Pharmaceutical composition of Anti-LIV-1 antibody-drug conjugate and use thereof

Abstract

A pharmaceutical composition of an anti-LIV-1 antibody-drug conjugate and use thereof. The pharmaceutical composition comprises the anti-LIV-1 antibody-drug conjugate, a buffer, a stabilizer, and a surfactant. By means of optimizing the composition ratio and the preparation process, the pharmaceutical composition of the anti-LIV-1 antibody-drug conjugate exhibits excellent physical and chemical stability, ensuring that said pharmaceutical composition can maintain a stable effect under long-term storage conditions.

Description

Pharmaceutical composition of anti-LIV-1 antibody-drug conjugate and application thereof TECHNICAL FIELD

[0001] The present disclosure belongs to the field of pharmacy, and specifically relates to a pharmaceutical composition of anti-LIV-1 antibody-drug conjugate and application thereof. BACKGROUND

[0002] LIV-1, also known as zinc transporter ZIP6, is a member of the ZIP (Zrt / Irt-like protein) family. It is mainly expressed on the cell membrane and is responsible for transporting zinc ions from the outside to the inside of the cell. It was first discovered in breast cancer cells and is closely related to the metastasis of estrogen receptor-positive breast cancer.

[0003] The LIV-1 protein is composed of multiple transmembrane domains that form zinc ion channels on the cell membrane. LIV-1 affects various physiological processes of cells such as proliferation, differentiation, and apoptosis by regulating the concentration of zinc ions inside and outside the cell. LIV-1 is highly expressed in breast cancer cells, especially in estrogen receptor-positive breast cancer. Estrogen can promote the growth and metastasis of cancer cells by up-regulating the expression of LIV-1. In addition to breast cancer, LIV-1 is also highly expressed in various cancers such as prostate cancer and gastric cancer, and is related to the invasion and metastasis of cancer.

[0004] LIV-1 is also expressed in immune cells, and studies have shown that LIV-1 affects the activation and function of T cells by regulating the concentration of zinc ions. This suggests that LIV-1 may play an important role in the tumor immune microenvironment by regulating the function of immune cells and affecting the immune escape of tumors.

[0005] Because LIV-1 is highly expressed in various cancers and is closely related to the invasion and metastasis of cancer cells, LIV-1 has become a potential anti-tumor target. By targeting LIV-1, it may inhibit the abnormal transport of zinc ions and affect the growth and metastasis of cancer cells. At the same time, targeting LIV-1 may also enhance the anti-tumor immune response by regulating the function of immune cells.

[0006] However, antibody-drug conjugates have poor stability and can undergo various chemical and physical degradation, especially the higher structure of antibodies is very fragile and prone to structural changes such as denaturation, aggregation, and precipitation. These degraded or unstable products can have a significant impact on the safety of biopharmaceuticals. In particular, some protein aggregates can trigger an immune response in the human body, which can reduce the efficacy of biological drugs, or even cause death in severe cases.

[0007] The multimers are considered as critical quality attributes (CQA) of the biopharmaceutical safety, which directly affect the medication safety of the anti-LIV-1 antibody-drug conjugate. The antibody-drug conjugate not only needs to obtain a product with high purity in production, but also needs to maintain structural stability during transportation, storage and use, which is particularly important for long-term medication products such as the anti-LIV-1 antibody-drug conjugate.

[0008] Therefore, it is still necessary to develop a new anti-LIV-1 antibody-drug conjugate preparation to improve the stability of the anti-LIV-1 antibody-drug conjugate, thereby improving the uniformity and consistency of the product quality, and improving the safety. SUMMARY

[0009] The inventors have found, through repeated experimental research, that the anti-LIV-1 antibody-drug conjugate preparation with high stability has a very broad market application prospect, by screening a large number of components and contents of various buffer systems.

[0010] The first aspect of the present disclosure discloses a pharmaceutical composition comprising an anti-LIV-1 antibody-drug conjugate, a buffer, a stabilizer and a surfactant; wherein:

[0011] The anti-LIV-1 antibody comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 in the sequence shown in SEQ ID NO: 2, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 in the sequence shown in SEQ ID NO: 4.

[0012] In some embodiments, the amino acid sequences of the HCDRs and the LCDRs are determined according to the Kabat, Chothia or IMGT numbering system. The correspondence between various numbering systems is well known to those skilled in the art. In other words, when a CDR sequence under a numbering system and its position in an antibody are provided, the skilled person is able to determine the corresponding CDR sequence under another numbering system and its position in an antibody. The technical solutions of the CDRs corresponding to different numbering systems are considered as equivalent technical solutions.

[0013] In some embodiments, the amino acid sequences of the HCDRs and the LCDRs are determined according to the Kabat numbering system.

[0014] In some embodiments, the heavy chain variable region of the anti-LIV-1 antibody comprises HCDR1 as set forth in SEQ ID NO: 5, HCDR2 as set forth in SEQ ID NO: 6, HCDR3 as set forth in SEQ ID NO: 7, and the light chain variable region comprises LCDR1 as set forth in SEQ ID NO: 8, LCDR2 as set forth in SEQ ID NO: 9, LCDR3 as set forth in SEQ ID NO: 10.

[0015] In some embodiments, the heavy chain variable region of the anti-LIV-1 antibody comprises an amino acid sequence as set forth in SEQ ID NO: 2 and the light chain variable region comprises an amino acid sequence as set forth in SEQ ID NO: 4.

[0016] In some embodiments, the heavy chain variable region of the anti-LIV-1 antibody comprises an amino acid sequence as set forth in SEQ ID NO: 2 and the light chain variable region comprises an amino acid sequence as set forth in SEQ ID NO: 4.

[0017] In some embodiments, the anti-LIV-1 antibody comprises a heavy chain comprising an amino acid sequence as set forth in SEQ ID NO: 1 and a light chain comprising an amino acid sequence as set forth in SEQ ID NO: 3.

[0018] In some embodiments, the anti-LIV-1 antibody comprises a heavy chain comprising an amino acid sequence as set forth in SEQ ID NO: 1 and a light chain comprising an amino acid sequence as set forth in SEQ ID NO: 3.

[0019] In some embodiments, the drug of the anti-LIV-1 antibody-drug conjugate is auristatin or a functional peptide analog or derivative thereof.

[0020] In some embodiments, the drug is monomethyl auristatin E (MMAE) or monomethyl auristatin F (MMAF).

[0021] In some embodiments, the average absolute number of drug moieties per anti-LIV-1 antibody is an integer or a fraction of 1-8. In some embodiments, the average absolute number of drug moieties per anti-LIV-1 antibody is an integer or a fraction of 1-5. In some embodiments, the average absolute number of drug moieties per anti-LIV-1 antibody is an integer or a fraction of 3-5.

[0022] In some embodiments, the drug of the anti-LIV-1 antibody-drug conjugate is linked to the anti-LIV-1 antibody by a linker-Val-Cit-PAB-, i.e., valine-citrulline-p-aminobenzoic acid. The -Val-Cit-PAB- is a degradable ADC linker having the structure shown below:

[0023] wherein a end is attached to a spacer unit of an anti-LIV-1 antibody-drug conjugate, and b end is attached to a drug. In some embodiments, the spacer unit is carboxyl.

[0024] In some embodiments, the anti-LIV-1 antibody-drug conjugate has a structure selected from the group consisting of:

[0025] wherein Ab represents an anti-LIV-1 antibody, and z is a number or an integer from 1 to 8.

[0026] In some embodiments, the z is from 3.5 to 4.5.

[0027] In some embodiments, the concentration of the anti-LIV-1 antibody-drug conjugate in the pharmaceutical composition is from 2 mg / mL to 40 mg / mL, such as from 2 mg / mL to 30 mg / mL, from 2 mg / mL to 20 mg / mL, from 5 mg / mL to 40 mg / mL, from 5 mg / mL to 30 mg / mL, or from 5 mg / mL to 20 mg / mL; non-limiting examples include about 5 mg / mL, about 10 mg / mL, about 11 mg / mL, about 12 mg / mL, about 13 mg / mL, about 14 mg / mL, about 15 mg / mL, about 16 mg / mL, about 17 mg / mL, about 20 mg / mL, about 25 mg / mL, about 30 mg / mL, about 35 mg / mL, or any value in between any two of the recited values. In some embodiments, the concentration of the anti-LIV-1 antibody-drug conjugate is from 5 mg / mL to 25 mg / mL. In some embodiments, the concentration of the anti-LIV-1 antibody-drug conjugate is from 10 mg / mL to 20 mg / mL. In some embodiments, the concentration of the anti-LIV-1 antibody-drug conjugate is 15 mg / mL.

[0028] In some embodiments, the buffer in the pharmaceutical composition is a histidine salt buffer. In some embodiments, the histidine salt buffer is a histidine-hydrochloric acid histidine buffer, a histidine-hydrochloric acid buffer, or a histidine-acetic acid buffer. In some embodiments, the buffer in the pharmaceutical composition is a histidine-hydrochloric acid histidine buffer.

[0029] In some embodiments, the concentration of the buffer is 10 mM to 50 mM. In some embodiments, the concentration of the buffer is, for example, 10 mM to 15 mM, 10 mM to 30 mM, 15 mM to 25 mM, 15 mM to 30 mM, 15 mM to 50 mM; non-limiting examples include about 15 mM, about 20 mM, about 25 mM, about 30 mM, about 35 mM, about 40 mM, about 45 mM, about 50 mM, or any value in between any two of these values. In some embodiments, the concentration of the buffer is 20 mM.

[0030] In some embodiments, the stabilizer in the pharmaceutical composition is sucrose, trehalose, mannitol, or a combination thereof. In some embodiments, the concentration of the stabilizer is 40 mg / mL to 100 mg / mL. In some embodiments, the concentration of the stabilizer is, for example, 40 mg / mL to 70 mg / mL, 70 mg / mL to 90 mg / mL, or 80 mg / mL to 100 mg / mL; non-limiting examples include about 40 mg / mL, about 50 mg / mL, about 60 mg / mL, about 65 mg / mL, about 70 mg / mL, about 75 mg / mL, about 80 mg / mL, about 85 mg / mL, about 90 mg / mL, about 95 mg / mL, about 100 mg / mL, or any value in between any two of these values. In some embodiments, the concentration of the stabilizer is 80 mg / mL.

[0031] In some embodiments, the surfactant in the pharmaceutical composition is a polysorbate. In some embodiments, the polysorbate is selected from polysorbate 20 or polysorbate 80. In some embodiments, the concentration of the surfactant is 0.01 mg / mL to 1.0 mg / mL. In some embodiments, the concentration of the surfactant is, for example, 0.01 mg / mL to 0.1 mg / mL, 0.1 mg / mL to 0.5 mg / mL, 0.1 mg / mL to 0.4 mg / mL, 0.1 mg / mL to 0.3 mg / mL, or 0.5 mg / mL to 1 mg / mL; non-limiting examples include about 0.02 mg / mL, about 0.05 mg / mL, about 0.1 mg / mL, about 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, or any value in between any two of these values. In some embodiments, the concentration of the surfactant is 0.2 mg / mL.

[0032] In some embodiments, the pharmaceutical composition has a pH of 5.0 to 7.0. In some embodiments, the pharmaceutical composition has a pH of, for example, 5.0 to 5.5, 5.0 to 6.0, 5.5 to 6.0, 6.0 to 6.5, or 6.5 to 7.0; non-limiting examples include 5.0, 5.2, 5.3, 5.4, 5.5, 5.6, 5.8, 6.0, 6.2, 6.3, 6.5, 7.0, or any value in between any two of the recited values.

[0033] In some embodiments, the pharmaceutical composition comprises:

[0034] a) 2 mg / mL to 40 mg / mL anti-LIV-1 antibody-drug conjugate;

[0035] b) 10 mM to 50 mM histidine salt buffer;

[0036] c) 70 mg / mL to 90 mg / mL stabilizer; and

[0037] d) 0.1 mg / mL to 0.5 mg / mL surfactant; and the pharmaceutical composition has a pH of 5.0 to 7.0.

