Pharmaceutical composition comprising Anti-PSMA antibody-drug conjugate

Abstract

The present disclosure relates to a pharmaceutical composition comprising an anti-PSMA antibody-drug conjugate. In particular, the present disclosure relates to a pharmaceutical composition comprising an anti-PSMA antibody-drug conjugate and a buffer.

Description

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

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

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

[0003] Prostate-specific membrane antigen (PSMA) is a type II transmembrane glycoprotein. Its expression level is very low in normal prostate tissue and non-prostate cancer tissues (such as the lacrimal gland, nervous system, and duodenum), but its expression in prostate cancer tissue is 100-1000 times higher than in normal tissue, making it an ideal diagnostic and therapeutic target for prostate cancer. In addition, PSMA is also expressed in other solid tumors, such as gastric cancer, pancreatic cancer, breast cancer, and lung cancer. However, in these tumors, PSMA is distributed in tumor blood vessels rather than tumor cells. Therefore, besides serving as a therapeutic target for prostate cancer, PSMA can also serve as an anti-angiogenic therapeutic target for many other tumors.

[0004] Ecinotecan toxoid is a camptothecin derivative that inhibits topoisomerase I, selectively inhibiting DNA replication in proliferating tumor cells. Furthermore, eccinotecan toxoid exhibits excellent membrane permeability, allowing it to penetrate killed cancer cells and continue killing adjacent cancer cells, demonstrating a clear bystander effect in clinical practice.

[0005] Antibody-drug conjugates (ADCs) for PSMA consist of an anti-PSMA monoclonal antibody linked to a DNA topoisomerase inhibitor. The mechanism of action of PSMA ADCs is as follows: when a PSMA ADC binds to PSMA on the surface of cancer cells, it is "internalized" (internalization refers to the process by which certain substances or molecules on the cell membrane move from the outside into the cell). Subsequently, the DNA topoisomerase inhibitor, the drug carrier responsible for its anti-cancer effects, is released through lysosomal metabolism, maximizing the killing of cancer cells while minimizing toxic side effects.

[0006] Antibody-drug conjugates (ADCs) are an important class of biological drugs. Due to their large molecular weight and complex structure, they are susceptible to degradation and polymerization caused by physical or chemical factors during production, storage, and use, leading to reduced activity or even inactivation. Therefore, the development of excellent antibody-immunodrug conjugate formulations is of paramount importance. Summary of the Invention

[0007] This disclosure provides a pharmaceutical composition comprising an anti-PSMA antibody-drug conjugate. The pharmaceutical composition exhibits good therapeutic activity, safety, pharmacokinetic properties, and drug-likeness (e.g., stability).

[0008] In some embodiments, this disclosure provides a pharmaceutical composition as shown in any of the following:

[0009] (1) A pharmaceutical composition comprising an anti-PSMA antibody-drug conjugate and a buffer;

[0010] (2) A pharmaceutical composition comprising an anti-PSMA antibody-drug conjugate and a surfactant; a pharmaceutical composition comprising an anti-PSMA antibody-drug conjugate and a sugar;

[0011] (3) A pharmaceutical composition comprising an anti-PSMA antibody-drug conjugate and excipients;

[0012] (4) A pharmaceutical composition comprising an anti-PSMA antibody-drug conjugate, a buffer, a surfactant, and a sugar;

[0013] (5) A pharmaceutical composition comprising an anti-PSMA antibody-drug conjugate, a buffer, a surfactant, a sugar, and excipients.

[0014] In some embodiments, the pharmaceutical composition provided in this disclosure, as described in any of the above, wherein the anti-PSMA antibody-drug conjugate has the structure shown below:

[0015] in:

[0016] n is between 1 and 10;

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

[0018] The heavy chain variable region includes HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO:5, SEQ ID NO:6 and SEQ ID NO:7 respectively; the light chain variable region includes LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO:8, SEQ ID NO:9 and SEQ ID NO:10 respectively.

[0019] In some embodiments, this disclosure provides a pharmaceutical composition comprising an anti-PSMA antibody-drug conjugate and a buffer, wherein:

[0020] The anti-PSMA antibody-drug conjugate has the following structure:

[0021] in:

[0022] n is between 1 and 10;

[0023] Pc is an anti-PSMA antibody, which contains a heavy chain variable region and a light chain variable region, wherein:

[0024] The heavy chain variable region includes HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO:5, SEQ ID NO:6 and SEQ ID NO:7 respectively; the light chain variable region includes LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO:8, SEQ ID NO:9 and SEQ ID NO:10 respectively.

[0025] The buffer is a histidine buffer, a citrate buffer, an acetate buffer, or a succinate buffer.

[0026] In some embodiments, the pharmaceutical composition as described above, wherein the buffer is a citrate-disodium hydrogen phosphate buffer, a histidine-histidine hydrochloride buffer, and an acetate-sodium acetate buffer.

[0027] In some embodiments, such as the pharmaceutical composition described in any of the preceding embodiments, the buffer is an acetate-sodium acetate buffer.

[0028] In some embodiments, the pharmaceutical composition as described in any of the preceding claims, wherein the anti-PSMA antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:12, or an amino acid sequence having at least 80% sequence identity with it; and the light chain variable region comprises the amino acid sequence of SEQ ID NO:14, or an amino acid sequence having at least 80% sequence identity with it.

[0029] In some embodiments, the pharmaceutical composition as described in any of the preceding claims, wherein the anti-PSMA antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:12 and the light chain variable region comprises the amino acid sequence of SEQ ID NO:14.

[0030] In some embodiments, the pharmaceutical composition as described in any of the preceding claims, wherein the anti-PSMA antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO: 19, or an amino acid sequence having at least 80% sequence identity therewith; and the light chain comprises the amino acid sequence of SEQ ID NO: 20, or an amino acid sequence having at least 80% sequence identity therewith.

[0031] In some embodiments, the pharmaceutical composition as described in any of the preceding claims, wherein the anti-PSMA antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO: 19, and the light chain comprises the amino acid sequence of SEQ ID NO: 20.

[0032] In some embodiments, in the pharmaceutical composition described in any of the preceding embodiments, wherein n is the average number of pharmaceutical modules per anti-PSMA antibody, and may be an integer or a decimal. In some embodiments, n is 1-10, or 2-10, or 3-10, or 4-10, or 5-10, or 6-10, or 7-10, or 8-10, or 1-9, or 2-9, or 3-9, or 4-9, or 5-9, or 6-9, or 7-9, or 1-8, or 2-8, or 3-8, or 4-8, or 5-8, or 6-8, or 1-7, or 2-7, or 3-7, or 4-7, or 5-7, or 1-6, or 2-6, or 3-6, or 4-6, or 1-5, or 2-5, or 3-5, or 1-4, or 2-4, or 1-3. In some embodiments, n is about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, or about 10.

[0033] In some embodiments, the pharmaceutical composition as described in any of the preceding embodiments, wherein n is 3 to 8. In some embodiments, n is 4 to 8. In some embodiments, n is 5 to 8. In some embodiments, n is 6 to 8. In some embodiments, n is about 6. In some embodiments, n is 6. In some embodiments, n is about 7. In some embodiments, n is 7. In some embodiments, n is about 7.5. In some embodiments, n is 7.5. In some embodiments, n is about 8. In some embodiments, n is 8.

[0034] In some embodiments, the pharmaceutical composition as described in any of the preceding embodiments, wherein the concentration of the anti-PSMA antibody-drug conjugate is from 1 mg / mL to 50 mg / mL. In some embodiments, the concentration of the anti-PSMA antibody-drug conjugate is from 5 mg / mL to 40 mg / mL. In some embodiments, the concentration of the anti-PSMA antibody-drug conjugate is from 5 mg / mL to 30 mg / mL. In some embodiments, the concentration of the anti-PSMA antibody-drug conjugate is from 15 mg / mL to 25 mg / mL. In some embodiments, the concentration of the anti-PSMA antibody-drug conjugate is from 18 mg / mL to 22 mg / mL. In some embodiments, the concentration of the anti-PSMA antibody-drug conjugate is about 1 mg / mL, about 5 mg / mL, about 10 mg / mL, about 15 mg / mL, about 16 mg / mL, about 18 mg / mL, about 20 mg / mL, about 22 mg / mL, about 24 mg / mL, about 25 mg / mL, about 30 mg / mL, about 35 mg / mL, about 40 mg / mL, about 45 mg / mL, or about 50 mg / mL. In some embodiments, the concentration of the anti-PSMA antibody-drug conjugate is about 20 mg / mL. In some embodiments, the concentration of the anti-PSMA antibody-drug conjugate is 1 mg / mL, 5 mg / mL, 10 mg / mL, 15 mg / mL, 16 mg / mL, 18 mg / mL, 20 mg / mL, 22 mg / mL, 24 mg / mL, 25 mg / mL, 30 mg / mL, 35 mg / mL, 40 mg / mL, 45 mg / mL, or 50 mg / mL, or any range between these values. In some embodiments, the concentration of the anti-PSMA antibody-drug conjugate is 20 mg / mL.

[0035] In some embodiments, the pharmaceutical composition as described in any of the preceding embodiments, wherein the concentration of the buffer is from 1 mM to 50 mM. In some embodiments, the concentration of the buffer is from 5 mM to 40 mM. In some embodiments, the concentration of the buffer is from 5 mM to 35 mM. In some embodiments, the concentration of the buffer is from 5 mM to 30 mM. In some embodiments, the concentration of the buffer is from 5 mM to 20 mM. In some embodiments, the concentration of the buffer is from 5 mM to 15 mM. In some embodiments, the concentration of the buffer is from 8 mM to 12 mM. In some embodiments, the concentration of the buffer is from 9 mM to 11 mM. In some embodiments, the concentration of the buffer is about 10 mM. In some embodiments, the concentration of the buffer is about 1 mM, about 5 mM, about 7 mM, about 8 mM, about 9 mM, about 9.2 mM, about 10 mM, about 11 mM, about 12 mM, about 15 mM, about 20 mM, about 22 mM, about 24 mM, about 25 mM, about 27 mM, about 27.8 mM, about 30 mM, about 33 mM, about 35 mM, about 36 mM, about 40 mM, about 45 mM, or about 50 mM. In some embodiments, the concentration of the buffer is 1 mM, 5 mM, 7 mM, 8 mM, 9 mM, 9.2 mM, 10 mM, 11 mM, 12 mM, 15 mM, 20 mM, 22 mM, 24 mM, 25 mM, 27 mM, 27.8 mM, 30 mM, 33 mM, 35 mM, 36 mM, 40 mM, 45 mM, or 50 mM, or any range between these values. In some embodiments, the concentration of the buffer is 10 mM.

[0036] In some embodiments, the pharmaceutical composition as described in any of the preceding embodiments, wherein the concentration of the acetate-sodium acetate buffer is from 1 mM to 50 mM. In some embodiments, the concentration of the acetate-sodium acetate buffer is from 5 mM to 40 mM. In some embodiments, the concentration of the acetate-sodium acetate buffer is from 5 mM to 35 mM. In some embodiments, the concentration of the acetate-sodium acetate buffer is from 5 mM to 30 mM. In some embodiments, the concentration of the acetate-sodium acetate buffer is from 5 mM to 20 mM. In some embodiments, the concentration of the acetate-sodium acetate buffer is from 5 mM to 15 mM. In some embodiments, the concentration of the acetate-sodium acetate buffer is from 8 mM to 12 mM. In some embodiments, the concentration of the acetate-sodium acetate buffer is from 9 mM to 11 mM. In some embodiments, the concentration of the acetate-sodium acetate buffer is about 10 mM. In some embodiments, the concentration of the acetate-sodium acetate buffer is about 1 mM, about 5 mM, about 7 mM, about 8 mM, about 9 mM, about 9.2 mM, about 10 mM, about 11 mM, about 12 mM, about 15 mM, about 20 mM, about 22 mM, about 24 mM, about 25 mM, about 27 mM, about 27.8 mM, about 30 mM, about 33 mM, about 35 mM, about 36 mM, about 40 mM, about 45 mM, or about 50 mM. In some embodiments, the concentration of the acetate-sodium acetate buffer is about 10 mM. In some embodiments, the concentration of the acetate-sodium acetate buffer is 1 mM, 5 mM, 7 mM, 8 mM, 9 mM, 9.2 mM, 10 mM, 11 mM, 12 mM, 15 mM, 20 mM, 22 mM, 24 mM, 25 mM, 27 mM, 27.8 mM, 30 mM, 33 mM, 35 mM, 36 mM, 40 mM, 45 mM, or 50 mM, or any range between these values. In some embodiments, the concentration of the acetate-sodium acetate buffer is 10 mM.

[0037] In some embodiments, the pharmaceutical composition as described in any of the preceding embodiments has a pH of 4.0 to 8.0. In some embodiments, the pharmaceutical composition has a pH of 4.6 to 7.4. In some embodiments, the pharmaceutical composition has a pH of 4.7 to 5.8. In some embodiments, the pharmaceutical composition has a pH of 5.0 to 5.8. In some embodiments, the pharmaceutical composition has a pH of 4.7 to 5.3. In some embodiments, the pharmaceutical composition has a pH of about 4.5, about 4.6, about 4.7, about 4.8, about 4.9, about 5.0, about 5.1, about 5.2, about 5.3, about 5.4, about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, about 6.0, about 6.1, about 6.2, about 6.3, about 6.4, about 6.5, about 7.0, about 7.4, about 7.5, or about 8.0. In some embodiments, the pH of the pharmaceutical composition is about 4.7. In some embodiments, the pH of the pharmaceutical composition is about 5.0. In some embodiments, the pH of the pharmaceutical composition is about 5.3. In some embodiments, the pH of the pharmaceutical composition is about 5.8. In some embodiments, the pH of the pharmaceutical composition is 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 7.0, 7.4, 7.5, or 8.0, or any range between these values. In some embodiments, the pH of the pharmaceutical composition is 4.7. In some embodiments, the pH of the pharmaceutical composition is 5.0. In some embodiments, the pH of the pharmaceutical composition is 5.3. In some embodiments, the pH of the pharmaceutical composition is 5.8. When point values ​​are mentioned in this disclosure, it should be understood that these point values ​​include a range of error. This range of error is due to factors such as laboratory environment, personnel operation, instrumentation, methodology, and measurement errors. For example, when measuring pH, a value of approximately 5.0 should be understood to include a range of error. As an example, when measuring formulations using an industrial pH meter, "approximately 5.0" means 5.5 ± 0.3 (i.e., pH from 4.7 to 5.3).

[0038] In some embodiments, the pharmaceutical composition as described in any of the preceding embodiments comprises a surfactant. In some embodiments, the surfactant is a nonionic surfactant. In some embodiments, the surfactant is selected from polysorbates (e.g., polysorbate 80, polysorbate 20), poloxamer (e.g., poloxamer 188, i.e., PF68 or P188), Triton, sodium lauryl sulfonate, sodium lauryl sulfonate, sodium octyl glycoside, lauryl-sulfobetaine, myristyl-sulfobetaine, linoleyl-sulfobetaine, stearyl-sulfobetaine, lauryl-sarcosine, myristyl-sarcosine, linoleyl-sarcosine, stearyl-sarcosine, linoleyl-saccharide, succinate ... Betaine, myristyl-betaine, cetyl-betaine, lauramidopropyl-betaine, cocarbamate-propyl-betaine, linoleamide-propyl-betaine, myristamidopropyl-betaine, palmitoamide-propyl-betaine, isostearamidopropyl-betaine, myristamidopropyl-dimethylamine, palmitoamide-propyl-dimethylamine, isostearamidopropyl-dimethylamine, sodium methyl cocoyl, sodium methyl oleate, polyethylene glycol, polypropylene glycol, copolymers of ethylene and propylene glycol, etc. In some embodiments, the surfactant is polysorbate. In some embodiments, the surfactant is polysorbate 80 (PS80), polysorbate 20 (PS20), and poloxamer 188. In some embodiments, the surfactant is polysorbate 80 (PS80).

[0039] In some embodiments, the pharmaceutical composition as described in any of the preceding embodiments, wherein the concentration of the surfactant is from 0.01 mg / mL to 2 mg / mL. In some embodiments, the concentration of the surfactant is from 0.02 mg / mL to 1 mg / mL. In some embodiments, the concentration of the surfactant is from 0.02 mg / mL to 0.8 mg / mL. In some embodiments, the concentration of the surfactant is from 0.02 mg / mL to 0.6 mg / mL. In some embodiments, the concentration of the surfactant is from 0.05 mg / mL to 0.5 mg / mL. In some embodiments, the concentration of the surfactant is from 0.05 mg / mL to 0.4 mg / mL. In some embodiments, the concentration of the surfactant is from 0.05 mg / mL to 0.35 mg / mL. In some embodiments, the concentration of the surfactant is from 0.1 mg / mL to 0.3 mg / mL. In some embodiments, the concentration of the surfactant is from 0.15 mg / mL to 0.25 mg / mL. In some embodiments, the surfactant concentration is about 0.01 mg / mL, about 0.02 mg / mL, about 0.05 mg / mL, about 0.1 mg / mL, 0.15 mg / mL, about 0.2 mg / mL, about 0.25 mg / mL, about 0.3 mg / mL, about 0.35 mg / mL, about 0.4 mg / mL, about 0.5 mg / mL, about 0.6 mg / mL, about 0.7 mg / mL, about 0.8 mg / mL, about 0.9 mg / mL, about 1.0 mg / mL, about 1.5 mg / mL, or about 2.0 mg / mL. In some embodiments, the surfactant concentration is about 0.2 mg / mL. In some embodiments, the surfactant concentration is 0.01 mg / mL, 0.02 mg / mL, 0.05 mg / mL, 0.1 mg / mL, 0.15 mg / mL, 0.2 mg / mL, 0.25 mg / mL, 0.3 mg / mL, 0.35 mg / mL, 0.4 mg / mL, 0.5 mg / mL, 0.6 mg / mL, 0.7 mg / mL, 0.8 mg / mL, 0.9 mg / mL, 1.0 mg / mL, 1.5 mg / mL, or 2.0 mg / mL, or any range between these values. In some embodiments, the surfactant concentration is 0.2 mg / mL.

