Pharmaceutical composition containing antibody drug conjugate and application thereof

Abstract

The invention relates to a pharmaceutical composition containing an antibody drug conjugate and application thereof. The present invention relates to a pharmaceutical composition comprising an antibody drug conjugate in a buffer. The pharmaceutical composition has good stability.

Description

A pharmaceutical composition containing an antibody-drug conjugate and its use Technical Field

[0001] The present disclosure belongs to the field of pharmaceutical preparations, and specifically relates to a pharmaceutical composition comprising an antibody-drug conjugate, and its use as an anti-cancer drug. Background Art

[0002] The statements herein merely provide background information related to the present disclosure and do not necessarily constitute prior art.

[0003] Antibody drug conjugates (ADCs) link monoclonal antibodies or antibody fragments to biologically active cytotoxins via stable chemical linker compounds. They fully leverage the specificity of antibodies for tumor cell surface antigen binding and the high efficacy of cytotoxins, while avoiding the drawbacks of the former, such as low efficacy and the latter's excessive toxic side effects. This means that compared to traditional chemotherapy drugs, ADCs can precisely bind to tumor cells while minimizing the effects on normal cells (Mullard A, (2013) Nature Reviews Drug Discovery, 12:329–332; DiJoseph JF, Armellino DC, (2004) Blood, 103:1807-1814).

[0004] There are several classes of cytotoxic small molecules used in antibody-drug conjugates, one of which is camptothecin derivatives, which have antitumor effects by inhibiting topoisomerase I. It has been reported that the camptothecin derivative exatecan (chemical name: (1S,9S)-1-amino-9-ethyl-5-fluoro-2,3-dihydro-9-hydroxy-4-methyl-1H,12H-benzo[de]pyrano[3 ， ,4 ， The application of [6,7]imidazo[1,2-b]quinoline-10,13(9H,15H)-dione in antibody-drug conjugates (ADCs) is reported in WO2014057687; Clinical Cancer Research (2016) 22(20): 5097-5108; Cancer Sci (2016) 107: 1039-1046. However, further development of ADC drugs with better efficacy is still needed.

[0005] However, ADCs have a more complex and heterogeneous structure than antibodies, thus posing greater challenges to ADC formulation for therapeutic purposes.

[0006] Summary of the Invention

[0007] The present disclosure provides a pharmaceutical composition comprising an antibody drug conjugate and a buffer, wherein the antibody drug conjugate has the structure shown below:

[0008] The antibody drug conjugate described above is prepared with reference to the antibody conjugate Example 9 FADC-2 on pages 45-47 of the specification of WO2020063673A1;

[0009] in:

[0010] h1702DS is an anti-B7H3 antibody, which is derived from antibody h1702DS on page 44 of WO2020063673A1, and comprises a heavy chain as shown in SEQ ID NO: 1 (SEQ ID NO: 14 in WO2020063673A1) and a light chain as shown in SEQ ID NO: 2 (SEQ ID NO: 16 in WO2020063673A1):

[0011] Heavy chain amino acid sequence of h1702DS: (SEQ ID NO: 1)

[0012] Light chain amino acid sequence of h1702DS: (SEQ ID NO: 2)

[0013] n is 1 to 10, preferably 1 to 8, more preferably 3 to 5, more preferably about 4;

[0014] The buffer of the pharmaceutical composition is a succinate buffer or a histidine buffer, preferably a succinic acid-succinic acid sodium salt, histidine-acetate or histidine-hydrochloride buffer.

[0015] The buffer concentration of the pharmaceutical composition is about 15 mM to about 50 mM, preferably about 20 mM to about 40 mM, and more preferably about 30 mM.

[0016] The pH of the pharmaceutical composition is from about 4.5 to about 6.5, preferably from about 5.0 to about 6.0, and more preferably from about 5.5 to about 5.6.

[0017] In an alternative embodiment, the pH of the buffer in the pharmaceutical composition is about 4.5 to about 6.5, non-limiting examples of which include about 4.5, about 4.6, about 4.7, about 4.8, about 4.9, about 5.0, about 5.1, about 5.2, about 5.3, about 5.4, about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, about 6.0, about 6.1, about 6.2, about 6.3, about 6.4, about 6.5, and any range therebetween; preferably, about 5.0 to about 6.0, and more preferably, about 5.5 to about 5.6.

[0018] In an optional embodiment, the succinate buffer in the pharmaceutical composition is succinic acid-sodium succinate, and the histidine salt buffer is preferably histidine-acetate or histidine-hydrochloride buffer.

[0019] In an optional embodiment, the pharmaceutical composition further comprises a surfactant. The surfactant may be selected from polysorbate, polysorbate 20, polysorbate 80, polyhydroxyalkylene, Triton, sodium dodecyl sulfate, sodium lauryl sulfate, sodium octyl glucoside, lauryl-sulfobetaine, myristyl-sulfobetaine, linoleyl-sulfobetaine, stearyl-sulfobetaine, lauryl-sarcosine, myristyl-sarcosine, linoleyl-sarcosine, stearyl-sarcosine, linoleyl-betaine, myristyl-betaine, cetyl-betaine, Cinnamyl-betaine, cocamidopropyl-betaine, linoleamidopropyl-betaine, myristamidopropyl-betaine, palmitamidopropyl-betaine, isostearamidopropyl-betaine, myristamidopropyl-dimethylamine, palmitamidopropyl-dimethylamine, isostearamidopropyl-dimethylamine, methyl cocoyl sodium, methyl oleyl taurate sodium, polyethylene glycol, polypropylene glycol and copolymer of ethylene and propylene glycol, etc. Preferred surfactants are polysorbate 80 or polysorbate 20, more preferably polysorbate 80.

[0020] In alternative embodiments, the concentration of the surfactant in the pharmaceutical composition is from about 0.01 mg / mL to about 1.0 mg / mL, preferably from about 0.05 mg / mL to about 0.5 mg / mL, more preferably from about 0.1 mg / mL to about 0.4 mg / mL or from about 0.2 mg / mL to about 0.3 mg / mL, and most preferably about 0.2 mg / mL, non-limiting examples of which include 0.02 mg / mL, 0.05 mg / mL, 0.1 mg / mL, 0.15 mg / mL, 0.2 mg / mL, 0.25 mg / mL, 0.3 mg / mL, 0.35 mg / mL, 0.4 mg / mL, 0.45 mg / mL, 0.5 mg / mL, 0.8 mg / mL, and any range therebetween.

[0021] In an optional embodiment, the aforementioned pharmaceutical composition further comprises sugar. The "sugar" disclosed herein comprises a conventional composition (CH2O) n and derivatives thereof, including monosaccharides, disaccharides, trisaccharides, polysaccharides, sugar alcohols, reducing sugars, non-reducing sugars, and the like. The sugar may be selected from glucose, sucrose, trehalose, lactose, fructose, maltose, dextran, glycerol, erythritol, glycerol, arabitol, sylitol, sorbitol, mannitol, milibiose, melezitose, raffinose, mannotriose, stachyose, maltose, lactulose, maltulose, sorbitol, maltitol, lactitol, iso-maltulose, and the like. Preferred sugars are non-reducing disaccharides, more preferably trehalose, mannitol, or sucrose, and most preferably sucrose or trehalose.

[0022] In an alternative embodiment, the concentration of sugar in the aforementioned pharmaceutical composition is from about 25 mg / mL to about 80 mg / mL, preferably from about 30 mg / mL to about 60 mg / mL, preferably about 40 mg / mL, non-limiting examples include 25 mg / mL, 30 mg / mL, 35 mg / mL, 40 mg / mL, 45 mg / mL, 50 mg / mL, 55 mg / mL, 60 mg / mL, 65 mg / mL, 70 mg / mL, 75 mg / mL, 80 mg / mL and any range between these points, preferably 40 mg / mL.

[0023] In an optional embodiment, the aforementioned pharmaceutical composition further comprises an amino acid and a salt thereof, wherein the amino acid and the salt thereof are preferably selected from glycine and arginine hydrochloride, more preferably glycine.

[0024] In an alternative embodiment, the glycine concentration in the aforementioned pharmaceutical composition is about 5 mg / mL to about 10 mg / mL, about 5.3 mg / mL to about 9.8 mg / mL, about 6 mg / mL to about 9 mg / mL, preferably about 7 mg / mL to about 8 mg / mL, non-limiting examples include 6 mg / mL, 6.5 mg / mL, 7 mg / mL, 7.2 mg / mL, 7.6 mg / mL, 7.8 mg / mL, 8 mg / mL, 8.5 mg / mL, 9 mg / mL and any range therebetween, most preferably about 7.6 mg / mL.