[0038] In some embodiments, the pharmaceutical composition comprises:

[0039] a) 2 mg / mL to 40 mg / mL anti-LIV-1 antibody-drug conjugate;

[0040] b) 10 mM to 50 mM histidine salt buffer;

[0041] c) 70 mg / mL to 90 mg / mL disaccharide; and

[0042] d) 0.1 mg / mL to 0.5 mg / mL polysorbate; and the pharmaceutical composition has a pH of 5.0 to 7.0.

[0043] In some embodiments, the pharmaceutical composition comprises:

[0044] a) 2 mg / mL to 40 mg / mL anti-LIV-1 antibody-drug conjugate;

[0045] b) 10 mM to 50 mM histidine salt buffer;

[0046] c) 70 mg / mL to 90 mg / mL disaccharide; and

[0047] d) 0.1 mg / mL to 0.5 mg / mL polysorbate; and the pharmaceutical composition has a pH of 5.0 to 7.0.

[0048] In some embodiments, the pharmaceutical composition comprises:

[0049] a) 5 mg / mL to 25 mg / mL anti-LIV-1 antibody-drug conjugate;

[0050] b) 15 mM to 30 mM histidine salt buffer;

[0051] c) 70 mg / mL to 90 mM mg / mL sucrose or trehalose; and

[0052] d) 0.1 mg / mL to 0.5 mg / mL polysorbate 20 or polysorbate 80, the pharmaceutical composition having a pH of 5.0 to 6.5.

[0053] In some embodiments, the pharmaceutical composition comprises:

[0054] a) 5 mg / mL to 25 mg / mL anti-LIV-1 antibody-drug conjugate;

[0055] b) 15 mM to 30 mM histidine salt buffer;

[0056] c) 70 mg / mL to 90 mM mg / mL sucrose; and

[0057] d) 0.1 mg / mL to 0.5 mg / mL polysorbate 20, the pharmaceutical composition having a pH of 5.0 to 6.5.

[0058] In some embodiments, the pharmaceutical composition comprises:

[0059] a) 5 mg / mL to 25 mg / mL anti-LIV-1 antibody-drug conjugate;

[0060] b) 15 mM to 30 mM histidine-histidine hydrochloride buffer;

[0061] c) 70 mg / mL to 90 mM mg / mL sucrose; and

[0062] d) 0.1 mg / mL to 0.5 mg / mL polysorbate 20, the pharmaceutical composition having a pH of 5.0 to 6.5.

[0063] In some embodiments, the pharmaceutical composition comprises:

[0064] 10 mg / mL to 20 mg / mL anti-LIV-1 antibody-drug conjugate, 20 mM histidine-histidine hydrochloride buffer, 80 mM sucrose, 0.2 mg / mL polysorbate 20, having a pH of 5.5 or 6.0.

[0065] In some embodiments, the pharmaceutical composition comprises:

[0066] 10 mg / mL to 20 mg / mL anti-LIV-1 antibody-drug conjugate, 20 mM histidine-hydrochloric acid buffer, 80 mM sucrose, 0.2 mg / mL polysorbate 20, pH 5.5 or 6.0.

[0067] In some embodiments, the pharmaceutical composition comprises:

[0068] 10 mg / mL to 20 mg / mL anti-LIV-1 antibody-drug conjugate, 10 mM histidine-hydrochloric acid buffer, 90 mM sucrose, 0.2 mg / mL polysorbate 80, pH 5.5 or 6.0.

[0069] In some embodiments, the pharmaceutical composition comprises:

[0070] 10 mg / mL to 20 mg / mL anti-LIV-1 antibody-drug conjugate, 10 mM histidine-hydrochloric acid buffer, 90 mM arginine, 0.2 mg / mL polysorbate 20, pH 5.5 or 6.0.

[0071] In some embodiments, the pharmaceutical composition comprises:

[0072] 10 mg / mL to 20 mg / mL anti-LIV-1 antibody-drug conjugate, 20 mM histidine-hydrochloric acid buffer, 80 mM trehalose, 0.2 mg / mL polysorbate 20, pH 5.5 or 6.0.

[0073] In some embodiments, the pharmaceutical composition comprises:

[0074] 10 mg / mL to 20 mg / mL anti-LIV-1 antibody-drug conjugate, 20 mM histidine-hydrochloric acid buffer, 80 mM trehalose, 0.2 mg / mL polysorbate 80, pH 5.5 or 6.0.

[0075] In some embodiments, the pharmaceutical composition comprises:

[0076] 10 mg / mL to 20 mg / mL anti-LIV-1 antibody-drug conjugate, 20 mM histidine-hydrochloric acid buffer, 80 mM sucrose, 0.2 mg / mL polysorbate 20, pH 5.5 or 6.0.

[0077] In some embodiments, the pharmaceutical composition comprises:

[0078] 10 mg / mL to 20 mg / mL anti-LIV-1 antibody-drug conjugate, 20 mM histidine- histidine hydrochloride buffer, 80 mM sucrose, 0.3 mg / mL polysorbate 20, at a pH of 5.5 or 6.0.

[0079] In some embodiments, the pharmaceutical composition comprises:

[0080] a) 15 mg / mL anti-LIV-1 antibody-drug conjugate;

[0081] b) 20 mM histidine-histidine hydrochloride buffer;

[0082] c) 80 mg / mL sucrose; and

[0083] d) 0.2 mg / mL polysorbate 20, the pharmaceutical composition having a pH of 5.0 to 6.0.

[0084] In some embodiments, the pharmaceutical composition comprises:

[0085] a) 15 mg / mL anti-LIV-1 antibody-drug conjugate;

[0086] b) 20 mM histidine-histidine hydrochloride buffer;

[0087] c) 80 mg / mL sucrose; and

[0088] d) 0.2 mg / mL polysorbate 20, the pharmaceutical composition having a pH of 5.5 to 6.0.

[0089] In some embodiments, the pharmaceutical composition comprises:

[0090] a) 15 mg / mL anti-LIV-1 antibody-drug conjugate;

[0091] b) 20 mM histidine-histidine hydrochloride buffer;

[0092] c) 80 mg / mL sucrose; and

[0093] d) 0.2 mg / mL polysorbate 20, the pharmaceutical composition having a pH of 5.5.

[0094] In some embodiments, the pharmaceutical composition comprises:

[0095] a) 15 mg / mL anti-LIV-1 antibody-drug conjugate;

[0096] b) 20 mM histidine-histidine hydrochloride buffer;

[0097] c) 80 mg / mL sucrose; and

[0098] d) 0.2 mg / mL polysorbate 20, the pH of the pharmaceutical composition is 6.0.

[0099] It should be understood that the pharmaceutical composition protected by the present disclosure is not limited to the above-mentioned combination. Any combination that can accomplish the technical solution of the present disclosure is within the protection scope of the present disclosure.

[0100] In some embodiments, the composition is a liquid formulation.

[0101] In a second aspect, a method for preparing the pharmaceutical composition of the present disclosure is provided, comprising accurately weighing each component according to the formulation of the composition, preparing with water for injection, and adjusting the pH. Optionally, the method comprises the step of replacing the anti-LIV-1 antibody-drug conjugate composition stock solution with a buffer, wherein the buffer is preferably histidine-hydrochloric acid buffer or histidine-hydrochloric acid buffer.

[0102] A third aspect of the present disclosure discloses a lyophilized formulation prepared by freeze-drying the above-mentioned pharmaceutical composition.

[0103] In some embodiments, the freeze-drying comprises the steps of pre-freezing, primary drying and secondary drying.

[0104] In some embodiments, the pre-freezing step comprises the following steps:

[0105] a. cooling the above-mentioned pharmaceutical composition to a temperature of -40°C or lower;

[0106] b. isothermal holding for at least 120 min;

[0107] c. warming to a temperature between -20°C and -5°C, preferably -10°C;

[0108] d. isothermal holding for at least 120 min;

[0109] e. cooling to a temperature of -40°C or lower; and

[0110] f. isothermal holding for at least 120 min.

[0111] It should be understood that before step a, between step a and b, between step b and c, between step c and d, between step d and e, between step e and f, and after step f, other additional steps can be included to accomplish the technical solution of the present disclosure, and are within the protection scope of the present disclosure.

[0112] In some embodiments, the rate of cooling in step a is from 0.1 °C / min to 1.0 °C / min. In some embodiments, the rate of cooling is, for example, from 0.1 °C / min to 0.2 °C / min, 0.2 °C / min to 0.5 °C / min, 0.4 °C / min to 0.5 °C / min, or 0.5 °C / min to 1.0 °C / min; non-limiting examples include about 0.2 °C / min, about 0.4 °C / min, about 0.45 °C / min, about 0.5 °C / min, about 0.6 °C / min, about 0.8 °C / min, about 1.0 °C / min, or any value between any two of these values. In some embodiments, the rate of cooling is 0.45 °C / min. In some embodiments, the time required for cooling in step a is 100 min.

[0113] In some embodiments, the rate of warming in step c is from 0.1 °C / min to 1.0 °C / min. In some embodiments, the rate of warming is, for example, from 0.1 °C / min to 0.2 °C / min, 0.2 °C / min to 0.5 °C / min, 0.4 °C / min to 0.6 °C / min, or 0.5 °C / min to 1.0 °C / min; non-limiting examples include about 0.2 °C / min, about 0.4 °C / min, about 0.45 °C / min, about 0.5 °C / min, about 0.6 °C / min, about 0.8 °C / min, about 1.0 °C / min, or any value between any two of these values. In some embodiments, the rate of warming is 0.5 °C / min. In some embodiments, the warming in step c is from -40 °C to -10 °C. In some embodiments, the time required for warming in step c is 60 min.

[0114] In some embodiments, the rate of cooling in step e is from 0.1 °C / min to 1.0 °C / min. In some embodiments, the rate of cooling is, for example, from 0.1 °C / min to 0.2 °C / min, 0.2 °C / min to 0.5 °C / min, 0.4 °C / min to 0.6 °C / min, or 0.5 °C / min to 1.0 °C / min; non-limiting examples include about 0.2 °C / min, about 0.4 °C / min, about 0.45 °C / min, about 0.5 °C / min, about 0.6 °C / min, about 0.8 °C / min, about 1.0 °C / min, or any value between any two of these values. In some embodiments, the rate of cooling is 0.5 °C / min. In some embodiments, the cooling in step e is from -10 °C to -40 °C. In some embodiments, the time required for cooling in step c is 60 min.

[0115] In some embodiments, the pharmaceutical composition described above is cooled to -40 °C in step a.

[0116] In some embodiments, the temperature is held isothermally for 120 min to 250 min in step b, preferably, the temperature is held isothermally for 120 min.

[0117] In some embodiments, the temperature is raised to -10°C in step c.

[0118] In some embodiments, the temperature is held isothermally for 120 min to 250 min in step d, preferably, the temperature is held isothermally for 120 min.

[0119] In some embodiments, the temperature is lowered to -40°C in step e.

[0120] In some embodiments, the temperature is held isothermally for 120 min to 250 min in step f, preferably, the temperature is held isothermally for 120 min.

[0121] In some embodiments, the vacuum level of the primary drying and the secondary drying is less than 0.12 mbar; non-limiting examples include about 0.04 mbar, about 0.06 mbar, about 0.08 mbar, about 0.10 mbar and about 0.12 mbar, in some specific embodiments, the vacuum level of the primary drying and the secondary drying is 0.08 mbar.

[0122] In some embodiments, the pre-freezing step is further preceded by a pre-cooling step. The pre-cooling step comprises pre-cooling the shelf to 5°C and maintaining at 5°C for 50 min to 80 min. Preferably, the pre-cooling step comprises pre-cooling the shelf to 5°C and maintaining at 5°C for 60 min.

