Pharmaceutical compositions of recombinant humanized anti-Nectin-4 monoclonal antibody-MMAE drug conjugates

Abstract

The present invention relates to a pharmaceutical composition comprising (i) an antibody-drug conjugate, (ii) a histidine buffer, (iii) trehalose, and (iv) a surfactant, and a method for producing the pharmaceutical composition.

Description

Detailed Description of the Invention

[0001] [Technical Field] The present invention relates to specific antibody-drug conjugate formulations and methods for producing such formulations.

[0002] [Background technology] Nectin-4, also known as poliovirus-like receptor 4 (PVRL4), is a type I transmembrane protein encoded by the Nectin-4 gene. It is primarily expressed in the placenta and weakly to moderately in normal human tissues, including skin, bladder, salivary gland, esophagus, breast, and stomach. However, it is overexpressed in various cancer tissues (e.g., breast, lung, and ovarian cancers), and has been found to be expressed in 62% of triple-negative breast cancers. Overexpression of Nectin-4 during cancer progression can promote intratumoral angiogenesis and tumor growth. Furthermore, the PI3K / AKT signaling pathway is involved in Nectin-4-mediated promotion of cancer cell proliferation. Overexpression of Nectin-4 is associated with poor prognosis in lung, breast, and ovarian cancers, and high Nectin-4 expression is a risk factor for lymph node metastasis in patients with papillary thyroid carcinoma (PTC). Depletion of Nectin-4 effectively inhibited the proliferation and invasion of two PTC cell lines (i.e., TPC1 and KTC-1) in vitro and induced apoptosis. Overexpression of Nectin-4 in human esophageal cancer tissues was closely associated with tumor size, tumor invasion depth, and poor patient prognosis. Interventions targeting Nectin-4 expression in esophageal cancer cell lines suggest that increased Nectin-4 expression can significantly promote cell activity, migration, invasion, and tumor formation. The difference in expression levels between tumor and normal tissues makes Nectin-4 a promising target for ADC drug development.

[0003] In the development of antibody and antibody-drug conjugate (ADC) formulations, aggregate formation and degradation product generation can cause pharmacologic undesirable side effects, resulting in increased risk of immunogenicity or venous disease in patients undergoing drug therapy. Based on these factors, it is necessary to suppress aggregate formation and degradation product generation when preparing related formulations. In light of these factors, researchers have investigated various pharmaceutical formulation forms and formulations (e.g., aqueous injection and lyophilized injection forms). Similarly, when developing antibody-drug conjugate drug formulations, more technical challenges are faced, and the specific properties of not only the antibody moiety but also the drug-linker moiety must be considered. For example, small molecule toxins may be shed during storage of antibody-drug conjugates, which can affect the efficacy and toxicity of the corresponding drug.

[0004] Thus, there remains a need in the art for stable antibody drug conjugate formulations, particularly those applicable to the antibody drug conjugates of the present invention.

[0005] Summary of the Invention The present invention provides an injectable recombinant humanized anti-Nectin-4 monoclonal antibody-MMAE drug conjugate. The antibody is an independently developed recombinant humanized anti-Nectin-4 monoclonal antibody, which is obtained by site-specific modification of glutamine on the antibody heavy chain using microbial transglutaminase (mTgase) catalytic means, followed by coupling with the small molecule microtubule inhibitor drug LND1002. LND1002 is a compound in which an amino-PEG-Val-Cit linker is linked to MMAE (monomethyl auristatin E) via p-amino-benzyloxycarbonyl (pABC). Val-Cit is a dipeptide that is sensitive to lysosomal proteases. After the ADC is internalized, Val-Cit is cleaved, the linker is self-degraded, and pABC is self-cleared, leaving MMAE as a free toxin without a linker. This inhibits tubulin polymerization during the cell division cycle, leading to G2 / M phase block and tumor cell apoptosis, ultimately resulting in tumor cell death and exerting an anti-tumor effect. The average drug-antibody ratio (DAR) is 2.0, with a DAR2 distribution exceeding 70%, ensuring high product uniformity and maintaining good batch-to-batch consistency. Furthermore, the inventors of the present invention conducted extensive experiments to optimize parameters such as pH, buffer salts, stabilizers, and surfactants to determine the optimal formulation of the enzymatic antibody-specific site-drug conjugate, and then used freeze-drying technology to prepare a freeze-dried powder of the Nectin-4 antibody-drug conjugate, which ensures the stability of the physicochemical properties of the antibody moiety while minimizing the loss of free drug and allowing for long-term storage at 2-8°C.

[0006] Specifically, the present invention relates to the following aspects.

[0007] In one aspect, the present invention relates to a pharmaceutical composition comprising: (i) an antibody drug conjugate; (ii) a buffering agent; (iii) a stabilizer; and (iv) a surfactant.

[0008] In some embodiments, the antibody-drug conjugate comprises a drug-linker represented by the following formula (Formula I) coupled to an anti-Nectin-4 monoclonal antibody:

[0009] [ka]

[0010] In some embodiments, the buffering agent includes, but is not limited to, acetate, succinate, gluconate, histidine, oxalate, lactate, phosphate, citrate, tartrate, fumarate, glycylglycine, and other organic acid buffers.

[0011] In some embodiments, the histidine salt buffer is a buffer containing histidine salt ions. Examples of histidine salt buffers include histidine-histidine hydrochloride, histidine acetate, histidine phosphate, histidine sulfate, etc., among which the histidine-acetate buffer is prepared from histidine and acetic acid, and the histidine-histidine hydrochloride buffer is prepared from histidine and histidine hydrochloride. Similarly, the succinate buffer may be prepared from succinic acid and sodium succinate, and the citrate buffer may be prepared from citric acid and sodium citrate.

[0012] In some embodiments, histidine-histidine hydrochloride buffer is preferred.

[0013] In some embodiments, the stabilizer includes, but is not limited to, sugars (such as sucrose and trehalose), polyols (such as mannitol and sorbitol), and amino acids (such as L-serine, monosodium glutamate, alanine, glycine, and sarcosine).

[0014] In some embodiments, the surfactant includes, but is not limited to, polysorbate 20 or polysorbate 80.

[0015] In some embodiments, the pH of the pharmaceutical composition is 5.0 to 7.4, preferably 5.0 to 6.5, and most preferably about pH 6.0.

[0016] In some embodiments, the pharmaceutical composition of the present invention further comprises water.

[0017] In some embodiments, the components of the pharmaceutical composition of the present invention include histidine-histidine hydrochloride at 5 to 30 mM, for example, about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, about 10 mM, about 11 mM, about 12 mM, about 13 mM, about 14 mM, about 15 mM, about 16 mM, about 17 mM, about 18 mM, about 19 mM, about 20 mM, about 21 mM, about 22 mM, about 23 mM, about 24 mM, about 25 mM, about 26 mM, about 27 mM, about 28 mM, about 29 mM, or about 30 mM, and polysorbate 20 or polysorbate 80 at 0.02 wt% to 0.08 wt%, for example, about 0.02 wt%, about 0.03 wt%, about 0.04 wt%, about 0.05 wt%, about 0.06 wt%, about 0.07 wt%, or about 100 wt%. about 0.08 wt%, about 0.08 wt%, trehalose at 4 wt% to 8 wt%, for example, about 4 wt%, about 5 wt%, about 5.5 wt%, about 6 wt%, about 6.5 wt%, about 7 wt%, about 7.5 wt%, or about 8 wt%, and an antibody-drug conjugate at a concentration of 5 to 50 mg / mL, for example, about 5 mg / mL, about 10 mg / mL, about 15 mg / mL, about 20 mg / mL, about 25 mg / mL, about 30 mg / mL, about 35 mg / mL, about 40 mg / mL, about 45 mg / mL, or about 50 mg / mL; and a pH of 5.0 to 6.5, for example, about 5.0, about 5.5, about 6.0, or about 6.5.

[0018] In some embodiments, the components of the drug formulation of the present invention include 5-30 mM histidine-histidine hydrochloride, 0.02 wt%-0.08 wt% polysorbate 20 or polysorbate 80, 5 wt%-7 wt% trehalose, and an antibody-drug conjugate at a concentration of 5-50 mg / mL, and the pH is 5.0-6.5.

[0019] In some embodiments, the components of the drug formulation of the present invention include 10-30 mM histidine-histidine hydrochloride, 0.02 wt%-0.04 wt% polysorbate 20 or polysorbate 80, 5 wt%-6 wt% trehalose, and an antibody-drug conjugate at a concentration of 10-20 mg / mL, and the pH is 5.0-6.0.

[0020] In some preferred embodiments, the components of the drug formulation of the invention include histidine-histidine hydrochloride 20 mM, polysorbate 20 0.02 wt%, trehalose 5.5 wt%, and an antibody drug conjugate at a concentration of 10 mg / mL, and the pH is 6.0.

