Methods for preparing CD20 antibody conjugate drugs, antibody conjugate drugs, and uses thereof

Abstract

The present application relates to a preparation method of a CD20 antibody conjugated drug, the antibody conjugated drug and the use thereof. Specifically, the present application discloses a CD20 antibody conjugated drug as shown in general formula I. The antibody conjugated drug has a significant anti-tumor effect.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine. Specifically, this invention relates to an antibody-drug conjugate targeting CD20, its preparation method, and its uses. Background Technology

[0002] Lymphoma is a group of malignant tumors originating from lymph nodes and other extranodal reticular tissues. It is diverse in type and has a high incidence rate, claiming tens of thousands of lives in my country each year. CD20 is a non-glycosylated tetramembrane phosphoprotein specifically expressed on the surface of B lymphocytes, playing a crucial regulatory role in B lymphocyte differentiation and proliferation. The stable and specific expression of CD20 on the surface of B cells makes it an ideal target for the treatment of B-cell lymphoma. Currently, FDA-approved anti-CD20 monoclonal antibodies for the treatment of B-cell lymphoma include Rituximab, Zevalin, and Bexxar.

[0003] Antibody-drug conjugates (ADCs) are a new type of anticancer drug developed in recent years. They combine antibodies and cytotoxic drugs through conjugates. The antibody targets the cytotoxic drug to the tumor site, and through endocytosis, the ADC drug enters the tumor cells and releases toxins to kill the target cells, thereby reducing the non-specific systemic toxicity of drugs commonly used in chemotherapy.

[0004] Therefore, those skilled in the art are dedicated to developing new and more effective antibody-drug conjugates that target CD20. Summary of the Invention

[0005] The purpose of this invention is to provide an antibody-drug conjugate targeting CD20.

[0006] Another object of the present invention is to provide a method for preparing the above-mentioned antibody-drug conjugate and its use.

[0007] In a first aspect, the present invention provides an antibody-drug conjugate or a pharmaceutically acceptable salt thereof, said antibody-drug conjugate having the structure shown in Formula I:

[0008] mAb-(LD)n I

[0009] in,

[0010] mAb represents a recombinant anti-CD20 monoclonal antibody, and the recombinant anti-CD20 monoclonal antibody is rituximab or its biosimilar;

[0011] D indicates a small molecule toxin, and D is one or more monomethyl auristatins;

[0012] L is a linker connecting antibodies and small molecule toxins;

[0013] n is the average number of small molecule toxins conjugated to the antibody, and n is an integer or non-integer of 4.2 ± 1; and

[0014] "-" is the key.

[0015] In another preferred embodiment, n is an integer or non-integer of 4.2 ± 0.5.

[0016] In another preferred embodiment, n is an integer or non-integer of 4.2 ± 0.3.

[0017] In another preferred embodiment, the small molecule toxin is linked to a thiol group formed by the reduction of disulfide bonds between the antibody chains via a linker.

[0018] In another preferred embodiment, D is monomethylaurestatin-E (MMAE), monomethylaurestatin-D (MMAD), monomethylaurestatin-F (MMAF), or a combination thereof.

[0019] In another preferred embodiment, L is maleimide hexanoyl-valine-citrulline-p-aminobenzyloxycarbonyl.

[0020] In another preferred embodiment, the structure of the antibody-drug conjugate is shown in the following formula:

[0021]

[0022] A second aspect of the present invention provides a pharmaceutical composition comprising the antibody-drug conjugate described in the first aspect of the present invention, and a pharmaceutically acceptable carrier.

[0023] A third aspect of the invention provides the use of an antibody-drug conjugate as described in the first aspect of the invention or a pharmaceutical composition as described in the second aspect of the invention for the preparation of an antitumor drug.

[0024] In another preferred embodiment, the tumor is lymphoma or leukemia.

[0025] In another preferred embodiment, the tumor is B-cell non-Hodgkin lymphoma or chronic lymphocytic leukemia.

[0026] A fourth aspect of the present invention provides a method for preparing the antibody-drug conjugate described in the first aspect of the present invention, the method comprising the steps of:

[0027] (1) A reaction system containing reduced recombinant anti-CD20 monoclonal antibody and a reducing agent is formed by a reduction reaction; and the molar ratio of the monoclonal antibody to the reducing agent is 1:2.9 to 1:3.1; and

[0028] (2) The reaction system of step (1) and the solution of small molecule toxin in acetonitrile and water are coupled to form the antibody-drug conjugate; and the molar ratio of monoclonal antibody to small molecule toxin in step (1) is 1:7.0 to 1:8.0.

