Patent Application
20240325564
The present disclosure belongs to the field of pharmaceutical formulations, and in particular relates to a pharmaceutical composition comprising an anti-TROP2 antibody drug conjugate and use thereof as an anti-cancer medicament.
The statements herein merely provide background information related to the present disclosure and may not necessarily constitute the prior art.
Antibody drug conjugate (ADC) links a monoclonal antibody or an antibody fragment to a biologically active cytotoxin via a stable chemical linker compound, fully exploiting the binding specificity of the antibody to surface antigens of tumor cells and the high-efficiency of the cytotoxin, and also avoiding the former's disadvantage of having a poor therapeutic effect, the latter's disadvantage of having serious toxic side effects, and the like. This means that the antibody drug conjugate can bind to tumor cells more precisely and has a reduced effect on normal cells compared to conventional chemotherapeutic drugs in the past (Mullard A, (2013) Nature Reviews Drug Discovery, 12:329-332; DiJoseph J F, Armellino D C, (2004) Blood, 103:1807-1814).
There are several classes of small molecules with cytotoxicity for antibody drug conjugates, one of which is camptothecin derivatives having an antitumor effect by inhibiting topoisomerase I. The camptothecin derivative, irinotecan (chemical name: (1S, 9S)-1-amino-9-ethyl-5-fluoro-2,3-dihydro-9-hydroxy-4-methyl-1H,12H-benzo[de]pyrano[3′,4′:6,7]imidazo[1,2-b]quinoline-10,13(9H,15H)-dione), was reported for use in antibody drug conjugates (ADCs) as described in WO2014057687; Clinical Cancer Research (2016)22 (20):5097-5108; Cancer Sci (2016) 107: 1039-1046. There is still a need to further develop ADC drugs with better therapeutic effects.
However, ADCs are of more complicated heterostructures than antibodies, and therefore, a greater challenge has been posed to ADC formulations for therapeutic purposes.
The present disclosure provides a pharmaceutical composition comprising an antibody drug conjugate and a buffer, wherein the antibody drug conjugate has a structure shown below:
The antibody drug conjugate described above is prepared according to Example 3-1 ADC-1 (an example for antibody conjugates) on page 41 of the description of PCT/CN2021/073279;
WINTYTGEPTYTQDFKGRFAFSLDTSVSTAYLQISSLKAEDTAVYYCAR
GGFGSSYWYFDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALG
SASYRYTGVPDRFSGSGSGTDFTLTISSLQPEDFAVYYCQQHYITPLTF
In an alternative embodiment, the buffer in the pharmaceutical composition has a pH of about 5.0 to about 6.5, and non-limiting examples include 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; the pH is preferably about 5.5 to about 6.5 and more preferably about 5.9 to about 6.2.
In alternative embodiment, the buffer in the pharmaceutical composition is at a concentration of about 5 mM to about 50 mM, and non-limiting examples include 10 mM, 12 mM, 15 mM, 16 mM, 17 mM, 18 mM, 19 mM, 20 mM, 30 mM, 40 mM, 50 mM, and any range therebetween; the buffer is preferably at a concentration of about 10 mM to about 50 mM, more preferably about 20 mM to about 40 mM, and most preferably about 30 mM.
In an alternative embodiment, the pharmaceutical composition further comprises a surfactant. The surfactant may be selected from the group consisting of polysorbate, polysorbate 20, polysorbate 80, poloxamer, Triton, sodium dodecyl sulfonate, sodium lauryl sulfonate, sodium octyl glycoside, lauryl-sulfobetaine, myristyl-sulfobetame, linoleyl-sulfobetaine, stearyl-sulfobetaine, lauryl-sarcosine, myristyl-sarcosine, linoleyl-sarcosine, stearyl-sarcosine, linoleyl-betaine, myristyl-betaine, cetyl-betaine, lauramido propyl-betaine, cocaramide propyl-betaine, linoleinamide propyl-betaine, myristylamide propyl-betaine, palmitamide propyl-betaine, isostearamide propyl-betame, myristylamide propyl-dimethylamine, palmitamide propyl-dimethylamine, isostearamide propyl-dimethylamine, sodium methyl cocoyl, sodium methyl oleyl taurate, polyethylene glycol, polypropylene glycol, copolymer of ethylene and propylene glycol, and the like. The surfactant is preferably polysorbate 80 or polysorbate 20, and more preferably polysorbate 80.
In an alternative embodiment, the surfactant in the pharmaceutical composition is at a concentration of about 0.01 mg/mL to about 1.0 mg/mL, preferably about 0.05 mg/mL to about 0.5 mg/mL, more preferably about 0.1 mg/mL to about 0.3 mg/mL or about 0.2 mg/mL to about 0.6 mg/mL, and most preferably about 0.2 mg/mL, and non-limiting examples include about 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, 0.6 mg/mL, 0.7 mg/mL, 0.8 mg/mL, and any range therebetween.
In an alternative embodiment, the aforementioned pharmaceutical composition further comprises a saccharide. “Saccharide” of the present disclosure includes the general composition (CH2O)n and derivatives thereof, including monosaccharides, disaccharides, trisaccharides, polysaccharides, sugar alcohols, reducing sugars, non-reducing sugars, etc. The saccharide may be selected from the group consisting of glucose, sucrose, trehalose, lactose, fructose, maltose, dextran, glycerin, erythritol, glycerol, arabitol, sylitol, sorbitol, mannitol, mellibiose, melezitose, raffinose, mannotriose, stachyose, maltose, lactulose, maltulose, glucitol, maltitol, lactitol, iso-maltulose, etc. The saccharide is preferably non-reducing disaccharide, is more preferably selected from the group consisting of trehalose dihydrate and sucrose, and is most preferably sucrose.
In an alternative embodiment, the saccharide in the aforementioned pharmaceutical composition is at a concentration of about 25 mg/mL to about 80 mg/mL and preferably about 30 mg/mL to about 50 mg/mL, and 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, 70 mg/mL, 80 mg/mL, and any range therebetween; the saccharide is preferably at a concentration of 40 mg/mL.
In an alternative embodiment, the aforementioned pharmaceutical composition further comprises an amino acid and a salt thereof, and the amino acid or the amino salt thereof is preferably selected from the group consisting of glycine and arginine hydrochloride and is more preferably glycine.
In an alternative embodiment, the glycine in the aforementioned pharmaceutical composition is at a concentration of about 6 mg/mL to about 15 mg/mL, about 7 mg/mL to about 11 mg/mL, preferably about 7 mg/mL to about 10 mg/mL, and more preferably about 7 mg/mL to about 9 mg/mL or about 8 mg/mL to about 9 mg/mL, and non-limiting examples include about 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, 10 mg/mL, 10.2 mg/mL, 11 mg/mL, 12 mg/mL, 13 mg/mL, 14 mg/mL, 15 mg/mL, and any range therebetween; the glycine is most preferably at a concentration of about 9 mg/mL.
In an alternative embodiment, the antibody drug conjugate in the pharmaceutical composition is at a protein concentration of about 1 mg/mL to about 100 mg/mL, and 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; the antibody drug conjugate is preferably at a protein concentration of about 10 mg/mL to about 30 mg/mL, more preferably about 18 mg/mL to about 22 mg/mL, and most preferably about 20 mg/mL. Specifically, non-limiting examples include 20.1 mg/mL, 20.2 mg/mL, 20.3 mg/mL, 20.4 mg/mL, 20.5 mg/mL, 20.6 mg/mL, 20.7 mg/mL, 20.8 mg/mL, 20.81 mg/mL, 20.82 mg/mL, 20.83 mg/mL, 20.84 mg/mL, 20.85 mg/mL, 20.86 mg/mL, 20.87 mg/mL, 20.88 mg/mL, 20.89 mg/mL, 20.9 mg/mL, 20.9 mg/mL, 20.91 mg/mL, 20.92 mg/mL, 20.93 mg/mL, 20.94 mg/mL, 20.95 mg/mL, 20.96 mg/mL, 20.97 mg/mL, 20.98 mg/mL, 20.99 mg/mL, 21 mg/mL, and any range therebetween. The protein concentration refers to the concentration of the antibody moiety in the antibody drug conjugate.
In an alternative embodiment, the range of drug loading (n) may be an average number of cytotoxic drugs bound per anti-Trop2 antibody, and non-limiting examples include an average number of cytotoxic drugs bound per antibody of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, and any range therebetween. Preferably, the drug loading is selected from the group consisting of 2-8, 2-7, 2-6, 2-5, 2-4, 3-4, 3-5, 5-6, 5-7, 5-8, and 6-8. Illustratively, the drug loading may be an average number of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. n is a decimal or an integer.