[0021] In some preferred embodiments, the drug formulation of the invention comprises 10 mg / mL antibody drug conjugate, 1.4 mg / mL histidine, 2.3 mg / mL histidine hydrochloride, 0.2 mg / mL polysorbate 20, 55 mg / mL trehalose, and has a pH of 6.0.

[0022] In some embodiments, the pharmaceutical composition of the present invention further comprises water.

[0023] In some preferred embodiments, the components of the drug formulation of the present invention consist of 20 mM histidine-histidine hydrochloride, 0.02 wt% polysorbate 20, 5.5 wt% trehalose, an antibody-drug conjugate at a concentration of 10 mg / mL, and water for injection, with a pH of 6.0.

[0024] In some preferred embodiments, the components of the drug formulation of the present invention consist of 20 mM histidine-histidine hydrochloride, 0.02 wt% polysorbate 20, 5.5 wt% trehalose, and an antibody-drug conjugate at a concentration of 10 mg / mL, and the pH is 6.0.

[0025] In some preferred embodiments, the liquid pharmaceutical composition can be prepared as a lyophilized formulation, a liquid formulation, or a powder injection formulation.

[0026] In another aspect, the present invention also relates to a method for producing the above-mentioned pharmaceutical composition, comprising the steps of producing an anti-Nectin-4 antibody-drug conjugate, liquid exchange of the antibody-drug conjugate with other components of a liquid pharmaceutical composition followed by sterile filtration and aseptic filling.

[0027] In some embodiments, the present invention also relates to a method for producing the above pharmaceutical composition, comprising the steps of:

[0028] (1) An aqueous solution containing predetermined amounts of substances such as (i) an antibody-drug conjugate, (ii) a buffer, (iii) a stabilizer, and (iv) a surfactant is prepared; (2) the pH of the aqueous solution is adjusted to a predetermined value, if necessary; (3) the solution is sterile filtered and then dispensed into injection bottles.

[0029] In some preferred embodiments, the present invention also provides a method for producing the above pharmaceutical composition, comprising:

[0030] (1) Ultrafiltration of antibody-drug conjugates: Prepare a buffer solution (e.g., histidine-histidine hydrochloride solution) with the same concentration as the final product, and use a 30 kD ultrafiltration membrane package to exchange the liquid. The original affinity chromatography solution is exchanged with the buffer solution (e.g., histidine-histidine hydrochloride solution). The exchange ratio of the liquid exchange by ultrafiltration is 100 times or more, and the pH of the exchange liquid is controlled to match that of the final product. At the same time, the concentration of the antibody-drug conjugate is equal to or greater than the product concentration (optimally ≥ 1.2 times). (2) Preparation of semi-finished dilution: Based on the formulation amount of this product and the drug concentration of the antibody-drug conjugate after liquid exchange by ultrafiltration, the final product preparation amount was calculated, and the required amount of each auxiliary agent was also calculated to prepare the semi-finished dilution solution. Approximately 80% of the water for injection was added to the preparation vessel, and buffer salts, surfactants, and stabilizers (e.g., histidine, histidine hydrochloride, polysorbate 20, trehalose) were weighed out and added to the preparation vessel in order, stirred and mixed. After each group of auxiliary agents was completely dissolved, water for injection was added to the final volume, stirred and mixed uniformly, and filtered for use.

[0031] (3) Regarding product manufacturing: The prepared diluted solution of the semi-finished product is added to the prepared solution containing the antibody-drug conjugate drug solution after liquid exchange by ultrafiltration, and the diluted solution is adjusted to the prepared amount of the semi-finished product. After uniform mixing, the solution is sampled to check the protein content, pH value, and microbial limit. After sterile filtration, the solution is dispensed into sterilized injection bottles in accordance with the product specifications, and then stoppered and capped to obtain the product.

[0032] In another aspect, the present invention further provides a freeze-dried formulation obtained by freeze-drying the liquid pharmaceutical composition. In some embodiments, the method for producing the freeze-dried formulation comprises the steps of pre-freezing, vacuuming, primary drying, and desorption drying.

[0033] In some embodiments, the desorption drying temperature is about 30°C.

[0034] In some embodiments, the conditions for the primary drying process are about −25° C. and 0.15 mbar.

[0035] In another aspect, the present invention provides the use of the pharmaceutical composition described above in the manufacture of a medicament for treating tumors (including cancer).

[0036] In another aspect, the present invention provides a method for treating tumors (including cancer), comprising administering to a subject in need thereof the pharmaceutical composition described above.

[0037] In another aspect, the present invention provides a pharmaceutical composition as described above for treating tumors (including cancer).

[0038] In some embodiments, the above-mentioned cancer is a Nectin-4 positive cancer.

[0039] In some embodiments, the above-mentioned tumors (including cancers) include, but are not limited to, hematological tumors or solid tumors.

[0040] In some embodiments, the above-described tumors (including cancers) are hematological tumors, preferably lymphomas or leukemias, including, but not limited to, myeloma, B-cell lymphoma, mantle cell lymphoma, non-Hodgkin's B-cell lymphoma, non-Hodgkin's T-cell lymphoma, cutaneous lymphoma, anaplastic large cell lymphoma, multiple myeloma, indolent non-Hodgkin's lymphoma, plasmacytoma, chronic lymphocytic leukemia, small lymphocytic lymphoma, and follicular lymphoma. In some embodiments, the hematological tumor is relapsed or refractory.

[0041] In some embodiments, the tumors (including cancers) described above are solid tumors, including, but not limited to, respiratory system tumors, gastrointestinal tumors, urinary system tumors, male organ tumors, female organ tumors, skin cancer, endothelial cell tumors, brain tumors, nervous system tumors, and endocrine organ tumors.

[0042] In some embodiments, such respiratory tumors include, but are not limited to, lung cancer, nasopharyngeal cancer, and laryngeal cancer.

[0043] In some embodiments, the gastrointestinal tumors include, but are not limited to, esophageal cancer, gastric cancer, colon cancer, liver cancer, pancreatic cancer, and bile duct cancer.

[0044] In some embodiments, the urinary system tumors include, but are not limited to, kidney cancer, renal pelvic and ureteral cancer, bladder cancer, and urethral cancer.

[0045] In some embodiments, the male organ tumors include, but are not limited to, penile cancer, prostate cancer, or testicular cancer.

[0046] In some embodiments, the female organ tumor includes, but is not limited to, breast cancer, vulvar cancer, vaginal cancer, cervical cancer, endometrial cancer, or ovarian cancer.

[0047] In some embodiments, the nervous system tumors include, but are not limited to, astrocytoma, oligodendroglioma, ependymoma, medulloblastoma, and meningioma.

[0048] In some embodiments, the brain tumor includes, but is not limited to, a glioma, a neurocytoma, an embryonal mesenchymal tumor, a stromal tumor, an epithelial tumor, a teratoma, and a pineal tumor.

[0049] In some embodiments, the skin cancer includes, but is not limited to, cutaneous melanoma or non-melanoma skin cancer.

[0050] In some embodiments, the tumor is a solid tumor, including, but not limited to, bladder cancer, brain cancer, breast cancer, cervical cancer, breast tumors, endometrial cancer, esophageal squamous cell carcinoma, gastric cancer, head tumors, pancreatic cancer, bile duct cancer, colorectal cancer, eye cancer, head and neck squamous cell carcinoma, urothelial carcinoma, kidney cancer, liver cancer, lymph node cancer, lung cancer, oral cancer, cervical tumors, ovarian cancer, prostate cancer, testicular cancer, laryngeal and uterine cancer, melanoma, salivary gland cancer, fibrosarcoma, soft tissue sarcoma, and osteosarcoma. In some embodiments, the cancer is recurrent or refractory.

[0051] In some embodiments, the breast cancer comprises ductal carcinoma, lobular carcinoma, medullary carcinoma, glioma, tubular carcinoma, inflammatory breast cancer, triple-negative breast cancer (TNBC).

[0052] In some embodiments, the ovarian cancer comprises epithelial ovarian tumors, such as adenocarcinomas in the ovaries and adenocarcinomas that migrate from the ovaries into the peritoneal cavity.

[0053] In some embodiments, the leukemia comprises acute myeloid leukemia (AML), acute lymphocytic leukemia, chronic lymphocytic leukemia, chronic myelogenous leukemia, hairy cell leukemia, myelodysplasia, myeloproliferative disorders, NK cell leukemia (e.g., blastic plasmacytoid dendritic cell neoplasm), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), mastocytosis, chronic lymphocytic leukemia (CLL), multiple myeloma (MM), and myelodysplastic syndromes (MDS).