[0029] In another preferred embodiment, in step (1), the reduction reaction is carried out in a buffer solution.

[0030] In another preferred embodiment, in step (1), the reduced recombinant anti-CD20 monoclonal antibody is a recombinant anti-CD20 monoclonal antibody in which the interchain disulfide bonds are reduced to thiol groups.

[0031] In another preferred embodiment, in step (1), the reducing agent is tris(2-carboxyethyl)phosphine.

[0032] In another preferred embodiment, in step (1), the buffer solution is PB reaction buffer (pH 7.6).

[0033] In another preferred embodiment, in step (1), the molar ratio of the monoclonal antibody to the reducing agent is 1:2.95 to 1:3.05; more preferably, it is 1:2.99 to 1:3.01.

[0034] In another preferred embodiment, in step (1), the reduction reaction is carried out at 25±1°C.

[0035] In another preferred embodiment, in step (1), the reduction reaction is carried out for 90 ± 10 minutes.

[0036] In another preferred embodiment, in step (2), the molar ratio of the monoclonal antibody to the small molecule toxin in step (1) is 1:7.2 to 1:7.7; more preferably, 1:7.4 to 1:7.6.

[0037] In another preferred embodiment, in step (2), the volume ratio of acetonitrile to water is 1:1.

[0038] In another preferred embodiment, in step (2), the coupling reaction is carried out at 4 ± 0.5 °C.

[0039] In another preferred embodiment, in step (2), the coupling reaction is carried out for 60 ± 10 minutes.

[0040] In another preferred embodiment, in step (2), the mixing is to add a solution of the small molecule toxin in acetonitrile and water dropwise to the reaction system of step (1).

[0041] A fifth aspect of the present invention provides a non-therapeutic method for inhibiting tumor cells, the method comprising the step of adding an antibody-drug conjugate as described in the first aspect of the present invention or a pharmaceutical composition as described in the second aspect of the present invention to a system containing tumor cells.

[0042] In another preferred embodiment, the tumor cells are CD20-positive tumor cells, such as Raji, Ramos, Daudi cells, etc.

[0043] The present invention also provides a method for treating or preventing tumors, the method comprising the steps of: administering to a desired subject an antibody-drug conjugate as described in the first aspect of the present invention or a pharmaceutical composition as described in the second aspect of the present invention.

[0044] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0045] Figure 1 Hydrophobic interaction chromatography was used to identify the drug loading of the antibody-drug conjugate TRS005.

[0046] Figure 2 Hydrophobic interaction chromatography was used to identify the drug loading of the antibody-drug conjugate TRS005.

[0047] Figure 3 Hydrophobic interaction chromatography was used to identify the drug loading of the antibody-drug conjugate TRS005.

[0048] Figure 4 Comparison of C4-RPLC chromatograms of three batches of TRS005.

[0049] Figure 5 A comparison of the hydrophobic interaction chromatograms of three batches of TRS005.

[0050] Figure 6A A scatter plot of flow cytometry data is shown.

[0051] Figure 6B A single-parameter histogram is displayed.

[0052] Figure 6C The endocytosis effect of the antibody-drug conjugate TRS005 of the present invention in Ramos cells was demonstrated.

[0053] Figure 6D The endocytosis effect of the antibody-drug conjugate TRS005 of the present invention in Raji cells was demonstrated.

[0054] Figure 7 The cell-inhibiting effect of the antibody-drug conjugate TRS005 of the present invention was demonstrated.

[0055] Figure 8Tumor volume-time curves are shown (6 injections over 21 days). Animal model: BALB / c nude mouse Ramos xenograft; DAR of monoclonal antibody and cytotoxic drug loading was 4.2.

[0056] Figure 9 A scatter plot of tumor weight is shown (6 injections over 21 days). Animal model: BALB / c nude mouse Ramos xenograft; the DAR of monoclonal antibody and cytotoxic drug loading was 4.2.

[0057] Figure 10 Tumor volume-time curves are shown (6 injections over 21 days). Animal model: BALB / c nude mouse Daudi xenograft; the DAR of monoclonal antibody and cytotoxic drug loading was 4.2.

[0058] Figure 11 A scatter plot of tumor weight is shown (6 injections over 21 days). Animal model: BALB / c nude mouse Daudi xenograft; the DAR of monoclonal antibody and cytotoxic drug loading was 4.2.