[0123] The fourth aspect of the present disclosure discloses a reconstituted solution, which is prepared by reconstituting the above-mentioned freeze-dried formulation.

[0124] In some embodiments, the reconstituted solution comprises:

[0125] a) 2 mg / mL to 40 mg / mL of anti-LIV-1 antibody-drug conjugate;

[0126] b) 10 mM to 50 mM of histidine salt buffer;

[0127] c) 70 mg / mL to 90 mg / mL of stabilizer; and

[0128] d) 0.1 mg / mL to 0.5 mg / mL of surfactant; the pH value of the reconstituted solution is 5.0 to 7.0.

[0129] In some embodiments, the reconstituted solution comprises:

[0130] a) 5 mg / mL to 25 mg / mL of anti-LIV-1 antibody-drug conjugate;

[0131] b) 15 mM to 30 mM histidine salt buffer;

[0132] c) 70 mg / mL to 90 mg / mL sucrose or trehalose; and

[0133] d) 0.1 mg / mL to 0.5 mg / mL polysorbate 20 or polysorbate 80, the reconstituted solution having a pH of 5.0 to 6.5.

[0134] In some embodiments, the reconstituted solution comprises:

[0135] a) 2 mg / mL to 40 mg / mL anti-LIV-1 antibody-drug conjugate;

[0136] b) 10 mM to 50 mM histidine salt buffer;

[0137] c) 70 mg / mL to 90 mg / mL disaccharide; and

[0138] d) 0.1 mg / mL to 0.5 mg / mL polysorbate, the reconstituted solution having a pH of 5.0 to 7.0.

[0139] In some embodiments, the reconstituted solution comprises:

[0140] a) 2 mg / mL to 40 mg / mL anti-LIV-1 antibody-drug conjugate;

[0141] b) 10 mM to 50 mM histidine salt buffer;

[0142] c) 70 mg / mL to 90 mg / mL disaccharide; and

[0143] d) 0.1 mg / mL to 0.5 mg / mL polysorbate, the reconstituted solution having a pH of 5.0 to 7.0.

[0144] In some embodiments, the pharmaceutical composition comprises:

[0145] a) 5 mg / mL to 25 mg / mL anti-LIV-1 antibody-drug conjugate;

[0146] b) 15 mM to 30 mM histidine salt buffer;

[0147] c) 70 mg / mL to 90 mg / mL sucrose; and

[0148] d) 0.1 mg / mL to 0.5 mg / mL polysorbate 20, the reconstituted solution having a pH of 5.0 to 6.5.

[0149] In some embodiments, the reconstituted solution comprises:

[0150] a) 5 mg / mL to 25 mg / mL anti-LIV-1 antibody-drug conjugate;

[0151] b) 15 mM to 30 mM histidine salt buffer;

[0152] c) 70 mg / mL to 90 mM mg / mL sucrose; and

[0153] d) 0.1 mg / mL to 0.5 mg / mL polysorbate 20, the reconstituted solution having a pH of 5.0 to 6.5.

[0154] In some embodiments, the reconstituted solution comprises:

[0155] a) 15 mg / mL anti-LIV-1 antibody-drug conjugate;

[0156] b) 20 mM histidine-hydrochloric acid histidine buffer;

[0157] c) 80 mg / mL sucrose; and

[0158] d) 0.2 mg / mL polysorbate 20, the reconstituted solution having a pH of 5.0 to 6.0.

[0159] In some embodiments, the reconstituted solution comprises:

[0160] a) 15 mg / mL anti-LIV-1 antibody-drug conjugate;

[0161] b) 20 mM histidine-hydrochloric acid histidine buffer;

[0162] c) 80 mg / mL sucrose; and

[0163] d) 0.2 mg / mL polysorbate 20, the reconstituted solution having a pH of 5.5 to 6.0.

[0164] In some embodiments, the reconstituted solution comprises:

[0165] a) 15 mg / mL anti-LIV-1 antibody-drug conjugate;

[0166] b) 20 mM histidine-hydrochloric acid histidine buffer;

[0167] c) 80 mg / mL sucrose; and

[0168] d) 0.2 mg / mL polysorbate 20, the reconstituted solution having a pH of 5.5.

[0169] In some embodiments, the reconstituted solution comprises:

[0170] a) 15 mg / mL anti-LIV-1 antibody-drug conjugate;

[0171] b) 20 mM histidine-histidine hydrochloride buffer;

[0172] c) 80 mg / mL sucrose; and

[0173] d) 0.2 mg / mL polysorbate 20, the reconstituted solution having a pH of 6.0.

[0174] The fifth aspect of the disclosure discloses a method of preparing an anti-LIV-1 antibody-drug conjugate reconstituted solution, comprising reconstituting a lyophilized formulation to obtain a reconstituted solution. In some embodiments, the lyophilized formulation is reconstituted with water for injection.

[0175] In a sixth aspect, there is provided an article of manufacture comprising a container containing a pharmaceutical composition, a lyophilized formulation or a reconstituted solution as described above.

[0176] For in vivo administration, the formulations of the disclosure should be sterile. The formulations of the disclosure can be sterilized by various methods known in the art, including sterile filtration, irradiation, etc. In one embodiment, the antibody formulation is sterilized by filtration through a pre-sterilized 0.22 micron filter. Sterile compositions for injection can be formulated according to conventional pharmaceutical practice as described in “Remington: The Science & Practice of Pharmacy”, 21stedition, Lippincott Williams & Wilkins, (2005). Formulations comprising antibody-drug conjugates, such as the formulations disclosed herein, can generally be stored in lyophilized form or in solution. It is contemplated that sterile compositions comprising antibody-drug conjugates are placed in containers having a sterile access, for example, an intravenous solution bag or vial having a stopper that can be pierced by a fitting such as a hypodermic needle to retrieve the formulation. In one embodiment, the pharmaceutical compositions of the disclosure are provided in a pre-filled syringe.

[0177] In a seventh aspect, there is provided a pharmaceutical composition, a lyophilized formulation or a reconstituted solution of the disclosure for use in therapy or as a medicament.

[0178] In an eighth aspect, there is provided use of a pharmaceutical composition, a lyophilized formulation or a reconstituted solution of the disclosure in the manufacture of a medicament for the treatment of a disease. In some embodiments, the disease is a tumor or a cancer.

[0179] In some embodiments, the tumor or cancer is selected from the group consisting of breast cancer, bladder cancer, ovarian cancer, pancreatic cancer, hepatocellular cancer, gastric cancer, non-Hodgkin's lymphoma, Hodgkin's lymphoma, acute lymphocytic leukemia, anaplastic large cell lymphoma, multiple myeloma, prostate cancer, non-small cell lung cancer, small cell lung cancer, malignant melanoma, squamous cell carcinoma, glioblastoma, renal cell carcinoma, gastrointestinal tumor, colorectal cancer, glioma, mesothelioma, cervical cancer, triple negative breast cancer, lung cancer, head and neck cancer, esophageal cancer, skin cancer, and endometrial cancer.

[0180] In a ninth aspect, there is provided a method of treating a disease, comprising administering to a subject in need thereof an effective amount of a pharmaceutical composition, a lyophilized formulation or a reconstituted solution of the present disclosure.

[0181] In some embodiments, the disease is a tumor or cancer.

[0182] In some embodiments, the tumor or cancer is selected from the group consisting of breast cancer, bladder cancer, ovarian cancer, pancreatic cancer, hepatocellular cancer, gastric cancer, non-Hodgkin's lymphoma, Hodgkin's lymphoma, acute lymphocytic leukemia, anaplastic large cell lymphoma, multiple myeloma, prostate cancer, non-small cell lung cancer, small cell lung cancer, malignant melanoma, squamous cell carcinoma, glioblastoma, renal cell carcinoma, gastrointestinal tumor, colorectal cancer, glioma, mesothelioma, cervical cancer, triple negative breast cancer, lung cancer, head and neck cancer, esophageal cancer, skin cancer, and endometrial cancer.

[0183] The pharmaceutical composition of the present disclosure has excellent physical and chemical stability, and is stable under high temperature, light and other conditions, and can be stably stored for a long time. BRIEF DESCRIPTION OF DRAWINGS

[0184] Figure 1 is a schematic diagram of the binding activity of antibody-drug conjugate A-BrAcMMAE to human LIV-1 detected by ELISA method.

[0185] Figure 2 shows the in vitro detection of the proliferation inhibition activity of ADC on cancer cell line Calu-6 cells.

[0186] Figure 3 shows the in vitro detection of the proliferation inhibition activity of ADC on human LIV-1 overexpressing cancer cell line MCF7-ATCC-LIV-1-#7 cells.

[0187] Figure 4 shows the in vitro detection of the proliferation inhibition activity of ADC on human LIV-1 overexpressing cancer cell line MCF7-ATCC-LIV-1-#12 cells.

[0188] Figure 5 shows the in vitro detection of the proliferation inhibition activity of ADC on human LIV-1 overexpressing cancer cell line MCF7-ATCC-LIV-1-#17 cells.

[0189] Figure 6 shows the results of the xenograft MCF7 breast cancer cell line into NSG mice, the dose and administration time are shown in the figure.

[0190] Figure 7 shows the results of the xenograft HCC1806 breast cancer cell line into NSG mice, the dose and administration time are shown in the figure.

[0191] Figure 8 shows the results of the xenograft PC3 prostate cancer cell line into nude mice, the dose and administration time are shown in the figure.

[0192] Figure 9 shows the results of the xenograft Calu-6 lung cancer cell line into nude mice, the dose and administration time are shown in the figure.

[0193] Figure 10 shows the results of the xenograft Calu-6 lung cancer cell line into nude mice, the dose and administration time are shown in the figure.

[0194] Figure 11 shows the results of the xenograft PC3 prostate cancer cell line into nude mice, the dose and administration time are shown in the figure.

[0195] Figure 12 shows the results of the xenograft PA-1 ovarian cancer cell line into nude mice, the dose and administration time are shown in the figure. DETAILED DESCRIPTION

[0196] The term "comprising" encompasses the meaning of "including" as well as "consisting of" and "consisting essentially of", e.g. a composition "comprising" X can consist exclusively of X or can include something additional, e.g. X + Y.

[0197] The singular forms "a", "an" and "the" include plural referents unless the content clearly dictates otherwise.

[0198] "Composition" refers to a mixture of one or more of the anti-LIV-1 antibody-drug conjugates described herein with other chemical components, such as physiologically / pharmaceutically acceptable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration of the active ingredient to an organism and to facilitate absorption of the active ingredient to thereby elicit a biological activity. As used herein, "pharmaceutical composition", "composition", "formulation" and "prescription" are used interchangeably. The solution form of the pharmaceutical composition described herein, if not otherwise specified, the solvent therein is water.

[0199] "Buffer" refers to a substance that resists changes in pH through the action of its acid-base conjugate components. Suitable buffering agents for use in the compositions of the present disclosure include, but are not limited to, histidine-hydrochloric acid buffer, histidine-hydrochloric acid histidine buffer, and histidine-acetic acid buffer.

[0200] A "histidine salt buffer" is a buffer that includes a histidine ion, such as a buffer that includes histidine and / or a histidine salt, where histidine includes histidine and / or a hydrate thereof, and a histidine salt includes a histidine salt and / or a hydrate thereof. Examples of a histidine salt buffer include a histidine-hydrochloric acid buffer, a histidine-acetic acid buffer, or a histidine-histidine hydrochloride buffer, with a preferred histidine salt buffer being a histidine-histidine hydrochloride buffer. Histidine-histidine hydrochloride includes histidine and histidine hydrochloride, where histidine includes histidine and / or a hydrate thereof, and histidine hydrochloride includes histidine hydrochloride and / or a hydrate thereof, and in one particular example, a histidine-histidine hydrochloride buffer includes histidine and histidine hydrochloride monohydrate (C6H9N3O2· HCI · H2O).