[0025] In an optional embodiment, the aforementioned pharmaceutical composition further comprises arginine hydrochloride at a concentration of about 40 mM to about 80 mM, preferably about 50 mM to about 70 mM, non-limiting examples of which include 40 mM, 45 mM, 50 mM, 55 mM, 58 mM, 60 mM, 65 mM, 70 mM, 75 mM and 80 mM, and most preferably about 58 mM.

[0026] In an alternative embodiment, the concentration of the antibody drug conjugate in the pharmaceutical composition is about 1 mg / mL to about 100 mg / mL, based on protein concentration, non-limiting examples include 1 mg / mL, 10 mg / mL, 11 mg / mL, 12 mg / mL, 13 mg / mL, 14 mg / mL, 15 mg / mL, 16 mg / mL, 17 mg / mL, 18 mg / mL, 19 mg / mL, 20 mg / mL, 21 mg / mL, 22 mg / mL, 23 mg / mL, 24 mg / mL, 25 mg / mL, 26 mg / mL, 27 mg / mL , 28 mg / mL, 29 mg / mL, 30 mg / mL, 40 mg / mL, 50 mg / mL, 60 mg / mL, 70 mg / mL, 80 mg / mL, 90 mg / mL, 100 mg / mL and any range therebetween, preferably about 10 mg / mL to about 70 mg / mL; more preferably about 20 mg / mL to about 50 mg / mL, further preferably about 20 mg / mL to about 30 mg / mL or about 30 mg / mL to about 50 mg / mL, even more preferably about 20 mg / mL or about 30 mg / mL or about 50 mg / mL. Specific, non-limiting examples include 50.1 mg / mL, 50.2 mg / mL, 50.3 mg / mL, 50.4 mg / mL, 50.5 mg / mL, 50.6 mg / mL, 50.7 mg / mL, 50.8 mg / mL, 50.81 mg / mL, 50.82 mg / mL, 50.83 mg / mL, 50.84 mg / mL, 50.85 mg / mL, 50.86 mg / mL, 50.87 mg / mL, g / mL, 50.88 mg / mL, 50.89 mg / mL, 50.9 mg / mL, 50.9 mg / mL, 50.91 mg / mL, 50.92 mg / mL, 50.93 mg / mL, 50.94 mg / mL, 50.95 mg / mL, 50.96 mg / mL, 50.97 mg / mL, 50.98 mg / mL, 50.99 mg / mL, 51 mg / mL, and any range therebetween. The term "protein concentration" refers to the concentration of the antibody portion of the antibody drug conjugate.

[0027] In an alternative embodiment, the buffer concentration in the pharmaceutical composition is about 15 mM to about 50 mM, non-limiting examples include 15 mM, 16 mM, 17 mM, 18 mM, 19 mM, 20 mM, 30 mM, 40 mM, 50 mM and any range therebetween, preferably about 15 mM to about 50 mM, more preferably about 20 mM to about 40 mM; most preferably about 30 mM.

[0028] In alternative embodiments, the drug loading (DAR) can range from an average number of cytotoxic drugs bound per antibody h1702DS, with non-limiting examples being approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, and any range therebetween. Preferably, the average number of cytotoxic drugs bound per antibody h1702DS is approximately 3 to 8, approximately 4 to 8, approximately 5 to 7, approximately 4 to 6, more preferably approximately 3 to 5, and most preferably 4.1. n is a decimal or integer.

[0029] In an alternative embodiment, the drug loading (DAR) is about 4.

[0030] In an alternative embodiment, the pharmaceutical composition comprises:

[0031] (a) about 10 mg / mL to about 70 mg / mL of the antibody drug conjugate, (b) about 0.1 mg / mL to about 0.8 mg / mL of polysorbate, (c) about 25 mg / mL to about 80 mg / mL of a saccharide, (d) about 5 mg / mL-10 mg / mL of glycine, and (e) about 15 mM to about 50 mM succinate buffer; the pH of the composition being about 5.0-6.0;

[0032] Preferably, the pharmaceutical composition comprises the following components:

[0033] (a) about 20 mg / mL to about 70 mg / mL of the antibody drug conjugate, (b) about 0.2 mg / mL to about 0.8 mg / mL of polysorbate, (c) about 25 mg / mL to about 80 mg / mL of a saccharide, (d) about 7 mg / mL-8 mg / mL of glycine, and (e) about 10 mM to about 40 mM succinate buffer; the pH of the composition being about 5.0-6.0;

[0034] More preferably, the pharmaceutical composition comprises the following components:

[0035] (a) about 20 mg / mL to about 50 mg / mL of the antibody drug conjugate, (b) about 0.2 mg / mL to about 0.8 mg / mL of polysorbate 80, (c) about 40 mg / mL to about 80 mg / mL of sucrose, (d) about 7 mg / mL to about 8 mg / mL of glycine, and (e) about 20 mM to about 40 mM of succinic acid-sodium salt, wherein the pH of the composition is about 5.0-6.0;

[0036] Further preferably, the pharmaceutical composition comprises the following components:

[0037] (a) about 20 mg / mL to about 30 mg / mL of the antibody drug conjugate, (b) about 0.2 mg / mL of polysorbate 80, (c) about 40 mg / mL of sucrose, (d) about 7.6 mg / mL of glycine, and (e) about 30 mM succinic acid-sodium salt, wherein the pH of the composition is about 5.5-5.6;

[0038] (a) about 30 mg / mL to about 50 mg / mL of the antibody drug conjugate, (b) about 0.2 mg / mL of polysorbate 80, (c) about 40 mg / mL of sucrose, (d) about 7.6 mg / mL of glycine, and (e) about 30 mM succinic acid-sodium salt, wherein the pH of the composition is about 5.5-5.6;

[0039] More preferably, the pharmaceutical composition comprises the following components:

[0040] (a) about 20 mg / mL of the antibody drug conjugate, (b) about 0.2 mg / mL of polysorbate 80, (c) about 40 mg / mL of sucrose, (d) about 7.6 mg / mL of glycine, and (e) about 30 mM succinic acid-sodium salt, wherein the pH of the composition is about 5.5-5.6.

[0041] (a) about 30 mg / mL of the antibody drug conjugate, (b) about 0.2 mg / mL of polysorbate 80, (c) about 40 mg / mL of sucrose, (d) about 7.6 mg / mL of glycine, and (e) about 30 mM succinic acid-sodium salt, wherein the pH of the composition is about 5.5-5.6.

[0042] (a) about 50 mg / mL of the antibody drug conjugate, (b) about 0.2 mg / mL of polysorbate 80, (c) about 40 mg / mL of sucrose, (d) about 7.6 mg / mL of glycine, and (e) about 30 mM succinic acid-sodium salt, wherein the pH of the composition is about 5.5-5.6.

[0043] In an alternative embodiment, any of the foregoing pharmaceutical compositions is a liquid formulation.

[0044] The present disclosure also provides a lyophilized formulation containing an antibody drug conjugate, characterized in that the formulation can form the pharmaceutical composition described above after reconstitution.

[0045] The present disclosure also provides a method for preparing a lyophilized formulation containing an antibody drug conjugate, which comprises the step of freeze-drying the pharmaceutical composition as described above.

[0046] In an optional embodiment, the freeze drying process comprises, in a method for preparing a lyophilized formulation comprising an antibody drug conjugate, the steps of pre-freezing, primary drying, and secondary drying. Freeze drying is performed by freezing the formulation and then subliming the 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 range for primary drying is about -30 to 25°C (assuming the product remains frozen during the primary drying process). The formulation, the size and type of container (e.g., glass vial) holding the sample, and the volume of 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 stage can be performed at about 0-40°C, depending primarily on the type and size of the container and the type of protein used. 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 the lyophilized formulation is less than about 5%, preferably less than about 3%. The pressure can be the same as that applied in the primary drying step, preferably, the pressure for secondary drying is lower than that for primary drying. Freeze drying conditions can vary with the formulation and vial size.

[0047] In an optional embodiment of the present disclosure, 5 mL of the composition stock solution is freeze-dried, and the freeze-drying procedure is as follows: the pre-freezing temperature is 5°C and -45°C, respectively; the primary drying temperature is -20°C, the vacuum degree is 10 Pa; the secondary drying temperature is 25°C, and the vacuum degrees are 10 Pa and 1 Pa, respectively.