[0054] In some embodiments, the pancreatic cancer is acinar cell adenocarcinoma of the pancreas, ductal cell adenocarcinoma of the pancreas, or phase IV pancreatic cancer.

[0055] In some embodiments, the lung cancer comprises non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC), in some embodiments, non-small cell lung cancer (NSCLC) includes, but is not limited to, squamous cell carcinoma, adenocarcinoma, and large cell carcinoma, in some embodiments, the lung cancer is recurrent, in some embodiments, the lung cancer is recurrent squamous cell lung cancer or (advanced) phase IV squamous cell lung cancer.

[0056] In some embodiments, the prostate cancer is metastatic castration-resistant prostate cancer (mCRPC).

[0057] In some embodiments, the pharmaceutical compositions of the present invention can be selectively used as drugs in chemotherapy, the primary method of treating cancer, to delay the onset of cancer cells, inhibit their growth, and kill cancer cells. These actions can achieve therapeutic effects by sparing cancer patients from symptoms caused by cancer or by improving their quality of life and preserving their lives. Even if the pharmaceutical compositions and treatment methods of the present invention do not achieve cancer cell death, they can inhibit or control cancer cell growth, thereby realizing a higher quality of life and enabling longer survival for cancer patients.

[0058] In another embodiment, the pharmaceutical composition of the present invention may be used alone or in combination with other therapies, and may be combined with surgery, radiation therapy, hormone therapy, and the like.

[0059] In some embodiments, the pharmaceutical composition of the present invention can be used in combination with other cancer therapeutic agents, thereby enhancing the anti-cancer effect. Examples of other cancer therapeutic agents for such purposes include, but are not limited to, 5-fluorouracil (5-FU), pertuzumab, trastuzumab, paclitaxel, carboplatin, cisplatin, gemcitabine, capecitabine, irinotecan (CPT-11), docetaxel, pemetrexed, sorafenib, vinblastine, vinorelbine, everolimus, tanespimycin, bevacizumab, oxaliplatin, and lapatinib, as long as they have anti-tumor activity.

[0060] In some embodiments, the antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises three CDR regions having the amino acid sequences set forth in SEQ ID NOs: 1, 2, and 3, respectively, and / or the light chain comprises three CDR regions having the amino acid sequences set forth in SEQ ID NOs: 4, 5, and 6, respectively.

[0061] In some embodiments, the heavy chain comprises a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 7, and / or the light chain comprises a light chain variable region having the amino acid sequence set forth in any one of SEQ ID NOs: 8 to 10.

[0062] In some embodiments, the heavy chain of the antibody comprises a heavy chain constant region having the amino acid sequence set forth in SEQ ID NO: 11 or 12, and / or the light chain of the antibody comprises a light chain constant region having the amino acid sequence set forth in SEQ ID NO: 13.

[0063] In some embodiments, the antibody is a monoclonal or bispecific antibody, preferably a monoclonal antibody, more preferably a humanized antibody, and most preferably a fully human antibody.

[0064] In some specific embodiments, the antibody or functional fragment thereof of the present invention has ADCC activity.

[0065] In some specific embodiments, the antibody or functional fragment thereof of the present invention has CDC activity.

[0066] In some specific embodiments, the antibody or functional fragment thereof of the present invention is an IgG1κ antibody.

[0067] The antibody drug conjugate (ADC) according to the present invention refers to a cytotoxic drug conjugated to an antibody, the antigen of which is expressed on the surface of cancer cells, and the antibody also binds to an antigen that can be internalized in cells, thereby selectively delivering the drug to cancer cells, thereby causing the accumulation of the drug in the cancer cells and killing the cancer cells.

[0068] The histidine hydrochloride according to the present invention is also called histidine hydrochloride, and analogously, the histidine-histidine hydrochloride according to the present invention is also called histidine-histidine hydrochloride (L-histidine, L-histidine monohydrochloride monohydrate).

[0069] The CDR coding system for the antibodies of the present invention is IMGT.

[0070] BRIEF DESCRIPTION OF THE DRAWINGS [Figure 1] Schematic diagram of the mTgase enzymatic ADC coupling reaction. FIG. 2 is a schematic diagram of the structure of an ADC. where LND1002 represents the linker + drug molecule portion, and the LND1002 on the right shows the complete structure, with the circle representing the connection between the linker in LND1002 and the amide bond of the antibody. The ADC in this example is a site-specific antibody-drug conjugate, in which one anti-Nectin-4 monoclonal antibody is coupled to one MMAE molecule via a linker (NH2-PEG3-Val-Cit-pABC) at amino acid Q294 of each heavy chain (i.e., Kabat number Q295), the antibody is connected to the linker via a stable amide bond (isopeptide bond), and the average drug-to-antibody ratio (DAR) is 2.0. [Figure 3] Endocytosis effect experiment of SWY2001-Ab1-LND1002 in SK-BR-3 cells. [Figure 4] Endocytosis effect experiment of SWY2001-Ab1-LND1002 in T47D cells. [Figure 5] In vivo inhibitory effect of SWY2001-Ab1-LND1002 on mouse PC3-Nectin-4 stable tumor line, mpk = mg / kg. [Fig. 6] In vivo inhibitory effect experiment of SWY2001-Ab1-LND1002 on mouse HT-1376 tumor.

[0071] [Example] Example 1: Preparation and efficacy evaluation of Nectin-4 antibody-drug conjugate 1.1. Preparation of antibody-drug conjugates LND1002 (1 g, dissolved in 5 mL of DMSO; structure shown in Figure 2), 10x reaction buffer, anti-Nectin-4 antibody (obtained by expressing a vector containing heavy and light chain variable region-related sequences in host cells using genetic engineering techniques and purifying the antibody; the related sequence refers to the sequence of humanized antibody SWY2001-Ab1 in Table 30, and the constant region is HCl+LC), mTgase (a transglutaminase used to catalyze transglutamination reactions at acceptor glutamines adjacent to N-glycosylation sites and capable of specifically catalyzing the transglutamination reaction at Kabat number Q295; its sequence is shown in SEQ ID NO: 14 (see Table 30)), and 20% HO were added in this order to an EP tube, sealed, mixed uniformly, and then reacted at 30°C for up to 72 hours. When the coupling rate reached 95%, the reaction was terminated and immediately purified to obtain the anti-Nectin-4 antibody-drug conjugate SWY2001-Ab1-LND1002. The reaction conditions were as follows:

[0072] [Table 1]

[0073] Experimental results confirmed that the transglutaminase catalytic site of the present antibody is located at position Q295, close to the N-glycosylation site.

[0074] Experimental measurements showed that the antibody-drug conjugate had a modification rate of 96.32% after 72 hours of reaction, a DAR value of approximately 2, and its main physicochemical properties met the requirements for further experiments.

[0075] 1.2. Endocytosis effect of antibody-drug conjugates SK-BR-3 cells and T47D cells (human breast cancer cells) were collected and resuspended in medium. The target cells were gently blown several times to resuspend them into a single-cell suspension. Cell activity and cell number were identified using trypan blue staining. The cell density was 1 × 10 5The solution was adjusted to a concentration of 1 × 10 cells / mL and inoculated into a 96-well confocal plate cell culture dish at 100 μL / well. 4 It was Zenon TM pHrodo TM iFL-labeled ADCs were added to a 96-well plate at a final concentration of 2 μg / mL and then continuously cultured for 24 hours at 37°C in a 5% CO2 incubator. All images were observed and captured using a 20x objective lens on a laser confocal microscope.

[0076] The experimental results, as shown in Figures 3 and 4, showed that SWY2001-Ab1-LND1002 underwent intracellular endocytosis and localized in lysosomes in an acidic environment, with an endocytosis rate much higher than that of the control drug PADCEV. For antibody-drug conjugates, the endocytosis rate was generally faster, the drug's ability to penetrate tumor cells was stronger, toxin release was better, and the drug's effectiveness was faster. Therefore, the tumor cell penetration ability of the antibody-drug conjugate obtained according to the present application was significantly superior to that of the control drug PADCEV.

[0077] 1.3. In-hydro efficacy studies of ADC Target cells were collected and resuspended to a single cell suspension, and cell viability and cell number were identified using trypan blue staining. A cell density of 1 x 10 5 The diluted sample was added to the plate at 20 μL / well to cells / mL and added at 100 μL / well. The plate was then placed in a cell incubator (37°C, 5% CO2) for 66 ± 3 hours. Resazurin sodium solution (0.03%) was added at 20 μL / well and incubated at 37°C for 3–4 hours. Fluorescence readings were measured at 550 nm / 610 nm using a microplate reader. The readings were plotted using Prism or similar graphics software, and the half-maximal inhibitory concentrations (IC50s) of the reference standard and samples were fitted. The output parameter, C, was the IC50 in ng / mL.