[0040] In some embodiments, the pharmaceutical composition as described in any of the preceding embodiments, wherein the concentration of polysorbate 80 is from 0.01 mg / mL to 2 mg / mL. In some embodiments, the concentration of polysorbate 80 is from 0.02 mg / mL to 1 mg / mL. In some embodiments, the concentration of polysorbate 80 is from 0.02 mg / mL to 0.8 mg / mL. In some embodiments, the concentration of polysorbate 80 is from 0.02 mg / mL to 0.6 mg / mL. In some embodiments, the concentration of polysorbate 80 is from 0.05 mg / mL to 0.5 mg / mL. In some embodiments, the concentration of polysorbate 80 is from 0.05 mg / mL to 0.4 mg / mL. In some embodiments, the concentration of polysorbate 80 is from 0.05 mg / mL to 0.35 mg / mL. In some embodiments, the concentration of polysorbate 80 is from 0.1 mg / mL to 0.3 mg / mL. In some embodiments, the concentration of polysorbate 80 is from 0.15 mg / mL to 0.25 mg / mL. In some embodiments, the concentration of polysorbate 80 is about 0.01 mg / mL, about 0.02 mg / mL, about 0.05 mg / mL, about 0.1 mg / mL, 0.15 mg / mL, about 0.2 mg / mL, about 0.25 mg / mL, about 0.3 mg / mL, about 0.35 mg / mL, about 0.4 mg / mL, about 0.5 mg / mL, about 0.6 mg / mL, about 0.7 mg / mL, about 0.8 mg / mL, about 0.9 mg / mL, about 1.0 mg / mL, about 1.5 mg / mL, or about 2.0 mg / mL. In some embodiments, the concentration of polysorbate 80 is about 0.2 mg / mL. In some embodiments, the concentration of polysorbate 80 is 0.01 mg / mL, 0.02 mg / mL, 0.05 mg / mL, 0.1 mg / mL, 0.15 mg / mL, 0.2 mg / mL, 0.25 mg / mL, 0.3 mg / mL, 0.35 mg / mL, 0.4 mg / mL, 0.5 mg / mL, 0.6 mg / mL, 0.7 mg / mL, 0.8 mg / mL, 0.9 mg / mL, 1.0 mg / mL, 1.5 mg / mL, or 2.0 mg / mL, or any range between these values. In some embodiments, the concentration of polysorbate 80 is 0.2 mg / mL.

[0041] In some embodiments, the pharmaceutical composition as described in any of the preceding embodiments comprises a sugar. In some embodiments, the sugar is selected from conventional compositions (CH2O). nAnd its derivatives, including monosaccharides, disaccharides, trisaccharides, polysaccharides, sugar alcohols, reducing sugars, non-reducing sugars, etc. In some embodiments, the sugar is selected from sucrose, trehalose, glucose, lactose, fructose, maltose, dextran, glycerol, erythritol, glycerol, arabinitol, sylitol, sorbitol, mannitol, melitriose, maltotriose, stachyose, maltose, lactulose, maltitol, maltitol, lactitol, isomaltulose, etc. In some embodiments, the sugar is sucrose, trehalose, mannitol, or sorbitol. In some embodiments, the sugar is sucrose.

[0042] In some embodiments, the pharmaceutical composition as described in any of the preceding embodiments, wherein the concentration of the sugar is from 10 mg / mL to 120 mg / mL. In some embodiments, the concentration of the sugar is from 40 mg / mL to 110 mg / mL. In some embodiments, the concentration of the sugar is from 50 mg / mL to 100 mg / mL. In some embodiments, the concentration of the sugar is from 60 mg / mL to 90 mg / mL. In some embodiments, the concentration of the sugar is from 70 mg / mL to 80 mg / mL. In some embodiments, the sugar concentration is about 10 mg / mL, about 20 mg / mL, about 30 mg / mL, about 33 mg / mL, about 36 mg / mL, about 40 mg / mL, about 44 mg / mL, about 48 mg / mL, about 50 mg / mL, about 60 mg / mL, about 64 mg / mL, about 65 mg / mL, about 70 mg / mL, about 75 mg / mL, about 80 mg / mL, about 88 mg / mL, about 90 mg / mL, about 95 mg / mL, about 96 mg / mL, about 100 mg / mL, about 110 mg / mL, or about 120 mg / mL. In some embodiments, the sugar concentration is about 75 mg / mL. In some embodiments, the sugar concentration is 10 mg / mL, 20 mg / mL, 30 mg / mL, 33 mg / mL, 36 mg / mL, 40 mg / mL, 44 mg / mL, 48 mg / mL, 50 mg / mL, 60 mg / mL, 64 mg / mL, 65 mg / mL, 70 mg / mL, 75 mg / mL, 80 mg / mL, 88 mg / mL, 90 mg / mL, 95 mg / mL, 96 mg / mL, 100 mg / mL, 110 mg / mL, or 120 mg / mL, or any range between these values. In some embodiments, the sugar concentration is 75 mg / mL.

[0043] In some embodiments, the pharmaceutical composition as described in any of the preceding embodiments, wherein the concentration of sucrose is from 10 mg / mL to 120 mg / mL. In some embodiments, the concentration of sucrose is from 40 mg / mL to 110 mg / mL. In some embodiments, the concentration of sucrose is from 50 mg / mL to 100 mg / mL. In some embodiments, the concentration of sucrose is from 60 mg / mL to 90 mg / mL. In some embodiments, the concentration of sucrose is from 70 mg / mL to 80 mg / mL. In some embodiments, the concentration of the sucrose is about 10 mg / mL, about 20 mg / mL, about 30 mg / mL, about 33 mg / mL, about 36 mg / mL, about 40 mg / mL, about 44 mg / mL, about 48 mg / mL, about 50 mg / mL, about 60 mg / mL, about 64 mg / mL, about 65 mg / mL, about 70 mg / mL, about 75 mg / mL, about 80 mg / mL, about 88 mg / mL, about 90 mg / mL, about 95 mg / mL, about 96 mg / mL, about 100 mg / mL, about 110 mg / mL, or about 120 mg / mL. In some embodiments, the concentration of the sucrose is about 75 mg / mL. In some embodiments, the concentration of the sucrose is 10 mg / mL, 20 mg / mL, 30 mg / mL, 33 mg / mL, 36 mg / mL, 40 mg / mL, 44 mg / mL, 48 mg / mL, 50 mg / mL, 60 mg / mL, 64 mg / mL, 65 mg / mL, 70 mg / mL, 75 mg / mL, 80 mg / mL, 88 mg / mL, 90 mg / mL, 95 mg / mL, 96 mg / mL, 100 mg / mL, 110 mg / mL, or 120 mg / mL, or any range between these values. In some embodiments, the concentration of the sucrose is 75 mg / mL.

[0044] In some embodiments, the pharmaceutical composition as described in any of the preceding embodiments further comprises an excipient. In some embodiments, the excipient is one or more selected from glycine, DTPA (diethylenetriaminepentaacetic acid), arginine hydrochloride, methionine, disodium edetate (EDTA-2Na), proline, histidine, phenylalanine, glutamic acid, aspartic acid, sodium chloride, or calcium chloride. In some embodiments, the excipient is methionine or disodium edetate. In some embodiments, the excipient is methionine.

[0045] In some embodiments, the pharmaceutical composition as described in any of the preceding claims, wherein the concentration of the excipient is from 0.01 mg / mL to 20 mg / mL. In some embodiments, the pharmaceutical composition as described in any of the preceding claims, wherein the concentration of the excipient is from 0.01 mg / mL to 15 mg / mL. In some embodiments, the pharmaceutical composition as described in any of the preceding claims, wherein the concentration of the excipient is from 0.01 mg / mL to 8 mg / mL. In some embodiments, the pharmaceutical composition as described in any of the preceding claims, wherein the concentration of the excipient is from 0.01 mg / mL to 5 mg / mL. In some embodiments, the concentration of the excipient is from 0.05 mg / mL to 15 mg / mL. In some embodiments, the concentration of the excipient is from 0.05 mg / mL to 13 mg / mL. In some embodiments, the concentration of the excipient is from 0.05 mg / mL to 10 mg / mL. In some embodiments, the concentration of the excipient is from 0.05 mg / mL to 8 mg / mL. In some embodiments, the concentration of the excipient is from 0.05 mg / mL to 6 mg / mL. In some embodiments, the concentration of the excipient is from 0.05 mg / mL to 4.5 mg / mL. In some embodiments, the concentration of the excipient is from 0.1 mg / mL to 12 mg / mL. In some embodiments, the concentration of the excipient is from 0.1 mg / mL to 8 mg / mL. In some embodiments, the concentration of the excipient is from 0.1 mg / mL to 5 mg / mL. In some embodiments, the concentration of the excipient is from 0.1 mg / mL to 4 mg / mL. In some embodiments, the concentration of the excipient is from 0.5 mg / mL to 3.5 mg / mL. In some embodiments, the concentration of the excipient is from 1 mg / mL to 12 mg / mL. In some embodiments, the concentration of the excipient is from 1 mg / mL to 10 mg / mL. In some embodiments, the concentration of the excipient is from 1 mg / mL to 9 mg / mL. In some embodiments, the concentration of the excipient is from 1 mg / mL to 8 mg / mL. In some embodiments, the concentration of the excipient is from 1 mg / mL to 7 mg / mL. In some embodiments, the concentration of the excipient is from 1 mg / mL to 6 mg / mL. In some embodiments, the concentration of the excipient is from 1 mg / mL to 5 mg / mL. In some embodiments, the concentration of the excipient is from 1 mg / mL to 4 mg / mL. In some embodiments, the concentration of the excipient is from 1 mg / mL to 3 mg / mL. In some embodiments, the concentration of the excipient is from 1.5 mg / mL to 2.5 mg / mL.In some embodiments, the concentration of the excipient is about 5 mg / mL, about 6 mg / mL, about 7 mg / mL, about 8 mg / mL, about 9 mg / mL, about 10 mg / mL, about 11 mg / mL, about 12 mg / mL, about 13 mg / mL, about 14 mg / mL, or about 15 mg / mL. In some embodiments, the concentration of the excipient is about 2 mg / mL. In some embodiments, the concentration of the excipient is 5 mg / mL, 6 mg / mL, 7 mg / mL, 8 mg / mL, 9 mg / mL, 10 mg / mL, 11 mg / mL, 12 mg / mL, 13 mg / mL, 14 mg / mL, or 15 mg / mL, or any range between these values. In some embodiments, the concentration of the excipient is 2 mg / mL.

[0046] In some embodiments, the pharmaceutical composition as described in any of the preceding claims, wherein the concentration of methionine is from 0.01 mg / mL to 20 mg / mL. In some embodiments, the pharmaceutical composition as described in any of the preceding claims, wherein the concentration of methionine is from 0.01 mg / mL to 15 mg / mL. In some embodiments, the pharmaceutical composition as described in any of the preceding claims, wherein the concentration of methionine is from 0.01 mg / mL to 10 mg / mL. In some embodiments, the pharmaceutical composition as described in any of the preceding claims, wherein the concentration of methionine is from 0.01 mg / mL to 8 mg / mL. In some embodiments, the pharmaceutical composition as described in any of the preceding claims, wherein the concentration of methionine is from 0.01 mg / mL to 5 mg / mL. In some embodiments, the concentration of methionine is from 0.05 mg / mL to 15 mg / mL. In some embodiments, the concentration of methionine is from 0.05 mg / mL to 13 mg / mL. In some embodiments, the concentration of methionine is from 0.05 mg / mL to 10 mg / mL. In some embodiments, the concentration of methionine is from 0.05 mg / mL to 8 mg / mL. In some embodiments, the concentration of methionine is from 0.05 mg / mL to 6 mg / mL. In some embodiments, the concentration of methionine is from 0.05 mg / mL to 4.5 mg / mL. In some embodiments, the concentration of methionine is from 0.1 mg / mL to 12 mg / mL. In some embodiments, the concentration of methionine is from 0.1 mg / mL to 8 mg / mL. In some embodiments, the concentration of methionine is from 0.1 mg / mL to 5 mg / mL. In some embodiments, the concentration of methionine is from 0.1 mg / mL to 4 mg / mL. In some embodiments, the concentration of methionine is from 0.5 mg / mL to 3.5 mg / mL. In some embodiments, the concentration of methionine is from 1 mg / mL to 12 mg / mL. In some embodiments, the concentration of methionine is from 1 mg / mL to 10 mg / mL. In some embodiments, the concentration of methionine is from 1 mg / mL to 9 mg / mL. In some embodiments, the concentration of methionine is from 1 mg / mL to 8 mg / mL. In some embodiments, the concentration of methionine is from 1 mg / mL to 7 mg / mL. In some embodiments, the concentration of methionine is from 1 mg / mL to 6 mg / mL. In some embodiments, the concentration of methionine is from 1 mg / mL to 5 mg / mL. In some embodiments, the concentration of methionine is from 1 mg / mL to 4 mg / mL. In some embodiments, the concentration of methionine is from 1 mg / mL to 3 mg / mL. In some embodiments, the concentration of methionine is from 1.5 mg / mL to 2.5 mg / mL.In some embodiments, the concentration of methionine is about 5 mg / mL, about 6 mg / mL, about 7 mg / mL, about 8 mg / mL, about 9 mg / mL, about 10 mg / mL, about 11 mg / mL, about 12 mg / mL, about 13 mg / mL, about 14 mg / mL, or about 15 mg / mL. In some embodiments, the concentration of methionine is about 2 mg / mL. In some embodiments, the concentration of methionine is 5 mg / mL, 6 mg / mL, 7 mg / mL, 8 mg / mL, 9 mg / mL, 10 mg / mL, 11 mg / mL, 12 mg / mL, 13 mg / mL, 14 mg / mL, or 15 mg / mL, or any range between these values. In some embodiments, the concentration of methionine is 2 mg / mL.

[0047] In some embodiments, the pharmaceutical composition as described in any of the preceding embodiments, wherein the concentration of the excipient is from 0.01 mg / mL to 1 mg / mL. In some embodiments, the concentration of the excipient is from 0.01 mg / mL to 0.5 mg / mL. In some embodiments, the concentration of the excipient is from 0.01 mg / mL to 0.1 mg / mL. In some embodiments, the concentration of the excipient is from 0.02 mg / mL to 0.08 mg / mL. In some embodiments, the concentration of the excipient is from 0.04 mg / mL to 0.06 mg / mL. In some embodiments, the concentration of the excipient is about 0.01 mg / mL, about 0.02 mg / mL, about 0.03 mg / mL, about 0.04 mg / mL, about 0.05 mg / mL, about 0.06 mg / mL, about 0.07 mg / mL, about 0.08 mg / mL, about 0.09 mg / mL, about 0.1 mg / mL, about 0.2 mg / mL, about 0.3 mg / mL, about 0.4 mg / mL, about 0.5 mg / mL, about 0.6 mg / mL, about 0.7 mg / mL, about 0.8 mg / mL, about 0.9 mg / mL, or about 1 mg / mL. In some embodiments, the concentration of the excipient is about 0.05 mg / mL. In some embodiments, the concentration of the excipient is 0.01 mg / mL, 0.02 mg / mL, 0.03 mg / mL, 0.04 mg / mL, 0.05 mg / mL, 0.06 mg / mL, 0.07 mg / mL, 0.08 mg / mL, 0.09 mg / mL, 0.1 mg / mL, 0.2 mg / mL, 0.3 mg / mL, 0.4 mg / mL, 0.5 mg / mL, 0.6 mg / mL, 0.7 mg / mL, 0.8 mg / mL, 0.9 mg / mL, or 1 mg / mL, or any range between these values. In some embodiments, the concentration of the excipient is 0.05 mg / mL.

[0048] In some embodiments, the pharmaceutical composition as described in any of the preceding embodiments, wherein the concentration of disodium edetate is from 0.01 mg / mL to 1 mg / mL. In some embodiments, the concentration of disodium edetate is from 0.01 mg / mL to 0.5 mg / mL. In some embodiments, the concentration of disodium edetate is from 0.01 mg / mL to 0.1 mg / mL. In some embodiments, the concentration of disodium edetate is from 0.02 mg / mL to 0.08 mg / mL. In some embodiments, the concentration of disodium edetate is from 0.04 mg / mL to 0.06 mg / mL. In some embodiments, the concentration of the disodium edetate is about 0.01 mg / mL, about 0.02 mg / mL, about 0.03 mg / mL, about 0.04 mg / mL, about 0.05 mg / mL, about 0.06 mg / mL, about 0.07 mg / mL, about 0.08 mg / mL, about 0.09 mg / mL, about 0.1 mg / mL, about 0.2 mg / mL, about 0.3 mg / mL, about 0.4 mg / mL, about 0.5 mg / mL, about 0.6 mg / mL, about 0.7 mg / mL, about 0.8 mg / mL, about 0.9 mg / mL, or about 1 mg / mL. In some embodiments, the concentration of the disodium edetate is about 0.05 mg / mL. In some embodiments, the concentration of the disodium edetate is 0.01 mg / mL, 0.02 mg / mL, 0.03 mg / mL, 0.04 mg / mL, 0.05 mg / mL, 0.06 mg / mL, 0.07 mg / mL, 0.08 mg / mL, 0.09 mg / mL, 0.1 mg / mL, 0.2 mg / mL, 0.3 mg / mL, 0.4 mg / mL, 0.5 mg / mL, 0.6 mg / mL, 0.7 mg / mL, 0.8 mg / mL, 0.9 mg / mL, or 1 mg / mL, or any range between these values. In some embodiments, the concentration of the disodium edetate is 0.05 mg / mL.