[0048] In some embodiments, the lyophilized formulation is stable at 2-8° C. for at least 16 days, at least 1 month, at least 3 months, at least 6 months, at least 12 months, at least 18 months, or at least 24 months. In some embodiments, the lyophilized formulation is stable at 40° C. for at least 7 days, at least 14 days, at least 28 days, or at least 30 days.

[0049] The present disclosure also provides a lyophilized formulation comprising an antibody drug conjugate, wherein the lyophilized formulation is obtained by freeze-drying the pharmaceutical composition of the antibody drug conjugate as described above.

[0050] The present disclosure also provides a reconstituted solution containing an antibody drug conjugate, characterized in that the reconstituted solution is prepared by reconstituted the lyophilized preparation as described above.

[0051] In an alternative embodiment, the reconstitution solution comprises:

[0052] (a) about 10 mg / mL to about 70 mg / mL of the antibody drug conjugate, (b) about 0.1 mg / mL to about 0.8 mg / mL of polysorbate, (c) about 25 mg / mL to about 80 mg / mL of a saccharide, (d) about 5 mg / mL-10 mg / mL of glycine, and (e) about 15 mM to about 50 mM of a succinate buffer; the pH of the reconstitution solution being about 5.0-6.0;

[0053] Preferably, the reconstitution solution comprises the following components:

[0054] (a) about 20 mg / mL to about 70 mg / mL of the antibody drug conjugate, (b) about 0.2 mg / mL to about 0.8 mg / mL of polysorbate, (c) about 25 mg / mL to about 80 mg / mL of a saccharide, (d) about 7 mg / mL to about 8 mg / mL of glycine, and (e) about 10 mM to about 40 mM succinate buffer; the pH of the composition being about 5.0-6.0;

[0055] More preferably, the reconstitution solution comprises the following components:

[0056] (a) about 20 mg / mL to about 50 mg / mL of the antibody drug conjugate, (b) about 0.2 mg / mL to about 0.8 mg / mL of polysorbate 80, (c) about 40 mg / mL to about 60 mg / mL of sucrose, (d) about 7 mg / mL to about 8 mg / mL of glycine, and (e) about 20 mM to about 40 mM of succinic acid-sodium salt, wherein the pH of the reconstituted solution is about 5.0-6.0;

[0057] Further preferably, the reconstitution solution comprises the following components:

[0058] (a) about 20 mg / mL to about 30 mg / mL of the antibody drug conjugate, (b) about 0.2 mg / mL of polysorbate 80, (c) about 40 mg / mL of sucrose, (d) about 7.6 mg / mL of glycine, and (e) about 30 mM succinic acid-sodium salt, wherein the pH of the composition is about 5.5-5.6; or

[0059] (a) about 30 mg / mL to about 50 mg / mL of the antibody drug conjugate, (b) about 0.2 mg / mL of polysorbate 80, (c) about 40 mg / mL of sucrose, (d) about 7.6 mg / mL of glycine, and (e) about 30 mM succinic acid-sodium salt, wherein the pH of the composition is about 5.5-5.6.

[0060] More preferably, the reconstitution solution comprises the following components:

[0061] (a) about 20 mg / mL of the antibody drug conjugate, (b) about 0.2 mg / mL of polysorbate 80, (c) about 40 mg / mL of sucrose, (d) about 7.6 mg / mL of glycine, and (e) about 30 mM succinic acid-sodium salt, wherein the pH of the composition is about 5.5-5.6;

[0062] (a) about 30 mg / mL of the antibody drug conjugate, (b) about 0.2 mg / mL of polysorbate 80, (c) about 40 mg / mL of sucrose, (d) about 7.6 mg / mL of glycine, and (e) about 30 mM succinic acid-sodium salt, wherein the pH of the composition is about 5.5-5.6;

[0063] (a) about 50 mg / mL of the antibody drug conjugate, (b) about 0.2 mg / mL of polysorbate 80, (c) about 40 mg / mL of sucrose, (d) about 7.6 mg / mL of glycine, and (e) about 30 mM succinic acid-sodium salt, wherein the pH of the composition is about 5.5-5.6.

[0064] The present disclosure also provides a method for preparing the aforementioned reconstituted solution, which comprises the step of reconstituted the aforementioned lyophilized preparation, wherein the solution used for reconstitution is selected from but not limited to water for injection, physiological saline or glucose solution.

[0065] The present disclosure also provides a product, which includes a container, wherein the container is filled with the pharmaceutical composition, lyophilized preparation or reconstituted solution as described above. In some embodiments, the container is a neutral borosilicate glass tube injection bottle.

[0066] The present disclosure also provides use of the aforementioned pharmaceutical composition, lyophilized preparation, reconstituted solution or product in the preparation of a drug for treating or preventing tumors.

[0067] The present disclosure also provides a method for treating a disease, comprising providing the aforementioned pharmaceutical composition, lyophilized preparation, reconstituted solution or product.

[0068] The present disclosure also provides the aforementioned pharmaceutical composition, or lyophilized preparation, or reconstituted solution, or product as a drug. Preferably, the drug is used to treat or prevent tumor diseases.

[0069] In an alternative embodiment, the disease or tumor is a cancer associated with HER2, HER3, B7H3 or EGFR expression.

[0070] In alternative embodiments, the cancer is selected from breast cancer, ovarian cancer, cervical cancer, uterine cancer, prostate cancer, kidney cancer, urethral cancer, bladder cancer, liver cancer, stomach cancer, endometrial cancer, salivary gland cancer, esophageal cancer, melanoma, glioma, neuroblastoma, sarcoma, lung cancer, colon cancer, rectal cancer, colorectal cancer, leukemia, bone cancer, skin cancer, thyroid cancer, pancreatic cancer, and lymphoma.

[0071] As is well known to those skilled in the art, one, some or all of the features of the various embodiments described in this disclosure may be further combined to form other embodiments of the present disclosure. The above embodiments of the present disclosure and other embodiments obtained by combination are further described in detail below.

[0072] For ADC formulation development, even at low concentrations, the interchain disulfide bonds opened during the coupling process are subject to oxidation, and the coupled strong hydrophobic toxins also affect the stability of the antibody to a certain extent. Increasing the concentration will inevitably exacerbate this effect, making the ADC more susceptible to aggregation / degradation and unable to stably exist in low-concentration formulations. Through reasonable formulation design and screening, we screened out suitable high-concentration formulations to maintain the stability of the ADC. In addition, the high-concentration ADC formulations of the present invention may have a larger safe treatment window in clinical practice, while greatly shortening production time, reducing consumables costs (vials, stoppers, aluminum caps, etc.), and improving the convenience of clinical drug administration for medical staff.

[0073] Detailed Description of the Invention

[0074] The present disclosure provides a pharmaceutical composition that is more convenient for production and administration and has stable performance. Undesirable instability may include any one or more of the following: aggregation, deamidation (e.g., Asn deamidation), oxidation (e.g., Met oxidation), isomerization (e.g., Asp isomerization), clipping / hydrolysis / fragmentation (e.g., hinge region fragmentation), succinimide formation, unpaired cysteine ​​residues, toxin dissociation, etc. Specifically, the pharmaceutical composition described herein comprises an antibody drug conjugate and a buffer.

[0075] the term

[0076] In order to make the present disclosure more easily understood, certain technical and scientific terms are specifically defined below. Unless otherwise explicitly defined herein, all other technical and scientific terms used herein have the meaning commonly understood by those skilled in the art to which the present disclosure belongs.

[0077] This disclosure incorporates the entire contents of application WO2020063673 into this application.

[0078] Antibody drug conjugates (ADCs) are antibodies that are linked to biologically active cytotoxins or small molecule drugs with cell-killing activity through a linker.

[0079] "Drug loading" is also called the drug-to-antibody ratio (DAR), which is the average number of drugs conjugated to each antibody in the ADC. It can be, for example, in the range of about 1 to about 10 drugs conjugated to each antibody, and in certain embodiments, in the range of about 1 to about 8 drugs conjugated to each antibody, preferably in the range of 2-8, 2-7, 2-6, 2-5, 2-4, 3-4, 3-5, 5-6, 5-7, 5-8 and 6-8. Exemplary, the drug loading can be an average of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10. The ADC formula disclosed herein includes a collection of antibodies conjugated to drugs within the aforementioned range. In embodiments of the present disclosure, the drug loading can be expressed as n. Drug loading can be determined by conventional methods such as UV / visible spectroscopy, mass spectrometry, ELISA assays and HPLC.