[0078] The experimental results, as shown in Table 2, showed that SWY2001-Ab1-LND1002 could inhibit the growth of 293T-Nectin-4, SK-BR-3, and PC3-Nectin-4 cancer cells in hydrolysis. The 293T-Nectin-4 stable cell in hydrolysis data showed that SWY2001-Ab1-LND1002 was slightly more potent than PADCEV. The SK-BR-3 in hydrolysis data showed that SWY2001-Ab1-LND1002 was four times more potent than PADCEV. The PC3-Nectin-4 in hydrolysis data showed that SWY2001-Ab1-LND1002 had no significant difference from PADCEV.

[0079] [Table 2]

[0080] 1.4. In vivo efficacy studies of ADCs 1. In vivo prostate cancer inhibitory efficacy experiment In this study, female NUNU mice of appropriate age were selected and inoculated with PC3-Nectin-4 stable cell line (a nude mouse model of human prostate cancer with high Nectin-4 expression). 3 Forty-two animals with good tumor growth were selected (day 19 after inoculation). They were divided into six groups (day 0) according to tumor volume: vehicle control, 1 mg / kg Nectin-4-mab (SWY2001-Ab1 monoclonal antibody), 1 mg / kg PADCEV, and 0.5, 1, and 2 mg / kg SWY2001-Ab1-LND1002 groups, with seven animals per group. After administration, mice were weighed and data were recorded. Tumor diameters were measured at various time points after administration to dynamically monitor tumor growth. The study was terminated on day 17 (day 17 after administration). After asphyxiation with carbon dioxide, the tumors were dissected and weighed.

[0081] Under these experimental conditions (see Figure 5), SWY2001-Ab1-LND1002 was able to inhibit tumor growth in a dose-dependent manner (P<0.05). At a dose of 1 mg / kg, the tumor inhibition rate of SWY2001-Ab1-LND1002 was significantly superior to that of PADCEV (58.3% vs. 39.0%). The tumor weight inhibition rates for 1 mg / kg Nectin-4-mab (Nectin-4 monoclonal antibody), 1 mg / kg PADCEV, and 0.5, 1, and 2 mg / kg SWY2001-Ab1-LND1002 groups were 28.2%, 39.0%, 22.1%, 58.3%, and 79.4%, respectively.

[0082] 2. In vivo bladder cancer inhibitory efficacy experiment In this experiment, female NUNU mice of the appropriate age were selected and inoculated with HT-1376 cells at a concentration of 5 × 10 6 The tumor volume was approximately 100 mm 3 Twelve animals with good tumor growth were selected and divided into three groups (D0) according to tumor volume. Group A consisted of four animals in the vehicle control group, administered 0.9% sodium chloride injection (0.9% INJ NS (physiological saline), the vehicle control group). Group B consisted of four animals in the PADCEV experimental group, administered 3 mg / kg of PADCEV (single dose). Group C consisted of four animals in the SWY2001-Ab1-LND1002 experimental group, administered 3 mg / kg of PADCEV (single dose). After administration, mice were weighed and data were recorded. Tumor diameters were measured at various time points after administration to dynamically monitor tumor growth. The study was terminated on D22, and the mice were asphyxiated with carbon dioxide, after which the tumors were dissected and weighed.

[0083] As shown in Figure 6, under these experimental conditions, the tumor inhibition rates of 3 mg / kg PADCEV and 3 mg / kg SWY2001-Ab1-LND1002 were 42.1% and 49.4%, respectively. Compared with the vehicle control group, the 3 mg / kg SWY2001-Ab1-LND1002 (single administration) group significantly inhibited tumor growth. Compared with the positive control 3 mg / kg PADCEV (single administration) group, the 3 mg / kg SWY2001-Ab1-LND1002 group showed significantly improved tumor inhibition.

[0084] 1.5. ADC stability experiment results Plasma stability experiments The stability of SWY2001-Ab1-LND1002 and MMAE in SD rat, cynomolgus monkey, and human plasma was measured using an in-water plasma incubation method at 37°C. PBST was used as a negative control to examine the reliability of the entire test system. Incubation samples were collected from each incubation group at 0 h, 24 h, 48 h (D2), 72 h (D3), 96 h (D4), 168 h (D7), and 336 h (D14). MMAE concentrations in the plasma of various species and PBST were measured using LC-MS / MS. The average percentage MMAE formation results after incubation of SWY2001-Ab1-LND1002 in the plasma of various species and PBST are shown in Table 3.

[0085] [Table 3]

[0086] The experimental results showed that within the test concentration range, after incubating three concentrations of the antibody-drug conjugate in human, monkey, and rat plasma for 336 hours, the amount of the small molecule MMAE produced accounted for approximately 1% of its theoretical amount, demonstrating its good stability.

[0087] Example 2: Screening experiment for optimal pH value pH is an important factor affecting the stability of biological product formulations. It regulates the charge distribution on the protein surface, affecting the inter- and intramolecular forces of proteins, as well as the conformational, colloidal, and chemical stability of proteins. Samples containing 10 mg / mL ADC at various pH values ​​were prepared using a citric acid-disodium hydrogen phosphate buffer system ranging from 4.5 to 8.0. The samples were dispensed into injection vials and stability studies were performed at temperatures ranging from 2 to 8°C and at a high temperature of 50°C to evaluate thermal stability, molecular size variant stability (SEC-HPLC), and charge variant stability (CEX-HPLC).

[0088] Inspection method Regarding DSC: The sample to be measured and the same buffer solution were diluted to a protein concentration of 1 mg / mL, and 350 μL of the sample and the corresponding buffer solution were taken and added to the corresponding positions of a 96-well plate. The 96-well plate was placed in a sample bath, and the temperature of the sample bath was set to 15°C. The instrument parameters were set as follows: starting temperature 20°C, ending temperature 100°C, and heating rate 90°C / min.

[0089] Regarding SEC-HPLC: In accordance with the 2020 edition of the Chinese Pharmacopoeia, Part 4, General Provisions 0512, High-Performance Liquid Chromatography, the analysis was performed using a column packed with a chromatography gel suitable for separating proteins with molecular weights of 10 to 500 kD. The proportions of the protein monomer peak, high molecular weight substance (HMWS) peak, and low molecular weight substance (HMWS) peak were calculated according to the area normalization method.

[0090] CEX-HPLC: According to the Chinese Pharmacopoeia, 2020 edition, Part 4, General Provisions 0512, High Performance Liquid Chromatography, the analysis was performed using a cation exchange column, and the proportions of the acidic peak, main peak, and basic peak of the sample were calculated according to the area normalization method.

[0091] Measurement results

[0092] [Table 4]

[0093] [Table 5]

[0094] [Table 6]

[0095] Based on the thermal stability results of the product at various pH values ​​and the decomposition tendency of the molecular size variants and charge variants of the product after forced high-temperature treatment, the preferred pH was 5.5-6.5.

[0096] Example 3: Screening experiments for buffer components Buffer salts have a significant impact on maintaining the pH value, physicochemical properties, and stability of the formulation. Buffer salts within a preferred pH range were screened to determine their effects. The buffer salts used included citric acid-sodium citrate, histidine-histidine hydrochloride, etc. Various buffer salt samples with an ADC concentration of 10 mg / mL were prepared and dispensed into injection vials. Stability studies were conducted at high temperature (40°C) to evaluate thermal stability, molecular size variant stability, charge variant stability, and changes in the content of free toxin LND1002. The methods for testing thermal stability, molecular size variant stability, and charge variant stability were the same as in Example 2.

[0097] Inspection method Regarding the content of free LND1002: In accordance with the 2020 edition of the Chinese Pharmacopoeia, Part 4, General Provisions 0512, high performance liquid chromatography was carried out using a column packed with octadecyl-bonded silica gel, precipitated with acetonitrile, and after an ice bath, the supernatant was collected and analyzed, and the content of free LND1002 in the sample was calculated using the external standard method.

[0098] Measurement results

[0099] [Table 7]

[0100] [Table 8]

[0101] [Table 9]

[0102] [Table 10]

[0103] As can be seen from the above test results, the thermal stability of the histidine buffer system was superior to that of the citrate buffer system, and the stability of molecular size variants and charge variants at relatively low pH values ​​was relatively higher than that at relatively high pH values. However, as the pH value increased, the content of free toxin LND1002 gradually decreased. Overall, pH 6.0 and the histidine buffer system were preferred.