[0059] Figure 12 The study shows the possible toxin binding sites and drug-carrying quantities of the resulting monoclonal antibody conjugate. Detailed Implementation

[0060] Through extensive and in-depth research, the inventors unexpectedly discovered a highly efficient antibody-drug conjugate targeting CD20 with optimized toxin loading. This invention was completed based on this discovery.

[0061] Antibody

[0062] The antibody applicable to this invention is an antibody targeting CD20, specifically a recombinant anti-CD20 monoclonal antibody. The recombinant anti-CD20 monoclonal antibody may be rituximab or its biosimilar.

[0063] Rituximab

[0064] The rituximab of this invention refers to the monoclonal antibody drug targeting CD20, developed by Roche and marketed under the brand name "Rituxan" by drug regulatory agencies. Only the original drug can be called rituximab.

[0065] Rituximab biosimilars

[0066] The rituximab biosimilars of this invention refer to monoclonal antibody drugs developed by other companies that, in addition to the original rituximab, have the same amino acid sequence as rituximab and similar physicochemical properties, efficacy, pharmacokinetics and safety as the original rituximab.

[0067] small molecule toxins

[0068] The small molecule toxins applicable to this invention are compounds with high cytotoxicity. Specifically, the small molecule toxin is one or more monomethylauristatins; more preferably, the small molecule toxin is monomethylauristatin-E (MMAE), monomethylauristatin-D (MMAD), monomethylauristatin-F (MMAF), or a combination thereof.

[0069] The molecular structure of MMAE is shown below:

[0070]

[0071] connector

[0072] The connector (L) suitable for this invention is used to connect the antibody and the small molecule toxin of this invention. Specifically, the connector is maleimide hexanoyl-valine-citrulline-p-aminobenzyloxycarbonyl, for example, as shown in the following formula:

[0073]

[0074] Antibody-drug conjugates

[0075] The present invention provides an antibody-drug conjugate comprising (a) a recombinant anti-CD20 monoclonal antibody and (b) a cytotoxic small molecule toxin linked together by a linker (L).

[0076] As used herein, the terms "antibody-drug conjugate of the present invention" or "ADC of the present invention" are used interchangeably and refer to a conjugate of an antibody and a small molecule toxin of the present invention bound together by a linker.

[0077] Specifically, the structure of the antibody-drug conjugate is shown in Formula I:

[0078] mAb-(LD)n I

[0079] in,

[0080] mAb represents the antibody of the present invention;

[0081] D represents the small molecule toxin of this invention;

[0082] L is a linker connecting antibodies and small molecule toxins;

[0083] n is the average number of small molecule toxins conjugated to the antibody, and n is an integer or non-integer of 4.2 ± 1; and

[0084] "-" is the key.

[0085] In another preferred embodiment, n is an integer or non-integer of 4.2 ± 0.5.

[0086] In another preferred embodiment, n is an integer or non-integer of 4.2 ± 0.3.

[0087] In another preferred embodiment, the structure of the antibody-drug conjugate is shown in the following formula:

[0088]

[0089] Preparation method

[0090] This invention provides a method for conjugating antibody-drug conjugates, which conjugates small molecule toxins to antibodies through specific linkers, thereby significantly improving the antibody's killing power against tumor cells without altering antibody affinity.

[0091] First, small molecule toxins and connectors can be linked using conventional chemical synthesis methods, or commercially available small molecule toxins with connectors can be used.

[0092] Then, the free thiol groups formed by the reduction of the recombinant monoclonal antibody molecules through four pairs of interchain disulfide bonds are linked to the maleimide groups in the linker that have been linked to the small molecule toxin.

[0093] Due to the heterogeneity of the reaction sites, the resulting monoclonal antibody-drug conjugates exist as a mixture of various drug-carrying quantities in solution. The possible toxin-binding sites and drug-carrying quantities of the resulting monoclonal antibody-drug conjugates are as follows... Figure 12 As shown.

[0094] Depend on Figure 12 It is known that, theoretically, there are five forms of monoclonal antibody-drug conjugates carrying different amounts of drug: 0, 2, 4, 6, and 8 drug molecules. To assess the drug loading, an internationally accepted evaluation method can be used, namely, calculating the average drug-to-antibody molar ratio (DAR).

[0095] The DAR value of the antibody-drug conjugate of the present invention is an integer or non-integer of 4.2±1; preferably, it is an integer or non-integer of 4.2±0.5.

[0096] use

[0097] The present invention also provides a method for treating mammalian diseases using the antibody-drug conjugate of the present invention. Preferably, the disease is a CD20-targeting disease, such as tumors, like lymphoma (e.g., B-cell non-Hodgkin lymphoma) or leukemia (chronic lymphocytic leukemia).