[0080] The term "linker unit" or "connection fragment" or "connection unit" refers to a chemical structure fragment or bond that is connected to an antibody or its antigen-binding fragment at one end and to a drug at the other end, and can also be connected to other linkers before being connected to the drug.

[0081] Linkers, including extenders, spacers, and amino acid units, can be synthesized by methods known in the art, such as those described in US 2005-0238649 A1. The linker can be a "cleavable linker" that facilitates release of the drug in the cell. For example, an acid-labile linker (e.g., a hydrazone), a protease-sensitive (e.g., a peptidase-sensitive) linker, a photolabile linker, a dimethyl linker, or a disulfide-containing linker can be used (Chari et al., Cancer Research 52: 127-131 (1992); U.S. Patent No. 5,208,020).

[0082] Cytotoxic drug loading can be controlled by the following non-limiting methods, including:

[0083] (1) Control the molar ratio of the linker and the monoclonal antibody,

[0084] (2) Control reaction time and temperature,

[0085] (3) Select different reaction reagents.

[0086] The three letter and one letter codes for amino acids used in this disclosure are as described in J. biol. chem, 243, p3558 (1968).

[0087] The term "antibody" as used herein is used in the broadest sense and encompasses various antibody structures, including but not limited to full-length antibodies and antibody fragments (or antigen-binding fragments, or antigen-binding portions), as long as they exhibit the desired antigen-binding activity. Typically, a natural intact antibody consists of a tetrapeptide chain structure consisting of two identical heavy chains and two identical light chains connected by interchain disulfide bonds.

[0088] The engineered antibodies or antigen-binding fragments disclosed herein can be prepared and purified using conventional methods. For example, cDNA sequences encoding heavy and light chains can be cloned and recombined into GS expression vectors. The recombinant immunoglobulin expression vector can be stably transfected into CHO cells. As a more recommended existing technology, mammalian expression systems will result in glycosylation of antibodies, especially at the highly conserved N-terminal site in the Fc region. Positive clones are expanded in serum-free culture medium in a bioreactor to produce antibodies. The culture fluid that secretes antibodies can be purified using conventional techniques. For example, purification can be performed using an A or G Sepharose FF column containing an adjusted buffer. Non-specifically bound components are washed away. The bound antibodies are then eluted using a pH gradient method, and the antibody fragments are detected by SDS-PAGE and collected. The antibodies can be filtered and concentrated using conventional methods. Soluble mixtures and polymers can also be removed using conventional methods, such as molecular sieves and ion exchange. The obtained product should be immediately frozen, such as at -70°C, or freeze-dried.

[0089] "Buffer" refers to a buffer that tolerates changes in pH through the action of its acid-base conjugate components. Examples of buffers that control pH within an appropriate range include acetate, succinate, gluconate, histidine, oxalate, lactate, phosphate, citrate, tartrate, fumarate, glycylglycine, and other organic acid buffers.

[0090] A "histidine salt buffer" is a buffer containing histidine ions. Examples of histidine salt buffers include histidine hydrochloride, histidine acetate, histidine phosphate, histidine sulfate, and the like. Histidine acetate buffer is preferred. Histidine acetate buffer is a mixture of histidine and acetic acid, while histidine hydrochloride buffer is a mixture of histidine and hydrochloric acid.

[0091] "Citrate buffer" is a buffer comprising citrate ions. Examples of citrate buffers include citric acid-sodium citrate, citric acid-potassium citrate, citric acid-calcium citrate, citric acid-magnesium citrate, and the like. A preferred citrate buffer is citric acid-sodium citrate.

[0092] A "succinate buffer" is a buffer comprising succinate ions. Examples of succinate buffers include succinic acid-sodium succinate, succinic acid-potassium succinate, succinic acid-calcium succinate, and the like. A preferred succinate buffer is succinic acid-sodium succinate. For example, the succinic acid-sodium succinate can be prepared from succinic acid and sodium hydroxide, or from succinic acid and sodium succinate.

[0093] "Phosphate buffer" is a buffer containing phosphate ions. Examples of phosphate buffers include disodium hydrogen phosphate-sodium dihydrogen phosphate, disodium hydrogen phosphate-potassium dihydrogen phosphate, disodium hydrogen phosphate-citric acid, and the like. A preferred phosphate buffer is disodium hydrogen phosphate-sodium dihydrogen phosphate.

[0094] "Acetate buffer" is a buffer containing acetate ions. Examples of acetate buffers include acetate-sodium acetate, histidine acetate, acetate-potassium acetate, acetate-calcium acetate, acetate-magnesium acetate, and the like. A preferred acetate buffer is acetate-sodium acetate.

[0095] A "pharmaceutical composition" refers to a mixture containing one or more antibody-drug conjugates described herein, or their physiologically / pharmaceutically acceptable salts or prodrugs, together with other chemical components, such as physiologically / pharmaceutically acceptable carriers and excipients. The purpose of a pharmaceutical composition is to maintain the stability of the antibody active ingredient, facilitate administration to an organism, and promote absorption of the active ingredient, thereby exerting its biological activity.

[0096] In the present disclosure, "pharmaceutical composition" and "formulation" are not mutually exclusive.

[0097] Unless otherwise specified, the solvent in the solution form of the pharmaceutical composition described in the present disclosure is water.

[0098] "Lyophilized formulation" refers to a pharmaceutical composition in liquid or solution form or a formulation or pharmaceutical composition obtained after a liquid or solution formulation has been subjected to a vacuum freeze-drying step.

[0099] As used herein, the terms "about" and "approximately" refer to values ​​that are within an acceptable error range for a particular value as determined by one of ordinary skill in the art, which depends in part on how it is measured or determined (i.e., the limits of the measurement system). For example, in each practice in the art, "about" can mean within 1 or more than 1 standard deviation. Alternatively, "about" or "substantially comprising" can mean a range of up to 20%. In addition, particularly for biological systems or processes, the term can mean up to an order of magnitude or up to 5 times the value. Unless otherwise indicated, when a specific value appears in the application and claims, the meaning of "about" or "substantially comprising" should be assumed to be within an acceptable error range for that specific value.

[0100] The pharmaceutical compositions disclosed herein can achieve a stable effect: the antibody drug conjugates therein substantially retain their physical stability and / or chemical stability and / or biological activity after storage. Preferably, the pharmaceutical compositions substantially retain their physical and chemical stability and their biological activity after storage. The storage period is generally selected based on the intended shelf life of the pharmaceutical composition. Currently, there are various analytical techniques for measuring protein stability after storage at a selected temperature for a selected period of time.

[0101] A stable formulation is one in which no significant change is observed after storage at refrigerated temperatures (2-8°C) for at least 3 months, preferably 6 months, more preferably 1 year, and even more preferably up to 2 years. In addition, a stable liquid formulation includes a liquid formulation that exhibits the desired characteristics after storage at a temperature of 25°C for a period of 1 month, 3 months, or 6 months. A typical example of stability is that no more than about 10%, preferably no more than about 5%, of the antibody monomers aggregate or degrade as measured by SEC-HPLC. By visual analysis, the formulation is a pale yellow, nearly colorless, clear liquid or colorless, or clear to slightly milky white. The concentration, pH, and weight-gram molecular osmotic pressure concentration of the formulation vary by no more than ±10%. A decrease of no more than about 10%, preferably no more than about 5%, is usually observed. Aggregates of no more than about 10%, preferably no more than about 5%, are usually formed.

[0102] The antibody drug conjugate "retains its physical stability" in the pharmaceutical formulation if it shows no significant increase in aggregation, precipitation, and / or denaturation as measured by visual inspection of color and / or clarity, or by UV light scattering, size exclusion chromatography (SEC), and dynamic light scattering (DLS). Changes in protein conformation can be assessed by fluorescence spectroscopy (which determines protein tertiary structure) and by FTIR spectroscopy (which determines protein secondary structure).

[0103] An antibody drug conjugate "retains its chemical stability" in a pharmaceutical formulation if the antibody drug conjugate shows no significant chemical changes. Chemical stability can be assessed by detecting and quantifying chemically altered forms of the protein. Degradation processes that often change the chemical structure of a protein include hydrolysis or truncation (assessed by methods such as size exclusion chromatography and CE-SDS), oxidation (assessed by methods such as peptide mapping in combination 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.).

[0104] An antibody drug conjugate "retains its biological activity" in a pharmaceutical formulation if the biological activity of the antibody drug conjugate at a given time is within a predetermined range of the biological activity exhibited when the pharmaceutical formulation is prepared.