[0201] A "stabilizer" refers to a substance that increases the stability of a pharmaceutical composition, and helps prevent protein oxidation and aggregation in a pharmaceutical composition, particularly a liquid pharmaceutical composition. Typical stabilizers for use in the present disclosure include, but are not limited to, sugars, including but not limited to monosaccharides, such as fructose, maltose, galactose, glucose, D-mannose, sorbose, and the like; disaccharides, such as lactose, sucrose, trehalose, cellobiose, and the like; polysaccharides, such as raffinose, melezitose, maltodextrin, dextran, starch, and the like; and sugar alcohols, such as mannitol, xylitol, maltitol, lactitol, xylitol, sorbitol (glucitol), and the like. The sugar can be a sugar alcohol or an amino sugar. Preferably, the sugar is not a reducing sugar. Reducing sugars include, but are not limited to, all monosaccharides, lactose, maltose, and cellobiose. Thus, the sugar is preferably a non-reducing sugar, such as sucrose, trehalose, raffinose, sorbitol, and mannitol. Sucrose is particularly useful in the present disclosure. In the present disclosure, a sugar includes the sugar and / or a hydrate thereof, such as trehalose includes trehalose and / or a hydrate thereof, such as trehalose dihydrate.

[0202] Surfactants suitable for use in the pharmaceutical compositions of the present disclosure include, but are not limited to, nonionic surfactants, ionic surfactants, zwitterionic surfactants, and combinations thereof. Typical surfactants for use in the present disclosure include, but are not limited to, sorbitan fatty acid esters (e.g., sorbitan monocaprylate, sorbitan monolaurate, sorbitan monopalmitate), sorbitan trioleate, glycerol fatty acid esters (e.g., glycerol monocaprylate, glycerol monomyristate, glycerol monostearate), polyglycerol fatty acid esters (e.g., decaglycerol monostearate, decaglycerol distearate, decaglycerol mono linoleate), polyoxyethylene sorbitan fatty acid esters (e.g., polyoxyethylene sorbitan monolaurate, polyoxyethylene sorbitan monooleate, polyoxyethylene sorbitan monostearate, polyoxyethylene sorbitan monopalmitate, polyoxyethylene sorbitan trioleate, polyoxyethylene sorbitan tristearate), polyoxyethylene sorbitol fatty acid esters (e.g., polyoxyethylene sorbitol tetra stearate, polyoxyethylene sorbitol tetraoleate), polyoxyethylene glycerol fatty acid esters (e.g., polyoxyethylene glyceryl monostearate), polyethylene glycol fatty acid esters (e.g., polyethylene glycol distearate), polyoxyethylene alkyl ethers (e.g., polyoxyethylene lauryl ether), polyoxyethylene polyoxypropylene alkyl ethers (e.g., polyoxyethylene polyoxypropylene glycol, polyoxyethylene polyoxypropylene propyl ether, polyoxyethylene polyoxypropylene cetyl ether), polyoxyethylene alkyl phenyl ethers (e.g., polyoxyethylene nonyl phenyl ether), polyoxyethylene hydrogenated castor oil (e.g., polyoxyethylene castor oil, polyoxyethylene hydrogenated castor oil), polyoxyethylene beeswax derivatives (e.g., polyoxyethylene sorbitol beeswax), polyoxyethylene lanolin derivatives (e.g., polyoxyethylene lanolin), and polyoxyethylene fatty acid amides (e.g., polyoxyethylene stearic acid amide); C10-C18 alkyl sulfates (e.g., sodium cetyl sulfate, sodium lauryl sulfate, sodium oleyl sulfate), polyoxyethylene C10-C18 alkyl ether sulfates with an average addition of 2 to 4 moles of ethylene oxide units (e.g., sodium polyoxyethylene lauryl sulfate), and C1-C18 alkyl sulfosuccinates (e.g., sodium lauryl sulfosuccinate); and natural surfactants such as lecithin, glycerophospholipids, sphingomyelin (e.g., sphingomyelin), and sucrose esters of C12-C18 fatty acids. The composition can comprise one or more of these surfactants. Preferred surfactants are polyoxyethylene sorbitan fatty acid esters, such as polysorbate 20, 40, 60, or 80. Polysorbate 20 or 80 (e.g., at a concentration of about 0.2 mg / mL) is particularly suitable.

[0203] As used herein, the terms “about” or “approximately” mean a numerical value within an acceptable margin of error for a specific value determined by a person skilled in the art, the numerical value depending in part on how it is measured or determined (i.e., the limits of the measurement system). For example, in every practice in the art, “about” may mean within or above a standard deviation of 1. Alternatively, “about” or “substantially comprises” may mean a range of up to ±20%, for example, about 5.5 pH means pH 5.5 ± 1.1. Furthermore, particularly for biological systems or processes, the term may mean up to an order of magnitude or up to five times the numerical value. Unless otherwise stated, when a specific value appears in this application and claims, the meaning of “about” or “substantially comprises” should be assumed to be within an acceptable margin of error for that specific value.

[0204] 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, full-length antibodies, and antigen-binding fragments, as long as they exhibit the desired antigen-binding activity. Typically, natural IgG antibodies are heterotetrameric proteins composed of two light chains and two heavy chains linked by disulfide bonds. From the N to C terminus, each heavy chain has one variable region (VH) and three constant domains (CH1, CH2, and CH3). From the N to C terminus, each light chain has one variable region (VL) and one constant light domain (CL). The specific meaning of "antibody" can be determined by a person skilled in the art based on the context.

[0205] An "antibody fragment" or "antigen-binding fragment" is different from a complete antibody molecule, but contains a portion of the complete antibody that retains the antigen-binding ability of the complete antibody. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2, single-domain antibodies, single-chain Fab (scFab), biantibodies, linear antibodies, single-chain antibodies (e.g., scFv); and multispecific antibodies formed from antibody fragments.

[0206] A "complementarity determining region" or "CDR region" or "CDR" is a region in an antibody variable domain that is hypervariable in sequence and forms structurally defined loops ("hypervariable loops") and / or contains antigen contact residues ("antigen contacts"). CDRs are primarily responsible for binding to an epitope of an antigen. The CDRs in a variable domain are typically referred to as CDR1, CDR2, and CDR3, numbered sequentially from the N-terminus. The precise amino acid sequence boundaries of each CDR in a given variable region amino acid sequence can be determined using any of a number of well-known antibody CDR assignment systems, or combinations thereof, including, for example: Chothia based on the three-dimensional structure of the antibody and the topology of the CDR loops (Chothia et al. (1989) Nature 342: 877-883, Al-Lazikani et al., "Standard conformations for the canonical structures of immunoglobulins", Journal of Molecular Biology, 273, 927-948 (1997)), Kabat based on antibody sequence variability (Kabat et al., Sequences of Proteins of Immunological Interest, 4th Ed., U.S. Department of Health and Human Services, National Institutes of Health (1987)), AbM (University of Bath), Contact (University College London), the international ImMunoGeneTics database (IMGT) (http: / / imgt.cines.fr / ), and North CDR definitions based on affinity propagation clustering with a large number of crystal structures. Correspondence between the various numbering systems is well known to those skilled in the art. For example, CDRs can be derived by using the AbYsis database (www.bioinf.org.uk / abysis / sequence_input / key_annotation / key_annotation.cg). In other words, when a CDR sequence under one numbering system and its position in an antibody are provided, the skilled person is able to determine the corresponding CDR sequence under another numbering system and its position in the antibody. The different numbering systems are considered as equivalent technical solutions. In one embodiment, the CDRs of the antibodies of the disclosure are determined according to the Kabat numbering scheme.

[0207] The term "antibody-drug conjugate" (ADC) refers to an antibody or antibody fragment linked to a biologically active toxic drug through a linker. The antibody or antibody fragment described in the present disclosure can be conjugated to an effector molecule by any means. For example, the antibody or antibody fragment can be attached to a toxic drug by chemical or recombinant means. Chemical means for preparing fusions or conjugates are known in the art. The method for conjugating the antibody or antibody fragment and the drug must be able to link the antibody to the toxic drug without interfering with the ability of the antibody or antibody fragment to bind to the target molecule.

[0208] The drug in the antibody-drug conjugate in the present disclosure is a cytotoxic drug or cytotoxic compound, which refers to a substance that inhibits or prevents the function of cells and / or causes cell death or destruction. Cytotoxic drugs can theoretically kill tumor cells at sufficiently high concentrations, but due to lack of specificity, they also cause apoptosis of normal cells, leading to serious side effects. Cytotoxic drugs include toxins such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, radioisotopes (e.g. At211, I131, I125, Y90, Re186, Re188, Sm153, Bi212, P32 and radioisotopes of Lu), chemotherapeutic drugs, antibiotics and nucleolytic enzymes.

[0209] The antibody of the present disclosure can be conjugated to the cytotoxic drug through a conjugating agent. Examples of the conjugating agent can be any one or several of non-selective conjugating agents, conjugating agents utilizing carboxyl groups, peptide chains, conjugating agents utilizing disulfide bonds. The non-selective conjugating agent refers to a compound that forms a covalent bond between the effector molecule and the antibody, such as glutaraldehyde and the like. The conjugating agent utilizing carboxyl groups can be any one or several of aconitic anhydride conjugating agents (such as aconitic anhydride), acylhydrazone conjugating agents (conjugating site is acylhydrazone).

[0210] The DAR value (Drug-Antibody Ratio), i.e. the ratio of drugs / payload molecules conjugated to each antibody, is typically an average value, and is typically in the range of 1-8, e.g. the DAR value can be 3-6, e.g. 3-5, 3.5-4.5, or 4-6; or can be 1, 2, 3, 4, 5, 6, 7 or 8, e.g. 3, 4 or 5; more preferably e.g. 4.

[0211] The spacer units of the present disclosure indirectly link the antibody to the drug moiety, and there are two main types of spacer units: non-cleavable or cleavable. Non-cleavable spacer units are those in which a portion or all of the spacer unit remains bound to the drug moiety after cleavage, particularly enzymatic cleavage, of the amino acid units of the antibody-drug conjugate. Cleavable spacer units release the drug moiety. In some embodiments, the spacer unit is -CH2-C(=0)-.

[0212] In some embodiments of the present disclosure, the anti-LIV-1 antibody is conjugated to a dolastatin derivative, such as auristatin. Auristatins or auristatin peptide analogs and derivatives have been shown to interfere with microtubule dynamics, GTP hydrolysis, and cell division, and have anti-cancer and anti-fungal activity, described, for example, in US5635483; US5780588; US5663149; all of which are incorporated herein by reference in their entirety. The auristatin drug moiety is typically linked to the antibody via a linker, either through the N (amino) terminus or C (carboxy) terminus of the peptide drug moiety. Exemplary auristatin embodiments include N-terminally linked monomethyl auristatin drug moieties DE and DF, disclosed in Senter et al., Proceedings of the American Association for Cancer Research. Vol. 45, Abstract No. 623, March 28, 2004, and described in US2005 / 0238649.

[0213] In one embodiment the auristatin is monomethyl auristatin E (MMAE):

[0214] wherein the wavy line indicates the site of attachment of the linker.

[0215] In one embodiment the auristatin is monomethyl auristatin F (MMAF):

[0216] wherein the wavy line indicates the site of attachment of the linker.

[0217] In one embodiment the linker is attached to a thiol residue of the antibody, such as an anti-LIV-1 antibody, which is obtained by (partial) reduction of the antibody.

[0218] The pharmaceutical compositions described herein can achieve a stable result in which the antibody substantially retains its physical stability and / or chemical stability and / or biological activity after storage, e.g., the pharmaceutical composition substantially retains its physical and chemical stability and its biological activity after storage. The storage period is generally selected based on the intended shelf life of the pharmaceutical composition. There are a variety of analytical techniques to measure protein stability, which can measure stability after storage for a selected period of time at a selected temperature.