[0104] Example 4: Stabilizer screening experiment The addition of a certain proportion of sugars (e.g., sucrose, trehalose) not only reduces the aggregation of protein-based drugs and stabilizes monoclonal antibody-based drugs, but also adjusts the osmotic pressure of the product and serves as an excipient for lyophilized products. A sample with an ADC concentration of 10 mg / mL was prepared using a histidine-histidine hydrochloride buffer solution, preferably at pH 6.0, with the addition of sucrose and trehalose. The sample was dispensed into injection vials and subjected to stability studies at high temperatures (40°C) to evaluate its thermal, physical, and chemical stability. Furthermore, pre-lyophilization experiments were performed on samples containing different stabilizers to evaluate the appearance and moisture content of the lyophilized products. The testing method was the same as in Example 2.

[0105] Measurement results

[0106] [Table 11]

[0107] [Table 12]

[0108] [Table 13]

[0109] [Table 14]

[0110] [Table 15]

[0111] As can be seen from the above test results, the thermal stability of the product after adding the stabilizer was slightly increased; the two types of saccharides showed no significant difference in the molecular size variants, charge variants, and free LND1002 content of the samples; the two types of saccharides showed no significant difference in the moisture content of the lyophilized powders as lyophilization excipients; the resolubility of the two types of lyophilized powders was good; and when observing the bottom morphology of the lyophilized powders, it was found that the bottom shrinkage of the sucrose lyophilized powder was slightly more severe than that of trehalose; therefore, trehalose is preferably used as the stabilizer.

[0112] Example 5: Surfactant screening experiment Biopolymer protein-drug systems are prone to aggregation and adsorption during storage. The addition of surfactants can reduce protein aggregation during stirring, shaking, freeze-thawing, and freeze-drying, and prevent protein adsorption to container surfaces. Based on a suitable pH, buffer, and stabilizer, various concentrations of surfactants, including polysorbate 20, were added to prepare samples with an ADC concentration of 10 mg / mL. The samples were dispensed into injection vials and stability studies were conducted under shaking and freeze-thawing conditions. A microfluidic imaging device (Flowcam) was used to detect changes in subvisible particles in the samples under different conditions.

[0113] [Table 16]

[0114] [Table 17]

[0115] The results showed that the addition of the surfactant polysorbate 20 could effectively control the increase in the number of fine particles, and the addition of 0.005% polysorbate 20 had a clear inhibitory effect on particle formation. Taking the above experimental data into consideration, 0.02% polysorbate 20 is preferably used as a component of ADC formulations to prevent ADC from forming aggregates during long-term storage.

[0116] Example 6: Freeze-drying process development experiment A preferred formulation of 10 mg / mL ADC, 20 mmol / L histidine-histidine hydrochloride, 0.02% polysorbate 20, 5.5% trehalose, pH 6.0 was used as the base formulation to develop a freeze-drying process. A sample of this formulation was tested for glass transition temperature and collapse temperature. Low-temperature DSC testing showed that the glass transition temperature of the sample was -29.08°C, and freeze-drying microscopy testing showed that the collapse temperature of the sample was -26.0°C.

[0117] Pre-freezing: The pre-freezing temperature was set at -40°C according to the glass transition temperature. The plate layer temperature was first cooled relatively quickly to -10°C and maintained at that temperature for 30 minutes. After that, the product was cooled rapidly (≥2°C / min) or slowly (≤0.5°C / min) to the pre-freezing temperature of -40°C. The pre-freezing temperature was maintained for 180 minutes or more, completing the freeze-drying process under the same primary and secondary drying conditions. For the freeze-drying process, samples were placed at different diagonal positions on the plate layer. (Positions A and E were primarily near the door or sidewalls, experiencing stronger heat radiation and lower moisture content; center point C was closest to the interior, primarily sublimated by heat conduction and exhibiting higher moisture content. Position A was located on the front door and sidewalls, receiving the most external heat radiation; positions B and D were closer to the interior than positions A and E.) The two pre-freezing methods did not show any significant difference in the moisture content of the freeze-dried powder, and although small ice crystals were formed during the rapid freezing process, the moisture content did not increase compared to slow freezing, so pre-freezing was preferably performed using the rapid freezing method.

[0118] [Table 18]

[0119] Regarding primary drying: The primary drying process removes more than 90% of the water in the product through a sublimation process, and this water is mainly free water. During the primary drying process, the product temperature decreases due to the heat absorption caused by sublimation, and the sample temperature is always lower than the plate layer temperature. Once sublimation is complete, the majority of the water has been removed, and the product temperature rises to the plate layer temperature, completing primary drying. To improve the product sublimation rate, the plate layer temperature should be set above the product's acceptable collapse temperature, but it is important to ensure that the product temperature of the unsublimated portion does not exceed its collapse temperature. Pre-freezing and rapid freezing processes are preferred. The effects of primary drying temperatures (-25°C, -20°C) and front tank pressures (0.15 mbar, 0.2 mbar) were considered, respectively. Different test combinations were designed, and the freeze-drying process was completed under the same pre-freezing and secondary drying conditions. The primary drying process was performed in triplicate. The results showed that the lower the set value of the plate layer temperature for primary drying and the lower the set value of the front tank pressure, the longer the primary drying time. Compared to 0.2 mbar, lowering the set value of the front tank pressure to 0.15 mbar can improve the shrinkage phenomenon of the bottom layer of the freeze-dried powder, and the appearance of the primary drying temperature at -25°C is better than that of -20°C. Taking all of the above factors into consideration, -25°C and 0.15 mbar are preferred for the primary drying process.

[0120] [Table 19]

[0121] Desorption drying: After primary drying, approximately 10% moisture remained in the product, adsorbed to the capillary walls and polar groups of the drying material. When the bound water reaches a certain content, it provides the conditions for microbial growth and proliferation and several chemical reactions. This moisture is bound to the drug through weak molecular forces such as van der Waals forces and hydrogen bonds. Removing this water requires more energy to overcome the intermolecular forces, so the plate temperature should be set below the maximum allowable temperature for the product. Desorption drying temperatures for biological products should not be too high; a desorption drying temperature of 30°C is preferred. Since the plate temperature is the main factor affecting the moisture content of freeze-dried powders during the desorption drying stage, the pressure in the front tank was set to 0.15 mbar, consistent with primary drying.

[0122] Comparing the key qualities of the freeze-dried samples of the preferred process before and after freeze-drying, the results showed that the product quality after freeze-drying did not change significantly, indicating that the freeze-drying process did not affect the product quality (Table 20).

[0123] [Table 20]

[0124] Example 7, Stability of the Formulation Using the optimized formulation (same as in Example 6) and freeze-drying process, three batches of samples were produced under GMP conditions, and stability studies were conducted on the three batches of samples. The detection methods for each research index were all methodologically verified, and the methods were accurate and reliable. Stability studies were conducted at 5±3°C on the raw solution before freeze-drying and the product after freeze-drying.

[0125] Inspection method SEC-HPLC: In accordance with the Chinese Pharmacopoeia, 2020, Part 4, General Provisions 0512, High-Performance Liquid Chromatography, a chromatography column packed with a chromatography gel suitable for separating proteins with molecular weights of 10 to 500 kD (e.g., XBridge® BEH200Å, 7.8 × 300 mm, 200Å, 3.5 μm, or other suitable chromatography column) was used. The mobile phase was 100 mmol / L phosphate buffer, 100 mmol / L NaCl, 10% isopropanol, pH 6.7 ± 0.1, the flow rate was 0.8 mL / min, and the detection wavelength was 280 nm. An appropriate amount of sample solution was injected into the liquid chromatograph, and the proportions of the protein monomer peak, high molecular weight species (HMWS), and low molecular weight species (LMWS) peaks were calculated using the area normalization method.

[0126] CEX-HPLC: According to the Chinese Pharmacopoeia, 2020, Part 4, General Rule 0512, High-Performance Liquid Chromatography, an ion exchange column (e.g., Thermo ProPac WCX-10, 4 x 250 mm or other suitable chromatography column) was used. Mobile phase A was 10 mmol / L PB, pH 6.5, and mobile phase B was 10 mmol / L PB, 300 mmol / L NaCl, pH 6.5. The flow rate was 0.8 mL / min, and the detection wavelength was 280 nm. An appropriate amount of sample solution was injected into the liquid chromatograph and gradient elution was performed. The proportions of the acidic peak, main peak, and basic peak of the sample were calculated according to the area normalization method.

[0127] Regarding R-CE-SDS: The test was performed in accordance with the Chinese Pharmacopoeia, 2020 edition, Part 4, General Principle 3127, Method for Determining Molecular Size Variants of Monoclonal Antibodies, and the proportions of heavy chain and light chain peaks and the proportion of non-glycosylated heavy chain peaks were calculated according to the area normalization method.