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

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

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

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

[0053] (e) A buffer of 1 mM to 50 mM, wherein the pH of the pharmaceutical composition is 4.6 to 7.4.

[0054] In some embodiments, the pharmaceutical composition as described in any of the preceding embodiments comprises the following components:

[0055] (a) The anti-PSMA antibody-drug conjugate at concentrations of 5 mg / mL to 40 mg / mL,

[0056] (b) Polysorbate 80 at concentrations ranging from 0.02 mg / mL to 1 mg / mL,

[0057] (c) sucrose at concentrations of 50 mg / mL to 100 mg / mL, and

[0058] (e) 5 mM to 30 mM of citrate-disodium hydrogen phosphate buffer, histidine-histidine hydrochloride buffer and acetate-sodium acetate buffer, wherein the pH of the pharmaceutical composition is 4.7 to 5.8.

[0059] In some embodiments, the pharmaceutical composition as described in any of the preceding embodiments comprises the following components:

[0060] (a) The anti-PSMA antibody-drug conjugate at concentrations of 5 mg / mL to 40 mg / mL,

[0061] (b) Polysorbate 80 at concentrations ranging from 0.02 mg / mL to 1 mg / mL,

[0062] (c) sucrose at concentrations of 50 mg / mL to 100 mg / mL, and

[0063] (e) 5 mM to 30 mM of citrate-disodium hydrogen phosphate buffer, histidine-histidine hydrochloride buffer and acetate-sodium acetate buffer, wherein the pH of the pharmaceutical composition is 5.0 to 5.8.

[0064] In some embodiments, the pharmaceutical composition as described in any of the preceding embodiments comprises the following components:

[0065] (a) The anti-PSMA antibody-drug conjugate at concentrations of 15 mg / mL to 25 mg / mL,

[0066] (b) Polysorbate 80 at concentrations ranging from 0.05 mg / mL to 0.4 mg / mL,

[0067] (c) sucrose at concentrations of 60 mg / mL to 90 mg / mL, and

[0068] (e) a 5 mM to 15 mM acetate-sodium acetate buffer, wherein the pH of the pharmaceutical composition is 4.7 to 5.3;

[0069] In some embodiments, the pharmaceutical composition as described in any of the preceding embodiments comprises the following components:

[0070] (a) The anti-PSMA antibody-drug conjugate at a concentration of approximately 20 mg / mL,

[0071] (b) Approximately 0.2 mg / mL of polysorbate 80,

[0072] (c) Approximately 75 mg / mL of sucrose, and

[0073] (e) about 10 mM of an acetate-sodium acetate buffer, wherein the pH of the pharmaceutical composition is about 5.0.

[0074] In some embodiments, the pharmaceutical composition as described in any of the preceding embodiments comprises the following components:

[0075] (a) 20 mg / mL of the aforementioned anti-PSMA antibody-drug conjugate,

[0076] (b) 0.2 mg / mL of polysorbate 80,

[0077] (c) 75 mg / mL sucrose, and

[0078] (e) 10 mM acetate-sodium acetate buffer, wherein the pH of the pharmaceutical composition is about 5.0.

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

[0080] (a) The anti-PSMA antibody-drug conjugate at concentrations from 1 mg / mL to 50 mg / mL,

[0081] (b) Surfactants ranging from 0.01 mg / mL to 2 mg / mL,

[0082] (c) Sugars ranging from 10 mg / mL to 120 mg / mL,

[0083] (d) Excipients ranging from 0.01 mg / mL to 20 mg / mL, and

[0084] (e) A buffer of 1 mM to 50 mM, wherein the pH of the pharmaceutical composition is 4.6 to 7.4.

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

[0086] (a) The anti-PSMA antibody-drug conjugate at concentrations of 5 mg / mL to 40 mg / mL,

[0087] (b) Polysorbate 80 at concentrations ranging from 0.02 mg / mL to 1 mg / mL,

[0088] (c) Sucrose at concentrations of 50 mg / mL to 100 mg / mL,

[0089] (d) Methionine at concentrations ranging from 0.1 mg / mL to 12 mg / mL, and

[0090] (e) 5 mM to 30 mM of citrate-disodium hydrogen phosphate buffer, histidine-histidine hydrochloride buffer and acetate-sodium acetate buffer, wherein the pH of the pharmaceutical composition is 4.7 to 5.8.

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

[0092] (a) The anti-PSMA antibody-drug conjugate at concentrations of 5 mg / mL to 40 mg / mL,

[0093] (b) Polysorbate 80 at concentrations ranging from 0.02 mg / mL to 1 mg / mL,

[0094] (c) Sucrose at concentrations of 50 mg / mL to 100 mg / mL,

[0095] (d) Methionine at concentrations ranging from 0.1 mg / mL to 12 mg / mL, and

[0096] (e) 5 mM to 30 mM of citrate-disodium hydrogen phosphate buffer, histidine-histidine hydrochloride buffer and acetate-sodium acetate buffer, wherein the pH of the pharmaceutical composition is 5.0 to 5.8.

[0097] In some embodiments, the pharmaceutical composition as described in any of the preceding embodiments comprises the following components:

[0098] (a) The anti-PSMA antibody-drug conjugate at concentrations of 15 mg / mL to 25 mg / mL,

[0099] (b) Polysorbate 80 at concentrations ranging from 0.05 mg / mL to 0.4 mg / mL,

[0100] (c) Sucrose at concentrations of 60 mg / mL to 90 mg / mL,

[0101] (d) Methionine at concentrations ranging from 1 mg / mL to 8 mg / mL, and

[0102] (e) a 5 mM to 15 mM acetate-sodium acetate buffer, wherein the pH of the pharmaceutical composition is 4.7 to 5.3;

[0103] In some embodiments, the pharmaceutical composition as described in any of the preceding embodiments comprises the following components:

[0104] (a) The anti-PSMA antibody-drug conjugate at concentrations of 18 mg / mL to 22 mg / mL,

[0105] (b) Polysorbate 80 at concentrations ranging from 0.05 mg / mL to 0.4 mg / mL,

[0106] (c) Sucrose at concentrations of 60 mg / mL to 90 mg / mL,

[0107] (d) Methionine at concentrations ranging from 1 mg / mL to 8 mg / mL, and

[0108] (e) a 5 mM to 15 mM acetate-sodium acetate buffer, wherein the pH of the pharmaceutical composition is 4.7 to 5.3;

[0109] In some embodiments, the pharmaceutical composition as described in any of the preceding embodiments comprises the following components:

[0110] (a) The anti-PSMA antibody-drug conjugate at a concentration of approximately 20 mg / mL,

[0111] (b) Approximately 0.2 mg / mL of polysorbate 80,

[0112] (c) Approximately 75 mg / mL of sucrose,

[0113] (d) Approximately 2 mg / mL of methionine, and

[0114] (e) about 10 mM of an acetate-sodium acetate buffer, wherein the pH of the pharmaceutical composition is about 5.0.

[0115] In some embodiments, the pharmaceutical composition as described in any of the preceding embodiments comprises the following components:

[0116] (a) 20 mg / mL of the aforementioned anti-PSMA antibody-drug conjugate,

[0117] (b) 0.2 mg / mL of polysorbate 80,

[0118] (c) 75 mg / mL sucrose,

[0119] (d) 2 mg / mL of methionine, and

[0120] (e) 10 mM acetate-sodium acetate buffer, wherein the pH of the pharmaceutical composition is about 5.0.

[0121] In some embodiments, the pharmaceutical composition as described in any of the preceding embodiments comprises the following components:

[0122] (a) The anti-PSMA antibody-drug conjugate at concentrations of 15 mg / mL to 25 mg / mL,

[0123] (b) Polysorbate 80 at concentrations ranging from 0.05 mg / mL to 0.4 mg / mL,

[0124] (c) Sucrose at concentrations of 60 mg / mL to 90 mg / mL,

[0125] (d) Methionine at concentrations ranging from 1 mg / mL to 8 mg / mL, and

[0126] (e) a 5 mM to 15 mM histidine-histidine hydrochloride buffer, wherein the pH of the pharmaceutical composition is 5.0 to 6.0;

[0127] In some embodiments, the pharmaceutical composition as described in any of the preceding embodiments comprises the following components:

[0128] (a) The anti-PSMA antibody-drug conjugate at a concentration of approximately 20 mg / mL,

[0129] (b) Approximately 0.2 mg / mL of polysorbate 80,

[0130] (c) Approximately 75 mg / mL of sucrose,

[0131] (d) Approximately 2 mg / mL of methionine, and

[0132] (e) about 10 mM histidine-histidine hydrochloride buffer, wherein the pH of the pharmaceutical composition is about 5.5 (5.2-5.8).

[0133] In some embodiments, the pharmaceutical composition as described in any of the preceding embodiments comprises the following components:

[0134] (a) 20 mg / mL of the aforementioned anti-PSMA antibody-drug conjugate,

[0135] (b) 0.2 mg / mL of polysorbate 80,

[0136] (c) 75 mg / mL sucrose,

[0137] (d) 2 mg / mL of methionine, and

[0138] (e) 10 mM histidine-histidine hydrochloride buffer, wherein the pH of the pharmaceutical composition is 5.5.

[0139] In some embodiments, the pharmaceutical composition as described in any of the preceding embodiments comprises the following components:

[0140] (a) The anti-PSMA antibody-drug conjugate at concentrations from 1 mg / mL to 50 mg / mL,

[0141] (b) Surfactants ranging from 0.01 mg / mL to 2 mg / mL,

[0142] (c) Sugars ranging from 10 mg / mL to 120 mg / mL,

[0143] (d) Disodium edetate at concentrations ranging from 0.01 mg / mL to 1 mg / mL, and

[0144] (e) A buffer of 1 mM to 50 mM, wherein the pH of the pharmaceutical composition is 4.6 to 7.4.

[0145] In some embodiments, the pharmaceutical composition as described in any of the preceding embodiments comprises the following components:

[0146] (a) The anti-PSMA antibody-drug conjugate at concentrations of 5 mg / mL to 40 mg / mL,

[0147] (b) Polysorbate 80 at concentrations ranging from 0.02 mg / mL to 1 mg / mL,

[0148] (c) Sucrose at concentrations of 50 mg / mL to 100 mg / mL,

[0149] (d) Disodium edetate at concentrations ranging from 0.01 mg / mL to 0.1 mg / mL, and

[0150] (e) 5 mM to 30 mM of citrate-disodium hydrogen phosphate buffer, histidine-histidine hydrochloride buffer and acetate-sodium acetate buffer, wherein the pH of the pharmaceutical composition is 4.7 to 5.8.

[0151] In some embodiments, the pharmaceutical composition as described in any of the preceding embodiments comprises the following components:

[0152] (a) The anti-PSMA antibody-drug conjugate at concentrations of 5 mg / mL to 40 mg / mL,

[0153] (b) Polysorbate 80 at concentrations ranging from 0.02 mg / mL to 1 mg / mL,

[0154] (c) Sucrose at concentrations of 50 mg / mL to 100 mg / mL,

[0155] (d) Disodium edetate at concentrations ranging from 0.01 mg / mL to 0.1 mg / mL, and

[0156] (e) 5 mM to 30 mM of citrate-disodium hydrogen phosphate buffer, histidine-histidine hydrochloride buffer and acetate-sodium acetate buffer, wherein the pH of the pharmaceutical composition is 5.0 to 5.8.

[0157] In some embodiments, the pharmaceutical composition as described in any of the preceding embodiments comprises the following components:

[0158] (a) The anti-PSMA antibody-drug conjugate at concentrations of 15 mg / mL to 25 mg / mL,

[0159] (b) Polysorbate 80 at concentrations ranging from 0.05 mg / mL to 0.4 mg / mL,

[0160] (c) Sucrose at concentrations of 60 mg / mL to 90 mg / mL,

[0161] (d) Sodium edetate at concentrations ranging from 0.02 mg / mL to 0.08 mg / mL, and

[0162] (e) a 5 mM to 15 mM acetate-sodium acetate buffer, wherein the pH of the pharmaceutical composition is 4.7 to 5.3;

[0163] In some embodiments, the pharmaceutical composition as described in any of the preceding embodiments comprises the following components:

[0164] (a) The anti-PSMA antibody-drug conjugate at a concentration of approximately 20 mg / mL,

[0165] (b) Approximately 0.2 mg / mL of polysorbate 80,

[0166] (c) Approximately 75 mg / mL of sucrose,

[0167] (d) Approximately 0.05 mg / mL of disodium edetate, and

[0168] (e) about 10 mM of an acetate-sodium acetate buffer, wherein the pH of the pharmaceutical composition is about 5.0.

[0169] In some embodiments, the pharmaceutical composition as described in any of the preceding embodiments comprises the following components:

[0170] (a) 20 mg / mL of the aforementioned anti-PSMA antibody-drug conjugate,

[0171] (b) 0.2 mg / mL of polysorbate 80,

[0172] (c) 75 mg / mL sucrose,

[0173] (d) 0.05 mg / mL of disodium edetate, and

[0174] (e) 10 mM acetate-sodium acetate buffer, wherein the pH of the pharmaceutical composition is about 5.0.

[0175] In some embodiments, the pharmaceutical composition described in any of the preceding claims is a solution obtained by reconstituted a lyophilized formulation with water for injection, said lyophilized formulation being obtained by freeze-drying the pharmaceutical composition described in any of the preceding claims.

[0176] In some embodiments, the pharmaceutical composition described in any of the preceding embodiments is an intravenous injection formulation, a subcutaneous injection formulation, an intraperitoneal injection formulation, or an intramuscular injection formulation.

[0177] In some embodiments, the pharmaceutical composition described in any of the preceding embodiments is an intravenous injection formulation.

[0178] This disclosure also provides a lyophilized formulation, characterized in that the lyophilized formulation, upon reconstitution, can form a pharmaceutical composition as described in any of the preceding claims.

[0179] This disclosure also provides a lyophilized formulation, which is a lyophilized form of the pharmaceutical composition as described in any of the preceding claims.

[0180] This disclosure also provides a lyophilized formulation obtained by freeze-drying a pharmaceutical composition as described in any of the preceding claims.

[0181] This disclosure also provides a method for preparing a lyophilized formulation, comprising the step of lyophilizing a pharmaceutical composition as described in any of the preceding claims. In some embodiments, the lyophilization as described in any of the preceding claims sequentially comprises the steps of a first pre-freezing, a second pre-freezing, a first drying, and a second drying.

[0182] In some implementations, freeze drying, as described in any of the preceding embodiments, sequentially includes the following steps:

[0183] 1) Pre-freezing is performed at -2℃ to -8℃ for 0.5 to 2 hours;

[0184] 2) The second pre-freezing involves pre-freezing at -40℃ to -45℃ for 3 to 4 hours;

[0185] 3) The first drying process involves drying at -15℃ to -20℃ and 0.10mbar to 0.15mbar for 40 to 45 hours.

[0186] 4) Secondary drying is carried out at 20℃~25℃ and 0.01mbar~0.02mbar for 8 to 10 hours.

[0187] In some implementations, freeze drying, as described in any of the preceding embodiments, sequentially includes the following steps:

[0188] 1) Pre-freezing is done at -5℃ for 1 hour;

[0189] 2) The second pre-freezing involves pre-freezing at -45℃ for 3 hours;

[0190] 3) The first drying step is to dry at -20℃ and 0.10mbar for 40 hours;

[0191] 4) The secondary drying process involves drying at 25℃ and 0.02mbar for 10 hours;

[0192] The above times are the holding times under the conditions described.

[0193] This disclosure also provides a reconstituted solution, characterized in that the reconstituted solution is prepared by reconstituted a lyophilized formulation as described in any of the preceding claims.

[0194] This disclosure also provides a reconstituted solution, which is a reconstituted form of the lyophilized formulation as described in any of the preceding claims.

[0195] In some embodiments, the reconstituted solution as described in any of the preceding embodiments has the same components and contents as the aforementioned pharmaceutical composition.

[0196] In some embodiments, the reconstituted solution as described in any of the preceding embodiments comprises the following components:

[0197] (a) The anti-PSMA antibody-drug conjugate at concentrations from 1 mg / mL to 50 mg / mL,

[0198] (b) Surfactants ranging from 0.01 mg / mL to 2 mg / mL,

[0199] (c) Sugars ranging from 10 mg / mL to 120 mg / mL,

[0200] (d) Excipients ranging from 0.01 mg / mL to 5 mg / mL, and

[0201] (e) A buffer of 1 mM to 50 mM, wherein the pH of the pharmaceutical composition is 4.6 to 7.4.

[0202] In some embodiments, the reconstituted solution as described in any of the preceding embodiments comprises the following components:

[0203] (a) The anti-PSMA antibody-drug conjugate at concentrations of 5 mg / mL to 40 mg / mL,

[0204] (b) Polysorbate 80 at concentrations ranging from 0.02 mg / mL to 1 mg / mL,

[0205] (c) Sucrose at concentrations of 50 mg / mL to 100 mg / mL,

[0206] (d) Methionine at concentrations ranging from 0.1 mg / mL to 4 mg / mL, and

[0207] (e) 5 mM to 30 mM of citrate-disodium hydrogen phosphate buffer, histidine-histidine hydrochloride buffer and acetate-sodium acetate buffer, wherein the pH of the pharmaceutical composition is 4.7 to 5.8.

