia (HCL), acute lymphocytic leukemia (ALL), and / or mantle cell lymphoma; use.

19. A pharmaceutical composition according to any one of claims 1 to 12 for treating a proliferative disease, a lyophilized preparation obtained by lyophilizing the pharmaceutical composition according to any one of claims 1 to 12, a lyophilized preparation which can form the pharmaceutical composition according to any one of claims 1 to 12 when reconstituted, or a reconstituted solution prepared by reconstituting the lyophilized preparation, Preferably, the proliferative condition is a cancer or tumor; More preferably, the cancer or tumor is selected from lymphoma, diffuse large B-cell lymphoma, non-Hodgkin's lymphoma (NHL), aggressive NHL, relapsed aggressive NHL, relapsed indolent NHL, refractory NHL, refractory indolent NHL, chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma, leukemia, hairy cell leukemia (HCL), acute lymphocytic leukemia (ALL), and / or mantle cell lymphoma; Pharmaceutical compositions, lyophilized formulations or reconstituted solutions.

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