In an alternative embodiment, the drug loading (n) is about 4.
In an alternative embodiment, the pharmaceutical composition comprises:
In an alternative embodiment, the pharmaceutical composition of any one of the aforementioned embodiments is a liquid formulation. The liquid formulation or the reconstituted formulation of the present invention has relatively good stability and can be stored at the refrigeration temperature (4° C.) for at least 3 months. In addition, stable liquid formulations include liquid formulations that exhibit desired features after storage at temperatures including 25° C. for periods including 1 month, 2 months, and 3 months. Further, stable liquid formulations include liquid formulations that exhibit desired features after storage at temperatures including 40° C. for 1 month.
The present disclosure further provides a lyophilized formulation comprising an antibody drug conjugate, wherein the formulation can be reconstituted to form the pharmaceutical composition described above.
The present disclosure further provides a method for preparing a lyophilized formulation comprising an antibody drug conjugate, which comprises a step of lyophilizing the pharmaceutical composition described above.
In an alternative embodiment, the lyophilizing in the method for preparing a lyophilized formulation comprising an antibody drug conjugate comprises steps of pre-freezing, primary drying, and secondary drying in sequence. The lyophilizing is performed by freezing the formulation and subsequently sublimating the water at a temperature suitable for primary drying. Under these conditions, the product is at a temperature lower than the eutectic point or the collapse temperature of the formulation. Typically, the temperature for the primary drying ranges from about −30° C. to 25° C. (assuming the product remains frozen during the primary drying). The formulation, the size and type of the container (e.g., glass vial) containing the sample, and the liquid volume determine the time required for drying, which may range from a few hours to several days (e.g., 40-60 hours). The secondary drying may be performed at about 0-40° C., which mainly depends on the type and size of the container and the type of the protein used. The time needed for the secondary drying is determined by the desired residual moisture content in the product, and it typically takes at least about 5 hours. Generally, the water content of the formulation lyophilized at low pressure is less than about 5%, preferably less than about 3%. The pressure may be the same as that applied to the step of primary drying; preferably, the pressure of the secondary drying is lower than that of the primary drying. The lyophilization conditions may vary with the formulation and vial size.
In an alternative embodiment of the present disclosure, 4.4 mL of a stock solution of the composition is lyophilized, and the lyophilization procedure is as follows: pre-freezing at temperatures of 5° C. and −45° C. successively; primary drying at a temperature of −20° C. and at a degree of vacuum of 10 Pa; and secondary drying at a temperature of 25° C. and at degrees of vacuum of 10 Pa and 1 Pa successively.
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.
The present disclosure further provides a lyophilized formulation comprising an antibody drug conjugate obtained by lyophilizing the pharmaceutical composition of the antibody drug conjugate described above.
The present disclosure further provides a reconstituted solution comprising an antibody drug conjugate, wherein the reconstituted solution is prepared by reconstituting the lyophilized formulation described above.
The present disclosure further provides a method for preparing the reconstituted solution described above, which comprises a step of reconstituting the aforementioned lyophilized formulation with a solution selected from the group consisting of, but not limited to, water for injection, normal saline, and glucose solution.
In an alternative embodiment, the reconstituted solution comprises the following components:
The present disclosure further provides an article of manufacture comprising a container containing the pharmaceutical composition, the lyophilized formulation or the reconstituted solution described above. In some embodiments, the container is a tubular injection vial made of neutral borosilicate glass.
The present disclosure further provides use of the aforementioned pharmaceutical composition, lyophilized formulation, reconstituted solution, or article of manufacture in preparing a medicament for treating or preventing a tumor.
The present disclosure further provides a method for treating a disease comprising providing the aforementioned pharmaceutical composition, lyophilized formulation, reconstituted solution, or article of manufacture.
The present disclosure further provides use of the aforementioned pharmaceutical composition, lyophilized formulation, reconstituted solution, or article of manufacture as a medicament in the treatment of a disease.
The present disclosure further provides the aforementioned pharmaceutical composition, lyophilized formulation, reconstituted solution, or article of manufacture as a medicament, which is preferably used for treating or preventing a tumor disease.
In an alternative embodiment, the disease or tumor is a TROP2-mediated disease or condition.
The present disclosure further provides use of the aforementioned pharmaceutical composition, lyophilized formulation, reconstituted solution, or article of manufacture as a medicament in preparing a medicament for the treatment and/or prevention of a tumor and cancer, wherein the tumor and the cancer are preferably head and neck squamous cell carcinoma, head and neck cancer, brain cancer, neuroglioma, glioblastoma multiforme, neuroblastoma, central nervous system carcinoma, neuroendocrine tumor, throat cancer, pharyngeal squamous cell carcinoma, oral squamous cell carcinoma, nasopharyngeal cancer, esophageal cancer, thyroid cancer, malignant pleural mesothelioma, lung cancer, breast cancer, liver cancer, hepatobiliary cancer, pancreatic cancer, stomach cancer, gastrointestinal cancer, intestinal cancer, colon cancer, colorectal cancer, kidney cancer, clear cell renal cell carcinoma, ovarian cancer, endometrial cancer, cervical cancer, bladder cancer, prostate cancer, testicular cancer, skin cancer, melanoma, leukemia, lymphoma, bone cancer, chondrosarcoma, myeloma, multiple myeloma, myelodysplastic syndrome, Krukenberg tumor, myeloproliferative tumor, squamous cell carcinoma, Ewing's sarcoma, urothelium carcinoma, and Merkel cell carcinoma; more preferably, the lymphoma is selected from the group consisting of Hodgkin's lymphoma, non-Hodgkin's lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, primary mediastinal large B-cell lymphoma, mantle cell lymphoma, small lymphocytic lymphoma, T-cell/histiocyte-rich large B-cell lymphoma, and lymphoplasmacytic lymphoma; the lung cancer is selected from the group consisting of non-small cell lung cancer and small cell lung cancer; the leukemia is selected from the group consisting of chronic myeloid leukemia, acute myeloid leukemia, lymphocytic leukemia, lymphoblastic leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, and myeloid cell leukemia.
As is well known to those skilled in the art, one, some or all of the features of the various embodiments described in the present 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 illustrated through the following detailed description.
The present disclosure provides a pharmaceutical composition that favors production and administration and is stable in properties. Undesirable instability may include any one or more of aggregation, deamidation (e.g., Asn deamidation), oxidation (e.g., Met oxidation), isomerization (e.g., Asp isomerization), clipping/hydrolysis/fragmentation (e.g., fragmentation of hinge region), formation of succinimide, unpaired cysteines, dissociation of toxins, and the like. In particular, the pharmaceutical composition described in the present disclosure comprises an antibody drug conjugate and a buffer.
In order to facilitate the understanding of the present disclosure, some technical and scientific terms are specifically defined below. Unless otherwise explicitly defined herein, all other technical and scientific terms used herein have the meanings generally understood by those of ordinary skill in the art to which the present disclosure belongs.
The content of the application WO2020063673 is incorporated herein by reference in its entirety.
“Antibody drug conjugate (ADC)” means that an antibody is linked to a biologically active cytotoxin or a small-molecule drug with cell killing activity by a linker unit.
“Drug loading”, also referred to as drug-to-antibody ratio (DAR), refers to the average number of drugs conjugated to each antibody in an ADC. It may range, for example, from about 1 to about 10 drugs conjugated to each antibody, and in certain embodiments, from about 1 to about 8 drugs conjugated to each antibody, preferably selected from the group consisting of 2-8, 2-7, 2-6, 2-5, 2-4, 3-4, 3-5, 5-6, 5-7, 5-8 and 6-8 drugs conjugated to each antibody. Illustratively, the drug loading may be an average number of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. The ADC general formulas of the present disclosure include a group of antibodies conjugated to drugs within a certain range as described above. In embodiments of the present disclosure, the drug loading may be represented as n. Drug loading can be determined by conventional methods such as UV/visible spectroscopy, mass spectrometry, ELISA assay and HPLC.
The term “linker unit” or “linker fragment” refers to a chemical structure fragment or bond, which is linked to an antibody or antigen-binding fragment thereof at one end and to a drug at the other end, and also may be linked to a drug after being linked to another linker.