[0219] A stable antibody pharmaceutical formulation is one in which no significant changes are observed: upon storage at refrigerated temperatures (2-8°C) for at least 3 months, at least 6 months, at least 1 year, up to 2 years. In addition, a stable liquid formulation includes a liquid formulation that exhibits desirable characteristics after storage at 25°C for a period of time including 1 month, 3 months, 6 months, or at 40°C for 1 month. Typical acceptable criteria for stability are as follows: generally no more than about 10%, for example no more than about 5% of the antibody monomer is degraded as measured by SEC-HPLC. The antibody pharmaceutical formulation is colorless, or clear to slightly opalescent by visual analysis. The concentration, osmolality of the formulation has no more than a ±10% change. Generally no more than about 10%, for example no more than about 5% truncation is observed, generally no more than about 10%, for example no more than about 5% aggregation is formed.

[0220] An antibody "retains its physical stability" in a pharmaceutical formulation if, upon visual inspection of color and / or clarity, or by UV light scattering, size exclusion chromatography (SEC), and dynamic light scattering (DLS), the antibody shows no significant increase in aggregation, precipitation, and / or denaturation. Changes in protein conformation can be assessed by fluorescence spectroscopy, which determines protein tertiary structure, and by FTIR spectroscopy, which determines protein secondary structure.

[0221] An antibody "retains its chemical stability" in a pharmaceutical formulation if the antibody shows no significant chemical changes. Chemical stability can be assessed by detecting and quantifying chemically altered forms of the protein. Degradation processes that frequently alter the chemical structure of proteins include hydrolysis or truncation (assessed by methods such as size exclusion chromatography and SDS-PAGE), oxidation (assessed by methods such as peptide mapping in conjunction with mass spectrometry or MALDI / TOF / MS), deamidation (assessed by methods such as ion exchange chromatography, capillary isoelectric focusing, peptide mapping, isoaspartate measurement), and isomerization (assessed by measuring isoaspartate content, peptide mapping, etc.).

[0222] An antibody "retains its biological activity" in a pharmaceutical formulation if the biological activity of the antibody at a given time is within a predetermined range of the biological activity exhibited at the time of preparation of the pharmaceutical formulation. The biological activity of an antibody can be determined, for example, by an antigen binding assay.

[0223] "Displacement" refers to the displacement of a solvent system in which an antibody protein is dissolved, e.g., a high salt or high osmotic solvent system containing an antibody protein is displaced by physical manipulation using the buffer system of the stable formulation, such that the antibody protein exists in the stable formulation. Physical manipulation includes, but is not limited to, ultrafiltration, dialysis, or resuspension following centrifugation.

[0224] "liquid composition" means an aqueous composition that is not reconstituted from a lyophilizate, which comprises at least one anti-LIV-1 antibody and at least one other excipient (e.g., a buffer). The liquid composition can comprise other excipients (stabilizers, surfactants) and other active ingredients. Such a formulation is also referred to as a "ready-to-use" formulation.

[0225] "lyophilized formulation" means a substantially water-free, dry (e.g., freeze-dried) pharmaceutical composition. Antibody lyophilization techniques are well known in the art, see, e.g., Rey and May (2004) Freeze-Drying / Lyophilization of Pharmaceutical & Biological Products ISBN 0824748689. Lyophilizates are reconstituted to obtain an aqueous composition (reconstitution solution), which is usually used immediately (e.g., within 1-10 days) because lyophilizates often have a limited shelf life after reconstitution.

[0226] Freeze-drying typically comprises the steps of pre-freezing, primary drying and secondary drying, in sequence. Freeze-drying is performed by freezing the formulation and subsequently subliming water at a temperature suitable for primary drying. Under these conditions, the product temperature is below the eutectic point or collapse temperature of the formulation. Typically, the temperature for primary drying ranges from about -40°C to 5°C (assuming that the product remains frozen during primary drying). The size and type of the formulation, the container holding the sample (e.g., a glass vial), and the volume of the liquid determine the time required for drying, which can range from a few hours to several days (e.g., 40-60 hours). The secondary drying phase can be performed at about 0°C to 40°C, depending mainly on the type and size of the container and the type of protein employed. The secondary drying time is determined by the desired residual moisture level in the product, and typically requires at least about 5 hours. Typically, the water content of a lyophilized formulation is less than about 5%, preferably less than about 3%. The pressure can be the same as applied in the primary drying step. Freeze-drying conditions can vary with the formulation and vial size.

[0227] "Pharmaceutically acceptable salt" refers to pharmaceutically acceptable organic or inorganic salts of an anti-LIV-1 antibody-drug conjugate or an agent administered with an anti-LIV-1 antibody-drug conjugate. Exemplary salts include sulfate, citrate, acetate, oxalate, chloride, bromide, iodide, nitrate, bisulfate, phosphate, acid phosphate, isonicotinate, lactate, salicylate, acid citrate, tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate, gluconate, glucaronate, saccharate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, and pamoate (i.e., 1,1' methylene bis-(2 hydroxy 3 naphthoate)) salts. A pharmaceutically acceptable salt can involve inclusion of another molecule such as an acetate ion, a succinate ion, or other counter ion. The counter ion can be any organic or inorganic moiety that stabilizes the charge on the parent compound. Furthermore, a pharmaceutically acceptable salt can have more than one charged atom in its structure. Instances where a parent compound has more than one charged atom are known as polyatomic ions. A pharmaceutically acceptable salt can have one or more charged atoms and / or one or more counter ions.

[0228] "Administering" and "treatment" when applied to an animal, human, experimental subject, cell, tissue, organ or biological fluid, means the contact of an exogenous drug, therapeutic agent, diagnostic agent or composition with the animal, human, subject, cell, tissue, organ or biological fluid. "Administering" and "treatment" can refer to, for example, therapeutic, pharmacokinetic, diagnostic, research and experimental methods. Treatment of a cell includes contact of the agent with the cell, as well as contact of the agent with a fluid that is in contact with the cell. "Administering" and "treatment" also mean in vitro and ex vivo treatment of, for example, a cell by an agent, diagnostic, binding composition or by another cell. "Treatment" when applied to a human, veterinary or research subject, means therapeutic treatment, prophylactic or preventative measures, research and diagnostic applications.

[0229] "Treatment" means the administration of an internal or external therapeutic agent, e.g., comprising any of the compositions disclosed herein, to a subject having one or more symptoms of a disease, where the therapeutic agent is known to have a therapeutic effect on those symptoms. Typically, the therapeutic agent is administered in an amount effective to alleviate one or more symptoms of the disease in the treated subject or population, to induce regression of such symptoms or to inhibit the progression of such symptoms to any clinically measurable extent. The amount of a therapeutic agent effective to alleviate any particular symptom of a disease (also referred to as a "therapeutically effective amount") can vary depending on factors such as the disease state, age, and weight of the subject, and the ability of the drug to elicit a desired effect in the subject. Whether a disease symptom has been alleviated can be assessed by any clinical detection method typically used by a physician or other professional health care provider to assess the severity or progression of the symptom. While embodiments disclosed herein (e.g., methods of treatment or articles of manufacture) can not be effective in alleviating every symptom of a target disease, it is determined that a statistically significant number of subjects should have alleviation of the target disease symptom according to any statistical test known in the art, such as the Student's t-test, the chi-square test, the U-test according to Mann and Whitney, the Kruskal-Wallis test (H-test), the Jonckheere-Terpstra test, and the Wilcoxon test.

[0230] "Effective amount" includes an amount which is enough to ameliorate or prevent the symptoms or conditions of a medical disease. An effective amount also means an amount which is enough to allow or facilitate diagnosis. An effective amount for a particular subject or veterinary subject can vary depending on factors such as the condition to be treated, the overall health status of the subject, the method route and dosage of administration, and the severity of side effects. An effective amount can be the maximum dose or administration regimen that avoids significant side effects or toxic effects.

[0231] "Optional" or "optionally" means that the subsequently described event or circumstance can or can not occur, and that the description includes situations where the event or circumstance occurs and situations where it does not.

[0232] "N / A" means not detected.

[0233] The present disclosure will be explained by specific examples below, but the scope of the present disclosure is not limited thereto.

[0234] Unless otherwise specified, the mice, proteins, cells used in the following examples are provided by Shanghai Reiviz Chemical Research Co., Ltd.

[0235] Example 1. Preparation of Anti-LIV-1 Antibodies

[0236] The present disclosure relates to a method of immunizing mice with a fragment of human LIV-1 extracellular region to prepare antibodies, and a method of humanizing murine antibodies against human LIV-1. Mice are injected with a composition comprising a fragment of human LIV-1 extracellular region, and the presence of antibodies is identified by taking serum samples. Lymphocytes in the lymph nodes of the mice are obtained, and specific B cells against human LIV-1 antigen are enriched, and single B cell sequencing and analysis are performed to obtain antibody sequences.

[0237] After obtaining the antibody sequences, the antibodies can be prepared by recombinant technology, and chimerization and humanization of the murine antibodies are performed. Specifically, the mouse complementarity determining regions are transferred from the heavy and light chain variable regions of the mouse immunoglobulin to the corresponding variable regions of the human antibody, and then the corresponding back mutations are performed on the human sequence framework region as needed, so as to prepare the humanized antibody. After humanization of the antibody, the CDR region can be optionally affinity matured or hotspot sites are removed to improve the performance of the antibody in certain aspects.

[0238] The antibodies prepared in this example include monoclonal antibodies A, B, and C, wherein antibody A is a humanized antibody, and antibodies B and C are chimeric antibodies, wherein the heavy chain sequence of antibody A is shown in SEQ ID NO: 1, the heavy chain variable region sequence is shown in SEQ ID NO: 2, the light chain sequence is shown in SEQ ID NO: 3, and the light chain variable region sequence is shown in SEQ ID NO: 4. The CDR sequences of antibody A are shown in Table 1 below. The mVH sequence of the murine antibody of antibody A is shown in SEQ ID NO: 11, and the mVL sequence is shown in SEQ ID NO: 12.

[0239] Table 1: CDR sequences of antibody A

[0240] Antibody A heavy chain:

[0241] Antibody A heavy chain variable region:

[0242] Antibody A light chain:

[0243] Antibody A light chain variable region:

[0244] Murine antibody mVH of antibody A (where the underlined part is the CDR):

[0245] Murine antibody mVL of antibody A (where the underlined part is the CDR):

[0246] Further, the binding affinity of the prepared antibody to human LIV-1 antigen (with His tag, amino acid sequence see NP_036451.1, comprising amino acids at positions Phe229-Ile323) was detected. The results showed that the binding activity of antibody A to human LIV-1 was significantly improved after humanization, as shown in Table 2. The binding affinity was detected by ForteBio.

[0247] Table 2: Binding activity of anti-LIV-1 antibody to LIV-1 protein

[0248] Example 2. Binding of anti-LIV-1 antibody to LIV-1-expressing cell lines

[0249] The specific binding ability of antibody A to cell surface-expressed LIV-1 antigen was detected using a flow cytometer.