[0098] The present invention also provides a pharmaceutical composition (e.g., an antitumor drug) containing the antibody-drug conjugate of the present invention.

[0099] The pharmaceutical composition comprises an effective amount of the antibody-drug conjugate according to the invention (as the active ingredient), and at least one pharmaceutically acceptable carrier, diluent, or excipient. In preparation, the active ingredient is typically mixed with, diluted with, or encapsulated in a carrier that may be in capsule or pouch form. When the excipient acts as a diluent, it may be a solid, semi-solid, or liquid material as a medium for the excipient, carrier, or active ingredient. Therefore, the composition may be a solution, a sterile injectable solution, etc.

[0100] Suitable excipients include: lactose, glucose, sucrose, sorbitol, mannitol, starch, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, etc.; formulations may also include: humectants, emulsifiers, preservatives (such as methylparaben and propylparaben), etc.

[0101] The pharmaceutical composition may be formulated into a single or multi-dosage form, each dosage form comprising a predetermined amount of the antibody-drug conjugate of the present invention calculated to produce the desired therapeutic effect, and a suitable pharmaceutical excipient.

[0102] The pharmaceutical composition can be administered via conventional routes, including (but not limited to): intramuscular, intraperitoneal, intravenous, subcutaneous, intradermal, local administration, etc.

[0103] When using this pharmaceutical composition, a safe and effective amount of the antibody-drug conjugate is administered to a human, preferably in the range of 0.001–3 mg / kg body weight, more preferably 0.01–2 mg / kg body weight. Of course, the specific dosage should also consider factors such as the route of administration and the patient's health condition, all of which are within the scope of a skilled physician's expertise.

[0104] Furthermore, the antibody-drug conjugate of the present invention can also be used in combination with other therapeutic agents, including (but not limited to): cyclophosphamide, doxorubicin, vincristine, prednisone, PD1 antibody, CTLA4 inhibitor, or combinations thereof.

[0105] Compared with the prior art, the main beneficial effects of the present invention include: the present invention provides an antibody-drug conjugate with optimized drug loading, which has high drug activity, good stability, low toxicity and good drug-like properties.

[0106] This invention provides a method for preparing monoclonal antibody-drug conjugates based on thiol coupling, which allows for precise control of drug loading. The product obtained by this method contains less than 3% naked antibody, and the efficacy of the drug has been demonstrated through in vitro and in vivo pharmacodynamic studies.

[0107] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. Percentages and parts are by weight unless otherwise stated. Unless otherwise specified, the raw materials, reagents, cell lines, or instruments used in the embodiments of the present invention are commercially available or conventional unless otherwise specified.

[0108] abbreviation

[0109] TCEP: Tris(2-carboxyethyl)phosphine; DTPA: Diethylaminetriaminepentaacetic acid; ACN: Acetonitrile; TFF: Tangential flow filtration; RT: Room temperature.

[0110] In this embodiment, unless otherwise specified, the reagents, instruments and cell lines are all commercially available or conventional.

[0111] Example 1: Preparation of antibody (TRS001)

[0112] The amino acid and nucleotide sequences of the light and heavy chains of rituximab were synthesized using fully synthesized coding sequences. Using suspension-acclimated CHO-K1 cells (from ATCC in the United States) as host cells, dual expression plasmids for the light chain (LC) and heavy chain (HC) of the monoclonal antibody, pcDNA3-RX-Neo and pcDNA3-RX-GS, were constructed using an expression vector based on pcDNA3.0. Both expression plasmids carried the light and heavy chain genes of the antibody (the light and heavy chain sequences of TRS001 are completely identical to those of commercially available rituximab) and corresponding selection markers. The two expression plasmids were co-transfected into host cells, and stable cells were screened using the dual selection markers. Multiple candidate monoclonal cell lines were then selected through a stepwise screening method using semi-solid culture medium. Finally, a monoclonal cell line for antibody (TRS001) production was screened using shake flask and reactor culture.

[0113] The monoclonal cell line was sequenced, and the sequencing results showed that the sequences encoding the light and heavy chains of TRS001 were completely identical to those of commercially available rituximab, and the molecular weight and other physicochemical properties of the produced rituximab biosimilar were the same as those of rituximab.

[0114] The monoclonal cell line selected in the above steps was used as the final production cell line. The recombinant anti-human CD20 monoclonal antibody TRS001 was prepared according to the production method provided in the Rituximab original drug development patent (CN93121424.6) for subsequent examples.