[0208] In some embodiments, the reconstituted solution as described in any of the preceding embodiments comprises the following components:

[0209] (a) The anti-PSMA antibody-drug conjugate at concentrations of 15 mg / mL to 25 mg / mL,

[0210] (b) Polysorbate 80 at concentrations ranging from 0.05 mg / mL to 0.4 mg / mL,

[0211] (c) Sucrose at concentrations of 60 mg / mL to 90 mg / mL,

[0212] (d) Methionine at concentrations ranging from 1 mg / mL to 3 mg / mL, and

[0213] (e) 5 mM to 15 mM acetate-sodium acetate buffer, wherein the pH of the pharmaceutical composition is 4.7 to 5.3.

[0214] In some embodiments, the reconstituted solution as described in any of the preceding embodiments comprises the following components:

[0215] (a) The anti-PSMA antibody-drug conjugate at a concentration of approximately 20 mg / mL,

[0216] (b) Approximately 0.2 mg / mL of polysorbate 80,

[0217] (c) Approximately 75 mg / mL of sucrose,

[0218] (d) Approximately 2 mg / mL of methionine, and

[0219] (e) about 10 mM of an acetate-sodium acetate buffer, wherein the pH of the pharmaceutical composition is about 5.0.

[0220] In some embodiments, the reconstituted solution as described in any of the preceding embodiments comprises the following components:

[0221] (a) The anti-PSMA antibody-drug conjugate at a concentration of approximately 20 mg / mL,

[0222] (b) Approximately 0.2 mg / mL of polysorbate 80,

[0223] (c) Approximately 75 mg / mL of sucrose,

[0224] (d) Approximately 2 mg / mL of methionine, and

[0225] (e) about 10 mM of an acetate-sodium acetate buffer, wherein the pH of the pharmaceutical composition is about 5.0.

[0226] In some embodiments, the reconstituted solution as described in any of the preceding embodiments comprises the following components:

[0227] (a) 20 mg / mL of the aforementioned anti-PSMA antibody-drug conjugate,

[0228] (b) 0.2 mg / mL of polysorbate 80,

[0229] (c) 75 mg / mL sucrose,

[0230] (d) 2 mg / mL of methionine, and

[0231] (e) 10 mM acetate-sodium acetate buffer, wherein the pH of the pharmaceutical composition is 5.0.

[0232] In some embodiments, the pharmaceutical composition or reconstituted solution as described in any of the preceding embodiments is an intravenous injection formulation, a subcutaneous injection formulation, an intraperitoneal injection formulation, or an intramuscular injection formulation. In some embodiments, the pharmaceutical composition or reconstituted solution as described in any of the preceding embodiments is an intravenous injection formulation.

[0233] In some embodiments, the pharmaceutical composition or reconstituted solution as described in any of the preceding embodiments is suitable for intravenous, subcutaneous, intraperitoneal, or intramuscular injection. In some embodiments, the pharmaceutical composition or reconstituted solution as described in any of the preceding embodiments is suitable for intravenous injection.

[0234] This disclosure also provides a medicine box comprising at least one container, each container independently containing a pharmaceutical composition as described in any of the preceding claims, a lyophilized formulation as described in any of the preceding claims, or a reconstituted solution as described in any of the preceding claims.

[0235] In some embodiments, the pharmaceutical composition, reconstituted solution, or lyophilized formulation described in any of the preceding embodiments is used to prepare a drug for intravenous, subcutaneous, intraperitoneal, or intramuscular injection. In some embodiments, the pharmaceutical composition, reconstituted solution, or lyophilized formulation described in any of the preceding embodiments is used to prepare a drug for intravenous injection.

[0236] On the other hand, this disclosure relates to the use of the pharmaceutical compositions, lyophilized formulations, or reconstituted solutions as described in any of the preceding claims in the preparation of medicaments for the prevention or treatment of diseases or conditions.

[0237] On the other hand, this disclosure relates to a method for preventing or treating a disease or condition, the method comprising administering to a subject a pharmaceutical composition as described in any of the preceding claims, a lyophilized formulation as described in any of the preceding claims, or a reconstituted solution as described in any of the preceding claims.

[0238] On the other hand, this disclosure relates to a pharmaceutical composition as described in any of the preceding claims, a lyophilized formulation as described in any of the preceding claims, or a reconstituted solution as described in any of the preceding claims, used as a medicament. In some embodiments, the medicament is used to prevent or treat a disease or condition.

[0239] In some implementations, the disease or condition is a PSMA-mediated disease or condition.

[0240] In some implementations, the disease or condition is cancer associated with high PSMA expression, medium PSMA expression, or low PSMA expression.

[0241] In some implementations, the disease or condition is a tumor or cancer.

[0242] In some implementations, the disease or condition is selected from prostate cancer, squamous cell carcinoma of the head and neck, head and neck cancer, brain cancer, glioma, glioblastoma multiforme, neuroblastoma, neuroendocrine tumor, pharyngeal cancer, nasopharyngeal cancer, esophageal cancer, thyroid cancer, malignant pleural mesothelioma, lung cancer, breast cancer, liver cancer, hepatobiliary cancer, pancreatic cancer, gastric cancer, colorectal cancer, kidney cancer, ovarian cancer, endometrial cancer, cervical cancer, bladder cancer, testicular cancer, melanoma, leukemia, lymphoma, chondrosarcoma, multiple myeloma, myelodysplastic syndrome, Kuckenberg tumor, squamous cell carcinoma, Ewing's sarcoma, urothelial carcinoma, and Merkel cell carcinoma. Attached Figure Description

[0243] Figure 1A shows the binding activity of the antibody to LNCaP cells.

[0244] Figure 1B shows the binding activity of the antibody to 22RV1 cells.

[0245] Figure 1C shows the binding activity of the antibody to PC-3 cells.

[0246] Figure 2 shows the pharmacokinetic (PK) of ADC molecules in SD rats.

[0247] Figure 3 shows the PK of ADC molecules in cynomolgus monkeys. Detailed Implementation

[0248] the term

[0249] To facilitate understanding of this disclosure, certain technical and scientific terms are specifically defined below. Unless otherwise expressly defined herein, all other technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this disclosure pertains.

[0250] The singular forms “a,” “an,” and “the” used in this disclosure include plural references unless the context clearly indicates otherwise.

[0251] Unless the context clearly requires otherwise, the words “comprising,” “having,” “including,” etc., in the patent specification and claims should be understood as “including but not limited to,” rather than as exclusive or exhaustive.

[0252] "Optional" or "optionally" means that the event or circumstances described below may, but do not have to, occur, including the circumstances in which the event or circumstances may or may not occur.

[0253] Those skilled in the art will understand that when used as a reference range, cutoff value, or specific value, "about" can mean within one or more standard deviations. Alternatively, "about" can mean a range with a difference of up to 20% (i.e., ±20%). Since many of the values ​​used herein were determined experimentally, those skilled in the art will understand that such determinations can vary between different experiments and are generally true across experiments. Due to this inherent variability, the values ​​used herein should not be unduly restricted. Therefore, the term "about" is used to cover variations of ±20%, ±10%, ±5%, ±1%, ±0.5%, or ±0.1% or less from a specified value.

[0254] Although this disclosure provides content ranges or content values, those skilled in the art will understand that the content ranges or content values ​​cover the acceptable range of error for the specific values ​​measured.

[0255] The three-letter and single-letter codes for amino acids used in this disclosure are as described in J. Biol. Chem., 243, p3558 (1968).

[0256] The term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimics that function in a manner similar to naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those that are subsequently modified, such as hydroxyproline, γ-carboxyglutamic acid, and O-phosphoserine. Amino acid analogs are compounds that have the same basic chemical structure as naturally occurring amino acids (i.e., the α-carbon bound to hydrogen, carboxyl, amino, and R groups), such as homoserine, ortholeucine, methionine sulfoxide, and methionine methylsulfonium. These analogs have modified R groups (e.g., ortholeucine) or modified peptide backbones but retain the same basic chemical structure as naturally occurring amino acids. Amino acid mimics are chemical compounds that have a structure different from the general chemical structure of amino acids but function in a manner similar to naturally occurring amino acids.

[0257] The term "antibody" is used in the broadest sense and encompasses a wide range of antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies; monospecific antibodies, multispecific antibodies (e.g., bispecific antibodies); full-length antibodies and antigen-binding fragments (or antigen-binding portions), as long as they exhibit the desired antigen-binding activity. "Natural antibody" refers to a naturally occurring immunoglobulin molecule. For example, a natural IgG antibody is a heterotetraglycoprotein of approximately 150,000 Daltons, composed of two identical light chains and two identical heavy chains bound by disulfide bonds. From the N to C terminus, each heavy chain has a variable region (VH), also called a variable heavy domain or heavy chain variable region, followed by a heavy chain constant region. The IgG heavy chain constant region (CH) typically contains three constant domains (CH1, CH2, and CH3); similarly, from the N to C terminus, each light chain has a variable region (VL), also called a variable light domain or light chain variable domain, followed by a constant light domain (light chain constant region, CL).

[0258] The term "variable region" or "variable domain" refers to the domain in the antibody heavy or light chain involved in antibody-antigen binding. In this paper, the antibody heavy chain variable region (VH) and light chain variable region (VL) each contain four conserved frame regions (FRs) and three complementarity-determining regions (CDRs). The term "complementarity-determining region" or "CDR" refers to the region within the variable domain that primarily facilitates antigen binding; "frame" or "FR" refers to the variable domain residues other than the CDR residues. The VH contains three CDR regions: HCDR1, HCDR2, and HCDR3; the VL contains three CDR regions: LCDR1, LCDR2, and LCDR3. Each VH and VL consists of three CDRs and four FRs arranged in the following order from the amino terminus (also called the N-terminus) to the carboxyl terminus (also called the C-terminus): FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. A single VH or VL may be sufficient to confer antigen-binding specificity.

[0259] The amino acid sequence boundaries of CDRs can be determined using various well-known schemes, such as the "Kabat" numbering rule, the "Chothia" numbering rule, the "ABM" numbering rule, the "contact" numbering rule (and the ImMunoGenTics (IMGT) numbering rule, etc.); the correspondence between various numbering systems is well known to those skilled in the art, and is exemplarily shown in Table 1 below.

[0260] Table 1. Relationship between CDR numbering systems

[0261] Unless otherwise stated, the variable regions and CDR sequences in this disclosure embodiment are subject to the "Kabat" numbering rule.

[0262] The term "monoclonal antibody" refers to a group of substantially homogeneous antibodies, meaning that the antibody molecules contained in this group have the same amino acid sequence, except for the possible small number of naturally occurring mutations. In contrast, polyclonal antibody formulations typically contain multiple different antibodies with different amino acid sequences in their variable structural domains, and they generally specifically target different epitopes. "Monoclonal" indicates the characteristic of an antibody obtained from a substantially homogeneous group of antibodies and should not be construed as requiring the antibody to be produced by any particular method. In some embodiments, the antibodies provided in this disclosure are monoclonal antibodies.

[0263] The antibodies disclosed herein may be derived from animals (such as antibodies from mice, birds, rabbits, camels, monkeys, etc.), chimeric antibodies, or humanized antibodies.

[0264] The term "chimeric" antibody refers to an antibody in which a portion of the heavy and / or light chain is derived from a specific source or species, while the remaining portion of the heavy and / or light chain is derived from another different source or species.

[0265] The term "humanized" antibody refers to an antibody that retains the reactivity of a non-human antibody while exhibiting lower immunogenicity in humans. For example, this can be achieved by retaining the non-human CDR region and replacing the rest of the antibody with its human counterpart (i.e., the frame region portion of the constant region and the variable region).

[0266] The term "affinity" refers to the overall strength of the non-covalent interaction between a single binding site of a molecule (e.g., an antibody) and its binding ligand (e.g., an antigen). Unless otherwise specified, as used herein, binding "affinity" refers to internal binding affinity, which reflects a 1:1 interaction between members of a binding pair (e.g., antibody and antigen). The affinity of molecule X for its ligand Y can typically be represented by the dissociation constant (KD). Affinity can be measured using conventional methods known in the art.

[0267] The terms "anti-PSMA antibody" and "PSMA-binding antibody" refer to antibodies capable of binding to PSMA or its epitopes with sufficient affinity. In one embodiment, the anti-PSMA antibody binds to unrelated proteins to a degree less than at least about 10% of the antibody's binding to PSMA, and this binding can be measured by BIACORE surface plasmon resonance assay.

[0268] The term "antigen" refers to a molecule or molecular moiety that can be bound by a selective binder of an antigen-binding protein (such as an antibody). An antigen may have one or more epitopes that can interact with different antigen-binding proteins (such as antibodies).

[0269] The term "epitope" refers to a region on an antigen that can specifically bind to an antibody or its antigen-binding fragment. Epitopes can be formed from consecutive amino acids (linear epitopes) or contain non-consecutive amino acids (conformal epitopes), for example, due to the folding of the antigen (i.e., tertiary folding of the antigen as a protein), which allows non-consecutive amino acids to be spatially close. The difference between conformational and linear epitopes is that antibody binding to a conformational epitope is lost in the presence of a denaturing solvent. An epitope contains at least 3, at least 4, at least 5, at least 6, at least 7, or 8-10 amino acids in a unique spatial conformation. Screening for antibodies that bind to a specific epitope (i.e., those that bind the same epitope) can be performed using methods routine in the art, such as, but not limited to, alanine scanning, Western blotting, peptide cleavage analysis, epitope excision, epitope extraction, chemical modification of the antigen, and cross-blocking.

[0270] The terms "capable of specific binding," "specific binding," or "binding" refer to the ability of an antibody to bind to a specific antigen or epitope with a higher affinity than other antigens or epitopes. Typically, antibodies bind at an affinity of approximately 1 × 10⁻⁶. -7 An equilibrium dissociation constant (KD) of M or less binds to an antigen or epitope. In some embodiments, the KD of antibody binding to an antigen is 10% or less (e.g., 1%) of the KD of the antibody binding to a nonspecific antigen (e.g., BSA, casein). KD can be measured using known methods, such as by FACS or surface plasmon resonance assays. However, antibodies that specifically bind to an antigen or an epitope within an antigen may be cross-reactive to other related antigens, for example, to corresponding antigens from other species (homologous), such as humans or monkeys, such as the cynomolgus (cyno), the chimpanzee (chimp), or the common marmoset (marmoset).

[0271] Antibody drug conjugates (ADCs) are conjugates obtained by linking an antibody (or its antigen-binding fragment) directly or through a linker to a drug.

[0272] A "drug" (abbreviated as D) is any substance that has biological or detectable activity (e.g., therapeutic agents, detectable markers, binders, etc.) and its prodrugs, which are metabolized in the body to become active agents. Examples of therapeutic agents include cytotoxic agents, chemotherapeutic agents, cell growth inhibitors, and immunomodulators. Chemotherapeutic agents are chemical compounds that can be used to treat cancer. Representative therapeutic agents include cytotoxins, cytotoxic agents, and cell growth inhibitors.

[0273] Cytotoxicity refers to the loss, elimination, and / or killing of target cells. Cytotoxic agents are drugs that have cytotoxic and / or cell growth-inhibiting effects on cells. Cell growth inhibition refers to the inhibition of cell proliferation. Cell growth inhibitors are drugs that have a cell growth-inhibiting effect on cells, thereby inhibiting the growth and / or expansion of specific subgroups of cells.

[0274] Additional representative therapeutic agents include radioisotopes, chemotherapeutic agents, immunomodulators, anti-angiogenic agents, antiproliferative agents, apoptosis-promoting agents, and cell-lysing enzymes (e.g., RNase). These drug descriptive terms are not mutually exclusive, and therefore, one or more of the aforementioned terms may be used to describe a therapeutic agent. For example, the selected radioisotope may also be a cytotoxic agent. Therapeutic agents can be prepared as pharmaceutically acceptable salts, acids, or derivatives of any of the above. Generally, conjugates containing a radioisotope as a drug are called radioimmunoconjugates, and those containing a chemotherapeutic agent as a drug are called chemoimmunoconjugates.

[0275] The terms "connector unit" and "connector" refer to a chemical structural fragment or bond that is linked to an antibody at one end and a drug at the other. Connectors can also be attached to other connectors before being linked to an antibody or drug. Connector attachment to antibodies can be accomplished in various ways, such as via surface lysine residues, reductive coupling to oxidized carbohydrates, release of cysteine ​​residues via reducing interchain disulfide bonds, modification of reactive cysteine ​​residues at specific sites, and tags containing acyl donor glutamine, or modification of peptides to make them reactive endogenous glutamine in the presence of transglutaminase and amines. Various ADC linker systems are known in the art, including hydrazone-, disulfide-, and peptide-based links.

[0276] The connector may comprise one or more connector elements. Exemplary connector elements include 6-maleiminohexanoyl (“MC”), maleiminopropionyl (“MP”), valine-citrulline (“val-cit” or “vc”), alanine-phenylalanine (“ala-phe”), p-aminobenzyloxycarbonyl (“PAB”), N-succinimino-4-(2-pyridylthio)valerate (“SPP”), N-succinimino-4-(N-maleiminomethyl)cyclohexane-1-carboxylate (“SMCC”, also referred to herein as “MCC”), N-succinimino-4-iodo-acetyl)aminobenzoate (“SIAB”), and GGFG (SEQ ID NO: 25).