[0128] Regarding NR-CE-SDS: The test was conducted in accordance with the Chinese Pharmacopoeia, 2020 edition, Part 4, General Provisions 3127, Method for Determining Molecular Size Variants of Monoclonal Antibodies, and the proportion of the monomer peak and the proportion of the low molecular weight substance (LMWS) peak were calculated according to the area normalization method.

[0129] Regarding icIEF: The test was conducted in accordance with the method for determining monoclonal antibody charge variants in the 2020 edition of the Chinese Pharmacopoeia, Part 4, General Rule 3129. Based on the isoelectric point (PI) characteristics of different charge variants, the samples were separated according to capillary electrophoresis (General Rule 0542), and the isoelectric points of the charge variants were measured to calculate the relative percentage content.

[0130] DAR distribution: According to the Chinese Pharmacopoeia, 2020, Part 4, General Rule 0512, high-performance liquid chromatography, a chromatography column packed with tetraalkyl-bonded silica gel (e.g., PLRP-S 1000A, 2.1 x 50 mm, 5 μm particle size, or other suitable chromatography column) was used. Mobile phase A was 0.1% (v / v) TFA in water, and mobile phase B was 0.1% (v / v) TFA in acetonitrile. The column temperature was 80°C, the flow rate was 0.25 mL / min, and the detection wavelength was 280 nm. An appropriate amount of sample solution was injected into the liquid chromatograph and gradient elution was performed to calculate the DAR value and DAR2 content.

[0131] Regarding the amount of uncoupled antibody: Similar to the DAR distribution detection method, the DAR0 peak content was reported to be the amount of uncoupled antibody.

[0132] Free MMAE content: According to the Chinese Pharmacopoeia, 2020 edition, Part 4, General Provisions 0512 for high performance liquid chromatography and 0431 for mass spectrometry, a chromatography column packed with octadecyl-bonded silica gel (e.g., Waters ACQUITY UPLC® BEH-C18, 2.1 × 50 mm, 1.7 μm particle size or other suitable chromatography column) was used, mobile phase A was a 0.1% TFA-containing aqueous solution, mobile phase B was a 0.1% TFA-containing acetonitrile solution, the column temperature was 40°C, the flow rate was 0.4 mL / min, and the detection wavelength was 250 nm. 50 μL (10 mg / mL) of the sample was taken, and the internal standard working solution was added. The mixture was precipitated with acetonitrile at a ratio of 1:3. The mixture was then placed at -20°C and centrifuged in an ice bath for 60 minutes. The supernatant was then sampled. MMAE of various concentrations was used as a control. The samples were subjected to gradient elution by liquid chromatography and then loaded into a mass spectrometer. After adjusting the parameters such as the mass spectrometer capillary and cone voltage, ion source temperature, collision energy, and atomization flow rate, the samples were analyzed. The content of free MMAE in the sample was calculated based on the standard curve.

[0133] Free MMAE content (%) = MMAE test concentration / ADC concentration × 100% Antigen binding activity was measured by enzyme-linked immunosorbent assay (ELISA).

[0134] Biological activity: Measured by cell growth inhibition assay.

[0135] The research results showed that the product quality after lyophilization was more stable than in the liquid state, the low molecular weight fragments of the product in the liquid state were significantly increased compared to the lyophilized state, the charge variants were more likely to produce basic variants and free MMAE in the liquid state, and the drop rate was more pronounced compared to the lyophilized powder, making the lyophilized dosage form more advantageous for long-term storage of the product.

[0136] [Table 21] JPEG2026507083000023.jpg26169

[0137] [Table 22] JPEG2026507083000025.jpg26169

[0138] [Table 23] JPEG2026507083000027.jpg26169

[0139] The above experimental results indicated that the main physicochemical properties of the ADC lyophilized powder were stable between different batches, and good stability and batch-to-batch consistency were maintained.

[0140] Example 8. Stability of liquid formulations at 40°C for 14 days Preparation of Ab1-ADC, Ab2-ADC, and Ab3-ADC molecules: Ab1 antibody molecule (see Table 30, variable region sequence SWY2001-Ab1, constant region HC1+LC), Ab2 antibody molecule (see Table 30, variable region sequence SWY2001-Ab2, constant region HC1+LC), and Ab3 antibody molecule (see Table 30, variable region sequence SWY2001-Ab3, constant region HC1+LC) were each mixed with LND1002, mTgase, and reaction buffer. After homogeneous mixing, the mixture was incubated at 30°C for 72 hours or less. (Specifically, preparation can be performed with reference to 1.1 of Example 1.) The reaction was terminated when the coupling rate reached 95%. Immediately after completion of the reaction, the mixture was purified to obtain Ab1-ADC, Ab2-ADC, and Ab3-ADC molecules, each with a DAR of approximately 2.0.

[0141] Regarding the preparation of the reference drug molecule: To prepare a molecule corresponding to the same toxin drug PADCEV, which is already on the market for the same target, the antibody was first constructed, and the corresponding antibody molecule was obtained by culturing and purifying. The antibody molecule was then liquid-exchanged into the corresponding buffer, and a TCEP reducing agent was added. After heating in a 37°C water bath for 1 hour, a corresponding amount of toxin (MC-VC-pABC-MMAE) was added and reacted at room temperature for 30 minutes. A corresponding amount of L-cysteine ​​was added, and the reaction was stopped after 20 minutes. After the reaction was completed, the reference molecule was immediately purified to obtain a DAR value of approximately 4.0.

[0142] To compare the stability of Ab1-ADC, Ab2-ADC, Ab3-ADC, and the reference molecule, four sets of ADC molecules were subjected to a liquid exchange with a buffer-free solution of water. The exchanged samples were then filtered through a 0.22 μm membrane filter, and 1.0 mL aliquots were dispensed into sterilized 2.0 mL injection bottles. The stability of the four molecules was analyzed at a high temperature of 40°C. Evaluation criteria included appearance, molecular size variants (SEC-HPLC), charge variants (CEX-HPLC), and free MMAE content (RP-HPLC / MS).

[0143] Analysis method For molecular size variants (SEC-HPLC): A chromatography column packed with a chromatography gel suitable for separating proteins with molecular weights between 10 and 500 kD (e.g., XBridge® BEH 200Å, 7.8 x 300 mm, 200Å, 3.5 μm, or other suitable chromatography column) was selected. The mobile phase was 100 mmol / L phosphate buffer, 100 mmol / L NaCl, 10% isopropanol, pH 6.7 ± 0.1, with a flow rate of 0.8 mL / min and a detection wavelength of 280 nm. An appropriate amount of sample solution was injected into the liquid chromatograph, and the proportions of the protein monomer peak, high molecular weight species (HMWS), and low molecular weight species (LMWS) peaks were calculated using the area normalization method.

[0144] Charge variant (CEX-HPLC): An ion exchange column (e.g., Thermo ProPac WCX-10, 4 x 250 mm, or other suitable chromatography column) was used as the chromatography column. Mobile phase A was 10 mmol / L PB, pH 6.5, and mobile phase B was 10 mmol / L PB, 300 mmol / L NaCl, pH 6.5. The flow rate was 0.8 mL / min, and the detection wavelength was 280 nm. An appropriate amount of sample solution was injected into the liquid chromatograph, and gradient elution was performed. The proportions of the acidic peak, main peak, and basic peak of the sample were calculated according to the area normalization method.

[0145] Free MMAE content (RP-HPLC / MS): Reversed-phase liquid chromatography mass spectrometry was used, using a reversed-phase chromatography column (e.g., Waters ACQUITY UPLC® BEH-C18, 2.1 × 50 mm, 1.7 μm particle size or other suitable chromatography column). Mobile phase A was a 0.1% TFA solution in water, and mobile phase B was a 0.1% TFA solution in acetonitrile. The column temperature was 40°C, the flow rate was 0.4 mL / min, and the detection wavelength was 250 nm. 50 μL (10 mg / mL) of the sample was taken, and the internal standard working solution was added. The mixture was precipitated with acetonitrile at a ratio of 1:3. The mixture was then placed at -20°C and centrifuged in an ice bath for 60 minutes. The supernatant was then sampled. MMAE of various concentrations was used as a control. The samples were subjected to gradient elution by liquid chromatography and then loaded into a mass spectrometer. After adjusting the parameters such as the mass spectrometer capillary and cone voltage, ion source temperature, collision energy, and atomization flow rate, the samples were analyzed. The content of free MMAE in the sample was calculated based on the standard curve.