[0250] The cell lines selected in this experiment were: Calu-6 (ATCC HTB-56, anaplastic lung cancer), HCC1806 (ATCC CRL-2335, human breast cancer TNM stage IIB level 2), PC3 (ATCC CRL-1435, prostate cancer), MCF7 (ATCC HTB-22, breast cancer metastatic site) overexpressing LIV-1, i.e. MCF7-ATCC-LIV-1 #7, #12, #17. Calu-6 and HCC1806 cells were cultured in RPMI-1640 medium containing 10% fetal bovine serum, PC3 cells were cultured in F12K medium containing 10% fetal bovine serum, and MCF7-ATCC-LIV-1 overexpressing cell lines were cultured in EMEM medium containing 10% fetal bovine serum and 1 μg / mL puromycin at 37°C in a 5% CO2 incubator. After dissociating each cell line and washing in staining buffer (Biolengend), the number of cells was counted and adjusted to 2 x 10 5 5 ​Cells were incubated in a solution of 100 μL cells / cell and reacted at 4°C for 15 minutes. After the cell reaction, cells were washed in staining buffer and single-cell readings on the PE channel were analyzed using a Novocyte 3000 (Agilent) device. Negative controls were treated with a non-specific isotype control commercial antibody IgG (BioXCell, BE0297) followed by treatment with a PE-labeled constant region (Fc)-specific antibody. To compare the binding of antibody A to different cancer cells, the quotient of the shifted reading of the experimental group treated with antibody A as disclosed herein divided by the shifted reading of the control group is expressed as the MFI ratio: MFI of anti-LIV-1 antibody / MFI of control antibody. The experimental results are shown in Table 3.

[0251] Experiments have demonstrated and confirmed that the anti-LIV-1 antibody disclosed herein specifically binds to LIV-1 expressed in various cancer cell lines of breast cancer, lung cancer, and prostate cancer.

[0252] Table 3: Staining readings of antibody A binding to various cell lines

[0253] Example 3. Synthesis of compounds used to prepare antibody-drug conjugates

[0254] In this embodiment, experimental methods without specific conditions are generally performed under standard conditions or as recommended by the raw material or product manufacturer. Reagents without a specific source are commercially available, standard reagents.

[0255] The structure of the compound was determined by nuclear magnetic resonance (NMR) and / or mass spectrometry (MS). NMR shifts (δ) were expressed in 10⁻¹⁰ ohms. -6 The unit (ppm) is given. 1 The H NMR measurements were performed using a Bruker AVANCE-400 NMR spectrometer, with deuterated dimethyl sulfoxide as the solvent and tetramethylsilane (TMS) as the internal standard.

[0256] MS was determined using a Shimadzu LCMS-2020 Single Quadrupole liquid chromatography-mass spectrometry system (manufacturer: Shimadzu, MS model: 2020 Single Quadrupole MS).

[0257] RP-HPLC analysis was performed using a Shimadzu LC-2030c Plus liquid chromatograph. Preparative chromatography was conducted using a Shimadzu Nexera preparative HPLC system with a Phenomenex Gemini NX 5μm, C18 column. 150 x 50 mm, mobile phase: 0.1% aqueous solution of trifluoroacetic acid / 0.1% acetonitrile (ACN) solution of trifluoroacetic acid.

[0258] Silica gel column chromatography was performed using SiliCycle (Canada) 200-300 mesh silica gel as the support.

[0259] 3.1 Synthesis of compound 11

[0260] The synthesis procedure is as follows:

[0261] To a solution of compound 1 (25 mg) in acetonitrile / water (6 / 4, v / v, 2 mL) was added saturated NaHC03solution (0.02 mL) and bromoacetic anhydride (10 mg). The reaction was stirred at room temperature for 10 min, and the mixture was directly purified by RP-HPLC to give compound 11 as a white powder (21 mg).

[0262] MS determination: m / z 1243.6 [M+H] + .

[0263] 1 H NMR (400 MHz, DMSO-d6) δ 10.00 - 10.01 (m, 1H), 8.02 - 8.32 (m, 3H), 7.56 - 7.89 (m, 3H), 7.15 - 7.35 (m, 7H), 5.97 (t, J = 6.0 Hz, 1H), 5.33 - 5.41 (m, 3H), 4.96 - 5.09 (m, 2H), 4.63 - 4.73 (m, 1H), 4.48 - 4.50 (m, 1H), 4.36 - 4.45 (m, 2H), 4.23 - 4.28 (m, 2H), 3.91 - 4.04 (m, 4H), 3.47 - 3.79 (m, 2H), 3.11 - 3.36 (m, 9H), 2.85 - 3.08 (m, 7H), 2.39 - 2.43 (m, 1H), 2.26 - 2.30 (m, 1H), 1.17 - 2.14 (m, 15H), 0.73 - 1.05 (m, 30H).

[0264] 3.2 Synthesis of compound 22 (MC-VC-PAB-MMAE)

[0265] The synthesis procedure is as follows:

[0266] To a solution of compound 1 (62 mg) in anhydrous DMF (2 mL) was added maleimide caproic acid (12 mg), followed by DIEA (0.02 mL) and HATU (20 mg). The reaction mixture was stirred at room temperature (22 °C). After 15 min, the crude reaction mixture was directly purified by RP-HPLC to give compound 22 as a white solid (62 mg, TFA salt) after lyophilization.

[0267] MS determination: m / z 1316.8 [M+H] + .

[0268] Preparation and physicochemical characterization of antibody-drug conjugates

[0269] The antibody prepared in Example 1 is treated with a reducing agent, such as tris(2-carboxyethyl)phosphine hydrochloride (TCEP) or dithiothreitol (DTT), to reduce some or all of the cysteine disulfide residues, forming highly nucleophilic cysteine thiol groups (-CH2SH). The partially or fully reduced antibody is then reacted with a drug linker or linker reagent and an electrophilic functional group, such as a maleimide.

[0270] For example, antibody A is dissolved in PBS buffer at pH 7.2, 2 mM EDTA is added, and the antibody is reduced with TCEP (molar ratio of TCEP: antibody is 2.8: 1). After incubation at 37 °C for about 120 minutes, drug linker compound 22 is added to the reduced antibody (molar ratio of drug linker: reduced antibody is 5: 1), and 5% (v / v) DMSO is added. After about 1 hour at room temperature, the ADC is purified by desalting with G25 resin into phosphate buffer (pH 7.4), filtered under sterile conditions with a 0.2 pm filter, and stored frozen. Analysis by hydrophobic interaction chromatography-high performance liquid chromatography determines the average DAR value of the ADC to be between 3.5 and 4.0.

[0271] For example, antibody A is dissolved in PBS buffer at pH 7.2, 2 mM EDTA is added, and the antibody is reduced with TCEP (molar ratio of TCEP: antibody is 2.8: 1). After incubation at 37 °C for about 120 minutes, drug linker compound 22 is added to the reduced antibody (molar ratio of drug linker: reduced antibody is 5: 1), and 5% (v / v) DMSO is added. After about 1 hour at room temperature, the ADC is purified by desalting with G25 resin into phosphate buffer (pH 7.4), filtered under sterile conditions with a 0.2 pm filter, and stored frozen. Analysis by hydrophobic interaction chromatography-high performance liquid chromatography determines the average DAR value of the ADC to be between 3.5 and 4.0.

[0272] Method for detecting DAR values:

[0273] The DAR values of the ADCs of the present disclosure were analyzed using hydrophobic interaction chromatography-high performance liquid chromatography (HIC-HPLC). The ADCs were separated in a chromatographic column using a MabPac HIC-Butyl analytical column (4.6 x 100 mm, 5 μm, Cat# 088558, ThermoFisher, USA). 25 mM sodium phosphate buffer (pH 6.8) containing 1.5 M ammonium sulfate was used as buffer solution A, 25 mM sodium phosphate buffer (pH 6.8) containing 25% acetonitrile was used as buffer B, and 85% buffer solution A and 15% buffer solution B were stabilized as initial conditions, using a linear gradient of 85% buffer solution A and 15% buffer solution B compared to 5% buffer solution A and 95% buffer solution B for 30 minutes, and an additional 5 minutes elution using 5% buffer solution A and 95% buffer solution B. The flow rate and temperature were set at 0.5 mL / min and 25 °C. The ADC drug profiles were detected at 214 nm and 280 nm for DAR value calculation.

[0274] Exemplarily, the prepared partial antibody drug conjugates (ADCs) are shown in Table 4.

[0275] Table 4: ADCs prepared in the present disclosure

[0276] Preparation of control L-McMMAE

[0277] Ladiratuzumab antibody, abbreviated as L, was prepared according to the reference patent US2013259860A1, the heavy chain sequence of which is shown in SEQ ID NO: 13, and the light chain sequence of which is shown in SEQ ID NO: 14; the antibody L was conjugated with MC-MMAE (compound 22) to obtain L-McMMAE with a DAR value of 3.5-4, which was used for subsequent in vivo efficacy test.

[0278] Heavy chain of Ladiratuzumab:

[0279] Light chain of Ladiratuzumab:

[0280] Example 5. Antibody-drug conjugate binding activity with LIV-1 antigen

[0281] Dilute hLIV-1 to 2 μg / mL with coating buffer, add to the enzyme-labeled plate, and coat at 4°C for 15-20 hours. Add 300 μL of blocking solution to each well, and block at room temperature for 1 hour. Dilute the antibody-drug conjugate to be tested with dilution solution, and incubate at room temperature for 1 hour. Dilute the mouse anti-toxin monoclonal antibody with dilution solution, and incubate at room temperature for 1 hour. Dilute the goat anti-mouse Fc domain secondary antibody with dilution solution, and incubate at room temperature for 1 hour. After the secondary antibody incubation is complete, incubate with TMB color developing solution at room temperature for 1-10 minutes. After the color development is complete, add 50 μL of stop solution (4 M sulfuric acid) to each well to stop the substrate reaction. Measure the absorbance on an enzyme-labeled instrument, read the absorbance value of each well at a detection wavelength of 450 nm, and analyze the data using Prism software, with the naked antibody A protein concentration as the horizontal coordinate, the corresponding average absorbance as the vertical coordinate, and the Sigmoidal, 4PL four-parameter equation to draw the dose response curve. The equation is Y=Bottom+(X^Hillslope)*(Top-Bottom) / (X^HillSlope+EC50^HillSlope).

[0282] The results are shown in FIG. 1, and the antibody-drug conjugate of the present disclosure can specifically bind to the LIV-1 antigen.

[0283] Example 6. In vitro killing effect of antibody-drug conjugate

[0284] The ADCs according to the present disclosure are tested in vitro for their potency and selectivity by determining their cytotoxicity in relevant cancer cell lines, such as those expressing the antigen corresponding to the antibody portion of the ADC and similar cancer cell lines lacking the antigen.

[0285] In this experiment, the inhibitory effect of anti-LIV-1 antibody-drug conjugates, such as A-BrAcMMAE, on the proliferation of each tumor cell line was studied.

[0286] In this experiment, the anti-proliferative effect of the drug was evaluated using the CellTiterGlo2 (Promega) reagent. The heat-resistant luciferase contained in the reagent can catalyze the monooxygenation of luciferin in the presence of Mg2+, ATP produced by living cells, and molecular oxygen, producing a stable “glowing” luminescence signal, thereby determining the number of living cells in the culture by quantifying the marker of metabolically active cells, ATP.

[0287] The cell lines selected in this experiment were: human lung cancer (undifferentiated) cell line Calu-6, human breast cancer cells MCF7-ATCC-LIV-1 #7, #12, #17 overexpressing LIV-1.

[0288] Calu-6 cells were cultured in RPMI-1640 medium containing 10% fetal bovine serum, and MCF7-ATCC-LIV-1 overexpressing cell line was cultured in EMEM medium containing 10% fetal bovine serum and 1 μg / mL puromycin at 37°C in a 5% CO2 incubator. Four kinds of cells were seeded into 96-well plates at a density of 2 x 10 3 -5 x 10 3 cells per well, 50 μL / well, and after 24 h of culture, different concentrations of A-BrAcMMAE or control antibody-MMAE conjugate (IgG1-BrAcMMAE) diluted in culture medium were added, 100 μL / well, and each concentration was set in duplicate wells, and solvent control and cell-free medium wells of the corresponding concentration were set, and after 96 h of culture at 37°C in a 5% CO2 incubator, 100 μL CellTiterGlo2 was added per well, and the mixture was placed on an orbital shaker at room temperature for 15 min, and the luminous value was determined, and the IC 50 values (nM) of A-BrAcMMAE conjugate on various cells were calculated. The calculation results of the inhibition of proliferation of the four tumor cells are shown in FIGS. 2, 3, 4 and 5 and Table 5.