[0277] The linker can be selected from the following elements or combinations thereof: extensions, spacers, and amino acid units. Linkers can be synthesized by methods known in the art, such as those described in US20050238649A1. The linker can be a “cleavable linker” that facilitates drug release into cells. For example, acid-labile linkers (e.g., hydrazones), protease-sensitive linkers (e.g., peptidase-sensitive linkers), photostable linkers, dimethyl linkers, or disulfide-containing linkers can be used (US Patent No. 5,208,020).

[0278] "Drug loading," also known as drug-to-antibody ratio (DAR), refers to the average number of drugs conjugated to each antibody in an ADC. It can range from about 1 to about 10 drugs per antibody, and in some embodiments, from about 1 to about 8 drugs per antibody, preferably from the ranges of 2-8, 2-7, 2-6, 2-5, 2-4, 1-3, 3-4, 3-5, 5-6, 5-7, 5-8, and 6-8. The general formula of the ADC disclosed herein includes a set of antibody-drug conjugates within the aforementioned range. In embodiments disclosed herein, drug loading may be expressed as n, which can be a decimal or an integer. Drug loading can be determined using conventional methods such as UV / visible spectroscopy, mass spectrometry, ELISA assays, HIC, and RP-HPLC.

[0279] "Surfactant" refers to a surface-active agent, preferably a nonionic surfactant. Surfactants can reduce protein aggregation and / or particle formation in formulations. The amount of surfactant added is such that it reduces protein aggregation and minimizes particle formation in the formulation.

[0280] Poloxamer is an α-hydro-ω-hydroxy poly(ethylene oxide)a-poly(propylene oxide)b-poly(ethylene oxide)a block copolymer. It is formed by reacting propylene oxide and propylene glycol to form polypropylene glycol, followed by the addition of ethylene oxide to form the block copolymer. Here, 'a' represents the number of ethylene oxide units, and 'b' represents the number of propylene oxide units. Examples of poloxamer include, but are not limited to, poloxamer 188 (P188 or PF68). Specifically, in poloxamer 188, the copolymer contains 75–85 ethylene oxide units (a), 25–30 propylene oxide units (b), an ethylene oxide (EO) content of 79.9%–83.7%, and an average molecular weight of 7680–9510.

[0281] "Viscosity modifier" is a conventional pharmaceutical excipient added to adjust the viscosity of a formulation. Viscosity modifiers can be inorganic salts and amino acid salts. Preferably, the inorganic salt is selected from sodium chloride, calcium chloride, and magnesium chloride. More preferably, the amino acid salt is selected from arginine hydrochloride, histidine hydrochloride, lysine hydrochloride, histidine acetate, etc.

[0282] "Buffer" refers to a buffering agent that tolerates pH changes through the action of its acid-base conjugate components. Examples of buffers that maintain pH within an appropriate range include acetate, succinate, gluconate, histidine, oxalate, lactate, phosphate, citrate (also known as tartrate), tartrate, fumarate, glycylglycine, and other organic acid buffers.

[0283] "Histidine buffer" is a buffer containing histidine. Examples of histidine buffers include histidine-histidine acetate, histidine-histidine hydrochloride, histidine-histidine phosphate, and histidine-histidine sulfate buffers, with histidine-histidine acetate buffers being preferred. Histidine-histidine acetate buffers can be prepared by reacting histidine with acetic acid, or by reacting histidine with histidine acetate.

[0284] "Succinate buffer" is a buffer containing succinate ions. Examples of succinate buffers include sodium succinate, potassium succinate, and calcium succinate. A preferred succinate buffer is sodium succinate. Exemplarily, the sodium succinate can be prepared from succinic acid and sodium hydroxide, or from succinic acid and sodium succinate.

[0285] Phosphate buffers are buffers that contain phosphate ions. Examples of phosphate buffers include disodium hydrogen phosphate-sodium dihydrogen phosphate, disodium hydrogen phosphate-potassium dihydrogen phosphate, and disodium hydrogen phosphate-citric acid.

[0286] "Acetate buffers" are buffers that contain acetate ions. Examples of acetate buffers include sodium acetate, potassium acetate, calcium acetate, and magnesium acetate.

[0287] "Citrate buffers" are buffers that contain citrate ions. Examples of citrate buffers include sodium citrate, potassium citrate, calcium citrate, magnesium citrate, etc.

[0288] "Citrate buffers" are buffers that contain citrate ions. Examples of citrate buffers include sodium citrate, potassium citrate, calcium citrate, disodium citrate, magnesium citrate, etc.

[0289] "Displacement" refers to the replacement of the solvent system in which antibody proteins are dissolved. For example, a buffer system containing antibody proteins may be physically replaced by a high-salt or hypertonic solvent system containing antibody proteins, thereby ensuring the presence of antibody proteins within the stable formulation. Such physical operations include, but are not limited to, ultrafiltration, dialysis, or centrifugation.

[0290] "Pharmaceutical composition" means a mixture containing one or more antibodies described herein or their physiologically / pharmacologically acceptable salts or prodrugs, along with other chemical components, such as physiologically / pharmacologically acceptable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration to a living organism, thereby promoting the absorption of the active ingredient and the exertment of its biological activity.

[0291] "Lyophilized formulation" refers to a pharmaceutical composition or formulation obtained by vacuum freeze-drying a liquid or solution preparation. Typically, freeze-drying includes pre-freezing, primary drying, and secondary drying. Pre-freezing aims to freeze the product to obtain a crystalline solid; in some embodiments, the pre-freezing temperature is set to -45°C, and the pre-freezing rate is set to 1°C / min. Primary drying, also known as main drying, is the main stage of sample freeze-drying and aims to remove ice from the product while maintaining its shape and minimizing damage. Improper selection of the temperature and vacuum level during primary drying can lead to product collapse; higher temperatures and vacuum levels increase freeze-drying efficiency but also increase the risk of product collapse. In some embodiments, the primary drying temperature can be a temperature conventional in the art, such as -30°C to 0°C. Secondary drying, also known as desorption drying, is the main step of removing bound water from the product by applying an ultimate vacuum (0.01 mbar) and increasing the temperature (20°C to 40°C). Because most biological products are temperature-sensitive, the secondary drying temperature is often chosen at the lower end of the temperature range, such as 25°C. The freeze-drying time depends on the freezer, the dosage of the freeze-dried formulation, and the container of the freeze-dried drug. Such adjustments are well known to those skilled in the art.

[0292] Unless otherwise specified, the solvent in the solution form of the pharmaceutical compositions described in this disclosure is water.

[0293] In this disclosure, "pharmaceutical composition" and "formulation" are not mutually exclusive.

[0294] The pharmaceutical compositions disclosed herein achieve a stable effect: the antibodies contained therein substantially retain their physical and / or chemical stability and / or biological activity after storage. Preferably, the pharmaceutical compositions substantially retain their physical and chemical stability and their biological activity after storage. The storage period is generally selected based on the intended shelf life of the pharmaceutical composition. Currently, there are various analytical techniques available for measuring protein stability, which can measure stability after storage at a selected temperature for a selected period of time.

[0295] Stable formulations include those in which no significant changes are observed when stored at refrigerated temperatures (2°C–8°C) for at least 1 month, at least 3 months, at least 6 months, preferably 1 year, and even more preferably up to 2 years. Stable liquid formulations also include those that exhibit the desired characteristics after storage at temperatures including 25°C for periods of 1 month, 3 months, or 6 months. Furthermore, stable liquid formulations also include those that exhibit the desired characteristics after storage at 40°C for periods of 4 weeks, 1 month, 3 months, or 6 months. Typical examples of stability include antibody aggregation or degradation typically not exceeding about 10%, preferably not exceeding about 5%, as determined by SEC-HPLC. Visually, the formulation is a pale yellow, nearly colorless, clear liquid or a colorless, clear liquid, or clear to slightly milky white. The concentration, pH, weight, and molecular osmotic pressure of the formulation exhibit aggregation of not more than about 10%, preferably not more than about 5%.

[0296] If, after visual inspection of color and / or clarity, or by means of UV light scattering, size exclusion chromatography (SEC), and dynamic light scattering (DLS), the antibody does not show significant increase in aggregation, precipitation, and / or denaturation, then the antibody “retains its physical stability” in the pharmaceutical formulation. Changes in protein conformation can be evaluated by fluorescence spectroscopy (which determines the tertiary structure of the protein) and by FTIR spectroscopy (which determines the secondary structure of the protein).

[0297] If an antibody does not exhibit significant chemical changes, then the antibody "retains its chemical stability" in the pharmaceutical formulation. Chemical stability can be assessed by detecting and quantifying the chemically altered form of the protein. Degradation processes that frequently alter the chemical structure of a protein include hydrolysis or truncation (evaluated by methods such as size exclusion chromatography and CE-SDS), oxidation (evaluated by methods such as peptide mapping combined with mass spectrometry or MALDI / TOF / MS), deamidation (evaluated by methods such as ion exchange chromatography, capillary isoelectric focusing, peptide mapping, and isofpartate measurement), and isomerization (evaluated by measuring isofpartate content, peptide mapping, etc.).

[0298] If the antibody's biological activity at a given time is within a predetermined range of the biological activity exhibited when the pharmaceutical formulation is prepared, then the antibody "retains its biological activity" in the pharmaceutical formulation.

[0299] "Administration," "giving," and "treatment," when applied to animals, humans, experimental subjects, cells, tissues, organs, or biological fluids, refer to the contact of an exogenous drug, therapeutic agent, diagnostic agent, or composition with the animal, human, subject, cell, tissue, organ, or biological fluid. "Administration," "giving," and "treatment" can refer to, for example, therapeutic, pharmacokinetic, diagnostic, research, and experimental methods. Cellular treatment includes contact between a reagent and cells, as well as contact between a reagent and a fluid, wherein the fluid is in contact with the cells. "Administration," "giving," and "treatment" also mean, by means of a reagent, diagnostic agent, conjugate composition, or by means of another cell in vitro and ex vivo, such as cells. "Treatment," when applied to humans, veterinary, or research subjects, refers to therapeutic treatment, preventative or prophylactic measures, research, and diagnostic applications.

[0300] "Treatment" means administering an oral or topical therapeutic agent, such as a pharmaceutical composition comprising any of the substances disclosed herein, to a patient who has symptoms of one or more diseases, and the therapeutic agent is known to have a therapeutic effect on these symptoms. Typically, a therapeutic agent is administered in a treated patient or population in an amount that effectively relieves symptoms of one or more diseases, in order to induce the regression of such symptoms or inhibit their progression to any clinically measurable degree. The amount of a therapeutic agent that effectively relieves any specific disease symptom (also referred to as a "therapeuticly effective amount") can vary depending on a variety of factors, such as the patient's disease state, age, and weight, and the drug's ability to produce the desired therapeutic effect in the patient. Whether the disease symptoms have been relieved can be evaluated using any clinical test method commonly used by a physician or other healthcare professional to assess the severity or progression of the symptoms. Although the embodiments disclosed herein (e.g., treatment methods or products) may be ineffective in alleviating symptoms of each target disease, they should reduce symptoms of the target disease in a statistically significant number of patients, as determined by any statistical test known in the art, such as the Student t-test, chi-square test, U-test according to Mann and Whitney, Kruskal-Wallis test (H-test), Jonckheere-Terpstra test, and Wilcoxon test.

[0301] "Effective amount" includes an amount sufficient to improve or prevent the symptoms or condition of a medical condition. Effective amount also means an amount sufficient to allow or facilitate a diagnosis. The effective amount used on a subject may vary depending on factors such as the condition to be treated, the subject's overall health, the route and dosage of administration, and the severity of side effects. Effective amount may be the maximum dose or administration regimen that avoids significant side effects or toxicity. Subjects disclosed herein may be animal or human subjects.

[0302] The pharmaceutical compositions disclosed herein may be administered by any suitable means, including parenteral, intrapulmonary, and intranasal administration, and, if local treatment is required, intralesional administration. Parenteral infusion includes intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. Administration may be carried out via any suitable route, such as by injection, such as intravenous or subcutaneous injection. Various dosing schedules are considered herein, including, but not limited to, single or multiple administrations at multiple time points, bolus administration, and pulsatile infusion. In some embodiments, the pharmaceutical compositions disclosed herein are administered by intravenous injection.

[0303] The pharmaceutical compositions disclosed herein will be formulated, administered, and applied in accordance with good medical practice. Factors considered in this context include the specific condition being treated, the specific mammal being treated, the individual patient's clinical condition, the cause of the condition, the site of delivery of the agent, the method of administration, the timing of administration, and other factors known to a medical practitioner. Optionally, the pharmaceutical composition may also be formulated with one or more other agents for the prevention or treatment of said condition. The effective amount of such other agents depends on the amount of antigen-binding molecules present in the pharmaceutical composition, the type of condition or treatment, and other factors. It may be used at the same dosage and route of administration as described herein, or at about 1% to 99% of the dosage described herein, or at any dosage and any route of administration determined empirically / clinically to be appropriate.

[0304] Details of one or more embodiments of this disclosure are set forth in the foregoing description. While any methods and materials similar to or the same as those described herein may be used to implement or test this disclosure, preferred methods and materials are described below. Other features, objects, and advantages of this disclosure will be apparent from the description and claims. In the description and claims, the singular form includes plural references unless the context clearly indicates otherwise. Unless otherwise defined, all technical and scientific terms used herein have their general meaning as understood by one of ordinary skill in the art to which this disclosure pertains. All patents and publications referenced in the description are incorporated herein by reference. The following embodiments are presented to illustrate preferred embodiments of this disclosure more fully. These embodiments should not be construed in any way as limiting the scope of this disclosure, which is defined by the claims.

[0305] Example

[0306] The present disclosure is further described below with reference to embodiments, but these embodiments are not intended to limit the scope of the invention.

[0307] Experimental methods not specifying specific conditions in the examples or test cases disclosed herein are generally performed under standard conditions or as recommended by the raw material or product manufacturer. Reagents not specifying their source are commercially available, standard reagents.

[0308] I. Antibody Preparation

[0309] PCT / CN2024 / 109472 (Application date: 2024-08-02; Priority patent application numbers: CN 202310976626.5, CN 202310990738.6, CN 202311485462.2) is included hereby disclosed by reference in its entirety.

[0310] Example 1: Construction of the PSMA-overexpressing cell line CHO-K1

[0311] The pCDH-huPSMA or pCDH-cynoPSMA lentiviral expression vector plasmid and the pVSV-G, pCMV-dR8.91 lentiviral system packaging vector were transfected into 293T cells using Lipofectamine 3000 transfection reagent. The virus-containing culture supernatant was collected, filtered, and ultracentrifuged. After discarding the supernatant, the cells were resuspended in 1 mL of sterile PBS. The concentrated virus was used to infect Chinese hamster ovary cells (CHO-K1). Cells were selected with puromycin for two to three weeks, followed by FACS single-cell sorting.

[0312] PSMA expression on the surface of lentivirally infected CHO-K1 cells was detected by FACS, and monoclonal cell lines with high PSMA expression levels, huPSMA-CHO-K1 and cynoPSMA-CHO-K1, were selected. The selected monoclonal cell lines were expanded and cryopreserved for future experiments. The relevant amino acid sequences are as follows:

[0313] Human PSMA amino acid sequence (UniProtKB-Q04609-1):

[0314] Monkey PSMA amino acid sequence (UniProtKB-A0A2K5VNZ0-1):

[0315] Example 2: Preparation of mouse anti-human PSMA monoclonal antibody

[0316] 2.1 Immunization and Fusion

[0317] Using human PSMA-His protein (ACRO, PSA-H52H3) and LNCaP cells (ATCC, CRL-1740) TM Mice were cross-immunized. The dosage for protein immunization was 25 μg, and for cell immunization, it was 5 × 10⁻⁶ mg per dose. 6Each cell was immunized weekly. After three immunizations, blood samples were collected to determine serum antibody titers. Mice with high and plateauing serum antibody titers were selected for spleen cell fusion. A PEG-mediated fusion procedure was used to fuse spleen lymphocytes with myeloma Sp2 / 0 cells (ATCC, CRL-8287). TM The fused hybridoma cells were then fused at a rate of 0.5-1 × 10⁻⁶ to obtain hybridoma cells. 6 Cells were resuspended at a density of [number] cells / mL in MC semi-solid complete medium (RPMI-1640 medium containing 20% ​​FBS, 1×HAT, 1×OPI, and 2% methylcellulose) and aliquoted into 35 mm cell culture dishes. Incubation was performed at 37°C with 5% CO2 for 7–9 days. On days 7–9 post-fusion, single-cell clones were picked according to clone size and transferred to 96-well cell culture plates containing 200 μL / well of HT complete medium (RPMI-1640 medium containing 20% ​​FBS, 1×HT, and 1×OPI). Cells were incubated at 37°C with 5% CO2 for 3 days before detection.

[0318] 2.2 Hybridoma cell screening

[0319] Based on the hybridoma cell growth density, the hybridoma culture supernatant was analyzed using a combined ELISA method. Hybridoma cells that strongly bind to huPSMA-CHO-K1 and cynoPSMA-CHO-K1 cells but do not bind to wild-type CHO-K1 cells were selected for timely expansion, cryopreservation, and two to three subcloning processes until a single-cell clone was obtained.

[0320] 2.3 Hybridoma antibody sequencing

[0321] The monoclonal hybridoma cell line mAb19-1 with good in vitro activity was selected, and its antibody sequence was cloned, then humanized, recombinantly expressed, and its activity was evaluated.