[0105] "Optional" or "optionally" means that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs or does not occur. For example, "optionally comprising 1-3 antibody heavy chain variable regions" means that the antibody heavy chain variable region of the specified sequence may but need not be present.

[0106] "Substituted" means that one or more hydrogen atoms, preferably up to 5, more preferably 1 to 3 hydrogen atoms, in a group are replaced independently of one another by a corresponding number of substituents. It goes without saying that the substituents are only in their possible chemical positions, and a person skilled in the art can determine (by experiment or theory) which substitutions are possible or impossible without undue effort. For example, an amino or hydroxyl group with free hydrogen may be unstable when combined with a carbon atom with an unsaturated (e.g., olefinic) bond.

[0107] The preparation of conventional pharmaceutical compositions can be found in the Chinese Pharmacopoeia.

[0108] The term "carrier" as used in the drugs disclosed herein refers to a system that can change the way a drug enters the human body and its distribution in the body, control the release rate of the drug, and deliver the drug to the targeted organ. Drug carrier release and targeting systems can reduce drug degradation and loss, reduce side effects, and improve bioavailability. For example, polymer surfactants that can be used as carriers can self-assemble to form various forms of aggregates due to their unique amphiphilic structure. Preferred examples include micelles, microemulsions, gels, liquid crystals, vesicles, etc. These aggregates have the ability to encapsulate drug molecules and have good permeability to membranes, making them excellent drug carriers.

[0109] "Administering" and "treating" as applied to an animal, a human, a laboratory subject, a cell, a tissue, an organ or a biological fluid, refers to the contacting of an exogenous drug, therapeutic agent, diagnostic agent or composition with an animal, a human, a subject, a cell, a tissue, an organ or a biological fluid. "Administering" and "treating" can refer to, for example, therapeutic, pharmacokinetics, diagnostics, research and experimental procedures. Treatment of cells includes contacting an agent with a cell, and contacting an agent with a fluid, wherein the fluid is in contact with the cell. "Administering" and "treating" also mean treating, for example, a cell in vitro and ex vivo, by an agent, a diagnostic, a binding composition or by another cell. "Treatment" as applied to a human, veterinary or research subject, refers to therapeutic treatment, prophylactic or preventative measures, research and diagnostic applications.

[0110] "Treatment" means administering an internal or external therapeutic agent, such as a composition comprising any of the binding compounds of the present disclosure, to a patient who has one or more symptoms of a disease for which the therapeutic agent is known to have a therapeutic effect. Typically, the therapeutic agent is administered in an amount effective to alleviate one or more symptoms of the disease in the treated patient or population to induce regression of such symptoms or inhibit the development of such symptoms to any clinically measurable degree. The amount of a therapeutic agent effective to alleviate any specific disease symptom (also referred to as a "therapeutically effective amount") can vary according to a variety of factors, such as the patient's disease state, age, and weight, and the ability of the drug to produce the desired therapeutic effect in the patient. Whether the symptoms of the disease have been alleviated can be assessed by any clinical test method commonly used by a physician or other health care professional to assess the severity or progression of the symptoms. Although embodiments of the present disclosure (e.g., methods of treatment or articles of manufacture) may not be effective in alleviating every symptom of the target disease, they should alleviate the target disease symptoms in a statistically significant number of patients as determined by any statistical test known in the art, such as Student's t-test, chi-square test, U test according to Mann and Whitney, Kruskal-Wallis test (H test), Jonckheere-Terpstra test, and Wilcoxon test.

[0111] An "effective amount" encompasses an amount sufficient to ameliorate or prevent the symptoms or conditions of a medical condition. An effective amount also means an amount sufficient to permit or facilitate diagnosis. The effective amount for a particular patient or veterinary subject may vary depending on factors such as the condition to be treated, the patient's overall health, the route and dosage of administration, and the severity of side effects. An effective amount can be the maximum dose or dosage regimen that avoids significant side effects or toxic effects.

[0112] "Displacement" refers to the replacement of the solvent system in which the antibody protein is dissolved. For example, a high-salt or hypertonic solvent system containing the antibody protein is replaced by a physical manipulation using a buffer system that stabilizes the formulation, thereby stabilizing the antibody protein in the formulation. Such physical manipulations include, but are not limited to, ultrafiltration, dialysis, or centrifugation followed by reconstitution. DETAILED DESCRIPTION

[0113] The present disclosure is further described below with reference to the following examples, but these examples are not intended to limit the scope of this disclosure. Experimental methods in the examples disclosed herein that do not specify specific conditions were generally performed according to conventional conditions, such as those in the Cold Spring Harbor Laboratory's "Antibody Techniques Laboratory Manual" and "Molecular Cloning Manual," or according to the conditions recommended by the raw material or product manufacturer. Reagents whose specific sources are not specified were commercially available.

[0114] Biological evaluation

[0115] 1. Preparation of anti-B7H3-ADC preparation ADC-1

[0116] The structure of the anti-B7H3-ADC disclosed herein is as follows:

[0117] First, the naked antibody solution h1702DS (6.43 L, 1.39 mmol) was diluted with pH 5.0 acetic acid-sodium acetate buffer (0.98 L, 30 mM), and then EDTA solution (0.57 L, 29 mmol), pH 8.2 histidine-acetate-Tris solution (1.47 L, 20 mM), and Tris solution (0.2 L, 1 M) were added in sequence. After thorough mixing, tris(2-carboxyethyl)phosphine (TCEP) solution (1.78 L, 3.06 mmol) was added and the mixture was reacted at 12°C for 2 hours. After the reaction, acetic acid was added to adjust the pH to 6.0.

[0118] First, 0.57L of DMSO solvent was added to the above reaction solution, and then the compound 5-A solution (prepared with reference to 5-A in Example 5 of the WO2020063673A1 specification, wherein the compound 5-A was dissolved in DMSO, 6.58 g, 6.12 mmol) was added dropwise to the reaction solution. After thorough mixing, the reaction was incubated at 12 ° C for 1 hour. After the reaction was completed, acetic acid was added to adjust the reaction solution to pH 5.0. The reaction solution was purified by cationic chromatography (column packing Capto S impAct, elution phase 50mM acetic acid-sodium acetate + 240mM NaCl, pH 5.5), and then the solution was exchanged by ultrafiltration to obtain an exemplary product ADC-1 of the B7H3-ADC antibody drug conjugate disclosed herein.

[0119] The DAR value was detected by RP-HPLC, and n was 4.1.

[0120] 2. Preparation

[0121] The equipment used in the preparation and testing process and the calculation method of the results are as follows:

[0122] 1)SEC size exclusion chromatography:

[0123] An analytical method that separates solutes based on the relative relationship between the pore size of the gel and the coil size of the polymer sample molecules.

[0124] SEC% (SEC monomer content percentage) = A monomer / A total * 100% (A monomer is the peak area of ​​the main peak monomer in the sample, and A total is the sum of all peak areas.)

[0125] SEC instrument: Agilent 1260; columns: waters, XBrige SEC (300×7.8mm3.5μm)

[0126] 2) CE capillary gel electrophoresis:

[0127] A method of electrophoresis in which gel is transferred to a capillary tube as a supporting medium and samples are separated according to their molecular weight at a certain voltage.

[0128] Reduction CE purity percentage = A main peak / A total * 100% (A main peak is the peak area of ​​the light chain main peak + the heavy chain main peak in the sample, and A total is the sum of all peak areas.

[0129] CE measurement instrument: Beckman model plus800

[0130] 3) Turbidity measurement:

[0131] The degree of obstruction to light passing through the water layer indicates the water's ability to scatter and absorb light. This is related not only to the suspended solids content but also to the particle composition, size, shape, and surface reflectivity. By comparing the absorbance values ​​of the same protein sample at the same concentration and wavelength (near-UV and visible wavelength regions), higher absorbance values ​​indicate greater turbidity and a more pronounced tendency for protein molecules in the sample to aggregate. The measurement instrument is a multifunctional microplate reader (Molecular Devices M5). Equal volumes of sample are added to a 96-well plate and the absorbance values ​​are read.

[0132] 4) Osmotic pressure measurement:

[0133] The freezing point method determines osmotic pressure, based on the proportional relationship between freezing point depression and the molar concentration of a solution. It uses a highly sensitive temperature sensor to measure the freezing point of a solution and converts the electrical charge into osmotic pressure. Instrument manufacturer: Loser, model OM815.

[0134] 5) Protein concentration determination:

[0135] The concentration of the antibody drug conjugate in the present disclosure is based on the concentration of the protein, that is, the concentration of the antibody portion in the antibody drug conjugate.