[0146] Free MMAE content (%) = MMAE test concentration / ADC concentration × 100%

[0147] [Table 24]

[0148] When Ab1-ADC, Ab2-ADC, Ab3-ADC, and the reference molecule were placed in an aqueous solution and treated at a high temperature of 40°C for 14 days, the appearance of the product clearly changed from a clear solution to a milky white solution. The SEC-HPLC results for Ab1-ADC, Ab2-ADC, Ab3-ADC, and the reference molecule showed a decrease in the monomer content, mainly manifested as an increase in the content of HMWS and LMWS. The SEC-HPLC results showed a decrease in the content of the main peak and an increase in the content of the corresponding acid peak and base peak, and the content of free MMAE tended to increase. The four groups of molecules had similar levels of instability in an aqueous system. Compared with Ab1-ADC, Ab2-ADC, and Ab3-ADC molecules, the reference molecule had a higher DAR value and a lower change in free MMAE content, indicating a slower dissociation rate of free MMAE under these conditions.

[0149] Although 0.9% saline is usable as a delivery system, it is not suitable as a drug storage system. The reasons for this incompatibility are that when drugs are stored in 0.9% saline, aggregation and particle formation occur due to the lack of relevant co-stabilizers, and at the same time, 0.9% saline may increase protein-protein adsorption due to electrostatic shielding, and at the same time, 0.9% saline is weakly acidic, which changes the charge state of ADC molecules, thereby reducing their solubility and increasing their tendency to aggregate.

[0150] Since neither aqueous nor 0.9% saline was suitable as a stock solution for the four sets of molecules, the four sets of molecules were formulated to examine their stability.

[0151] The preferred formulation for the Ab1-ADC formulation was 10 mg / mL ADC, 20 mmol / L histidine-histidine hydrochloride, 0.02% polysorbate 20, and 5.5% trehalose (pH 6.0). After coupling of Ab1, Ab2, and Ab3 molecules with LND1002, the ADC molecules and the reference molecule were subjected to ultrafiltration. The resulting samples were filtered through a 0.22 μm membrane filter. After filtration, 1.0 mL aliquots were dispensed into sterilized 2.0 mL injection vials. The stability of the four molecules was analyzed at a high temperature of 40°C. Evaluation parameters included appearance, molecular size variants (SEC-HPLC), charge variants (CEX-HPLC), and free MMAE content (RP-HPLC / MS). The analytical methods were the same as those described above.

[0152] [Table 25]

[0153] Ab1-ADC, Ab2-ADC, Ab3-ADC, and the reference molecule were each placed in the preferred formulation of Ab1-ADC preparation and treated at 40°C for 14 days. The product appearance remained unchanged, and SEC-HPLC results showed a decrease in the monomer content, CEX-HPLC results showed a decrease in the main peak content, and the free MMAE content tended to increase. In the preferred formulation, the changes in the SEC-HPLC monomer content, CEX-HPLC main peak content, and free MMAE content of the four sets of molecules were all smaller than in the aqueous system, demonstrating that the formulation had a protective effect on all four sets of molecules. A comprehensive analysis of the changes in molecular size variants, charge variants, and free MMAE content in the four groups of molecules showed that in this formulation system, the stability of Ab1-ADC, Ab2-ADC, and Ab3-ADC, as well as the improvement in stability in aqueous systems, were significantly superior to the reference molecules. There was no significant difference in stability between Ab1-ADC, Ab2-ADC, and Ab3-ADC. Among them, the monomer content of Ab3-ADC determined by SEC-HPLC was slightly higher than that of Ab1-ADC and Ab2-ADC, and its stability was slightly better. Compared with the reference molecule, the corresponding molecular size variant SEC-HPLC indicators, charge variant CEX-HPLC indicators, and free MMAE content of Ab1-ADC, Ab2-ADC, and Ab3-ADC all showed significant differences. The decrease in SEC-HPLC monomer content and the increase in HMWS and LMWS content were significantly lower than those of the reference molecule. The decrease in the content of the charge variant CEX-HPLC main peak and the increase in the content of the acid peak and base peak were significantly lower than those of the reference molecule. The increase in free MMAE content was significantly lower than those of the reference molecule. Ab1-ADC, Ab2-ADC, and Ab3-ADC were significantly more stable than the reference molecule in pharmaceutical formulations.

[0154] Example 9: Liquid stability for 6 months at 2-8°C and -20°C The preferred formulation for the Ab1-ADC formulation was 10 mg / mL ADC, 20 mmol / L histidine-histidine hydrochloride, 0.02% polysorbate 20, and 5.5% trehalose (pH 6.0). Ab1, Ab2, and Ab3 molecules were coupled with LND1002, and the ADC molecules and reference molecules were subjected to ultrafiltration. The resulting samples were filtered through a 0.22 μm membrane filter. After filtration, 1.0 mL aliquots were dispensed into sterilized 2.0 mL injection bottles. The stability of the four molecules was analyzed at 2-8°C and -20°C. Evaluation parameters included appearance, molecular size variants (SEC-HPLC), charge variants (CEX-HPLC), and free MMAE content (RP-HPLC / MS). Analytical methods were the same as in Example 8.

[0155] [Table 26] JPEG2026507083000031.jpg51169

[0156] [Table 27] JPEG2026507083000033.jpg56169

[0157] Ab1-ADC, Ab2-ADC, Ab3-ADC, and the reference molecule were each placed in the preferred formulation of Ab1-ADC formulation and stored at 2-8°C and -20°C for 6 months. The product appearance remained unchanged, and the molecular size variant SEC-HPLC, charge variant CEX-HPLC, and free MMAE content indicators all changed. However, as the storage temperature of the product decreased, the rate of change of the relevant testing indicators slowed and the stability increased.

[0158] Under storage conditions at -20°C, the SEC-HPLC monomer content of Ab1-ADC, Ab2-ADC, Ab3-ADC, and the reference molecule decreased, which mainly corresponded to an increase in the HMWS content; the CEX-HPLC main peak content decreased, the corresponding acid peak and base peak contents increased, and the free MMAE content increased. The decrease in the SEC-HPLC monomer content of Ab1-ADC, Ab2-ADC, and Ab3-ADC was smaller than that of the reference molecule; the increase in the HMWS content was also smaller than that of the reference molecule; the decrease in the CEX-HPLC main peak content and the increase in the corresponding acid peak and base peak contents were smaller than that of the reference molecule; and the increase in the free MMAE content was also smaller than that of the reference molecule.

[0159] Under conditions of 2 to 8°C, the decreases in the SEC-HPLC monomer content and CEX-HPLC main peak content of Ab1-ADC, Ab2-ADC, and Ab3-ADC were significantly lower than those of the reference molecule, and the corresponding increases in HMWS and LMWS content and in the acid peak and base peak contents were all significantly lower than those of the reference molecule, while the increases in free MMAE content were significantly lower than those of the reference molecule. Thus, Ab1-ADC, Ab2-ADC, and Ab3-ADC were significantly more stable than the reference molecule in formulations.

[0160] Comparing the evaluation indexes of the products at the two temperatures, it was found that temperature affected Ab1-ADC, Ab2-ADC, Ab3-ADC, and the reference molecule, and stability increased when the storage temperature was lowered. Under both conditions, Ab1-ADC, Ab2-ADC, and Ab3-ADC were all more stable than the reference molecule. Considering the convenience of long-term storage of the products, freeze-drying was performed on the four sets of molecules to evaluate the stability of the products.

[0161] Example 10: Lyophilized powder, stability at 2-8°C and 25°C for 6 months The preferred formulation of Ab1-ADC was 10 mg / mL ADC, 20 mmol / L histidine-histidine hydrochloride, 0.02% polysorbate 20, and 5.5% trehalose (pH 6.0). LND1002 was coupled to Ab1, Ab2, and Ab3 molecules, and the ADC molecules and reference molecules were then exchanged by ultrafiltration. The exchanged samples were filtered through a 0.22 μm membrane filter, and the filtered samples were dispensed into washed and sterilized 10 mL injection bottles at 30 mg per bottle. Four sets of Ab1-ADC molecules were freeze-dried using the freeze-drying process (specifically, the parameters were the same as in Example 6, with primary drying at -25°C and 0.15 mbar, and secondary drying at 30°C and 0.15 mbar). After freeze-drying, the stability of the four molecules was analyzed at 25°C and 2-8°C. Before the stability analysis, approximately 3.2 mL of water was slowly added to the injection vial using a sterile syringe and gently rotated to reconstitute and mix. Evaluation parameters included appearance, molecular size variants (SEC-HPLC), charge variants (CEX-HPLC), and free MMAE content (RP-HPLC / MS). The analytical methods were the same as in Example 8.