[0115] Example 2 Preparation of antibody-drug conjugate (TRS005: DAR approximately 4.2)

[0116] 1. Preparation of solutions and materials

[0117] Buffer A: 0.05 mol / L PB reaction buffer (pH 7.6)

[0118] A 0.2 mol / L PB storage buffer (pH 7.6) was prepared using a solution of 0.2 mol / L NaH2PO4·H2O and 0.2 mol / L Na2HPO4·7H2O (using Millipore purified water). The buffer was filtered through a 0.22 μm membrane (Nalgene Rapid-Flow unit) and stored at 4 °C. The buffer was then equilibrated at RT and diluted to 0.05 mol / L as a reduction buffer.

[0119] Reducing agent: 100 mmol / L TCEP solution

[0120] Dissolve 1.43 g TECP in 50 mL of pure water to obtain a 100 mmol / L solution, aliquot 1 mL / bottle and store at -80 °C.

[0121] Buffer B: 10 mg / mL L-cysteine ​​termination buffer

[0122] Dissolve 1 g of L-cysteine ​​in 100 ml of 0.1 mM DTPA solution, filter through a 0.22 μm membrane, and store at -80 °C.

[0123] 50% ACN solution

[0124] A 50% acetonitrile solution was prepared by mixing equal volumes of ACN and ultrapure water, filtered through a 0.22 μm membrane, and stored at 4 °C.

[0125] Buffer C: HT formulation buffer

[0126] 1L of HT preparation buffer contains 3.18g of L-histidine, 70g of trehalose dihydrate and 0.2mL of Tween 80. The pH is adjusted to 6.5 with 0.5mol / L hydrochloric acid, filtered through a 0.22μm membrane, and stored at 4℃.

[0127] 2. Reduction of TRS001 (molar ratio: mAb:TCEP≈1:3)

[0128] The TRS001 stock solution (55 mg / mL) prepared in Example 1 was thawed overnight (stabilization chamber) at 25°C. 1616 mL of buffer A was added to a reactor (pre-filled with 0.1 mol / L NaOH for over 24 hours and then washed clean), followed by 2 mL of 100 mmol / L TCEP solution and mixing (100 rpm, 5 min). 182 mL of the TRS001 stock solution was added, and the mixture was stirred at 100 rpm. The reduction reaction was maintained at 25°C for 90 min.

[0129] 3. Combined (molar ratio: mAb:vcMMAE≈1:7.5)

[0130] Set the temperature control reaction bath to 4°C (addition with ice cooling). Maintain a stirring speed of 100-200 rpm. Add 13.2 mL of vcMMAE (50 mg / mL) (purchased from Levena Biopharma) to 186.8 mL of 50% ACN, then carefully add and mix very slowly through a sample addition tube (controlling the sample addition time to within 10 min). Gently add all materials dropwise to the reaction system from the previous step. After the vcMMAE solution has been completely added, maintain the reaction time for 1 h.

[0131] 4. Stop the reaction

[0132] Add 10 mL of buffer B to the reactor within 10 minutes to stop the coupling reaction. After stopping the coupling reaction, proceed to the next step of buffer replacement.

[0133] 5. Buffer replacement

[0134] Transfer the coupling solution to a TFF system (pre-soaked and washed with 0.1 mol / L NaOH). Replace buffer A with buffer C and then filter using a 0.22 μL Rapid-Flow apparatus. The residual amount of buffer A in the final solution should be less than 0.25%. The replaced TRS005 stock solution can be stored at 2–8 °C.

[0135] The final antibody-drug conjugate TRS005 had a DAR of approximately 4.2. The drug loading determination results for the antibody-drug conjugate are as follows: Figure 1 As shown.

[0136] Example 3 Preparation of antibody-drug conjugate (TRS005: DAR approximately 6.2)

[0137] 1. Preparation of solutions and materials

[0138] Buffer A: 0.05 mol / L PB reaction buffer (pH 7.6)

[0139] A 0.2 mol / L PB storage buffer (pH 7.6) was prepared using a solution of 0.2 mol / L NaH2PO4·H2O and 0.2 mol / L Na2HPO4·7H2O (using Millipore purified water). The buffer was filtered through a 0.22 μm membrane (Nalgene Rapid-Flow unit) and stored at 4 °C. The buffer was then equilibrated at RT and diluted to 0.05 mol / L as a reduction buffer.