[0322] The process of cloning sequences from hybridomas is as follows: Log-growing hybridoma cells are collected, and RNA is extracted using Trizol (Invitrogen, 15596-018) (following the kit instructions). Reverse transcription (PrimeScript) is then performed. TM Reverse Transcriptase (Takara, cat#2680A). The cDNA obtained from reverse transcription was amplified by PCR using a mouse Ig-Primer Set (Novagen, TB326 Rev.B 0503) and then sent to a sequencing company for sequencing. The resulting antibody sequence is as follows:

[0323] mAb19-1 Heavy Chain Variable Region:

[0324] mAb19-1 light chain variable region: Note: The underlined part is the CDR area determined according to the Kabat numbering rules.

[0325] Table 2. mAb19-1 antibody CDR sequence (according to Kabat numbering rules)

[0326] Table 3. mAb19-1 antibody CDR sequence (according to IMGT numbering rules)

[0327] Table 4. mAb19-1 antibody CDR sequence (according to Chothia numbering rules)

[0328] Example 3: Humanization of mouse-derived anti-human PSMA monoclonal antibody

[0329] By comparing the Kabat human antibody heavy and light chain variable region germline gene database and MOE software, germline genes with high homology in the heavy and light chain variable regions were selected as templates. The CDRs of the murine antibody were transplanted into the corresponding human templates to form variable region sequences in the order FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Then, partial amino acid reversion mutations were performed on the FR region, followed by recombination with constant regions (exemplarily, with the human IgG1 heavy chain constant region shown in SEQ ID NO: 15 or 16 and the human κ light chain constant region shown in SEQ ID NO: 17 or 18) to obtain humanized antibodies.

[0330] The human light chain template for mAb19-1 antibody is IGKV1-39*01, and the human heavy chain template is IGHV3-7*01.

[0331] Table 5. Humanization design of mAb19-1 antibody

[0332] The variable region sequence of the humanized antibody is as follows:

[0333] hu19-1 VH1

[0334] hu19-1 VH2

[0335] hu19-1 VL1

[0336] hu19-1 VL2 Note: Single-strikethrough areas represent CDRs, while double-strikethrough areas represent reversion mutation sites.

[0337] The constant region of the antibody is as follows:

[0338] heavy chain constant region of human IgG1

[0339] Human IgG1 heavy chain constant region variants:

[0340] κ chain constant region:

[0341] κ chain constant region variant:

[0342] An example combination of the heavy and light chain variable regions of a humanized antibody is as follows:

[0343] Table 6. Humanized antibodies against mAb19-1 Note: 19L1H1 indicates that the antibody contains the heavy chain variable region hu19-1VH1 and the light chain variable region hu19-1VL1, and the sequence of its heavy chain constant region is SEQ ID NO: 15, the sequence of its light chain constant region is SEQ ID NO: 17, and so on.

[0344] The antibodies were cloned, expressed, and purified separately. After affinity testing, the humanized antibodies with good activity were finally selected.

[0345] For example, the heavy and light chain amino acid sequences of the humanized antibody P19 are as follows, derived from SEQ ID NO: 19 and SEQ ID NO: 20 in patent PCT / CN2024 / 109472 (application date: 2024-08-02; priority patent application numbers: CN 202310976626.5, CN 202310990738.6, CN 202311485462.2):

[0346] P19(19L2H2) heavy chain:

[0347] P19(19L2H2) Light Chain: Note: The underlined portion in the sequence represents the variable region.

[0348] This disclosure uses the positive control antibody H-AB-P1 (full name AB-PG1-XG1-006, prepared according to WO2003034903A2) to prepare the antibody-drug conjugate, the sequence of which is as follows:

[0349] H-AB-P1 heavy chain:

[0350] H-AB-P1 light chain:

[0351] This disclosure uses the negative control antibody hIgG1 to prepare antibody-drug conjugates, the sequence of which is as follows:

[0352] hIgG1 heavy chain:

[0353] hIgG1 light chain:

[0354] II. Preparation of ADC

[0355] ADC drug loading analysis

[0356] The method for calculating the DAR value of the ADC disclosed herein uses RP-HPLC (reversed-phase high-performance liquid chromatography), as detailed below:

[0357] Determination methods

[0358] The naked antibody and the ADC sample (concentration 1 mg / mL) were reduced with 4 μL of 0.25 M DDT (sigma) and incubated at 37℃ for 1 hour. After the reduction, the sample was transferred to an inner tube. Detection was performed using an Agilent 1200 high-performance liquid chromatograph (HPLC). The column used was an Agilent PLRP-S1000A 8μm 4.6*250mm, with a column temperature of 80℃; the DAD detector wavelength was 280nm; the flow rate was 1 mL / min; and the injection volume was 40 μL. The positions of the light and heavy chains were then distinguished by comparing the spectra of the sample and the naked antibody. The DAR value was calculated by integrating the spectra of the detected sample.

[0359] Solution preparation

[0360] 1) 0.25M DTT solution:

[0361] Preparation example: Take 5.78 mg of DTT, add 150 μL of purified water to dissolve it completely, and prepare a 0.25 MDTT solution. Store at -20 °C.

[0362] 2) Mobile phase A (0.1% TFA aqueous solution):

[0363] Preparation example: Measure 1000mL of purified water with a graduated cylinder, add 1mL of TFA (sigma), mix thoroughly before use, and store at 2-8℃ for 14 days.

[0364] 3) Mobile phase B (0.1% TFA acetonitrile solution):

[0365] Preparation example: Measure 1000 mL of acetonitrile with a graduated cylinder, add 1 mL of LTFA, mix thoroughly before use, and store at 2-8℃ for 14 days.

[0366] Data Analysis

[0367] By comparing the spectra of the sample with those of the bare antibody, the positions of the light and heavy chains are distinguished. Then, the DAR value is calculated by integrating the spectrum of the tested sample.

[0368] The calculation formula is as follows:

[0369] Table 7

[0370] Total LC peak area = LC peak area + LC+1 peak area

[0371] Total HC peak area = HC peak area + HC+1 peak area + HC+2 peak area + HC+3 peak area

[0372] LC DAR = Σ(number of linked drugs * percentage of peak area) / total LC peak area

[0373] HC DAR = Σ(number of linked drugs * percentage of peak area) / total HC peak area

[0374] DAR = LC DAR + HC DAR.

[0375] Preparation Examples of PSMA Antibody-Drug Conjugate P19-9-A with Different DAR Values

[0376] The following examples illustrate the preparation process of the ADCs disclosed herein. In Examples 4-1, 4-2, 4-3, and 4-4, antibody P19 is coupled to drug 9-A with a linker unit via a thiol group on cysteine ​​residue to prepare antibody-drug conjugates. In Example 4-5, antibody H-AB-P1 is coupled to drug 9-A with a linker unit via a thiol group on cysteine ​​residue to prepare a control ADC: H-AB-P1-9-A (hereinafter referred to as ADC-5). In Example 4-6, control antibody hIgG1 is coupled to drug 9-A with a linker unit via a thiol group on cysteine ​​residue to prepare a control ADC: hIgG1-9-A.

[0377] By adjusting the ratio of antibody to drug, the scale of the reaction, and other conditions, antibody-drug conjugates with different DAR values ​​(n) can be obtained. The preferred DAR value is 1 to 8, more preferably 3 to 8, and most preferably 5 to 8.

[0378] (I) Preparation of antibody-drug conjugation with P19-9-A with different DAR values

[0379] Example 4-1: ADC-1

[0380] At 37°C, a prepared aqueous solution of tris(2-carboxyethyl)phosphonic acid hydrochloride (TCEP.HCl) (10 mM, 312 μL, 3120 nM) was added to the PBS buffered aqueous solution of antibody P19 (pH = 6.5, 0.05 M PBS buffered aqueous solution; 10.0 mg / mL, 14 mL, 946 nM). The solution was placed in a water bath and shaken at 37°C for 3 hours, after which the reaction was stopped. The reaction solution was then cooled to 25°C in a water bath.

[0381] Compound 9-A (see WO2020063676A1, pages 58-60, Preparation of 9-A in Example 9, 12.19 mg, 11.35 μM) was dissolved in an appropriate amount of dimethyl sulfoxide and added to the above reaction solution. The mixture was placed in a water bath shaker and shaken at 25°C for 3 hours, after which the reaction was stopped. The reaction solution was purified by desalting using a Sephadex G25 gel column (manufacturer: Cytiva; catalog number: 17003201) (elution phase: 0.05 M PBS buffered aqueous solution at pH 6.5 containing 0.001 M EDTA) to obtain the exemplary product ADC-1 of the conjugate shown in formula P19-9-A in PBS buffer (3.48 mg / mL, 35.3 mL), which was stored at 4°C.

[0382] RP-HPLC calculated average: n = 5.55.

[0383] Example 4-2: ADC-2

[0384] At 37°C, a prepared aqueous solution of tris(2-carboxyethyl)phosphonic acid hydrochloride (TCEP.HCl) (10 mM, 1.621 mL, 16.21 μM) was added to the PBS buffered aqueous solution of antibody P19 (pH = 6.5, 0.05 M PBS buffered aqueous solution; 10.0 mg / mL, 40 mL, 2.703 μM). The solution was placed in a water bath and shaken at 37°C for 3 hours, after which the reaction was stopped. The reaction solution was then cooled to 25°C in a water bath.

[0385] Compound 9-A (43.54 mg, 40.54 μM) was dissolved in an appropriate amount of dimethyl sulfoxide and added to the above reaction solution. The mixture was placed in a water bath and shaken at 25 °C for 3 hours, after which the reaction was stopped. The reaction solution was desalted and purified by Sephadex G25 gel column chromatography (elution phase: 0.05 M PBS buffer solution at pH 6.5 containing 0.001 M EDTA) to obtain PBS buffer (4.25 mg / mL, 80.3 mL) of the exemplary product ADC-2 of the conjugate shown in formula P19-9-A, which was stored at 4 °C.

[0386] RP-HPLC calculated average: n = 7.41.

[0387] Example 4-3: ADC-3

[0388] At 37°C, a prepared aqueous solution of tris(2-carboxyethyl)phosphonic acid hydrochloride (TCEP.HCl) (10 mM, 32.4 μL, 324 nM) was added to the PBS buffered aqueous solution of antibody P19 (pH = 6.5, 0.05 M PBS buffered aqueous solution; 10.0 mg / mL, 1.5 mL, 101.3 nM). The solution was placed in a water bath and shaken at 37°C for 3 hours, after which the reaction was stopped. The reaction solution was then cooled to 25°C in a water bath.

[0389] Compound 9-A (1.31 mg, 1215 nM) was dissolved in an appropriate amount of dimethyl sulfoxide and added to the above reaction solution. The mixture was placed in a water bath shaker and shaken at 25 °C for 3 hours, after which the reaction was stopped. The reaction solution was desalted and purified by Sephadex G25 gel column chromatography (elution phase: 0.05 M PBS buffer solution at pH 6.5 containing 0.001 M EDTA) to obtain PBS buffer (0.85 mg / mL, 12.5 mL) of the exemplary product ADC-3, the conjugate shown in formula P19-9-A, which was stored at 4 °C.

[0390] RP-HPLC calculated average: n = 5.5.

[0391] Example 4-4: ADC-4

[0392] At 37°C, a prepared aqueous solution of tris(2-carboxyethyl)phosphonic acid hydrochloride (TCEP.HCl) (10 mM, 60.8 μL, 608 nM) was added to the PBS buffered aqueous solution of antibody P19 (pH = 6.5, 0.05 M PBS buffered aqueous solution; 10.0 mg / mL, 1.5 mL, 101.3 nM). The solution was placed in a water bath and shaken at 37°C for 3 hours, after which the reaction was stopped. The reaction solution was then cooled to 25°C in a water bath.

[0393] Compound 9-A (1.63 mg, 1520 nM) was dissolved in an appropriate amount of dimethyl sulfoxide and added to the above reaction solution. The mixture was placed in a water bath shaker and shaken at 25 °C for 3 hours, after which the reaction was stopped. The reaction solution was desalted and purified by Sephadex G25 gel column chromatography (elution phase: 0.05 M PBS buffer solution at pH 6.5 containing 0.001 M EDTA) to obtain PBS buffer (0.89 mg / mL, 12.9 mL) of the exemplary product ADC-4, the conjugate shown in formula P19-9-A, which was stored at 4 °C.

[0394] RP-HPLC calculated average: n = 7.34.

[0395] (II) Preparation of control antibody-drug conjugate H-AB-P1-9-A

[0396] Examples 4-5: ADC-5

[0397] At 37°C, a prepared aqueous solution of tris(2-carboxyethyl)phosphonic acid hydrochloride (TCEP.HCl) (10 mM, 60.8 μL, 608 nmol) was added to the PBS buffered aqueous solution of antibody H-AB-P1 (pH = 6.5, 0.05 M PBS buffered aqueous solution; 10.0 mg / mL, 1.5 mL, 101 nmol). The solution was placed in a water bath and shaken at 37°C for 3 hours, after which the reaction was stopped. The reaction solution was then cooled to 25°C in a water bath.

[0398] Compound 9-A (1.63 mg, 1.517 μmol) was dissolved in 75 μL of dimethyl sulfoxide and added to the above reaction solution. The mixture was placed in a water bath and shaken at 25 °C for 3 hours, after which the reaction was stopped. The reaction solution was then purified by desalting using a Sephadex G25 gel column (elution phase: 0.05 M PBS buffer solution at pH 6.5 containing 0.001 M EDTA) to obtain the PBS buffer of the title product ADC-5, which was stored at 4 °C.

[0399] RP-HPLC calculated average value: y = 7.25.

[0400] (III) Preparation of control antibody-drug conjugate hIgG1-9-A

[0401] Examples 4-6: hIgG1-9-A

[0402] At 37°C, a prepared aqueous solution of tris(2-carboxyethyl)phosphonic acid hydrochloride (TCEP.HCl) (10 mM, 162.2 μL, 1622 nm) was added to a PBS buffered aqueous solution of antibody hIgG1 (pH = 6.5, 0.05 M PBS buffered aqueous solution; 10.0 mg / mL, 4 mL, 270.2 nM). The solution was placed in a water bath and shaken at 37°C for 3 hours, after which the reaction was stopped. The reaction solution was then cooled to 25°C in a water bath.

[0403] Compound 9-A (4.35 mg, 4050 nM) was dissolved in an appropriate amount of dimethyl sulfoxide and added to the above reaction solution. The mixture was placed in a water bath and shaken at 25 °C for 3 hours, after which the reaction was stopped. The reaction solution was purified by desalting using a Sephadex G25 gel column (elution phase: 0.05 M PBS buffer solution at pH 6.5 containing 0.001 M EDTA) to obtain PBS buffer (1.95 mg / mL, 16.7 mL) of the title product hIgG1-9-A, which was stored at 4 °C.

[0404] RP-HPLC calculated average: n = 7.60.

[0405] III. Examples of Antibody and ADC Tests

[0406] Test Example 1: FACS Binding Assay at the Cellular Level of Antibody

[0407] This experiment evaluates antibody binding by detecting the fluorescence signal of antibodies on the cell surface and by assessing the intensity of the fluorescence signal.

[0408] LNCaP (human prostate cancer cells, ATCC, CRL-1740), 22RV1 (human prostate cancer cells, ATCC, CRL-2505), and PC-3 (human prostate cancer cells, ATCC, CRL-1435) cells were prepared into 1×10⁻⁶ cells using FACS buffer (2% fetal bovine serum (Gibco, 10099141) and pH 7.4 PBS). 6 100 μL / well of cell suspension was added to each well of a 96-well round-bottom plate. After centrifugation to remove the supernatant, 50 μL / well of different concentrations of the test antibody diluted with FACS buffer was added, and the plates were incubated at 4°C in the dark for 1 hour. After washing three times with FACS buffer at 500g, the working concentration of FITC-labeled goat anti-human IgG (H+L) secondary antibody (Jackson Immuno Research, 109-095-003) was added, and the plates were incubated at 4°C in the dark for 40 minutes. After washing three times with FACS buffer at 500g, the geometric mean fluorescence intensity was detected on a BD FACSCantoII flow cytometer to calculate the antibody binding to EC2 cells expressing PSMA. 50 Values. The results are shown in Table 8 and Figures 1A, 1B and 1C below.

[0409] Table 8. In vitro binding activity of antibody and PSMA at different expression levels (ECG) 50 (nM) Note: + indicates the expression level of PSMA on the cell, and more + indicates higher expression level; - indicates no expression of PSMA.

[0410] The results showed that P19 had good binding activity with tumor cells expressing PSMA.

[0411] Test Example 2: Biacore detection of antibody affinity

[0412] IgG antibodies were affinity-captured using a Protein A biosensor chip (Cat.#29127556, GE). Human PSMA-His (ACRO, PSA-H52H3), monkey PSMA-His (ACRO, PSA-C5247), and mouse PSMA-His (ACRO, PSA-M5245) antigens diluted in HBS-EP buffer at pH 7.4 (Cat.#BR-1001-88, GE) were then passed through the chip surface. Antigen-antibody binding kinetics were tracked for 3 minutes, and dissociation kinetics for 10 minutes. Reaction signals were monitored in real-time using a Biacore T200 instrument (GE) to obtain binding and dissociation curves. After dissociation in each experimental cycle, the biosensor chip was regenerated by washing with 10 mM Gly-HCl at pH 1.5 (Cat.#BR-1003-54, GE). The data obtained by using GE's BIAevaluation software with a 1:1 (Langmuir) model were analyzed to determine the values ​​of ka (kon), kd (koff), and KD. The results are shown in Table 9 below.