The linker includes a stretcher unit, a spacer unit and an amino acid unit, and may be synthesized by methods known in the art, such as those described in US2005-0238649A1. The linker may be a “cleavable linker” favoring the release of drugs in cells. For example, acid-labile linkers (e.g., hydrazones), protease-sensitive (e.g., peptidase-sensitive) linkers, photolabile linkers, dimethyl linkers or disulfide-containing linkers can be used (Chari et al., Cancer Research, 52: 127-131(1992); U.S. Pat. No. 5,208,020).
The loading of the cytotoxic drug can be controlled using the following non-limiting methods, including:
The three-letter and single-letter codes for amino acids used in the present disclosure are as described in J. Biol. Chem., 243, p 3558 (1968).
The “antibody” described herein is used in the broadest sense and encompasses a variety of antibody structures, including but not limited to full-length antibodies, and antibody fragments (or antigen-binding fragments, or antigen-binding moieties) so long as they exhibit the desired antigen-binding activity. In general, a natural intact antibody is of a tetrapeptide chain structure formed by connection between two identical heavy chains and two identical light chains by interchain disulfide bonds.
The engineered antibody or antigen-binding fragment of the present disclosure can be prepared and purified by conventional methods. For example, cDNA sequences encoding the heavy and light chains can be cloned and recombined into a GS expression vector. Recombinant immunoglobulin expression vectors can be stably transfected into CHO cells. As a more recommended prior art, mammalian expression systems will result in glycosylation of antibodies, particularly at the highly conserved N-terminal site of the Fc region. Positive clones are expanded in a serum-free medium of a bioreactor to produce antibodies. The culture medium with the secreted antibody can be purified by conventional techniques. For example, purification is performed using an A or G Sepharose FF column containing an adjusted buffer. Non-specifically bound fractions are washed away. The bound antibody is eluted by the pH gradient method, and the antibody fragments are detected by SDS-PAGE and collected. The antibody can be filtered and concentrated by conventional methods. Soluble mixtures and polymers can also be removed by conventional methods, such as molecular sieves and ion exchange. The resulting product needs to be immediately frozen, e.g., at −70° C., or lyophilized.
“Buffer” refers to a buffer that resists changes in pH by the action of its acid-base conjugate components. Examples of buffers that control the pH in an appropriate range include acetate, succinate, gluconate, histidine, oxalate, lactate, phosphate, citrate, tartrate, fumarate, glycylglycine and other organic acid buffers.
“Histidine buffer” is a buffer comprising histidine. Examples of histidine buffers include histidine-histidine hydrochloride, histidine-histidine acetate, histidine-histidine phosphate, histidine-histidine sulfate, and the like, and the histidine-histidine hydrochloride buffer is preferred. The histidine-histidine hydrochloride buffer can be prepared from histidine and hydrochloric acid or histidine and histidine hydrochloride.
“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. The preferred citrate buffer is citric acid-sodium citrate.
“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. The preferred succinate buffer is succinic acid-sodium succinate. Illustratively, the succinic acid-sodium succinate may be prepared with succinic acid and sodium hydroxide or with succinic acid and sodium succinate.
“Phosphate buffer” is a buffer comprising 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. The preferred phosphate buffer is disodium hydrogen phosphate-sodium dihydrogen phosphate.
“Acetate buffer” is a buffer comprising acetate ions. Examples of acetate buffers include acetic acid-sodium acetate, histidine-histidine acetate, acetic acid-potassium acetate, acetic acid-calcium acetate, acetic acid-magnesium acetate, and the like. The preferred acetate buffer is acetic acid-sodium acetate.
“Pharmaceutical composition” refers to a mixture containing one or more of the antibody drug conjugates described herein or physiologically/pharmaceutically acceptable salts or prodrugs thereof, and other chemical components, wherein the other components are, for example, physiologically/pharmaceutically acceptable carriers and excipients. The purpose of the pharmaceutical composition is to maintain the stability of the active ingredient of the antibody and promote the administration to an organism, which facilitates the absorption of the active ingredient, thereby exerting biological activity.
As used herein, “pharmaceutical composition” and “formulation” are not mutually exclusive.
Unless otherwise stated, the solvent in the pharmaceutical composition described herein in solution form is water.
“Lyophilized formulation” refers to a formulation or a pharmaceutical composition obtained by lyophilizing a pharmaceutical composition or a formulation in liquid or solution form in vacuum.
The terms “about” and “approximately” as used herein mean that a numerical value is within an acceptable error range for the particular value determined by one of ordinary skill in the art, and the numerical value depends in part on how the value is measured or determined (i.e., the limits of the measurement system). For example, “about” may mean a standard deviation within 1 or more than 1 in each practice in the art. Or, “about” or “substantially comprising” may mean a range of up to 20%. Furthermore, particularly for biological systems or processes, the term may mean up to an order of magnitude or up to 5-fold of a numerical value. Unless otherwise stated, when a specific value is provided in the present application and claims, the meaning of “about” or “substantially comprising” should be assumed to be within an acceptable error range for that specific value.
The pharmaceutical composition of the present disclosure can achieve a stable effect: a pharmaceutical composition in which the antibody drug conjugate substantially retains its physical and/or chemical stability and/or biological activity after storage; preferably, the pharmaceutical composition substantially retains its physical and chemical stability as well as its biological activity after storage. The storage period is generally selected based on a predetermined shelf life of the pharmaceutical composition. There are a variety of analytical techniques currently available for measuring protein stability, and the stability after storage for a selected period of time at a selected temperature can be measured.
Stable formulations are formulations in which no significant change is observed under the following conditions: storage at the refrigeration temperature (2-8° C.) for at least 3 months, preferably 6 months, and more preferably 1 year. In addition, stable liquid formulations include liquid formulations that exhibit desired features after storage at temperatures including 25° C. for periods including 1 month, 2 months, and 3 months. Further, stable liquid formulations include liquid formulations that exhibit desired features after storage at temperatures including 40° C. for periods including 10 days, 20 days, and 1 month. Typical examples for stability are as follows: generally, no more than about 10%, preferably no more than about 5%, of antibody monomers aggregate or are degraded as measured by SEC-HPLC. The formulation is a pale yellow, nearly colorless and clear liquid, or colorless, or clear to slightly opalescent, by visual analysis. The concentration, pH, and osmolality of the formulation have a change of no more than ±10%. Generally, a decrease of no more than about 10%, preferably no more than about 5% is observed. Generally, aggregation of no more than about 10%, preferably no more than about 5% is formed.
An antibody drug conjugate “retains its physical stability” in a pharmaceutical formulation if it shows no significant increase in aggregation, precipitation and/or denaturation upon visual examination of color and/or clarity, or as measured by UV light scattering, size exclusion chromatography (SEC) and dynamic light scattering (DLS). Changes in protein conformation can be evaluated by fluorescence spectroscopy (which determines the protein tertiary structure) and by FTIR spectroscopy (which determines the protein secondary structure).
An antibody drug conjugate “retains its chemical stability” in a pharmaceutical formulation if it shows no significant chemical change. Chemical stability can be evaluated by detecting and quantifying chemically changed forms of the protein. Degradation processes that often change the chemical structure of proteins include hydrolysis or clipping (evaluated by methods such as size exclusion chromatography and CE-SDS), oxidation (evaluated by methods such as peptide mapping in conjunction with mass spectrometry or MALDI/TOF/MS), deamidation (evaluated by methods such as ion-exchange chromatography, capillary isoelectric focusing, peptide mapping, and isoaspartic acid measurement), and isomerization (evaluated by measuring the isoaspartic acid content, peptide mapping, etc.).
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 at the time the pharmaceutical formulation was prepared.
“Optional” or “optionally” means that the event or circumstance subsequently described may, but not necessarily, 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 a particular sequence may, but does not necessarily, exist.
“Substituted” means that one or more, preferably up to 5, more preferably 1 to 3 hydrogen atoms in the group are independently substituted with a corresponding number of substituents. It goes without saying that a substituent is only in its possible chemical position, and those skilled in the art will be able to determine (experimentally or theoretically) possible or impossible substitution without undue effort. For example, it may be unstable when amino or hydroxy having free hydrogen is bound to a carbon atom having an unsaturated (e.g., olefinic) bond.
For the preparation of conventional pharmaceutical compositions, refer to Chinese Pharmacopoeia.
The term “carrier” for the drug of the present disclosure refers to a system that can alter how the drug gets into a human body and the distribution of the drug in the human body, control the release rate of the drug, and deliver the drug to a targeted organ. The drug carrier release and targeted system can reduce drug degradation and loss, reduce side effects and improve bioavailability. For example, polymeric surfactants that can be used as carriers can self-assemble due to their unique amphiphilic structures to form various forms of aggregates, such as micelles, microemulsions, gels, liquid crystals and vesicles, as preferred examples. The aggregates have the capability of encapsulating drug molecules and have good permeability for membranes, and therefore can be used as excellent drug carriers.