[0136] Because the toxin in the antibody drug conjugate absorbs at the protein characteristic absorption wavelength of 280nm, and the toxin also absorbs at 370nm, the following formula is used to calculate the above protein concentration: A 280nm =(C drug ×E drug-280 +C mAb ×E mAb-280 )×l A 370nm =C drug ×E drug-370 ×l

[0137] Pick

[0138] Right now:

[0139] Where A 280nm : The average absorbance of a single sample of the test solution at a wavelength of 280 nm when the optical path length is 1 cm;

[0140] A 370nm : The average absorbance of a single sample of the test solution at a wavelength of 370 nm when the optical path length is 1 cm;

[0141] E mAb-280 : The mass extinction coefficient of the protein at a wavelength of 280nm is 1.532g -1 cm -1 L;

[0142] E drug-280 : Mass extinction coefficient of toxin at 280nm wavelength, 5.17g -1 cm -1 L;

[0143] E drug-370 : Mass extinction coefficient of toxin at 370nm wavelength, 17.89g -1 cm -1 L;

[0144] R: the ratio of the toxin extinction coefficient at 370 nm to that at 280 nm, which is 3.46;

[0145] C mAb : protein concentration, mg / mL;

[0146] l: optical path length, cm (the optical path here is 1 cm).

[0147] If the test solution is diluted, the protein concentration is: C (mg / mL) = C mAb ×N, where N is the dilution multiple.

[0148] Protein concentration determination instrument: UV-visible spectrophotometer, model: Nano Drop One.

[0149] 6) Drug loading calculation (reverse-phase high performance liquid chromatography, RP-HPLC)

[0150] 1. Reagents and instruments:

[0151] HPLC system: Waters H-Class ultra-high performance liquid chromatography UPLC system

[0152] Detector: TUV detector (measurement wavelength: 280nm)

[0153] Column: BioResolve RP mAb Polyphenyl (2.7 μm 4.6*150 mm)

[0154] 2. Testing conditions:

[0155] Column temperature: 80°C

[0156] Flow rate: 1.0 mL / min

[0157] Mobile phase A: 0.1% formic acid + 0.025% trifluoroacetic acid (TFA) in water.

[0158] Mobile phase B: 0.1% FA + 0.025% trifluoroacetic acid (TFA) in acetonitrile.

[0159] Gradient program: 27.0% B-45.0% B (0.00 min-12.00 min), 45.0% B-80.0% B (12.00 min-13.00 min), 80.0% B-80.0% B (13.00 min-15.00 min), 27.0% B-27.0% B (15.04 min-20.00 min),

[0160] Injection sample volume: 5.0 μL

[0161] 3. Data Analysis

[0162] Compared to the antibody light chain (L0) and antibody heavy chain (H0) that do not bind to the drug, the hydrophobicity of the drug-bound light chain (light chain that binds one drug: L1) and the drug-bound heavy chain (heavy chain that binds one drug: H1, heavy chain that binds two drugs: H2, heavy chain that binds three drugs: H3, heavy chain that binds four drugs: H4) increases in direct proportion to the number of drugs bound to them, and their retention time is prolonged. Therefore, they can be eluted in the order of L0, L1, H0, H1, H2, H3, and H4. As a result of comparing the retention times between L0 and H0, the detection peak is assigned to any one of L0, L1, H0, H1, H2, H3, and H4.

[0163] Since the drug linker absorbs UV, the peak area obtained is corrected for the number of bound drugs using the molar absorptivity of the light chain, heavy chain, and drug linker according to the following expression. The calculation formula is as follows:

[0164] Light chain (εLC-280) / (εLC-280+number of linked drugs*εdrug-280)

[0165] Heavy chain (εHC-280) / (εHC-280+number of linked drugs*εdrug-280)

[0166] Note: εLC-280: molar extinction coefficient of light chain at 280 nm;

[0167] εHC-28: molar extinction coefficient of heavy chain at 280 nm;

[0168] εdrug-280: molar extinction coefficient of the toxin at 280 nm.

[0169] Table 1 Calculation of drug loading capacity of reversed phase chromatography

[0170] Note: Sum of LC corrected peak areas = L0 corrected peak area + L1 corrected peak area

[0171] The sum of HC corrected peak areas = H0 corrected peak area + H1 corrected peak area + H2 corrected peak area + H3 corrected peak area + H4 corrected peak area. The drug loading capacity of the ADC drug is calculated as follows: Drug loading capacity n = 2 × ∑ (number of connected drugs × corrected peak area percentage) / 100

[0172] Example 1: Anti-B7H3-ADC Antibody Preparation Buffer System and pH Screening

[0173] An anti-B7H3-ADC formulation containing 60 mg / mL sucrose and 0.2 mg / mL polysorbate 80 (PS80) was prepared using the following buffer, with the ADC-1 protein concentration being 20 mg / mL:

[0174] 1) 10 mM citric acid-sodium citrate (CA), pH 5.0;

[0175] 2) 10 mM succinic acid-sodium succinate (SA), pH 5.0;

[0176] 3) 10 mM SA, pH 5.5;

[0177] 4) 10 mM histidine hydrochloride (abbreviated as: His), pH 5.5;

[0178] 5) 10 mM His, pH 6.0;

[0179] 6) 10 mM His, pH 6.5;

[0180] 7) 10 mM histidine-acetate (abbreviated as: His-AA), pH 5.0;

[0181] 8) 10 mM His-AA, pH 5.5;

[0182] 9) 10 mM sodium dihydrogen phosphate (PB), pH 6.5.

[0183] The prepared formulation was filtered, filled, stoppered, and capped. The stability of the sample was evaluated under forced degradation conditions (40°C M1, i.e., 1 month at 40°C; 300 rpm shaking D10, i.e., 10 days). The stability of the formulation was evaluated using appearance, SEC, and reduced CE-SDS. The experimental results are shown in Table 2.

[0184] Under forced degradation conditions of D10 with shaking, the appearance of protein preparations from SA and His-AA buffer systems was superior to those from CA, His, and PB systems. There was no significant difference in purity between the SA, His-AA, CA, and His buffer systems, and all were superior to the PB system. Under forced degradation conditions of M1 at 40°C, the appearance of protein preparations from SA and His-AA buffer systems was superior to that of CA and PB systems. Based on these considerations, SA and His-AA were preferred as buffer systems for B7H3-ADC preparations for subsequent formulation screening, with a preferred pH range of 5.0-5.5, more preferably 5.5.

[0185] Table 2 Results of pH and buffer system screening experiments

[0186] Note: D0 means the start of the experiment, "M" means month, for example, M1 means one month

[0187] Example 2: Screening of surfactants in anti-B7H3-ADC antibody preparations

[0188] Using a 10 mM SA, pH 5.0 buffer system, anti-B7H3-ADC formulations containing 60 mg / ml sucrose and 20 mg / mL ADC-1 protein concentration were prepared using different surfactant types and concentrations as follows:

[0189] 1) 0.2 mg / mL polysorbate 80 (PS80);

[0190] 2) 0.4 mg / mL PS80;

[0191] 3) 0.2 mg / mL polysorbate 20 (abbreviated as PS20).

[0192] The prepared formulation was filtered, filled, stoppered, and capped. The sample was subjected to shaking (25°C, 300 rpm, 6 days), five cycles of freeze-thaw (-35°C to 2-8°C), and high temperature (40°C / min). The stability of the formulation was evaluated using appearance, SEC, and reduced R-CE. The results are shown in Table 3.

[0193] The experimental results showed that under storage conditions, there was no significant difference in appearance and purity among the 0.2mg / mL PS80, 0.4mg / mL PS80 and 0.2mg / mL PS20 groups.

[0194] Table 3 Results of surfactant screening experiments in preparations

[0195] Note: In the table, “D” means day, for example, D6 means 6 days; D0 means the start of the experiment; “M” means month, for example, M1 means 1 month; and “FT 5Cycle” means 5 freeze-thaw cycles.

[0196] Example 3: Screening of sugars for anti-B7H3-ADC antibody preparations

[0197] An anti-B7H3-ADC formulation was prepared using 10 mM SA, pH 5.0 buffer, 0.2 mg / mL polysorbate 80, and 20 mg / mL ADC-1 protein concentration. The different sugars were as follows:

[0198] 1) 60 mg / mL sucrose;

[0199] 2) 60 mg / mL trehalose;

[0200] 3) 50 mg / mL mannitol.