[0413] Table 9. Affinity of antibodies to PSMA protein

[0414] The results showed that the antibody disclosed in this patent has a high affinity for both human and cynomolgus monkey PSMA, but does not bind to mouse PSMA.

[0415] Test Example 3: Antibody DT3C Internalization Assay

[0416] The purpose of this experiment is to indirectly reflect the endocytosis of PSMA antibodies by observing the cytotoxic effects of activated DT3C protein after it enters the cell. This is based on the IC50 assay. 50 The in vitro endocytic activity of antibodies is evaluated using the maximum killing value.

[0417] DT3C is a recombinant fusion protein composed of fragment A (toxin only) of diphtheria toxin and fragment 3C (IgG binding part) of group G streptococcus. This protein has a high affinity for the IgG portion of an antibody and enters the cell along with the antibody during endocytosis. Under the action of intracellular furin protease, it releases the toxic DT3C, which inhibits EF2-ADP ribosylation activity, blocks protein translation, and ultimately leads to cell death. DT3C that does not enter the cell does not possess cell-killing activity. The endocytic activity of the antibody is evaluated based on its cell-killing effect.

[0418] LNCaP cell (ATCC, CRL-1740) suspension was prepared using fresh cell culture medium containing 20% ​​low IgG FBS, and the cell density was 5 × 10⁶ cells / year. 4 Cells / mL, 50 μL / well added to cell culture plates, incubated at 37°C for 16 hours with 5% CO2. DT3C (Sigma, catalog number D0564) was diluted to 1.6 μM with serum-free medium, and the antibody was diluted to 133.2 nM with serum-free medium. 80 μL of DT3C and 80 μL of antibody solution were mixed at a 1:1 volume ratio and incubated at room temperature for 30 minutes. The molar concentration of DT3C was 6 times the molar concentration of the antibody.

[0419] The DT3C and antibody mixture was serially diluted 4-fold with serum-free medium, resulting in 8 gradients. The 9th spot was pure medium. hIgG1 served as the isotype IgG1 negative control. 50 μL of the diluted mixture was added to 50 μL of cells and incubated for three days. 50 μL of CTG was added to each well, and the cells were incubated at room temperature in the dark for 10 minutes. A white backing membrane was then attached to the bottom of the cell culture plate, and the chemiluminescence values ​​were read using a Victor 3 microplate reader. The results are shown in Table 10 below.

[0420] Table 10. In vitro endocytic activity of antibodies

[0421] The results showed that P19 could be endocytosed by LNCaP cells expressing PSMA, and the endocytic activity of P19 was significantly better than that of the control antibody H-AB-P1.

[0422] Test Example 4: In vitro cell binding assay of ADC

[0423] This experiment evaluates antibody binding by detecting the fluorescence signal of cell surface antibodies and assessing the intensity of the fluorescence signal. Prepared ADC-1, ADC-2, control ADC hIgG1-9-A, and antibody P19 were used together for in vitro binding detection.

[0424] Serially diluted ADC-1, ADC-2, control ADC hIgG1-9-A, and antibody P19 were mixed with 1×10 5 Cells (LNCaP / CRL-1740, 22Rv1 / CRL-2505, PC-3 / CRL-1435) were incubated at 4°C for 60 minutes, after which excess ADC or antibody was washed away. Cells were then incubated with FITC-labeled goat anti-human IgG (H+L) secondary antibody (Jackson Immuno Research, 109-095-003) at 4°C for 30 minutes. After washing away excess antibody, the fluorescence signal on the cell surface was read using BD CantoII. The results are shown in Table 11.

[0425] Table 11. In vitro cell binding activity of ADCs (ECG) 50 (nM) Note: + indicates the expression level of PSMA on the cell, and more + indicates higher expression level; - indicates no expression of PSMA.

[0426] The results showed that ADC-1, ADC-2, the control ADC hIgG1-9-A, and antibody P19 exhibited correspondingly strong or weak binding abilities to cells with different PSMA antigen expression levels. After conjugation into ADCs, the binding affinity of the P19 antibody to PSMA-expressing tumor cells did not change significantly.

[0427] Test Example 5: ADC Molecular Cellular Activity Assay

[0428] The purpose of this experiment was to detect the cytotoxic effect of compound 2-B and its ADC molecules on PSMA-expressing cells, based on IC50. 50 and I max To evaluate the in vitro activity of PSMA-ADC.

[0429] LNCaP (ATCC, CRL-1740), 22RV1 (ATCC, CRL-2505), and PC-3 (ATCC, CRL-1435) cells were digested with trypsin, neutralized with fresh culture medium, centrifuged at 1000 rpm, and resuspended in culture medium. After counting, the cell suspension density was adjusted to 1.48 × 10⁻⁶ cells / mL. 4 Cells were seeded into 96-well plates at a density of 135 μL / mL, with 1000 cells per well. Only the central 60 wells were used for seeding, and six wells in the twelfth column were used as cell-free pure culture medium controls. An equal volume of PBS was added to the side wells. The plates were incubated at 37°C with 5% CO2 for 16 hours.

[0430] Prepare the test ADCs (ADC-1, ADC-2, and control ADC: hIgG1-9-A) and antibody P19 to a starting concentration (1000 nM), and serially dilute 9 wells with PBS at a 1:5 ratio. Add 20 μL of each solution to the cell culture plate and incubate for 5 days (37°C, 5% CO2). For compound 2-B (i.e., the free toxin of 9-A, refer to WO2020063676A1, page 50, Example 2, 2-B preparation), first dilute with DSMO to prepare a stock solution and prepare 100 nM with PBS as the starting concentration. Then serially dilute 9 wells with PBS at a 1:3 ratio. Add 1 μL of each solution to 20 μL of RPMI-1640 and transfer to a 96-well cell culture plate. Incubate for 5 days (37°C, 5% CO2). Add 80 μL of CellTiter-Glo reagent to each well of a 96-well cell culture plate, incubate at room temperature in the dark for 10-15 minutes, and read the chemiluminescence signal value in a Victor3. The data are processed using GraphPad software.

[0431] On the fifth day, remove the culture plate and allow it to return to room temperature. Add 75 μL of CellTiter-Glo detection solution to each well and incubate at room temperature in the dark for 10 minutes. Attach the backseal membrane to the bottom of the cell culture plate and place it on a Victor3 microplate reader to read the luminescence signal value. Use Graphpad Prism software to perform curve fitting based on the logarithmic values ​​of each concentration of the test compound and the corresponding signal values, and calculate the IC50 value. The maximum killing value is defined as the maximum killing rate (Imax) of the compound, and the killing rate is calculated as follows: Kill rate (%) = (RLU) / (Imax / Imax) 无药对照读值 -RLU 样品读值 ) / (RLU 无药对照读值 -RLU 溶媒对照读值 )×100%

[0432] The results are shown in Table 12.

[0433] Table 12. Killing effect of ADCs on LNCaP, 22RV1, and PC-3 cells Note: + indicates the expression level of PSMA on the cell, and more + indicates higher expression level; - indicates no expression of PSMA.

[0434] The results showed that PSMA-ADC had a significant killing effect on cells with high PSMA expression, but its killing effect on cells with low PSMA expression was significantly reduced, and it almost did not kill cells that did not express PSMA. That is, the ADC molecule disclosed in this paper has selective killing effect on cells. In contrast, the cytotoxic compound 2-B (i.e., the free toxin of 9-A) did not have selective killing effect on cells.

[0435] Test Example 6: In vivo efficacy evaluation of a CDX expression model in the ADC molecule PSMA

[0436] 22RV1 cells were divided into 2×10 6 Cells / mouse / 200μL were subcutaneously injected into the right rib area of ​​50 male NU / NU nude mice. Tumors in the tumor-bearing mice reached a tumor volume of 188 mm². 3 Around the time of the experiment, after removing those that were too large, too small, or too light, 35 animals were randomly selected and divided into 5 groups of 7 animals each. The day of grouping was defined as day 0 of the experiment. The drugs were administered intraperitoneally on day 0 (hereinafter referred to as D0), day 7 (hereinafter referred to as D7), and day 14 (hereinafter referred to as D14), for a total of 3 administrations.

[0437] Tumor volume and body weight were measured twice weekly, and the data were recorded. Experimental data are expressed as mean ± standard error (SEM), and graphs and statistical analyses were performed using GraphPad Prism software.

[0438] The formula for calculating tumor volume (V) is: V = 1 / 2 × a × b 2 , where a and b represent length and width, respectively.

[0439] Relative tumor proliferation rate T / C (%) = (T - T0) / (C - C0) × 100%, where T and C are the tumor volumes of the treatment group and control group at the end of the experiment; T0 and C0 are the tumor volumes at the beginning of the experiment. Tumor inhibition rate TGI (%) = 1 - T / C (%).

[0440] The results are shown in Table 13.

[0441] Table 13. The therapeutic effects of ADC molecules on 22RV1 tumors in NU / NU nude mice

[0442] The results showed that both ADC-3 and ADC-4 had significant tumor-suppressing effects in the mouse 22RV1 tumor model. The tumor-suppressing effect was positively correlated with the administered dose, and the tumor-suppressing effect of ADCs with high DAR values ​​(n=7.34) was superior to that of ADCs with low DAR values ​​(n=5.5). No significant changes were observed in mouse body weight.

[0443] Test Example 7: PK Evaluation of ADC Molecules

[0444] 1. Study the single-dose pharmacokinetics of ADC molecules in adult SD rats.

[0445] Adult SD rats (Zhejiang Vital River) were randomly divided into groups of four, each containing one ADC molecule, and intravenously injected with 3 mpk of the ADC molecule (n = 4 rats / group). Serum samples were collected at 0.083, 8, 24, 48, 96, 168, 240, 336, 504, and 672 hours after intravenous injection for bioanalytical measurements. The HTRF method was used to determine the content of the analytes in the rat serum samples, and the content was quantitatively analyzed using a four-parameter model curve of the standards. Pharmacokinetic parameters were analyzed using a standard non-compartmental model with WinNonlin software (6.1). The results are shown in Table 14 and Figure 2.

[0446] Table 14. PK of ADC molecules in SD rats Note: iv indicates intravenous injection; TAb indicates total antibody; Intact ADC indicates complete ADC; the same applies below.

[0447] At an intravenous dose of 3 mpk, the half-lives of total antibody and intact ADC-4 in rats were 8.2 ± 0.5 days and 6.9 ± 0.1 days, respectively. In contrast, the half-lives of total antibody and intact ADC-5 in rats were 5.3 ± 0.3 days and 4.8 ± 0.3 days, respectively. Clearly, ADC-4 exhibits a significantly longer half-life than ADC-5 in rats, indicating greater stability in vivo.

[0448] 2. Study the single-dose pharmacokinetics of ADC molecules in adult male cynomolgus monkeys.

[0449] Cynomolgus monkeys (Suzhou Guochen) were divided into groups of three per ADC molecule. After intravenous injection of 10 mpk of the ADC molecule (n = 3 monkeys / group), serum samples were collected at 0.083, 1, 6, 12, 24, 48, 96, 168, 240, 336, 504, and 672 hours for bioanalytical measurements. The HTRF method was used to determine the content of the analytes in the cynomolgus monkey serum samples, and the content of the analytes was quantitatively analyzed using a four-parameter model curve of the standards. Pharmacokinetic parameters were analyzed using a standard non-compartmental model in WinNonlin software (6.1). The results are shown in Table 15 and Figure 3.

[0450] Table 15. PK of ADC molecules in cynomolgus monkeys

[0451] At an intravenous dose of 10 mpk, the half-lives of total antibody and intact ADC-2 in cynomolgus monkeys were 6.3 ± 1.3 days and 5.7 ± 0.9 days, respectively. ADC-2 exhibited normal pharmacokinetic activity and good stability in cynomolgus monkeys.

[0452] IV. Preparation Examples - Anti-PSMA Antibody-Drug Conjugate Formulation

[0453] 1) SEC size exclusion chromatography:

[0454] An analytical method for separating solutes based on the relative relationship between the pore size of the gel and the coil size of the polymer sample molecules.

[0455] SEC% polymer (SEC polymer content percentage) = A polymer / A total * 100% (A polymer is the peak area of ​​the polymer peak in the sample, and A total is the sum of the peak areas of all peaks.)

[0456] △SEC aggregate% represents the difference between the measured value of this test item after placement under various conditions and the initial placement value.

[0457] Instrument used for SEC determination: Agilent HPLC 1260;

[0458] Pillars: Waters, BioResolve TM SEC mAb 2.5μm 7.8×300mm Column

[0459] 2) R-CE capillary gel electrophoresis:

[0460] An electrophoresis method in which a gel is transferred into a capillary as a supporting medium and then separated according to the molecular weight of the sample under a certain voltage.

[0461] R-CE% fragments (R-CE fragment content percentage) = A fragments / A total * 100% (A fragments is the peak area of ​​fragments in the sample, and A total is the sum of the peak areas of all peaks).

[0462] Instrument used for R-CE determination: Beckman capillary electrophoresis apparatus, model PA 800plus

[0463] △R-CE fragments% represents the difference between the value of this test item after placement under each condition and the value at the beginning of placement.

[0464] 3) Osmotic pressure measurement:

[0465] The freezing point method for determining osmotic pressure is based on the principle that the freezing point depression is directly proportional to the molar concentration of the solution. It uses a highly sensitive temperature sensing element to measure the freezing point of the solution and converts the electrical charge into osmotic pressure.

[0466] Instrument used for osmotic pressure measurement: Loser, model OM815.

[0467] 4) Protein concentration determination:

[0468] The concentrations of antibody-drug conjugates disclosed herein are expressed as protein concentrations, i.e., the concentrations of the antibody portion within the antibody-drug conjugate.

[0469] Because the toxin in the antibody-drug conjugate absorbs at the characteristic absorption wavelength of proteins at 280 nm, and also absorbs at 370 nm, the protein concentration is calculated using the following formula: A 280nm =(C drug ×E drug-280 +C mAb ×E mAb-280 )×l A 370nm =C drug ×E drug-370 ×l

[0470] Pick

[0471] Right now:

[0472] In the formula, A 280nm The average absorbance of a single sample of the test solution at a wavelength of 280 nm when the optical path length is 1 cm;

[0473] A 370nm The average absorbance of a single sample of the test solution at a wavelength of 370 nm when the optical path length is 1 cm;

[0474] E mAb-280 The mass extinction coefficient of the protein at a wavelength of 280 nm is 1.429 g. -1 cm -1 L;

[0475] E drug-280 The mass extinction coefficient of the toxin at a wavelength of 280 nm is 5.17 g. -1 cm -1 L;

[0476] E drug-370 The mass extinction coefficient of the toxin at a wavelength of 370 nm is 17.89 g. -1 cm -1 L;

[0477] R: The ratio of the toxin extinction coefficient at 370nm to 280nm is 3.46;

[0478] C mAb Protein concentration, mg / mL;

[0479] l: Optical path length, cm (the optical path length here is 1 cm).

[0480] If the test solution is diluted, the protein concentration is: C (mg / ml) = C mAb ×N, where N is the dilution factor.

[0481] Protein concentration measurement instrument: UV-Vis spectrophotometer, model: Nano Drop 2000.

[0482] The concentrations of the anti-PSMA antibody-drug conjugates (DAR: approximately 7.5 (7.5 ± 0.5), such as ADC-2 or ADC-4; hereinafter referred to as "ADC") disclosed herein are calculated based on protein concentrations, i.e., based on the concentration of the anti-PSMA antibody portion in the anti-PSMA antibody-drug conjugate.

[0483] Formulation Example 1. pH Screening of the Formulation Composition

[0484] Eight formulations with an antibody-drug conjugate (ADC) concentration of 20.0 mg / mL and pH 4.6–7.4 were prepared using the following buffer solutions. The stability of the formulations under high temperature (40℃) conditions was investigated by detecting the appearance of the samples, SEC-HPLC, and RCE purity.

[0485] 1) 10mM citrate-disodium hydrogen phosphate, pH 4.6, 20.0 mg / mL ADC

[0486] 2) 10mM citrate-disodium hydrogen phosphate, pH 5.0, 20.0 mg / mL ADC

[0487] 3) 10mM citrate-disodium hydrogen phosphate, pH 5.4, 20.0 mg / mL ADC

[0488] 4) 10mM citrate-disodium hydrogen phosphate, pH 5.8, 20.0 mg / mL ADC

[0489] 5) 10mM citrate-disodium hydrogen phosphate, pH 6.2, 20.0 mg / mL ADC

[0490] 6) 10mM citrate-disodium hydrogen phosphate, pH 6.6, 20.0 mg / mL ADC

[0491] 7) 10mM citrate-disodium hydrogen phosphate, pH 7.0, 20.0 mg / mL ADC

[0492] 8) 10mM citrate-disodium hydrogen phosphate, pH 7.4, 20.0 mg / mL ADC

[0493] Table 16. Comparison of pH screening appearance

[0494] Table 17. Comparison of purity in pH screening

[0495] The results showed that under high temperature (40℃) conditions, ADC had relatively fewer protein particles in formulations 2 (pH 5.0) and 3 (pH 5.4), while other formulations contained a large number of protein particles. After one week of high temperature (40℃) observation, the SEC polymer content in each formulation tended to increase with increasing pH, exceeding 2.0% when pH was above 6.2. In conclusion, ADC performed better in the pH range of 5.0–5.8.