“Giving” and “treating”, when applied to animals, humans, experimental subjects, cells, tissues, organs or biological fluids, refer to contact of an exogenous drug, a therapeutic agent, a diagnostic agent or a composition with the animals, humans, subjects, cells, tissues, organs or biological fluids. “Giving” and “treating” can refer to, for example, therapeutic, pharmacokinetic, diagnostic, research and experimental methods. The treatment of cells comprises contacting the reagent with the cells and contacting the reagent with fluid, where the fluid is in contact with the cells. “Giving” and “treating” also refer to treating, e.g., cells by reagents, diagnosis, binding compositions or by another cell in vitro and ex vivo. “Treating”, when applied to humans, veterinary or research subjects, refers to therapeutic treatment, preventive or prophylactic measures, and research and diagnostic applications.
“Treatment” refers to administering a therapeutic agent, such as a composition comprising any one of the conjugation compounds of the present disclosure, either internally or externally to a patient with one or more symptoms of a disease on 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 patient or population being treated to induce regression of such symptoms or to inhibit the development of such symptoms to any clinically measurable degree. The amount of therapeutic agent effective to alleviate any particular symptom of the disease (also known as a “therapeutically effective amount”) may vary depending on a variety of factors, such as the disease state, age, and weight of the patient, and the ability of the drug to produce a desired therapeutic effect in the patient. Whether a symptom of a disease has been alleviated can be evaluated by any clinical testing methods commonly used by doctors or other health care professionals to evaluate the severity or progression of the symptom. Although the embodiments of the present disclosure (for example, treatment methods or articles of manufacture) may not be effective in alleviating the symptoms of each disease of interest, they shall reduce the symptoms of the disease of interest in a statistically significant number of patients, as determined according to any statistical testing methods known in the art, such as Student t-test, chi-square test, Mann and Whitney's U test, Kruskal-Wallis test (H test), Jonckheere-Terpstra test and Wilcoxon test.
“Effective amount” comprises an amount sufficient to ameliorate or prevent a symptom or condition of a medical disease. An effective amount also refers to an amount sufficient to allow or facilitate diagnosis. The effective amount for a particular patient or veterinary subject may vary depending on the factors such as the condition to be treated, the general health of the patient, the method and route and dosage of administration, and the severity of side effects. An effective amount may be the maximum dose or administration regimen to avoid significant side effects or toxic effects.
“Exchange” refers to the exchange of a solvent system that solubilizes an antibody protein. For example, a high-salt or hypertonic solvent system comprising the antibody protein is exchanged, by physical operations, with a buffer system of a stable formulation, such that the antibody protein is present in the stable formulation. The physical operations include, but are not limited to, ultrafiltration, dialysis or reconstitution following centrifugation.
The present disclosure is further described below with reference to examples, which, however, are not intended to limit the scope of the present disclosure. The experimental methods in the examples of the present disclosure in which specific conditions are not specified are generally performed under conventional conditions, for example, by referring to Antibodies: A Laboratory Manual and Molecular Cloning: A Laboratory Manual by Cold Spring Harbor Laboratory, or under conditions recommended by the manufacturer of the raw material or the goods. Reagents without specific origins indicated are commercially available conventional reagents.
The control molecule hRS7 used in the present disclosure is constructed according to patent application WO03074566, and the TINA antibody is constructed according to patent application WO2015098099A1, the sequences of which are as follows:
QVQLQQSGSELKKPGASVKVSCKASGYTFTNYGMNWVKQAPGQGLKWMG
WINTYTGEPTYTDDFKGREFAFSLDTSVSTAYLQISSLKADDTAVYFCA
RGGFGSSYWYFDVWGQGSLVTVSSASTKGPSVFPLAPSSKSTSGGTAAL
DIQLTQSPSSLSASVGDRVSITCKASQDVSIAVAWYQQKPGKAPKLLIY
SASYRYTGVPDRFSGSGSGTDFTLTISSLQPEDFAVYYCQQHYITPLTF
GAGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQ
QVQLVQSGAEVKKPGASVKVSCKASGYTFTTAGMQWVRQAPGQGLEWMG
WINTHSGVPKYAEDFKGRVTISADTSTSTAYLQLSSLKSEDTAVYYCAR
SGFGSSYWYFDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALG
DIQMTQSPSSLSASVGDRVTITCKASQDVSTAVAWYQQKPGKAPKLLIY
SASYRYTGVPSRFSGSGSGTDFTLTISSLQPEDFAVYYCQQHYITPLTF
GQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQ
An ADC is an antibody cross-linked drug, and the mechanism of treating diseases thereof is to transport toxin molecules into cells depending on the targeting performance of the antibody so as to kill the cells. The drug loading plays a decisive role in the drug efficacy.
The drug loading of the ADC stock solution was determined using the UV method.
Cuvettes containing a sodium succinate buffer were separately placed into the reference cell and sample cell, and the absorbance value of the solvent blank was subtracted. Then, a cuvette containing the test sample solution was placed into the sample cell, and the absorbance values at 280 nm and 370 nm were determined.
The loading capacity of the ADC stock solution was determined by ultraviolet spectrophotometry (instrument: a Thermo nanodrop2000 ultraviolet spectrophotometer), based on the principle that the total absorbance value of the ADC stock solution at a certain wavelength was the sum of the absorbance value of the cytotoxic drug and the monoclonal antibody at the certain wavelength, namely:
Similarly, an equation for the total absorbance value of the sample at 370 nm can be given as:
The drug loading can be calculated using both equations (1) and (2) as well as the extinction coefficients of the monoclonal antibody and the drug at both wavelengths and their concentrations.
The adopted was SDS-Mw Analysis Kit produced by Beckman, Cat. #390953, and containing SDS-MW gel separation buffer, SDS-MW sample buffer, acidic cleaning solution (0.1 mol/L hydrochloric acid solution), basic cleaning solution (0.1 mol sodium hydroxide solution) and internal standard substance (10 kDa). The adopted was also the SDS kit produced by Beijing BioCEart Technology Institute, Cat. #BSYK018, and containing CE-SDS gel buffer and CE-SDS sample buffer.
Alkylation solution (0.25 mo iodoacetamide solution): about 0.046 g of iodoacetamide was weighed, 1 mL of ultrapure water was added to dissolve and mix well, and the resulting solution was stored at 2-8° C. for 7 days in dark.
Capillary electrophoresis apparatus: PA800plus from SCIEX.
Capillary: uncoated fused-silica capillary (with an internal diameter of 50 μm), cut to a total length of 30.2 cm and an effective separation length by a high-resolution method of 20 cm.
The test sample was diluted to 1 mg/mL with SDS sample buffer. 95 μL of test sample solution (1 mg/mL) was taken and added with 5 μL of iodoacetamide aqueous solution (0.8 mol/L), and the resulting solution was vortexed and mixed well. 95 μL of blank control was taken and added with 5 μL of 0.8 mol/L iodoacetamide aqueous solution, and the resulting solution was vortexed and mixed well. 75 μL of samples were taken from sample tubes and added into the sample vials, and subjected to analysis immediately.
1) Pretreatment of capillary: 0.1 mol/L sodium hydroxide solution was washed under 60 psi pressure for 3 min, then washed with 0.1 mol/L hydrochloric acid solution under 60 psi pressure for 2 min, and finally washed with pure water under 70 psi pressure for 1 min. The above pretreatment should be performed before each operation.
2) Pre-filling of capillary: SDS gel separation buffer was washed under 50 psi pressure for 15 min. The above pretreatment should be performed before each operation.
Sample injection: electrokinetic sample injection was performed at 10 kV of reversed polarity, and the reduced sample was injected for 20 s.
Separation: separation was performed at 15 kV of reversed polarity for 40 min.
Temperature of sample chamber: 18° C. to 25° C.
Temperature of capillary: 18° C. to 25° C.
Data were analyzed by using software from Beckman based on the coupling of corresponding drug on sulfydryl liberated from an opened disulfide bond in the antibody, and the proportion of the corrected peak area such as a heavy chain, a nonglycosylated heavy chain and a light chain in the sum of all corrected peak areas was calculated. Calculation formula: DAR=[4×heavy chain (H) peak area+2×half antibody (H-L) peak area+4×double heavy chain (H-H) peak area+2×heavy-heavy-light chain (H-H-L) peak area]/[heavy chain (H) peak area/2+half antibody (H-L) peak area/2+double heavy chain (H-H) peak area+heavy-heavy-light chain (H-H-L) peak area+full antibody peak area], and the weighted average value of the ADC was finally calculated.