[0162] [Table 28]

[0163] Ab1-ADC, Ab2-ADC, Ab3-ADC, and the reference molecule were each placed in the preferred formulation of Ab1-ADC, lyophilized, and stored at 25°C for 6 months, with no change in the product appearance. The molecular size variant SEC-HPLC, charge variant CEX-HPLC, and free MMAE content indicators all showed changes, primarily as follows: the SEC-HPLC monomer content decreased, corresponding to an increase in HMWS content; the CEX-HPLC main peak content decreased; the corresponding acid and base peak contents increased; and the free MMAE content increased. The decrease in the monomer content of Ab1-ADC, Ab2-ADC, and Ab3-ADC molecules by SEC-HPLC was smaller than that of the reference molecule; the decrease in the content of the main peak of CEX-HPLC and the increase in the content of the corresponding acid peak and base peak were significantly smaller than those of the reference molecule; the increase in the MMAE content was significantly smaller than that of the reference molecule; and the change in the charge variant CEX-HPLC indicators and free MMAE content of Ab1-ADC, Ab2-ADC, and Ab3-ADC molecules were significantly different from those of the reference molecule. After the same freeze-drying process and storage at 25°C, the stability of Ab1-ADC, Ab2-ADC, and Ab3-ADC molecules was significantly superior to that of the reference molecule.

[0164] [Table 29] JPEG2026507083000036.jpg22169

[0165] Ab1-ADC, Ab2-ADC, Ab3-ADC, and the reference molecule were placed in the preferred formulation of Ab1-ADC and lyophilized, then refrigerated at 2-8°C for 6 months. Compared with a liquid formulation of the same formulation before lyophilization and stored under the same conditions, the decrease in SEC-HPLC monomer content, the decrease in CEX-HPLC main peak content, and the increase in free MMAE content were significantly smaller, showing significant differences from the stability of the liquid formulation. The stability of the lyophilized samples was significantly superior to that of the liquid formulation. After lyophilization and storage at 2-8°C, the decrease in CEX-HPLC main peak content and the increase in the corresponding acid peak content of Ab1-ADC, Ab2-ADC, and Ab3-ADC molecules were lower than those of the reference molecule, and the increase in MMAE content was lower than those of the reference molecule. Compared with storage at 25°C, storage at 2-8°C significantly slowed the increase in free MMAE of the four groups, reduced the content of molecular size variant HMWS, and significantly reduced the changes in the charge variant CEX-HPLC main peak, acid peak, and base peak. Refrigeration at 2-8°C is suitable for long-term storage of samples.

[0166] The above description is merely a preferred embodiment and does not limit the combination of features necessary to practice the present invention, but is merely illustrative. The provided headings are not meant to limit the various embodiments of the present invention. Terms such as "comprises," "containing," and "including" are not intended to be limiting. Furthermore, unless otherwise specified, the plural is included when not modified by a numeral, and "or" means "and / or." Unless otherwise defined herein, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art.

[0167] All publications and patents mentioned in this application are incorporated herein by reference. Various modifications and variations to the methods and compositions described herein will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in terms of specific preferred embodiments, it should be understood that the invention as claimed is not unduly limited to these specific embodiments. Indeed, various variations of the described modes for carrying out the invention that are apparent to those skilled in the relevant fields are intended to be within the scope of the appended claims.

[0168] [Table 30] JPEG2026507083000038.jpg245169 [Brief explanation of the drawings]

[0169] [Figure 1] FIG. 1 is a schematic diagram of the mTgase enzymatic ADC coupling reaction. [Figure 2] 1 shows a schematic diagram of the ADC structure, where LND1002 represents the linker + drug molecule portion; LND1002 on the right shows the complete structure, and the circle represents the connection between the linker in LND1002 and the amide bond of the antibody. The ADC in this example is a site-specific antibody-drug conjugate, where each molecule contains one anti-Nectin-4 monoclonal antibody coupled to one MMAE molecule via a linker (NH2-PEG3-Val-Cit-pABC) at amino acid Q294 of each heavy chain (i.e., Kabat number Q295). The antibody and linker are connected via a stable amide bond (isopeptide bond), and the average drug-to-antibody ratio (DAR) is 2.0. [Figure 3] This is an experiment on the endocytosis effect of SWY2001-Ab1-LND1002 in SK-BR-3 cells. [Figure 4] This is an experiment on the endocytosis effect of SWY2001-Ab1-LND1002 in T47D cells. [Figure 5]In vivo inhibitory effect of SWY2001-Ab1-LND1002 on mouse PC3-Nectin-4 stable tumor line, mpk=mg / kg. [Figure 6] This is an in vivo inhibitory effect experiment of SWY2001-Ab1-LND1002 on mouse HT-1376 tumors.

Claims

1. A pharmaceutical composition comprising a Nectin-4 antibody-drug conjugate, (i) an antibody-drug conjugate, (ii) a buffer, (iii) a stabilizer, and (iv) a surfactant, wherein the antibody-drug conjugate is formed by coupling a drug-linker represented by Formula I with an anti-Nectin-4 monoclonal antibody; 【Chemistry 1】 The antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises three CDR regions having the amino acid sequences set forth in SEQ ID NOs: 1, 2, and 3, respectively, and / or the light chain comprises three CDR regions having the amino acid sequences set forth in SEQ ID NOs: 4, 5, and 6, respectively; Pharmaceutical compositions.

2. (a) the surfactant is polysorbate 20 or polysorbate 80, preferably polysorbate 20; (b) the stabilizer is sucrose or trehalose, preferably trehalose; and (c) the buffer is histidine-histidine hydrochloride, succinic acid-sodium succinate, or citric acid-sodium citrate, preferably histidine-histidine hydrochloride. The pharmaceutical composition of claim 1.

3. histidine-histidine hydrochloride 5-30 mM, polysorbate 20 or polysorbate 80 0.02 wt%-0.08 wt%, trehalose 4 wt%-8 wt%, and 5-50 mg / mL of an antibody-drug conjugate, and the pH is 5.0-6.

5. The pharmaceutical composition according to any one of claims 1 to 2.

4. histidine-histidine hydrochloride 10-30 mM, polysorbate 20 or polysorbate 80 0.02 wt%-0.04 wt%, trehalose 5 wt%-7 wt%, and antibody-drug conjugate 10-20 mg / mL, having a pH of 5.0-6.0; The pharmaceutical composition according to any one of claims 1 to 3.

5. histidine-histidine hydrochloride 20 mM, polysorbate 20 0.02 wt %, trehalose 5.5 wt %, and 10 mg / mL antibody drug conjugate, pH 6.0; The pharmaceutical composition according to any one of claims 1 to 4.

6. Further comprising water, The pharmaceutical composition according to any one of claims 1 to 5.

7. It may be a liquid formulation, a lyophilized formulation, or a powder injection formulation, preferably a liquid formulation or a lyophilized formulation; The pharmaceutical composition according to any one of claims 1 to 6.

8. Use of the pharmaceutical composition of any one of claims 1 to 7 in the manufacture of a medicament for treating cancer.

9. The cancer is a Nectin-4 positive cancer, including, but not limited to, bladder cancer, brain cancer, breast cancer, cervical cancer, breast tumor, endometrial cancer, esophageal squamous cell carcinoma, gastric cancer, head tumor, pancreatic cancer, bile duct cancer, colorectal cancer, eye cancer, head and neck squamous cell carcinoma, urothelial carcinoma, kidney cancer, liver cancer, lymph node cancer, lung cancer, oral cancer, cervical tumor, ovarian cancer, prostate cancer, testicular cancer, laryngeal cancer and uterine cancer, melanoma, salivary gland cancer, fibrosarcoma, soft tissue sarcoma and osteosarcoma, 9. The use according to claim 8.

10. the heavy chain of the antibody comprises a heavy chain variable region of the amino acid sequence shown in SEQ ID NO: 7, and / or the light chain of the antibody comprises a light chain variable region of the amino acid sequence shown in any one of SEQ ID NOs: 8 to 10; The pharmaceutical composition according to any one of claims 1 to 9.

11. the heavy chain of the antibody comprises a heavy chain constant region of the amino acid sequence shown in SEQ ID NO: 11 or 12, and / or the light chain of the antibody comprises a light chain constant region of the amino acid sequence shown in SEQ ID NO: 13; The pharmaceutical composition according to any one of claims 1 to 10.

12. 12. A method for producing the pharmaceutical composition according to any one of claims 1 to 11, characterized in that the method comprises the steps of producing an anti-Nectin-4 antibody-drug conjugate, liquid exchange of the antibody-drug conjugate with other components of a liquid pharmaceutical composition, sterile filtration, and aseptic filling. method.

13. A method for producing a freeze-dried preparation of the pharmaceutical composition according to any one of claims 1 to 11, characterized in that the method comprises the steps of pre-freezing the pharmaceutical composition according to any one of claims 1 to 10, vacuuming, primary drying and desorption drying. method.

14. The primary drying conditions are about −25° C. and 0.15 mbar. The method of claim 13.

15. The desorption drying temperature is about 30°C. The method of claim 13.

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