[0289] Table 5: IC 50 values (nM) of MMAE conjugate on the proliferation of each cell line

[0290] As can be seen from Table 5 and FIGS. 2, 3, 4 and 5, A-BrAcMMAE has obvious killing effect on three kinds of tumor cells with different expression amounts of LIV-1, and the killing effect is proportional to the expression amount of LIV-1. And it has obvious inhibition effect on the proliferation of breast cancer and lung cancer cells.

[0291] Example 7. In vivo efficacy study of antibody drug conjugate

[0292] CD1 athymic nude mice were implanted with tumor cells grown in culture: Calu-6 from ATCC (2.5 x 10 6 cells in 50% matrigel), PC3 from ATCC (2.5 x 10 6 cells), PA-1 from ATCC (5 x 10 6 cells in 50% matrigel). NSG mice were implanted with tumor cells grown in culture: HCC1806 from ATCC (2.5 x 10 6 cells in 50% matrigel), MCF-7 from NCI (10 x 10 6 cells, and in order to make MCF-7 grow in vivo, 8.5 μg / mL estradiol was added to the drinking water of female mice).

[0293] When the tumor grew to 150 mm3 At this time, start using an anti-LIV-1 ADC or an unbound control ADC (1, 3, 6, or 10 mg / kg), administered intravenously once every four days for a total of four doses (Q4D×4), or once weekly for a total of two doses (Q1W×2), or as a single intravenous dose. Monitor tumor volume using calipers; when the tumor volume reaches approximately 2000 mmHg... 3 Animals were euthanized as needed. Tumor volume was measured in each group until one or more animals were euthanized. All animal procedures were performed in facilities accredited by the Laboratory Animal Care Assessment and Accreditation Association, according to protocols approved by the Institutional Animal Care and Use Committee. The TGI calculation formula is as follows:

[0294] TGI(%)=1-[(Td-T0) / (Cd-C0)]×100%

[0295] Where Td and Cd are the average tumor volumes of the treatment group and the control group on the day of tumor volume measurement, and T0 and C0 are the average tumor volumes of the treatment group and the control group on day 0.

[0296] The results are shown in Figures 6-10. In the efficacy study for MCF7 xenografts, tumors completely regressed in 4 mice on day 22 after administration. The TGI was 126.3% on day 36 after administration. In the efficacy study for HCC1806 xenografts, the TGI was 102.99% on day 21 after administration. In the efficacy study for PC3 xenografts, the TGI was 101.5% on day 11 after administration. In the efficacy study for Calu-6 xenografts, in the multiple-dose groups, the TGI was 75.14% in the 3 mg / kg group and 111.56% in the 6 mg / kg group on day 25 after administration; and completely regressed in 7 mice in the 6 mg / kg group on day 28 after administration. In the single-dose groups, the TGI was 52.72% in the 6 mg / kg group and 76.96% in the 10 mg / kg group on day 25 after administration. These results indicate that A-BrAcMMAE exhibits significant antitumor efficacy in different tumor models.

[0297] In addition, this disclosure also tested the tumor-suppressing effects of A-BrAcMMAE and the existing LIV-1-targeting ADC ladiratuzumab vedotin (abbreviated as L-McMMAE) in PC3 and PA-1 mouse tumor models, and the results are shown in Figures 11 and 12 and Tables 6-1 and 6-2.

[0298] Table 6-1: Tumor inhibition rate of different ADCs on subcutaneous tumors in PC3 mice

[0299] Table 6-2: Tumor inhibition rates of different ADCs against PA-1 mouse subcutaneous tumors

[0300] The above results show that A-BrAc-MMAE can delay or prevent tumor growth in LIV-1 expressing prostate cancer (PC3) and ovarian cancer (PA-1) models, showing significant anti-tumor activity. Although L-McMMAE reported in the previous literature also has anti-tumor activity, A-BrAcMMAE is significantly more effective than L-McMMAE in mouse tumor models. Therefore, A-BrAcMMAE antibody-drug conjugates can be used to treat a variety of LIV-1 expressing cancers.

[0301] Example 8. Screening of stable formulations of anti-LIV-1 antibody-drug conjugates

[0302] The solutions of different compositions of A-BrAcMMAE were prepared with water for injection, and after being freeze-dried into freeze-dried formulations by freeze-drying process 2 (see Example 9), each formulation was subjected to a stability test. The experimental conditions of the stability test are shown in Table 7 below.

[0303] Table 7: Stability test conditions

[0304] Reagents and testing instruments

[0305] Reagents:

[0306] Histidine (purchased from Pfanstiehl), hydrochloric acid histidine (purchased from Pfanstiel), sucrose (purchased from Merck KGaA), polysorbate 20 (purchased from Croda), citric acid (purchased from Merck KGaA), sodium hydroxide (purchased from Hunan Erkang Pharmaceutical Co., Ltd.), sodium dihydrogen phosphate (purchased from Hunan Jiudian Hongyang Pharmaceutical Co., Ltd.), mannitol (purchased from Merck KGaA).

[0307] Instruments:

[0308] Agilent 1200 liquid chromatograph (purchased from Agilent, USA), nanometer laser particle size instrument Zetasier Nano-ZS ZEN3600 (purchased from Malvern Instruments), Agilent 7100 capillary electrophoresis instrument (purchased from Agilent, USA).

[0309] Experimental method:

[0310] SEC-HPLC method: Column: TSK-GEL G3000 SWXL, 7.8 x 300 mm, 5 pm; Mobile phase: 0.0188 mol / L sodium dihydrogen phosphate, 0.5 mol / L sodium chloride buffer, pH 7.0; Flow rate: 0.5 mL / min; Column temperature: 25 °C; Run time: 35 min; Wavelength: 280 nm.

[0311] Non-reduced CE-SDS method: Instrument: Agilent 7100 capillary electrophoresis; Capillary effective length: 225 mm, total length: 310 mm; Injection voltage: -10.0 kv for 40 s; Run voltage: -15.0 kv; Run time: 40 min; Detection wavelength: 214 nm; Column temperature: 20 °C.

[0312] iCIEF method: The sample mixture to be analyzed was loaded into the isoelectric focusing capillary, and the capillary isoelectric focusing detection had the cathode as the sample detection inlet end and the anode as the outlet end, with the cathode solution being 100 mM NaOH (containing 0.1% methyl cellulose solution) and the anode solution being 80 mM H3PO4(containing 0.1% methyl cellulose solution). The focusing voltage and time used were 1.5 kV for 1 min and then 3 kV for 10 min.

[0313] HIC-HPLC method: Column: TOSOH TSKgel Butyl-NPR, 4.6 mm x 10 cm, 2.5 pm; Flow rate: 0.5 mL / min; Column temperature: maintained at 30 °C; Injection volume: 10 pL; Sample concentration: 4 mg / mL; Detection wavelength: 280 nm; Mobile phase A: ammonium sulfate 1.5 mol / L, sodium dihydrogen phosphate 26.3 mM, pH 6.8; Mobile phase B: sodium dihydrogen phosphate 26.3 mM, 25% isopropanol; Gradient elution run for 30 min, according to the following Table 8 gradient, where A(%) and B(%) refer to the volume percentage of A phase and B phase, respectively.

[0314] Table 8

[0315] Protein content detection method: The protein content was determined using the high-throughput protein detection equipment Lunatic of Unchained Labs. The absorbance of the sample at 280 nm was determined, and the extinction coefficient 1.46 L*g -1 *cm -1 was used to calculate the protein content. The concentration of the ADC of the present disclosure was calculated according to the antibody protein concentration.

[0316] 8.1. Screening of buffers for anti-LIV-1 antibody-drug conjugate formulations

[0317] The anti-LIV-1 antibody-drug conjugate A-BrAcMMAE obtained by the conjugation was ultrafiltration-replaced with 5 mM histidine-hydrochloric acid to replace the original buffer in the conjugation product. The protein content in the concentrated solution after ultrafiltration-replacement was determined, and according to the protein content in the concentrated solution, the following preparations were prepared:

[0318] Table 9: Preparation composition of buffer screening experiment

[0319] After the above preparation combinations were prepared into solutions and prepared into freeze-dried preparations by freeze-drying process 2 (see Example 9), they were stored at 5°C±3°C as 0-time samples, high-temperature and light tests were carried out according to Table 7, and appearance inspection was carried out on preparation combinations F1-F6. Visible foreign matter, purity (SEC-HPLC, non-reducing CE-SDS, icIEF, HIC-HPLC) detection was carried out on preparation combinations F1-F2.

[0320] The appearance and visible foreign matter results are as follows:

[0321] In addition to prescriptions F1 and F2, different degrees of collapse occurred in the remaining prescriptions, and even unqualified samples were separated from the container. The reason may be that the collapse temperatures of different prescriptions are different, resulting in cracking and other phenomena under the same freeze-drying process. The freeze-dried preparations of combinations F1 and F2 were reconstituted with sterile injection water, and the composition of the reconstituted solution is shown in Table 10:

[0322] Table 10: Composition of reconstituted solution

[0323] Under the conditions of 0-time, high-temperature and light tests, each reconstituted solution sample of preparation combinations F1 and F2 had no visible foreign matter.

[0324] Purity (SEC-HPLC, non-reducing CE-SDS, icIEF, HIC-HPLC) detection was carried out on the reconstituted solutions of preparation combinations F1 and F2, and the results are as follows:

[0325] SEC-HPLC detection results:

[0326] Table 11: SEC-HPLC detection results

[0327] The SEC-HPLC results show that under the conditions of high-temperature test for 28 days, high-temperature test for 14 days and light test for 10 days, the main peak content and the aggregate peak content of preparation combinations F1 and F2 are basically unchanged, and the fragment content slightly increases at high temperature for 28 days, all of which meet the preset quality reference standard. The above results show that the preparation combinations F1 and F2 of the present disclosure have good stability under high-temperature and light conditions.

[0328] Non-reduced CE-SDS detection results: x

[0329] Table 12: Non-reduced CE-SDS detection results

[0330] The results show that the main peak of the anti-LIV-1 antibody-drug conjugate detected by non-reduced CE-SDS is composed of peaks 1-6, and under high temperature conditions and light test conditions, the contents of peaks 1-6 of F1 and F2 are basically unchanged. The above results show that the disclosed formulation combinations F1 and F2 have good stability under high temperature and light conditions.

[0331] icIEF, HIC-HPLC detection results: x

[0332] Table 13: icIEF, HIC-HPLC detection results

[0333] Isoelectric point analysis: Under high temperature and light test conditions, the isoelectric points of combinations F1 and F2 did not change significantly, indicating that the overall charge distribution of the protein remained stable.

[0334] icIEF acid-base peak analysis: Under high temperature and light test conditions, the contents of the acidic peaks and main peaks of icIEF changed slightly, while the basic peaks were basically unchanged, further confirming that the charge variant distribution of the product was relatively stable.

[0335] HIC-HPLC analysis: Under high temperature and light test conditions, the DAR values were basically unchanged, all meeting the preset quality reference standard (within the range of 3.8±0.5), indicating that the drug-antibody binding ratio was relatively stable.

[0336] Based on the comprehensive stability study results, combinations F1 and F2 have good appearance and high stability, and the histidine-hydrochloric acid buffer can be used as the buffer for the disclosed antibody-drug conjugate A-BrAcMMAE, with a pH range of 5.5-6.0.

[0337] 8.2 Stability detection of anti-LIV-1 antibody-drug conjugate formulations

[0338] To improve the efficiency of adjusting the pH of the formulation during production, the 20 mM histidine-hydrochloric acid buffer system was replaced with an equivalent 20 mM histidine-hydrochloric acid histidine buffer system, and the remaining components remained unchanged, and the stability of the formulation was checked. The formulation components are shown in Table 14 below.