HPLC system: Waters H-Class ultra-high performance liquid chromatograph UPLC system
Detector: TUV detector (measuring wavelength: 280 nm)
Chromatographic column: BioResolve RP mAb Polyphenyl (2.7 μm 4.6×150 mm)
Column temperature: 80° C.
Flow rate: 1.0 mL/min
Mobile phase A: 0.1% formic acid+0.025% aqueous trifluoroacetic acid (TFA) solution
Mobile phase B: 0.1% FA+0.025% trifluoroacetic acid (TFA) in acetonitrile
Gradient program: 27.0% B-45.0% B (0.00 min to 12.00 min), 45.0% B-80.0% B (12.00 min to 13.00 min), 80.0% B-80.0% B (13.00 min to 15.00 min), 27.0% B-27.0% B (15.04 min to 20.00 min)
Sample injection amount: 5.0 μL
Compared to an antibody light chain (L0) and an antibody heavy chain (H0) not binding to drugs, the hydrophobicity of the drug-bound light chain (light chain bound to 1 drug: L1) and the drug-bound heavy chains (heavy chain bound to 1 drug: H1, heavy chain bound to 2 drugs: H2, heavy chain bound to 3 drugs: H3, heavy chain bound to 4 drugs: H4) increased in direct proportion to the number of drugs bound thereto, and the retention time was prolonged. Therefore, elution can be performed in the order of L0, L1, H0, H1, H2, H3, and H4. To compare the retention time of L0 and H0, a detection peak was assigned to any one of L0, L1, H0, H1, H2, H3, and H4.
Since the drug linkers absorbed UV, the resulting peak area was corrected according to the number of bound drugs using molar absorption coefficients of the light chains, the heavy chains, and the drug linkers according to the following expression. The calculation formula is as follows:
Note: ε LC-280 is molar extinction coefficient of light chain at 280 nm;
Note: total LC corrected peak area=L0 corrected peak area+L1 corrected peak area.
Total HC corrected peak area=H0 corrected peak area+H1 corrected peak area+H2 corrected peak area+H3 corrected peak area+H4 corrected peak area, and the drug loading of the ADC is calculated as follows:
To an aqueous PBS buffer of antibody PD3 (0.05 M aqueous PBS buffer at pH 6.5; 10.0 mg/mL, 4.0 mL, 270 nmol) was added at 37° C. a prepared aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 67.5 μL, 675 nmol). The reaction mixture was shaken on a water bath shaker at 37° C. for 3 h before the reaction was terminated. The reaction mixture was cooled to 25° C. in a water bath.
Compound 9-A (prepared according to 9-A of Example 9 in description of WO2020063676A1, 2.9 mg, 2700 nmol) was dissolved in 180 μL of DMSO, and the resulting solution was added to the above reaction mixture, which was then shaken on a water bath shaker at 25° C. for 3 h before the reaction was terminated. The reaction mixture was desalted and purified through a Sephadex G25 gel column (elution phase: 0.05 MPBS buffer at pH 6.5, containing 0.001 M EDTA) to give example product ADC-1 of PD3-9-A in PBS buffer (1.93 mg/mL, 18.4 mL), which was then stored at 4° C.
Average value calculated by UV-Vis: n=3.77.
To an aqueous PBS buffer of antibody PD3 (0.05 M aqueous PBS buffer at pH 6.5; 10.0 mg/mL, 4.0 mL, 270 nmol) was added at 37° C. a prepared aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 143.1 μL, 1431 nmol). The reaction mixture was shaken on a water bath shaker at 37° C. for 3 h before the reaction was terminated. The reaction mixture was cooled to 25° C. in a water bath.
Compound 9-A (4.35 mg, 4050 nmol) was dissolved in 270 μL of DMSO, and the resulting solution was added to the above reaction mixture, which was then shaken on a water bath shaker at 25° C. for 3 h before the reaction was terminated. The reaction mixture was desalted and purified through a Sephadex G25 gel column (elution phase: 0.05 MPBS buffer at pH 6.5, containing 0.001 M EDTA) to give example product ADC-2 of PD3-9-A in PBS buffer (1.69 mg/mL, 17.8 mL), which was then stored at 4° C.
Average value calculated by UV-Vis: n=6.59.
An antibody stock solution containing 145.24 g of PD3 (1.00 mmol, diluted with 10 mM histidine-acetic acid to an antibody concentration of 17.5 mg/mL) and 0.6308 g of tris(2-carboxyethyl)phosphine hydrochloride (Sigma, 2.20 mmol) were stirred in a 10 mM histidine-acetic acid-Tris buffer (pH 7.2) containing 2.5 mM EDTA at 12° C. for 100 min under a constant temperature water bath to give an intermediate I solution. 2 M acetic acid was added dropwise to adjust the pH of the reaction mixture to 5.0.
Compound 9-A (4.834 g, 4.50 mmol) was dissolved in 0.456 L of DMSO to give a solution of compound 9-A in DMSO. 0.373 L of DMSO was added to the above intermediate I solution, and the above DMSO solution of compound 9-A was added to the resulting solution, which was then stirred under a water bath at 12° C. for 90 min before the reaction was terminated.
After being filtered by a 0.22 μm filter, the reaction mixture described above was first subjected to buffer exchange to 10% DMSO-30 mM histidine-acetic acid buffer (pH=5.0) by 15-fold volume equal-volume ultrafiltration (30 kd ultrafiltration membrane) and then subjected to buffer exchange to 30 mM histidine-hydrochloric acid buffer (pH=6.0) by 18-fold volume equal-volume ultrafiltration, followed by addition of sucrose (to a final concentration of 40 g/L), glycine (to a final concentration of 9 g/L), and tween-80 (to a final concentration of 0.2 g/L), thus obtaining the example product ADC-3 (20 g/L, 132.20 g, yield: 91.0%, average value calculated by reversed phase chromatography: n=4.0) shown as PD3-9-A; finally lyophilized powder at 80 mg/bottle was prepared.
The synthesis was made according to Example 19 in WO2015098099.
To an aqueous PBS buffer of antibody TINA (0.05 M aqueous PBS buffer at pH 6.5; 10.0 mg/mL, 2.0 mL, 135.4 nmol) was added at 37° C. a prepared aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 33.8 μL, 338 nmol). The reaction mixture was shaken on a water bath shaker at 37° C. for 3 h before the reaction was terminated. The reaction mixture was cooled to 25° C. in a water bath.
Compound 58 (1.4 mg, 1354 nmol) was dissolved in 70 μL of DMSO, and the resulting solution was added to the above reaction mixture, which was then shaken on a water bath shaker at 25° C. for 3 h before the reaction was terminated. The reaction mixture was desalted and purified through a Sephadex G25 gel column (elution phase: 0.05 MPBS buffer at pH 6.5, containing 0.001 M EDTA) to give the example title product ADC-4 of TINA-58 in PBS buffer (1.13 mg/mL, 15.1 mL), which was then stored at 4° C.
Average value calculated by UV-Vis: n=3.99.
To an aqueous PBS buffer of antibody PD3 (0.05 M aqueous PBS buffer at pH 6.5; 10.0 mg/mL, 1.5 mL, 101.4 nmol) was added at 37° C. a prepared aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 25.3 μL, 253 nmol). The reaction mixture was shaken on a water bath shaker at 37° C. for 3 h before the reaction was terminated. The reaction mixture was cooled to 25° C. in a water bath.
Compound 58 (1.05 mg, 1014 nmol, according to Example 58 on page 163 of patent application CN104755494A) was dissolved in 60 μL of DMSO, and the resulting solution was added to the above reaction mixture, which was then shaken on a water bath shaker at 25° C. for 3 h before the reaction was terminated. The reaction mixture was desalted and purified through a Sephadex G25 gel column (elution phase: 0.05 MPBS buffer at pH 6.5) to give the example title product ADC-5 of PD3-58 in PBS buffer (0.82 mg/mL, 13.5 mL), which was then stored at 4° C.
Average value calculated by UV-Vis: n=3.88.
To an aqueous PBS buffer of antibody hRS7 (0.05 M aqueous PBS buffer at pH 6.5; 10.0 mg/mL, 1.4 mL, 94.60 nmol) was added at 37° C. a prepared aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 50.1 μL, 501 nmol). The reaction mixture was shaken on a water bath shaker at 37° C. for 3 h before the reaction was terminated. The reaction mixture was cooled to 25° C. in a water bath.