[0201] The prepared formulation was filtered, filled, stoppered, and capped. The stability of the sample was investigated under forced degradation conditions (shaking D6, i.e., 6 days of shaking; FT 5 Cycle, i.e., 5 freeze-thaw cycles at -35°C to -2-8°C). SEC was used to evaluate the formulation stability. The experimental results are shown in Table 4.

[0202] Experimental data showed that under the forced degradation conditions of shaking D6 and FT5C, the SEC purity of the sucrose and trehalose groups was superior to that of the mannitol group. Subsequently, an anti-B7H3-ADC antibody formulation containing 20 mg / mL antibody, 60 mg / mL sucrose, and 0.2 mg / mL PS80 was prepared in 10 mM SA pH 5.0 buffer. The formulation sample was lyophilized and showed a collapsed bottom appearance after lyophilization. Further optimization was performed, and the sucrose content was reduced from 60 mg / mL to 40 mg / mL.

[0203] Table 4 Excipient screening test results

[0204] Note: “D” in the table means day, and D0 means the start of the experiment.

[0205] Example 4: Screening of osmotic pressure regulators in anti-B7H3-ADC antibody preparations

[0206] An anti-B7H3-ADC formulation was prepared using a 10 mM SA, pH 5.5 buffer system containing 40 mg / mL sucrose, 0.2 mg / mL PS80, and a protein concentration of 20 mg / mL ADC-1. The osmotic pressure of the formulation was controlled within the isotonic range (270-330 mOsm) by increasing the ionic strength and screening osmotic pressure regulators.

[0207] Using a 30 mM SA, pH 5.5 buffer system, anti-B7H3-ADC formulations containing 40 mg / mL sucrose, 0.2 mg / mL PS80, and ADC-1 protein concentration of 20 mg / mL were prepared with different types of isotonic osmotic pressure regulators. The details are as follows:

[0208] 1) 58 mM arginine hydrochloride;

[0209] 2) 7.6 mg / mL glycine;

[0210] The prepared preparation is filtered, filled, semi-stoppered, and freeze-dried. The freeze-drying procedure is shown in Table 5:

[0211] Table 5 Freeze-drying procedure

[0212] The stability of the lyophilized samples was investigated under forced degradation conditions (40°C D23, i.e., 23 days at 40°C). Appearance, SEC, and reduced R-CE were used as evaluation indicators to assess the stability of the formulation. The experimental results are shown in Table 6.

[0213] Under forced degradation conditions at 40°C (D23), no significant differences in purity were observed between the groups. The glycine group exhibited significantly better appearance than the arginine hydrochloride group. The osmotic pressure of a formulation containing 40 mg / mL sucrose and 7.6 mg / mL glycine was approximately 300 mOsm, so a glycine concentration of 7.6 mg / mL was preferred. Using glycine as an osmotic pressure regulator, concentrations between 5.3 mg / mL and 9.8 mg / mL maintained the formulation's osmotic pressure within the isotonic range.

[0214] Table 6 Results of osmotic pressure regulator screening experiments in preparations

[0215] Note: “D” in the table stands for day, for example, D23 stands for 23 days.

[0216] Example 5: Screening of protein concentration in anti-B7H3-ADC antibody preparations

[0217] Using a 30mM SA, pH 5.5 buffer system, anti-B7H3-ADC formulations with different protein concentrations containing 40mg / mL sucrose, 0.2mg / mL PS80, and 7.6mg / mL glycine were prepared. The details are as follows:

[0218] 1) 20 mg / mL ADC-1

[0219] 2) 50 mg / mL ADC-1

[0220] 3) 60 mg / mL ADC-1

[0221] 4) 70 mg / mL ADC-1

[0222] The prepared formulation was filtered, filled, stoppered, and capped. The sample was subjected to shaking (25°C, 300 rpm, 6 days), five cycles of freeze-thaw (-35°C to 2-8°C), and high temperature (40°C / min). The stability of the formulation was evaluated using appearance, SEC, and DAR values. The experimental results are shown in Table 7.

[0223] The experimental results showed that under storage conditions, there was no significant difference in appearance, purity and toxin load among the 20 mg / mL ADC-1, 50 mg / mL ADC-1, 60 mg / mL ADC-1 and 70 mg / mL ADC-1 groups.

[0224] Table 7 Results of surfactant screening experiments in preparations

[0225] Note: In the table, “D” means day, D0 means the start of the experiment; “M” means month, for example, M1 means 1 month, and “FT 5Cycle” means 5 freeze-thaw cycles.

[0226] Example 6: Anti-B7H3-ADC Antibody Preparation Stability Experiment

[0227] An anti-B7H3-ADC formulation was prepared containing ADC-1 at a protein concentration of 20 mg / mL, 30 mM succinic acid-sodium succinate (SA) pH 5.5, 40 mg / mL sucrose, 7.6 mg / mL glycine, and 0.2 mg / mL PS80.

[0228] The prepared formulation was filtered, filled, stoppered, and capped. The stability of the formulation was evaluated under forced degradation conditions (freeze-thaw at -35°C to 2-8°C for five cycles, or shaking for eight days) and long-term storage (2-8°C for 16 days). Appearance, SEC, and reduced R-CE were used as evaluation criteria. The experimental results are shown in Table 8.

[0229] The experimental results show that the product remains clear under various forced degradation conditions, with no significant difference in purity, indicating good stability.

[0230] Table 8 Preparation stability test results

[0231] Note: In the table, “D” means day, D0 means the beginning of the experiment, and FT5C means 5 freeze-thaw cycles.

[0232] Example 7: B7H3-ADC formulation optimization experiment

[0233] Optimization experiment 1:

[0234] A DOE design was conducted for an anti-B7H3-ADC formulation containing 7.6 mg / mL glycine and 40 mg / mL sucrose, using 30 mM succinic acid (SA)-sodium succinate (pH), ADC-1 protein concentration, and PS80 concentration as variables. The DOE, experimental factors, and levels were set as follows: pH 5-6, ADC-1 protein concentration 10-30 mg / mL, and PS80 concentration 0.1-0.3 mg / mL, resulting in a series of formulations (see Table 9). Lyophilized samples were subjected to a forced degradation test at 40°C for one month, with appearance and SEC evaluation as the evaluation criteria. The results are shown in Table 9.

[0235] Results showed that after one month at 40°C, all formulations remained clear, with no significant differences in purity between SEC groups. Therefore, B7H3-ADC samples exhibited excellent stability within a pH range of 5-6, ADC-1 protein concentrations of 10-30 mg / mL, and PS80 concentrations of 0.1-0.3 mg / mL.

[0236] Table 9 DOE prescription screening experiment prescription design

[0237] Table 10 DOE screening test results

[0238] Note: In the table, “D” represents day, D0 represents the start of the experiment, and “M” represents month, for example, M1 represents one month.

[0239] Optimization experiment 2:

[0240] Based on an anti-B7H3-ADC formulation in 30 mM SA, pH 5.5 succinate-sodium succinate buffer, an ADC-1 protein concentration of 50 mg / mL, 40 mg / mL sucrose, 7.6 mg / mL glycine, and 0.2 mg / mL PS80, a series of formulations were designed (see Table 11) with buffer concentration, pH, sucrose concentration, glycine concentration, PS80 concentration, and ADC-1 protein concentration as variables. Lyophilized samples were subjected to a forced degradation test at 40°C for one month, with appearance, SEC purity, and DAR values ​​as evaluation criteria. The results are shown in Table 12.

[0241] Results showed that after one month at 40°C, all formulations remained clear, with no significant differences in purity, SEC, or DAR toxin loading between groups. Therefore, B7H3-ADC samples exhibited excellent stability within the range of SA concentrations of 20-30 mM, pH values ​​of 5.0-5.5, ADC-1 protein concentrations of 20-70 mg / mL, sucrose concentrations of 40-80 mg / mL, and PS80 concentrations of 0.2-0.8 mg / mL.

[0242] Table 11 DOE prescription screening experiment prescription design

[0243] Table 12 DOE screening test results

[0244] Note: In the table, “D” stands for day, “W” stands for week, D0 stands for the beginning of the experiment, and W4 stands for four weeks.

[0245] Example 8: Lyophilization of anti-B7H3-ADC antibody formulation

[0246] An anti-B7H3-ADC antibody formulation containing ADC-1 at a protein concentration of 20 mg / mL, 40 mg / mL sucrose, 7.6 mg / mL glycine, and 0.2 mg / mL PS80 was prepared in 30 mM SA, pH 5.5 succinate-sodium succinate buffer. The formulation samples were lyophilized using a pre-freeze, primary drying, and secondary drying procedure (see Table 13). After the lyophilization process, the formulations were stoppered under vacuum. Samples before and after lyophilization were evaluated using appearance, SEC, and R-CE (see Table 14). The results demonstrate that the reconstituted solution maintains the excellent performance of the liquid formulation.