[0496] Formulation Example 2. Buffer System Screening

[0497] A formulation containing 20.0 mg / mL ADC was prepared using either a 10 mM acetate-sodium acetate system (pH 5.0) or a 10 mM histidine-histidine hydrochloride system (pH 5.5), 75.0 mg / mL sucrose as a stabilizer, and 0.2 mg / mL polysorbate 80 as a surfactant. The stability of the two formulations under high temperature (40℃), room light (460 lx / room temperature), and freeze-thaw cycles (-35℃ / room temperature) conditions was investigated by measuring sample appearance, SEC-HPLC, R-CE purity, free toxins, and DAR.

[0498] 1) 10mM acetate-sodium acetate, 75.0mg / mL sucrose, 0.2mg / mL polysorbate 80, pH 5.0, 20.0mg / mL ADC

[0499] 2) 10 mM histidine-histidine hydrochloride, 75.0 mg / mL sucrose, 0.2 mg / mL polysorbate 80, pH 5.5, 20.0 mg / mL ADC

[0500] Table 1. Buffer System Screening - Results 1

[0501] Table 19. Buffer System Screening - Results 2 Note: NT indicates not detected; under ambient light conditions, the DAR value cannot be integrated due to peak shape variations.

[0502] The results showed that, under ambient light conditions for 5 days, compared with Group 1: acetic acid system at pH 5.0, the SEC-HPLC polymer content of Group 2: histidine system at pH 5.5 increased more significantly than that at pH 0.

[0503] Under high temperature (40℃) conditions for 4 weeks, compared with Group 1: acetic acid system at pH 5.0, Group 2: histidine system at pH 5.5 showed a greater decrease in RCE purity.

[0504] In summary, the results show that the stability of ADC in the acetic acid system at pH 5.0 is significantly better than that in the histidine system at pH 5.5. Further studies will be conducted using a 10 mM acetic acid-sodium acetate system at pH 5.0.

[0505] Formulation Example 3. Stabilizer Screening

[0506] A 10 mM acetate-sodium acetate buffer system at pH 5.0 was selected. 75.0 mg / mL sucrose, 0.05 mg / mL disodium edetate, or 2.0 mg / mL methionine were chosen as stabilizers. 0.2 mg / mL polysorbate 80 was selected as a surfactant. A formulation containing 20.0 mg / mL ADC was prepared. The stability of the four formulations under high temperature (40℃), room light (460 lx / room temperature), freeze-thaw (-35℃ / room temperature), cryopreservation (-35℃), and shaking (250 rpm / 25℃) conditions was investigated by measuring sample appearance, SEC-HPLC, and R-CE purity.

[0507] 1) 10mM acetate-sodium acetate, 75.0mg / mL sucrose, 0.2mg / mL polysorbate 80, pH 5.0, 20.0mg / mL ADC

[0508] 2) 10mM acetate-sodium acetate, 75.0mg / mL sucrose, 0.05mg / mL disodium edetate, 0.2mg / mL polysorbate 80, pH 5.0, 20.0mg / mL ADC

[0509] 3) 10mM acetate-sodium acetate, 75.0mg / mL sucrose, 2.0mg / mL methionine, 0.2mg / mL polysorbate 80, pH 5.0, 20.0mg / mL ADC

[0510] Table 20. Screening Results of ADC Stabilizers Note: NT indicates no detection.

[0511] Table 21. Screening Results of ADC Stabilizers Note: NT indicates no detection.

[0512] The results showed that, under ambient light (460 lx / room temperature) and high temperature (40 °C) conditions, the SEC-HPLC polymer content of formulations 1 and 2 increased more significantly compared with formulation 3.

[0513] Under ambient light (460 lx / room temperature) conditions, the RCE purity of formulations 1 and 2 decreased more significantly compared to formulation 3.

[0514] In summary, formulation 3 showed the best stability. 2.0 mg / mL methionine and 75.0 mg / mL sucrose were selected as stabilizers for further studies.

[0515] Formulation Example 4. Effect of pH on ADC stability

[0516] Two formulations containing 20.0 mg / mL ADC were prepared using a 10 mM acetate-sodium acetate buffer system at pH 4.7 or pH 5.3, with 75.0 mg / mL sucrose, 2.0 mg / mL methionine, or 0.2 mg / mL polysorbate 80. The stability of the two formulations under shaking (250 rpm, 25℃), high temperature (40℃), frozen storage (-35℃), 25℃, and freeze-thaw cycle (-35℃ / room temperature) conditions was investigated by measuring sample appearance, SEC-HPLC, and RCE purity.

[0517] 1) 10mM acetate-sodium acetate, 75.0mg / mL sucrose, 2.0mg / mL methionine, 0.2mg / mL polysorbate 80, pH 4.7, 20.0mg / mL ADC

[0518] 2) 10mM acetate-sodium acetate, 75.0mg / mL sucrose, 2.0mg / mL methionine, 0.2mg / mL polysorbate 80, pH 5.3, 20.0mg / mL ADC

[0519] Table 22. Effect of pH on ADC stability - Results 1

[0520] Table 23. Effect of pH on ADC stability - Results 2 Note: NT indicates no detection.

[0521] The results showed that, compared with pH 0, both Formulation 1 (pH 4.7) and Formulation 2 (pH 5.3) exhibited good ADC stability under shaking (250 rpm, 25℃), frozen storage (-35℃), 25℃, and freeze-thaw cycles (-35℃ / room temperature). However, under high temperature (40℃) conditions, the SEC-HPLC purity and RCE purity of both Formulation 1 (pH 4.7) and Formulation 2 (pH 5.3) decreased to some extent, while the free toxins increased to some extent. However, the trends were consistent, indicating that the difference in the effect of pH 4.7 and pH 5.3 on ADC stability was not significant. Therefore, the pH range of the ADC formulation was set at 4.7–5.3, with the midpoint 5.0 taken as the target pH value.

[0522] Formulation Example 5. Stability of Lyophilized Product

[0523] A 10 mM acetate-sodium acetate pH 5.0 buffer system was prepared, along with a stock solution of 75.0 mg / mL sucrose, 2.0 mg / mL methionine, 0.2 mg / mL polysorbate 80, and 20.0 mg / mL ADC. The solution was filtered through a 0.22 μm filter and filled into 20 mL vials, with a volume of 5.35 mL per vial. Lyophilization was performed according to the parameters in Table 25. The stability of the lyophilized product under light (4500 ± 500 lx, 25 °C) and high temperature (40 °C) conditions was investigated by measuring sample appearance, reconstitution time, SEC-HPLC, RCE purity, and free toxins.

[0524] Table 24. Stability of Freeze-dried Products - Freeze-drying Process Parameters

[0525] Table 25. Stability of freeze-dried products - Results 1 Note: NT indicates no detection.

[0526] Table 26. Stability of freeze-dried products - Results 2 Note: NT indicates no detection.

[0527] The results showed that, compared with the time at 0°C, after 7 days of light exposure, there were no significant changes in the appearance, reconstitution time, iCIEF, DAR, free toxins, SEC, and RCE of the samples; after 4 weeks of storage at 40°C, compared with the time at 0°C, there were no significant changes in the appearance, reconstitution time, iCIEF, DAR, free toxins, SEC, and RCE, indicating that the freeze-dried products have good stability.

[0528] Formulation Example 6. Optional ADC Formulation

[0529] This invention provides an antibody-drug conjugate formulation comprising, but not limited to, "18.0–22.0 mg / mL antibody-drug conjugate, 75.0 mg / mL sucrose, 2.0 mg / mL methionine, 10 mM acetate-sodium acetate pH 4.7–5.3, and 0.2 mg / mL polysorbate 80", including, but not limited to:

[0530] (1) 20.0 mg / mL ADC, 75.0 mg / mL sucrose, 2.0 mg / mL methionine, 0.2 mg / mL polysorbate 80, 10 mM acetate-sodium acetate buffer pH 4.7;

[0531] (2) 20.0 mg / mL ADC, 75.0 mg / mL sucrose, 2.0 mg / mL methionine, 0.2 mg / mL polysorbate 80, 10 mM acetate-sodium acetate buffer pH 5.0;

[0532] (3) 20.0 mg / mL ADC, 75.0 mg / mL sucrose, 2.0 mg / mL methionine, 0.2 mg / mL polysorbate 80, 10 mM acetate-sodium acetate buffer pH 5.3.

[0533] Formulation Example 7. ADC lyophilization process can be selected.

[0534] The freeze-drying process for the ADC formulation is as follows:

[0535] (1) Pre-freeze at -2℃ to -8℃ for 0.5 to 2 hours;

[0536] (2) Pre-freeze at -40℃ to -45℃ for 180 to 240 minutes;

[0537] (3) Dry for 2400 to 2700 minutes at -15℃ to -20℃ and 0.10 to 0.15mbar; (4) Dry for 500 to 600 minutes at 20℃ to 25℃ and 0.01 to 0.02mbar.

Claims

1. A pharmaceutical composition comprising an anti-PSMA antibody-drug conjugate and a buffer, wherein: The anti-PSMA antibody-drug conjugate has the following structure: in: n is between 1 and 10; Pc is an anti-PSMA antibody, which contains a heavy chain variable region and a light chain variable region, wherein: The heavy chain variable region includes HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO:5, SEQ ID NO:6 and SEQ ID NO:7 respectively; the light chain variable region includes LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO:8, SEQ ID NO:9 and SEQ ID NO:10 respectively. The buffer is a histidine buffer, a citrate buffer, an acetate buffer, or a succinate buffer. Preferably, the buffer is a citrate-disodium hydrogen phosphate buffer, a histidine-histidine hydrochloride buffer, or an acetate-sodium acetate buffer. More preferably, the buffer is an acetate-sodium acetate buffer.

2. The pharmaceutical composition according to claim 1, wherein the anti-PSMA antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:12, or an amino acid sequence having at least 80% sequence identity with it; and the light chain variable region comprises the amino acid sequence of SEQ ID NO:14, or an amino acid sequence having at least 80% sequence identity with it; Preferably, the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:12, and the light chain variable region comprises the amino acid sequence of SEQ ID NO:14; More preferably, the anti-PSMA antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO: 19, or an amino acid sequence having at least 80% sequence identity with it; and the light chain comprises the amino acid sequence of SEQ ID NO: 20, or an amino acid sequence having at least 80% sequence identity with it; More preferably, the anti-PSMA antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO: 19, and the light chain comprises the amino acid sequence of SEQ ID NO:

20.

3. The pharmaceutical composition according to claim 1 or 2, wherein n is 3 to 8; preferably, n is 6 to 8.

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

5. The pharmaceutical composition according to any one of claims 1 to 4, wherein the concentration of the buffer is from 1 mM to 50 mM; Preferably, the concentration of the buffer is from 5 mM to 30 mM; More preferably, the concentration of the buffer is 5 mM to 20 mM; Most preferably, the concentration of the buffer is about 10 mM.

6. The pharmaceutical composition according to any one of claims 1 to 5, wherein the pH of the pharmaceutical composition is 4.6 to 7.4; Preferably, the pH of the pharmaceutical composition is 4.7 to 5.8; More preferably, the pH of the pharmaceutical composition is 4.7 to 5.3; More preferably, the pH of the pharmaceutical composition is about 5.

0.

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

8. The pharmaceutical composition according to claim 7, wherein the concentration of the surfactant is from 0.01 mg / mL to 2.0 mg / mL; Preferably, the concentration of the surfactant is from 0.02 mg / mL to 1 mg / mL; More preferably, the concentration of the surfactant is from 0.05 mg / mL to 0.4 mg / mL; Most preferably, the concentration of the surfactant is about 0.2 mg / mL.

9. The pharmaceutical composition according to any one of claims 1 to 8, wherein the pharmaceutical composition comprises sugar; Preferably, the sugar is sucrose, trehalose, mannitol, or sorbitol; More preferably, the sugar is sucrose.

10. The pharmaceutical composition according to claim 9, wherein the concentration of the sugar is from 10 mg / mL to 120 mg / mL; Preferably, the concentration of the sugar is from 50 mg / mL to 100 mg / mL; More preferably, the concentration of the sugar is from 60 mg / mL to 90 mg / mL; Most preferably, the concentration of the sugar is about 75 mg / mL.

11. The pharmaceutical composition according to any one of claims 1 to 10, wherein the pharmaceutical composition comprises an excipient; the excipient is selected from one or more of glycine, DTPA, arginine hydrochloride, disodium edetate, methionine, proline, histidine, phenylalanine, glutamic acid, aspartic acid, sodium chloride, and calcium chloride; Preferably, the excipient is methionine or disodium edetate; More preferably, the excipient is methionine.

12. The pharmaceutical composition according to claim 11, wherein the concentration of the excipient is from 0.01 mg / mL to 20 mg / mL; Preferably, its concentration is from 0.1 mg / mL to 12 mg / mL; More preferably, its concentration is from 1 mg / mL to 8 mg / mL; Most preferably, its concentration is about 2 mg / mL.

13. The pharmaceutical composition according to claim 1, comprising the following components: (a) The anti-PSMA antibody-drug conjugate at concentrations from 1 mg / mL to 50 mg / mL, (b) Surfactants ranging from 0.01 mg / mL to 2 mg / mL, (c) Sugars ranging from 10 mg / mL to 120 mg / mL, (d) Excipients ranging from 0.01 mg / mL to 20 mg / mL, and (e) a buffer of 1 mM to 50 mM, wherein the pH of the pharmaceutical composition is 4.6 to 7.4; Preferably, the pharmaceutical composition comprises the following components: (a) The anti-PSMA antibody-drug conjugate at concentrations of 5 mg / mL to 40 mg / mL, (b) Polysorbate 80 at concentrations ranging from 0.02 mg / mL to 1 mg / mL, (c) Sucrose at concentrations of 50 mg / mL to 100 mg / mL, (d) Methionine at concentrations ranging from 0.1 mg / mL to 12 mg / mL, and (e) 5 mM to 30 mM of citrate-disodium hydrogen phosphate buffer, histidine-histidine hydrochloride buffer and acetate-sodium acetate buffer, wherein the pH of the pharmaceutical composition is 4.7 to 5.8; More preferably, the pharmaceutical composition comprises the following components: (a) The anti-PSMA antibody-drug conjugate at concentrations of 15 mg / mL to 25 mg / mL, (b) Polysorbate 80 at concentrations ranging from 0.05 mg / mL to 0.4 mg / mL, (c) Sucrose at concentrations of 60 mg / mL to 90 mg / mL, (d) Methionine at concentrations ranging from 1 mg / mL to 8 mg / mL, and (e) a 5 mM to 20 mM acetate-sodium acetate buffer, wherein the pH of the pharmaceutical composition is 4.7 to 5.3; Most preferably, the pharmaceutical composition comprises the following components: (a) The anti-PSMA antibody-drug conjugate at a concentration of approximately 20 mg / mL, (b) Approximately 0.2 mg / mL of polysorbate 80, (c) Approximately 75 mg / mL of sucrose, (d) Approximately 2 mg / mL of methionine, and (e) about 10 mM of an acetate-sodium acetate buffer, wherein the pH of the pharmaceutical composition is about 5.

0.

14. The pharmaceutical composition according to any one of claims 1-13, wherein it is a solution obtained by reconstituted a lyophilized preparation with water for injection, said lyophilized preparation being obtained by freeze-drying the pharmaceutical composition according to any one of claims 1-13.

15. The pharmaceutical composition according to any one of claims 1 to 14, wherein it is an intravenous injection formulation, a subcutaneous injection formulation, an intraperitoneal injection formulation, or an intramuscular injection formulation; preferably an intravenous injection formulation.

16. A lyophilized formulation obtained by freeze-drying the pharmaceutical composition according to any one of claims 1 to 13.

17. A method for preparing lyophilized formulations, wherein, The method includes the step of freeze-drying the pharmaceutical composition according to any one of claims 1 to 13, wherein the freeze-drying sequentially includes the steps of a first pre-freezing, a second pre-freezing, a first drying, and a second drying. Preferably, the pre-freezing is performed at -2℃ to -8℃ for 0.5 to 2 hours; The aforementioned secondary pre-freezing involves pre-freezing at -40℃ to -45℃ for 3 to 4 hours. The first drying process is carried out at -15℃ to -20℃. Dry at 0.10 mbar to 0.15 mbar for 40 to 45 hours; The secondary drying process involves drying at 20℃~25℃ and 0.01mbar~0.02mbar for 8 to 10 hours. More preferably, The aforementioned pre-freezing refers to pre-freezing at -5°C for 1 hour; The aforementioned secondary pre-freezing involves pre-freezing at -45℃ for 3 hours. The first drying process is carried out at -20°C. Dry at 0.10 mbar for 40 hours; The secondary drying process involves drying at 25°C and 0.02 mbar for 10 hours.

18. Use of the pharmaceutical composition according to any one of claims 1 to 15, or the lyophilized formulation according to claim 16, in the preparation of a medicament for treating tumors or cancer; preferably, wherein the tumor or cancer is preferably prostate cancer, squamous cell carcinoma of the head and neck, head and neck cancer, brain cancer, glioma, glioblastoma multiforme, neuroblastoma, neuroendocrine tumor, pharyngeal cancer, nasopharyngeal cancer, esophageal cancer, thyroid cancer, malignant pleural mesothelioma, lung cancer, breast cancer, liver cancer, hepatobiliary cancer, pancreatic cancer, gastric cancer, colorectal cancer, kidney cancer, ovarian cancer, endometrial cancer, cervical cancer, bladder cancer, testicular cancer, melanoma, leukemia, lymphoma, chondrosarcoma, multiple myeloma, myelodysplastic syndrome, Kuckenberg tumor, squamous cell carcinoma, Ewing's sarcoma, urothelial carcinoma, and Merkel cell carcinoma.

Images