Compound SN38 (synthesized according to Example 1 on Page 59 of patent application CN105407891A, 2.1 mg, 1419 nmol) was dissolved in 50 μL of DMSO, and the resulting solution was added to the above reaction mixture, which was then shaken on a water bath shaker at 25° C. for 3 h before the reaction was terminated. The reaction mixture was desalted and purified through a Sephadex G25 gel column (elution phase: 0.05 M PBS buffer at pH 6.5) to give the example title product ADC-6 of hRS7-SN38 in PBS buffer (1.03 mg/mL, 11.5 mL), which was then stored at 4° C.
Average value calculated by CE-SDS: n=7.56.
To an aqueous PBS buffer of antibody hRS7 (0.05 M aqueous PBS buffer at pH 6.5; 10.0 mg/mL, 2.18 mL, 147.3 nmol) was added at 37° C. a prepared aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 36.8 μL, 368 nmol). The reaction mixture was shaken on a water bath shaker at 37° C. for 3 h before the reaction was terminated. The reaction mixture was cooled to 25° C. in a water bath.
Compound 9-A (1.58 mg, 1471 nmol) was dissolved in 100 μL of DMSO, and the resulting solution was added to the above reaction mixture, which was then shaken on a water bath shaker at 25° C. for 3 h before the reaction was terminated. The reaction mixture was desalted and purified through a Sephadex G25 gel column (elution phase: 0.05 MPBS buffer at pH 6.5) to give the example title product ADC-7 of hRS7-9-A in PBS buffer (1.10 mg/mL, 16.4 mL), which was then stored at 4° C.
Average value calculated by UV-Vis: n=3.72.
To an aqueous PBS buffer of antibody hRS7 (0.05 M aqueous PBS buffer at pH 6.5; 10.0 mg/mL, 2.18 mL, 147.3 nmol) was added at 37° C. a prepared aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 36.8 μL, 368 nmol). The reaction mixture was shaken on a water bath shaker at 37° C. for 3 h before the reaction was terminated. The reaction mixture was cooled to 25° C. in a water bath.
Compound 58 (1.52 mg, 1473 nmol, according to Example 58 on page 163 of patent application CN104755494A) was dissolved in 100 μL of DMSO, and the resulting solution was added to the above reaction mixture, which was then shaken on a water bath shaker at 25° C. for 3 h before the reaction was terminated. The reaction mixture was desalted and purified through a Sephadex G25 gel column (elution phase: 0.05 M PBS buffer at pH 6.5) to give the example title product ADC-8 of hRS7-58 in PBS buffer (1.02 mg/mL, 16.8 mL), which was then stored at 4° C.
Average value calculated by UV-Vis: n=3.93.
To an aqueous PBS buffer of antibody TINA (0.05 M aqueous PBS buffer at pH 6.5; 10.0 mg/mL, 0.95 mL, 64.2 nmol) was added at 37° C. a prepared aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 16.0 μL, 160 nmol). The reaction mixture was shaken on a water bath shaker at 37° C. for 3 h before the reaction was terminated. The reaction mixture was cooled to 25° C. in a water bath.
Compound 9-A (0.69 mg, 642 nmol) was dissolved in 30 μL of DMSO, and the resulting solution was added to the above reaction mixture, which was then shaken on a water bath shaker at 25° C. for 3 h before the reaction was terminated. The reaction mixture was desalted and purified through a Sephadex G25 gel column (elution phase: 0.05 MPBS buffer at pH 6.5) to give the example title product ADC-9 of TINA-9-A in PBS buffer (0.99 mg/mL, 7.0 mL), which was then stored at 4° C.
Average value calculated by UV-Vis: n=3.99.
The activity of the antibodies and conjugates of the present disclosure is verified with biochemical test methods.
hTROP-2 protein was diluted to 1 μg/mL with PBS buffer at pH 7.4 (Shanghai BasalMedia Technologies Co., LTD., B320), added into a 96-well microplate at 100 μL per well, and incubated at 4° C. overnight. After the liquid was discarded, 300 μL of 5% skim milk (BD, 232100) diluted with PBS was added to each well for blocking, and the mixture was incubated at 37° C. for 2 h. After the blocking was completed, the blocking solution was discarded, and the plate was washed 3 times with a PBST buffer (pH 7.4, PBS containing 0.1% tween-20). 100 μL of the antibody solution diluted in a gradient was added to each well, and the mixture was incubated at 37° C. for 1 h. After the incubation was completed, the plate was washed 3 times with PBST. 100 μL of mouse anti-human IgG (H+L) (Jackson ImmunoResearch, 209-035-088, 1:8000 dilution) was added to each well, and the mixture was incubated at 37° C. for 1 h. After the plate was washed 3 times with PBST buffer, 100 μL of TMB chromogenic substrate (KPL, 5120-0077) was added into each well and incubated at room temperature for 10-15 min, and the reaction was terminated by adding 50 μL of 1 M H2SO4 into each well. The plate was detected for the absorbance value at 450 nm using a microplate reader, the binding curves of antibodies to antigen were fitted with software, and the EC50 value was calculated. The binding activity of the antibodies to the proteins is shown in Table 2.
The result shows that the PD3 antibody in the present application has higher binding activity to hTROP-2 protein.
The stably transfected TROP-2-expressing CHOK1 cells were suspended in FACS buffer (2% fetal bovine serum (Gibco, 10099141) pH 7.4 PBS (Sigma, P4417-100TAB)) to give a 1×106/mL cell suspension, which was then added to a 96-well round-bottom plate at 100 μL/well. After centrifugation and removal of the supernatant, the test antibodies diluted with FACS buffer to different concentrations were added at 50 μL/well. The plate was incubated in the dark in a 4° C. refrigerator for 1 h. The plate was washed 3 times with FACS buffer by centrifugation at 300 g, and Alexa Fluor 488 goat anti-human IgG (H+L) (invitrogen, A-11013) at working concentration was added. The plate was incubated in the dark in a 4° C. refrigerator for 40 min. The plate was washed 3 times with FACS buffer by centrifugation at 300 g and tested on a BD FACSCantoII flow cytometer for geometric mean fluorescence intensity (MFI). The binding EC50 value of the antibody to the stably transfected TROP-2-expressing cells was calculated. The binding activity of the antibodies to the cells is shown in Table 3.
The result shows that the PD3 antibody in the present application has higher binding activity to TROP-2 protein-expressing cells.
The purpose of the assay is that the activated DT kills cells after the DT3C protein enters the cells, indirectly reflecting endocytosis of the anti-TROP-2 antibody. The in vitro endocytic activity of the antibody is evaluated according to EC50 and Emax.
DT3C is a recombinantly expressed fusion protein and is formed by fusing fragment A of diphtheria toxin (toxin part only) and fragment 3C of group G streptococcus (IgG binding part). The protein can have a high affinity for IgG part of antibody, enter cells together with the IgG part when the antibody is endocytosed, and release toxic DT under the action of intracellular furin protease. The DT can inhibit the activity of EF2-ADP ribosylation, block the protein translation process and finally cause cell death. DT3C that does not enter the cell has no activity of cell killing. The endocytic activity of the antibody was evaluated according to cell killing.
Cell suspensions were prepared with fresh cell medium containing 20% low IgG FBS at a cell density of 2×104 cells/mL, and added into the cell culture plates at 50 μL/well and incubated with 5% carbon dioxide at 37° C. for 16 h. DT3C at 4× concentration was formulated in serum-free medium and filtered through a 0.22 μm filter to obtain a sterile solution. Antibody at 4× concentration was prepared in serum-free medium, and 80 μL of DT3C and 80 μL of antibody were mixed at a volume of 1:1, and incubated at room temperature for 30 min. 50 μL of the diluted antibody-DT3C mixture was added to 50 μL of cells and incubated in an incubator for three days. 50 μL of CTG was added into each well and incubated for 10 min at room temperature in dark, and the chemiluminescence values were detected using Victor3. The endocytic activity of the antibody is shown in Table 4.
The results show that the PD3 antibody in the present application has higher endocytosis efficiency.
The affinity of antibody for TROP-2 was detected in a form of a capture antibody. The antibody was affinity-captured by a Protein A (Cat. #29127556, GE) biosensor chip conjugated with an anti-human IgG antibody (Cat. #BR-1008-39, Lot. #10260416, GE), then an antigen hTROP-2 flowed on the surface of the chip, and a Biacore T200 instrument was used for detecting reaction signals in real time to obtain association and dissociation curves. After dissociation was completed for each assay cycle, the chip was washed clean and regenerated with regeneration buffer, Glycine1.5 (Cat. #BR100354, GE) or 3 M MgCl2 (from Human antibody capture kit, Cat. #BR100839, GE). After the assay was completed, the data were fitted with (1:1) Langmuir model using GE Biacore T200 Evaluation version 3.0 to obtain affinity values. The affinity of the antibody for the proteins is shown in Table 5.