[0247] Table 13 Freeze-drying procedure

[0248] Table 14 Experimental results before and after freeze-drying

Claims

1. A pharmaceutical composition comprising an antibody drug conjugate and a buffer, characterized in that: The antibody drug conjugate has the following structure: in: h1702DS is an anti-B7H3 antibody comprising a heavy chain as shown in SEQ ID NO: 1 and a light chain as shown in SEQ ID NO: 2; n is 1 to 10, preferably 1 to 8, more preferably 3 to 5, more preferably about 4; The buffer of the pharmaceutical composition is a succinate buffer or a histidine buffer, preferably succinic acid-succinic acid sodium salt or histidine-acetate.

2. The pharmaceutical composition according to claim 1, characterized in that The concentration of the buffer is about 10 mM to about 50 mM, preferably about 20 mM to about 40 mM, and more preferably about 30 mM.

3. The pharmaceutical composition according to claim 1 or 2, characterized in that The pH of the pharmaceutical composition is about 4.5 to 6.5, preferably about 5.0 to 6.0, more preferably about 5.5 to 5.

6.

4. The pharmaceutical composition according to any one of claims 1 to 3, characterized in that The pharmaceutical composition further comprises a surfactant, which is preferably polysorbate, more preferably polysorbate 80 or polysorbate 20, and most preferably polysorbate 80.

5. The pharmaceutical composition according to claim 4, characterized in that The surfactant concentration is from about 0.01 mg / mL to about 1.0 mg / mL, preferably from about 0.2 mg / mL to about 0.8 mg / mL, and more preferably about 0.2 mg / mL.

6. The pharmaceutical composition according to any one of claims 1 to 5, characterized in that The pharmaceutical composition further comprises sugar; preferably, the sugar is selected from sucrose, mannitol and trehalose; more preferably, the sugar is sucrose or trehalose.

7. The pharmaceutical composition according to claim 6, characterized in that The sugar concentration is about 25 mg / mL to about 80 mg / mL, preferably about 40 mg / mL to about 60 mg / mL, and more preferably about 40 mg / mL.

8. The pharmaceutical composition according to any one of claims 1 to 7, characterized in that The pharmaceutical composition further comprises an amino acid or an amino acid salt, wherein the amino acid or the amino acid salt is selected from glycine and arginine hydrochloride, preferably glycine.

9. The pharmaceutical composition according to claim 8, characterized in that The concentration of the amino acid or amino acid salt is about 5 mg / mL to about 10 mg / mL; preferably about 7 mg / mL to about 8 mg / mL; more preferably about 7.6 mg / mL.

10. The pharmaceutical composition according to any one of claims 1 to 9, characterized in that The concentration of the antibody drug conjugate is, based on protein concentration, about 10 mg / mL to about 70 mg / mL, preferably about 20 mg / mL to about 50 mg / mL, more preferably about 20 mg / mL to about 30 mg / mL or about 30 mg / mL to about 50 mg / mL, further preferably about 20 mg / mL, about 30 mg / mL or about 50 mg / mL.

11. The pharmaceutical composition according to any one of claims 1 to 10, characterized in that Contains the following components: (a) about 20 mg / mL to about 70 mg / mL of the antibody drug conjugate, based on protein concentration, (b) about 0.2 mg / mL to about 0.8 mg / mL of polysorbate, (c) about 25 mg / mL to about 80 mg / mL of a saccharide, (d) about 7 mg / mL to about 8 mg / mL of glycine, and (e) about 10 mM to about 40 mM succinate buffer; the pH of the composition being about 5.0-6.0; Preferably, the pharmaceutical composition comprises the following components: (a) about 20 mg / mL to about 50 mg / mL of the antibody drug conjugate, based on protein concentration, (b) about 0.2 mg / mL to about 0.8 mg / mL of polysorbate 80, (c) about 40 mg / mL to about 80 mg / mL of sucrose, (d) about 7.6 mg / mL of glycine, and (e) about 30 mM succinic acid-sodium salt, wherein the pH of the composition is about 5.5-5.6; More preferably, the pharmaceutical composition comprises the following components: (a) about 20 mg / mL to about 30 mg / mL of the antibody drug conjugate, based on protein concentration, (b) about 0.2 mg / mL of polysorbate 80, (c) about 40 mg / mL of sucrose, (d) about 7.6 mg / mL of glycine, and (e) about 30 mM succinic acid-sodium salt, wherein the pH of the composition is about 5.5-5.6; (a) about 30 mg / mL to about 50 mg / mL of the antibody drug conjugate, based on protein concentration, (b) about 0.2 mg / mL of polysorbate 80, (c) about 40 mg / mL of sucrose, (d) about 7.6 mg / mL of glycine, and (e) about 30 mM succinic acid-sodium salt, wherein the pH of the composition is about 5.5-5.

6.

12. A lyophilized preparation containing an antibody drug conjugate, characterized in that: The lyophilized preparation can be reconstituted to form the pharmaceutical composition according to any one of claims 1 to 11.

13. A method for preparing a lyophilized preparation containing an antibody drug conjugate, characterized in that: The method comprises the step of freeze-drying the pharmaceutical composition according to any one of claims 1 to 11.

14. A lyophilized preparation containing an antibody drug conjugate, characterized in that: The lyophilized preparation is obtained by freeze-drying the pharmaceutical composition according to any one of claims 1 to 11.

15. A reconstitution solution containing an antibody drug conjugate, characterized in that: The reconstituted solution is prepared by reconstituted the lyophilized preparation according to claim 12 or 14, and the reconstituted solution comprises the following components: (a) about 20 mg / mL to about 70 mg / mL of the antibody drug conjugate, based on protein concentration, (b) about 0.2 mg / mL to about 0.8 mg / mL of polysorbate, (c) about 25 mg / mL to about 80 mg / mL of a saccharide, (d) about 7 mg / mL to about 8 mg / mL of glycine, and (e) about 10 mM to about 40 mM succinate buffer; the pH of the composition being about 5.0-6.0; Preferably, the reconstitution solution comprises the following components: (a) about 20 mg / mL to about 50 mg / mL of the antibody drug conjugate, based on protein concentration, (b) about 0.2 mg / mL to about 0.8 mg / mL of polysorbate 80, (c) about 40 mg / mL to about 80 mg / mL of sucrose, (d) about 7.6 mg / mL of glycine, and (e) about 30 mM succinic acid-sodium salt, wherein the pH of the composition is about 5.5-5.6; More preferably, the reconstitution solution comprises the following components: (a) about 20 mg / mL to about 30 mg / mL of the antibody drug conjugate, based on protein concentration, (b) about 0.2 mg / mL of polysorbate 80, (c) about 40 mg / mL of sucrose, (d) about 7.6 mg / mL of glycine, and (e) about 30 mM succinic acid-sodium salt, wherein the pH of the composition is about 5.5-5.6; (a) about 30 mg / mL to about 50 mg / mL of the antibody drug conjugate, based on protein concentration, (b) about 0.2 mg / mL of polysorbate 80, (c) about 40 mg / mL of sucrose, (d) about 7.6 mg / mL of glycine, and (e) about 30 mM succinic acid-sodium salt, wherein the pH of the composition is about 5.5-5.

6.

16. A product, characterized in that The invention comprises a container containing the pharmaceutical composition according to any one of claims 1 to 11, the lyophilized preparation according to claim 12 or 14, or the reconstituted solution according to claim 15.

17. Use of the pharmaceutical composition according to any one of claims 1 to 11, the lyophilized formulation according to claim 12 or 14, the reconstituted solution according to claim 15, or the product according to claim 16 in the preparation of a medicament for treating a disease; preferably, the disease is a cancer associated with B7H3 expression; more preferably, the cancer is selected from breast cancer, ovarian cancer, cervical cancer, uterine cancer, prostate cancer, kidney cancer, urethral cancer, bladder cancer, liver cancer, gastric cancer, endometrial cancer, salivary gland cancer, esophageal cancer, melanoma, glioma, neuroblastoma, sarcoma, lung cancer, colon cancer, rectal cancer, colorectal cancer, leukemia, bone cancer, skin cancer, thyroid cancer, pancreatic cancer, and lymphoma.