[0339] Table 14: Formulation prescription composition

[0340] The solution formulation containing the above ingredients was prepared, and after being prepared into a lyophilized formulation by lyophilization process 2 (see Example 9), it was stored at 5°C ± 3°C as a 0-time sample. High-temperature, light, and accelerated tests were carried out according to Table 7, and appearance and visible foreign matter inspection were performed. The lyophilized formulation was reconstituted with water for injection, and the composition and content of the reconstituted solution were the same as those of the solution formulation before lyophilization.

[0341] The reconstituted solution was subjected to protein content and purity (SEC-HPLC, non-reducing CE-SDS, icIEF, HIC-HPLC) tests, and the results are shown in Table 15 below.

[0342] Appearance and visible foreign matter inspection results: x

[0343] The lyophilized formulation and the reconstituted solution samples were good in appearance at 0 time and under each test condition, and there were no visible foreign matters in the reconstituted solution.

[0344] Table 15: Stability results

[0345] The results show that:

[0346] After being subjected to light for 10 days or high temperature for 14 or 28 days or accelerated test, the SEC-HPLC main peak content, non-reducing CE-SDS main peak content, charge isomer, DAR value, and isoelectric point did not change significantly compared with 0 time. As can be seen from the above, the formulation containing 15 mg / mL anti-LIV-1 antibody-drug conjugate A-BrAcMMAE, 20 mM histidine-histidine hydrochloride, 80 mg / mL sucrose, and 0.2 mg / mL polysorbate 20, pH 5.5 is good in stability.

[0347] 8.3 Long-term stability test of anti-LIV-1 antibody-drug conjugate formulation

[0348] A solution formulation containing 15 mg / mL anti-LIV-1 antibody-drug conjugate A-BrAcMMAE, 20 mM histidine-histidine hydrochloride, 80 mg / mL sucrose, and 0.2 mg / mL polysorbate 20, pH 5.5 was prepared, and after being prepared into a lyophilized formulation by lyophilization process 2 (see Example 9), it was stored at 5°C ± 3°C as a 0-time sample. The formulation was placed in a low-temperature refrigerator (5°C ± 3°C) for 18 months to carry out a long-term stability test. Appearance and visible foreign matter inspection were performed, and the protein purity (SEC-HPLC, IEC-HPLC, non-reducing CE-SDS) of the reconstituted solution was tested, wherein the composition and content of the reconstituted solution were the same as those of the solution formulation before lyophilization.

[0349] Table 16: Long-term stability results

[0350] The results show that the appearance of the samples is good and no visible foreign matter is observed at 0 hour and under each test condition.

[0351] SEC-HPLC shows no significant change under long-term stability conditions; IEC-HPLC shows no significant change under long-term stability conditions; non-reduced CE-SDS shows no significant change under long-term stability conditions; reduced CE-SDS shows no significant change under long-term stability conditions. It can be seen that the long-term stability of the preparation is good.

[0352] Example 9. Lyophilization process optimization of anti-LIV-1 antibody-drug conjugate preparation

[0353] The antibody-drug conjugate A-BrAcMMAE is mixed with a buffer, a stabilizer and a surfactant to obtain a liquid preparation; the components of the liquid preparation are shown in Table 17 below.

[0354] Table 17: Components of the liquid preparation of anti-LIV-1 antibody-drug conjugate

[0355] The above liquid preparation is lyophilized in a lyophilizer (Model Martin Christ Epsilon 2-6D LSCplus) using Process 1 and Process 2, respectively, and the lyophilization steps include pre-freezing, primary drying and secondary drying in sequence. Process 2 increases the pre-freezing annealing step on the basis of Process 1, which helps the sample to form a larger and uniform ice crystal structure, and reduces the vacuum requirement in the primary drying and secondary drying stages, and increases the mass transfer rate in the lyophilization process. The parameters and experimental steps of Process 1 and Process 2 are shown in Table 18 below.

[0356] Table 18: Lyophilization process parameters

[0357] After obtaining the lyophilized preparation, it is stored at 5°C±3°C, and the appearance of the samples prepared by lyophilization processes 1 and 2 is checked.

[0358] The results show that the appearance of the samples prepared by lyophilization process 1 shows bottom edge shrinkage, and the overall appearance of the samples prepared by lyophilization process 2 is good without obvious shrinkage at the bottom edge. Therefore, lyophilization process 2 is more suitable for lyophilization of anti-LIV-1 antibody-drug conjugate.

Claims

1. A pharmaceutical composition comprising an anti-LIV-1 antibody-drug conjugate, a buffer, a stabilizer and a surfactant; wherein: the anti-LIV-1 antibody comprises a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 in the sequence shown in SEQ ID NO: 2, and a light chain variable region comprising LCDR1, LCDR2 and LCDR3 in the sequence shown in SEQ ID NO: 4; preferably, the heavy chain variable region comprises HCDR1 shown in SEQ ID NO: 5, HCDR2 shown in SEQ ID NO: 6 and HCDR3 shown in SEQ ID NO: 7, and the light chain variable region comprises LCDR1 shown in SEQ ID NO: 8, LCDR2 shown in SEQ ID NO: 9 and LCDR3 shown in SEQ ID NO: 10; more preferably, the heavy chain variable region of the anti-LIV-1 antibody comprises the amino acid sequence shown in SEQ ID NO: 2, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 4; most preferably, the anti-LIV-1 antibody comprises a heavy chain comprising the amino acid sequence shown in SEQ ID NO: 1, and a light chain comprising the amino acid sequence shown in SEQ ID NO:

3. 2.The pharmaceutical composition of claim 1, wherein the drug of the anti-LIV-1 antibody-drug conjugate is auristatin or a functional peptide analogue or derivative thereof; preferably, the drug is monomethyl auristatin E (MMAE) or monomethyl auristatin F (MMAF) ; more preferably, the average absolute number of drug moieties per anti-LIV-1 antibody-drug conjugate is an integer or a decimal number in the range of 1-8. wherein Ab represents the anti-LIV-1 antibody, and z is a decimal number or an integer in the range of 1-8; more preferably, z is in the range of 3.5-4.

5. 4.The pharmaceutical composition of any one of claims 1-3, wherein the concentration of the anti-LIV-1 antibody-drug conjugate is in the range of 2 mg / mL-40 mg / mL, preferably in the range of 5 mg / mL-25 mg / mL, more preferably 15 mg / mL. 5.The pharmaceutical composition of any one of claims 1-4, wherein the buffer is a histidine salt buffer; preferably, the histidine salt buffer is a histidine-hydrochloric acid buffer, a histidine-hydrochloric acid buffer or a histidine-acetic acid buffer; preferably, the concentration of the buffer is in the range of 10 mM-50 mM, more preferably in the range of 15 mM-30 mM, most preferably, the concentration of the buffer is 20 mM. 6.The pharmaceutical composition of any one of claims 1-5, wherein the pH value of the pharmaceutical composition is in the range of 5.0-7.0, preferably in the range of 5.0-6.5; more preferably, the pH value of the pharmaceutical composition is in the range of 5.5-6.

0.

3. The pharmaceutical composition according to claim 1 or 2, wherein the anti-LIV-1 antibody-drug conjugate is linked to the anti-LIV-1 antibody through a linker-Val-Cit-PAB-; preferably, the anti-LIV-1 antibody-drug conjugate has the structure shown below: ​ ​ ​ ​ 7. The pharmaceutical composition of any one of claims 1 to 6, wherein the stabilizer is sucrose, trehalose, mannitol or a combination thereof; preferably, the concentration of the stabilizer is 40 mg / mL to 100 mg / mL, more preferably 70 mg / mL to 90 mg / mL; most preferably, the concentration of the stabilizer is 80 mg / mL.

8. The pharmaceutical composition of any one of claims 1 to 7, wherein the surfactant is polysorbate; preferably, the polysorbate is selected from polysorbate 20 or polysorbate 80; preferably, the concentration of the surfactant is 0.01 mg / mL to 1.0 mg / mL, more preferably 0.1 mg / mL to 0.5 mg / mL, most preferably 0.2 mg / mL.

9. The pharmaceutical composition of any one of claims 1 to 8, comprising: a) 2 mg / mL to 40 mg / mL anti-LIV-1 antibody-drug conjugate; b) 10 mM to 50 mM histidine salt buffer; c) 70 mg / mL to 90 mg / mL stabilizer; and d) 0.1 mg / mL to 0.5 mg / mL surfactant; and the pH of the pharmaceutical composition is 5.0 to 7.0; preferably, the pharmaceutical composition comprises: a) 5 mg / mL to 25 mg / mL anti-LIV-1 antibody-drug conjugate; b) 15 mM to 30 mM histidine salt buffer; c) 70 mg / mL to 90 mg / mL sucrose or trehalose; and d) 0.1 mg / mL to 0.5 mg / mL polysorbate 20 or polysorbate 80, and the pH of the pharmaceutical composition is 5.0 to 6.5; more preferably, the pharmaceutical composition comprises: a) 15 mg / mL anti-LIV-1 antibody-drug conjugate; b) 20 mM histidine-histidine hydrochloride buffer; c) 80 mg / mL sucrose; and d) 0.2 mg / mL polysorbate 20, and the pH of the pharmaceutical composition is 5.0 to 6.

0.

10. A lyophilized formulation prepared by lyophilizing the pharmaceutical composition of any one of claims 1 to 9.

11. The lyophilized formulation of claim 10, wherein the lyophilization comprises the steps of pre-freezing, primary drying and secondary drying; preferably, the pre-freezing step comprises the steps of: a. cooling the pharmaceutical composition of any one of claims 1 to 9 to a temperature of -40 °C or lower; b. isothermally holding for at least 120 min; c. warming to a temperature between -20 °C and -5 °C, preferably -10 °C; d. isothermally holding for at least 120 min; e. cooling to a temperature of -40 °C or lower; and f. isothermally holding for at least 120 min.

12. The lyophilized formulation of claim 11, wherein the vacuum of the primary drying and the secondary drying is lower than 0.12 mbar; preferably, the vacuum of the primary drying and the secondary drying is 0.08 mbar. ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ 13. A reconstituted solution prepared by reconstituting the lyophilized formulation of any one of claims 10 to 12; preferably, the reconstituted solution comprises: a) 5 mg / mL to 25 mg / mL of the anti-LIV-1 antibody-drug conjugate; b) 15 mM to 30 mM of a histidine salt buffer; c) 70 mg / mL to 90 mg / mL of sucrose or trehalose; and d) 0.1 mg / mL to 0.5 mg / mL of polysorbate 20 or polysorbate 80, the pharmaceutical composition having a pH of 5.0 to 6.

5.

14. An article of manufacture comprising a container housing the pharmaceutical composition of any one of claims 1 to 9, the lyophilized formulation of any one of claims 10 to 12, or the reconstituted solution of claim 13.

15. Use of the pharmaceutical composition of any one of claims 1 to 9, the lyophilized formulation of any one of claims 10 to 12, or the reconstituted solution of claim 13 in the manufacture of a medicament for treating a tumor or cancer; preferably, the tumor or cancer is selected from the group consisting of breast cancer, bladder cancer, ovarian cancer, pancreatic cancer, hepatocellular cancer, gastric cancer, non-Hodgkin’s lymphoma, Hodgkin’s lymphoma, acute lymphocytic leukemia, anaplastic large cell lymphoma, multiple myeloma, prostate cancer, non-small cell lung cancer, small cell lung cancer, malignant melanoma, squamous cell carcinoma, glioblastoma, renal cell carcinoma, gastrointestinal tumor, colorectal cancer, glioma, mesothelioma, cervical cancer, triple negative breast cancer, lung cancer, head and neck cancer, esophageal cancer, skin cancer, and endometrial cancer.

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