The results show that the PD3 antibody in the present application has a higher affinity for hTROP-2.
The cells used in this assay were as follows: FaDu(+++) purchased from ATCC, Cat. #HTB-43™; HCC827(+++), purchased from ATCC, Cat. #CRL-2868; Colo205(++), purchased from Cell Bank, Chinese Academy of Sciences, Cat. #TCHu102; DMS53(++), purchased from ATCC, Cat. #CRL-2062™; SK-OV-3(+), purchased from ATCC, Cat. #HTB-77; CHO-K1(−), purchased from ATCC, Cat. #CCL-61™; wherein “+” represents the expression amount of TROP-2 in the cell population, more “+” means that the expression amount of TROP-2 is higher, and “−” means that TROP-2 is not expressed.
Cell suspensions were prepared with fresh cell medium containing 10% FBS at a density of 3703 cells/mL, and added into the 96-well cell culture plate at 135 μL/well and incubated with 5% carbon dioxide at 37° C. for 16 h. ADC samples were formulated to be 5 μM with PBS. This concentration was taken as the initial concentration, and the sample was 5-fold diluted with PBS to a total of 8 concentrations. 15 μL of the above ADC solution was added into each well. The plate was cultured at 37° C. in 5% carbon dioxide for 6 days. 70 μL of CTG was added into each well and incubated for 10 min at room temperature in dark, the chemiluminescence values were detected using Victor3, and data analysis was performed using the GraphPad Prism software.
The result shows that ADC-1 has stronger cell killing effect, and the killing effect is positively correlated with the TROP-2 expression level on the surface of the tumor cell.
A second parallel comparison assay was performed in the method as described above, and the obtained results are as follows:
BxPC3 (human pancreatic cancer cells, ATCC, CRL-1687) and MiaPaCa2 cells (human pancreatic cancer cells, biocytogen, B-HCL-014) were cultured with RPMI1640+10% FBS and DMEM/high glucose+10% FBS, respectively; cells were trypsinized, neutralized with fresh medium, and centrifuged at 1000 rpm for 3 min; supernatant was discarded, and cells were resuspended with RPMI1640+10% FBS. After the cells were counted, the cell density of BxPC3 was adjusted to 6×104 cells/mL and the cell density of MiaPaCa2-luc was adjusted to 1.5×104 cells/mL. 500 μL of BxPC3 cells and 500 μL of MiaPaCa2-luc cells were added into each well of a 12-well plate 1. 500 μL of MiaPaCa2-luc cells and 500 μL of RPMI1640 medium containing 10% FBS serum were added into a 12-well plate 2. The plates were cultured at 37° C. in 5% carbon dioxide for 24 h.
The ADC samples were formulated into 40× concentration of intermediate solutions (0.2 μM). 25 μL of the above samples were taken and added into the corresponding well of the 12-well plates. Solvent control group was set. The plates were cultured at 37° C. in 5% carbon dioxide for 6 days. The cells in the 12-well plates were trypsinized, neutralized with fresh medium, and centrifuged at 1000 rpm for 3 min. The supernatant was discarded, and the cells were resuspended in 1 mL of FACS buffer (PBS+2.5% FBS). 20 μL of the cells were taken and stained with 20 μL of trypan blue and counted. The cells in the plate 1 were centrifuged at 1000 rpm for 3 min, the supernatant was discarded, the cells were resuspended in 100 μL of FACS Buffer, 2 μL of TROP-2 (EGP-1) monoclonal antibody (MR54) was added, and the cells were incubated on ice for 30 min. The cells were centrifuged at 2000 rpm at 4° C. for 1 min, the supernatant was discarded, and the cells were resuspended in 150 μL of FACS buffer. Detection was performed using BD FACSVerse. Data was analyzed by Flowjo 7.6. The results of the bystander killing activity study are shown in
The results show that ADC-1 in the present disclosure has a clear bystander killing effect, and ADCs do not kill the TROP-2 negative MiaPaCa2 cells, but ADC-1 also kills TROP-2 negative cells when the TROP-2 expressing BxPC3 cells are mixed with the negative cells MiaPaCa2.
Fadu cells (3×106) were subcutaneously inoculated in the right flank of Balb/c nude mice, after 10 days of inoculation, the nude mice with heavy weight, too big tumor and too small tumor were removed after the tumor volume was about 245 mm3 and the remaining mice were randomized into 5 groups of 8 mice according to tumor volume.
Each mouse was intraperitoneally injected with an ADC at 0.1 mL/10 g body weight at days 0 and 8, making a total of 2 injections with a dose of 1 mg/kg. The tumor volumes and body weights were measured twice a week and the results were recorded for 21 days.
Data were recorded using Excel statistical software: the average values were calculated as avg; the SD values were calculated as STDEV; the SEM values were calculated as STDEV/SQRT (number of animals per group); GraphPad Prism software was used for plotting, and statistical analysis of the data was performed using Two-way ANOVA or One-way ANOVA.
Relative tumor proliferation rate T/C (%)=(T−T0)/(C−C0)×100, wherein T and C are the tumor volumes of animals at the end of the experiment in the treatment group and the control group, respectively; To and C0 are the tumor volumes of animals at the beginning of the experiment in the treatment group and the control group, respectively.
The results are shown in Table 8, which indicates that ADC-1 has a strong tumor-inhibiting effect on FaDu xenograft tumors at a dose of 1 mpk.
SKOV3 cells (5×106) were subcutaneously inoculated in the right flank of Balb/c nude mice, after 23 days of inoculation, the nude mice with heavy weight, too big tumor and too small tumor were removed after the tumor volume was about 180 mm3, and the remaining mice were randomized into 5 groups of 8 mice according to tumor volume.
Each mouse was intraperitoneally injected with an ADC at 0.1 mL/10 g body weight for a total of 2 injections, and the dose is shown in following table. The tumor volumes and body weights were measured twice a week and the results were recorded. Data were recorded using Excel statistical software: the average values were calculated as avg; the SD values were calculated as STDEV; the SEM values were calculated as STDEV/SQRT (number of animals per group); GraphPad Prism software was used for plotting, and statistical analysis of the data was performed using Two-way ANOVA or One-way ANOVA.
Relative tumor proliferation rate T/C (%)=(T−T0)/(C−C0)×100, wherein T and C are the tumor volumes of animals at the end of the experiment in the treatment group and the control group, respectively; To and C0 are the tumor volumes of animals at the beginning of the experiment in the treatment group and the control group, respectively.
The results are shown in Table 9, which indicates that ADC-1 has a strong tumor-inhibiting effect on SKOV3 xenograft tumors at different doses, and the tumor-inhibiting effect is dose-dependent.
The effect is dose-dependent.
Colo205 cells (5×106) were subcutaneously inoculated in the right flank of Balb/c nude mice, after 10 days of inoculation, the nude mice with heavy weight, too big tumor and too small tumor were removed after the tumor volume was about 245 mm3, and the remaining mice were randomized into 6 groups of 8 mice according to tumor volume.
Each mouse was intraperitoneally injected with an ADC at 0.1 mL/10 g body weight at day 0 (DO) and day 10, making a total of 2 injections with a dose of 10 mg/kg. The tumor volumes and body weights were measured twice a week and the results were recorded for 28 days (D28).
Data were recorded using Excel statistical software: the average values were calculated as avg; the SD values were calculated as STDEV; the SEM values were calculated as STDEV/SQRT (number of animals per group); GraphPad Prism software was used for plotting, and statistical analysis of the data was performed using Two-way ANOVA or One-way ANOVA.
Relative tumor proliferation rate T/C (%)=(T−T0)/(C−C0)×100, wherein T and C are the tumor volumes of animals at the end of the experiment in the treatment group and the control group, respectively; To and C0 are the tumor volumes of animals at the beginning of the experiment in the treatment group and the control group, respectively.
The results are shown in Table 10, which indicates that ADC-9 and ADC-7 prepared by conjugation to compound 9-A have a strong tumor-inhibiting effect on Colo205 xenograft tumors at a dose of 1 mpk.
The equipment used in the formulation preparation and determination and the calculation method for results are shown below:
This is a method for analyzing the separation of a solute by the relative relationship between the pore size of the gel pores and the size of the polymer sample molecule coil.
SEC % (SEC monomer content percentage)=A monomer/A total×100% (A monomer represents the peak area of the main peak monomer in the sample, and A total represents the sum of all peak areas).
Instrument for SEC measurement: Agilent 1260; column: waters, XBrige BEH200 Å SEC (300×7.8 mm 3.5 μm)
This is a method of moving the gel into a capillary as a supporting medium for electrophoresis and performing separation according to the molecular weight of the sample under a certain voltage.
R-CE purity percentage=A main peak/A total×100% (A main peak represents the light chain main peak area+the heavy chain main peak area in the sample, and A total represents the sum of all peak areas).
Instrument for R-CE determination: Beckman model plus800
The degree to which light is hindered from passing through a water layer indicates the ability of the water layer to scatter and absorb light. It is related not only to the suspended matter content but also to the particle composition, size and shape as well as the reflection performance of its surface. In comparing the absorption values of the same protein sample at the same concentration at the same wavelength (within near ultraviolet and visible wavelength regions), the greater the absorption value is, the greater the turbidity is, the more the protein molecules in the sample tend to aggregate. The instrument for measurement was a multifunctional microplate reader (Molecular Devices M5). Samples in the same volume were added to a 96-well plate and the absorbance values were read.
The freezing point method is used for determining the osmotic pressure. The freezing point of a solution is determined by using a high-sensitivity temperature-sensing element on the basis of the proportional relation between the freezing point depression value and the molar concentration of the solution, and then converted into the osmotic pressure through electric quantity. Manufacturer of instrument: Loser, model: OM815.
In the present disclosure, the concentration of the antibody drug conjugate is expressed in protein concentration, i.e., the concentration of the antibody moiety in the antibody drug conjugate.
Since the toxin in the antibody drug conjugate absorbs light at a protein characteristic absorption wavelength of 280 nm and also at 370 nm, the protein concentration is calculated by the following formula:
In the formula, A280 nm: the average value of the absorbance of a single piece of the test sample solution at the wavelength of 280 nm when the optical path is 1 cm;
If the test sample solution is diluted, the protein concentration is as follows: C (mg/mL)=CmAb×N, and N is a dilution factor.
Instrument for protein concentration determination: ultraviolet-visible spectrophotometer, model: Nano Drop One.
The following buffers were used to prepare anti-Trop2-ADC antibody formulations with the protein concentration of ADC-3 being 10-30 mg/mL. Specific formula composition is as follows:
The prepared formulations were filtered sterilely and placed in containers, which were plugged and capped. The stability of the samples under a forced degradation condition (40° C. high temperature) was examined, and the appearance, SEC, and R-CE were used as evaluation indexes to examine the stability of the formulations. The experimental results are shown in Table 11.
At 40° C. and M, the particle number of the His-HCl system was less than those of the CA and SA systems according to appearance inspection, the purity of the His system was higher than those of the CA and SA systems at the same pH value and the same protein concentration, and the SEC monomer peak and the R-CE main peak decrease value became smaller along with the increase of the pH value in the same buffer system. In the 10 mM pH5.5 His system, there was a slight increase in SEC polymers along with increasing protein concentration, and there was no significant change in the R-CE purity. In summary, the buffer system is preferably His-HC, the pH range is preferably 5.5-6.5, and the protein concentration is preferably 10 mg/mL to 30 mg/mL.
Anti-Trop2-ADC antibody formulations containing 80 mg/mL sucrose and polysorbate 80 (PS80 for short) at different concentrations with the protein concentration of ADC-3 being 20 mg/mL were prepared by using a 10 mM pH 6.0 His-HCl buffer system. Specific formula composition is as follows:
The prepared formulations were filtered sterilely and placed in containers, which were plugged and capped. The samples were subjected to shaking (25° C., 300 rpm, 3 days) and high temperature (40° C. M1) conditions, and the appearance, SEC, and R-CE were used as evaluation indexes to examine the stability of the formulations. The experimental results are shown in Table 12.
Experimental results show that the appearance of the 0.2-0.6 mg/mL PD80 groups is better than that of the group free of P980 under all the examining conditions, and for the R-CE purity, the decrease in main peak of the 0.2 mg/mL PS80 group after 1 month of high temperature was lower than those of the 0.4 mg/mL and 0.6 mg/mL groups. In summary, the P980 concentration is preferably 0.2 mg/mL.
Anti-Trop2-ADC antibody formulations containing 0.4 mg/mL polysorbate 80 and different types of saccharides with the protein concentration of ADC-3 being 20 mg/mL were prepared by using a 10 mM pH 6.0 His-HCl buffer system. The different saccharides are as follows, and the saccharides content is the concentration when the osmotic pressure of the solution is isotonic:
The prepared formulations were filtered sterilely and placed in containers, which were plugged and capped. The samples were subjected to shaking (25° C., 300 rpm, 3 days), FT5C (5 freeze-thaw cycles between −35° C. and 2-8° C.), and high temperature (40° C. M1) conditions, and the appearance, SEC, and R-CE were used as evaluation indexes to examine the stability of the formulations. The experimental results are shown in Table 13.
The experimental results show that the appearance and purity of the formulations containing sucrose and trehalose dihydrate exhibited no significant difference under different conditions.
Anti-Trop2-ADC antibody formulations containing 0.2 mg/mL PS80, sucrose at different concentrations, and glycine at different concentrations with the protein concentration of ADC-3 being 20 mg/mL were prepared by using a pH 6.0 His-HCl buffer system. Specific formula composition is as follows:
Two formulation modes, i.e., solution and lyophilized formulation, were used for examination.
Solutions: the prepared formulations were filtered sterilely and placed in containers, which were plugged and capped. The solutions were subjected to high temperature (40° C. D10) conditions, and the SEC and R-CE were used as evaluation indexes to examine the stability of the formulations. The experimental results are shown in Table 14. There was no significant difference in purity after storage at 40° C. D10 conditions for all groups.
Lyophilized formulation: the prepared formulations were filtered sterilely, placed in containers, half-plugged, lyophilized, plugged, and capped. The appearance and the reconstitution of the lyophilized products were examined. The lyophilization procedure was as follows:
The appearance of the lyophilized powder cake showed that the bottom of the group with high sucrose concentration slightly shrank, and when the sucrose concentration was reduced to 40 mg/mL and glycine was added, the powder cake obtained under the same lyophilization conditions was uniform, full, and collapse-free, indicating that the primary drying time required when the sucrose concentration was reduced and glycine was added was shorter than that required for formulations with high sucrose concentration.
When the sucrose concentration was 40 mg/mL, samples prepared using buffers at different concentrations were uniform, full, and collapse-free in appearance after the lyophilization process.
An anti-Trop2-ADC formulation containing 40 mg/mL sucrose, 9.0 mg/mL glycine, and 0.2 mg/mL PS80 with the protein concentration of ADC-3 being 20 mg/mL were prepared by using a 30 mM pH 6.0 His-HCl buffer system.
The prepared formulation was filtered sterilely and placed in containers, which were plugged and capped. The samples were subjected to forced degradation conditions of shaking (25° C., 300 rpm, 10 days), FT5C (5 freeze-thaw cycles between −35° C. and 2-8° C.), and high temperature (40° C. M1), acceleration at 25° C. (25° C. M3), and 2-8° C. long-term conditions (4° C. M3), and the appearance, SEC, and R-CE were used as evaluation indexes to examine the stability of the formulations. The experimental results are shown in Table 17.
The experimental results show that the appearance of the final formula formulation was clear and transparent under all the forced degradation conditions, and the final formula formulation has good stability.
An anti-Trop2-ADC antibody formulation containing 40 mg/mL sucrose, 9 mg/mL glycine, and 0.2 mg/mL PS80 with the protein concentration of ADC-3 being 20 mg/mL was prepared in the 30 mM pH 6.0 His-HCl buffer, and the formulation sample was lyophilized by using a lyophilization procedure of pre-freezing, primary drying, and secondary drying, as shown in Table 18. After the lyophilization procedure was completed, plugging was performed in vacuum. The lyophilized sample was white uniform cake in appearance, met the standards in terms of the moisture content, and was easy to reconstitute, and it met the standards for the SEC and NR-CE purity after the reconstitution. The results are shown in Table 19. The pH after reconstitution was 6.1.
| Number | Date | Country | Kind |
|---|---|---|---|
| 202110823295.2 | Jul 2021 | CN | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CN2022/107134 | 7/21/2022 | WO |