[0140] Reducing agent: 100 mmol / L TCEP solution

[0141] Dissolve 1.43 g TECP in 50 mL of pure water to obtain a 100 mmol / L solution, aliquot 1 mL / bottle and store at -80 °C.

[0142] Buffer B: 10 mg / mL L-cysteine ​​termination buffer

[0143] Dissolve 1 g of L-cysteine ​​in 100 ml of 0.1 mM DTPA solution, filter through a 0.22 μm membrane, and store at -80 °C.

[0144] 50% ACN solution

[0145] A 50% acetonitrile solution was prepared by mixing equal volumes of ACN and ultrapure water, filtered through a 0.22 μm membrane, and stored at 4 °C.

[0146] Buffer C: HT formulation buffer

[0147] 1L of HT preparation buffer contains 3.18g of L-histidine, 70g of trehalose dihydrate and 0.2mL of Tween 80. The pH is adjusted to 6.5 with 0.5mol / L hydrochloric acid, filtered through a 0.22μm membrane, and stored at 4℃.

[0148] 2. Reduction of TRS001 (molar ratio: mAb:TCEP≈1:3.6)

[0149] The TRS001 stock solution (55 mg / mL) prepared in Example 1 was thawed overnight (stabilization chamber) at 25°C. 1616 mL of buffer A was added to a reactor (pre-filled with 0.1 mol / L NaOH for over 24 hours and then thoroughly washed), followed by 2.4 mL of 100 mmol / L TCEP solution and mixing (100 rpm, 5 min). 182 mL of the TRS001 stock solution was added, and the mixture was stirred at 100 rpm. The reduction reaction was maintained at 25°C for 90 min.

[0150] 3. Combine (molar ratio: mAb:vcMMAE≈1:9)

[0151] Set the temperature control reaction bath to 4°C (addition with ice cooling). Maintain a stirring speed of 100-200 rpm. Add 15.8 mL of vcMMAE (50 mg / mL) (purchased from Levena Biopharma) to 184.2 mL of 50% ACN, then carefully add and mix very slowly through a sample addition tube (controlling the sample addition time to within 10 min). Gently add all materials dropwise to the reaction system from the previous step. After the vcMMAE solution has been completely added, maintain the reaction time for 1 h.

[0152] 4. Stop the reaction

[0153] Add 10 mL of buffer B to the reactor within 10 minutes to stop the coupling reaction. After stopping the coupling reaction, proceed to the next step of buffer replacement.

[0154] 5. Buffer replacement

[0155] Transfer the coupling solution to a TFF system (pre-soaked and washed with 0.1 mol / L NaOH). Replace buffer A with buffer C and then filter using a 0.22 μL Rapid-Flow apparatus. The residual amount of buffer A in the final solution should be less than 0.25%. The replaced TRS005 stock solution can be stored at 2–8 °C.

[0156] The final antibody-drug conjugate TRS005 had a DAR of approximately 6.2. The drug loading determination results for the antibody-drug conjugate are as follows: Figure 2 As shown.

[0157] Example 4 Preparation of antibody-drug conjugate (TRS005: DAR approximately 3.2)

[0158] 1. Preparation of solutions and materials

[0159] Buffer A: 0.05 mol / L PB reaction buffer (pH 7.6)

[0160] A 0.2 mol / L PB storage buffer (pH 7.6) was prepared using a solution of 0.2 mol / L NaH2PO4·H2O and 0.2 mol / L Na2HPO4·7H2O (using Millipore purified water). The buffer was filtered through a 0.22 μm membrane (Nalgene Rapid-Flow unit) and stored at 4 °C. The buffer was then equilibrated at RT and diluted to 0.05 mol / L as a reduction buffer.

[0161] Reducing agent: 100 mmol / L TCEP solution

[0162] Dissolve 1.43 g of TECP in 50 mL of pure water to obtain a 100 mmol / L solution. Divide the solution into 1 mL aliquots and store at -80°C.

[0163] Buffer B: 10 mg / mL L-cysteine ​​termination buffer

[0164] Dissolve 1 g of L-cysteine ​​in 100 ml of 0.1 mM DTPA solution, filter through a 0.22 μm membrane, and store at -80 °C.

[0165] 50% ACN solution

[0166] A 50% acetonitrile solution was prepared by mixing equal volumes of ACN and ultrapure water, filtered through a 0.22 μm membrane, and stored at 4 °C.

[0167] Buffer C: HT formulation buffer

[0168] 1L of HT preparation buffer contains 3.18g of L-histidine, 70g of trehalose dihydrate and 0.2mL of Tween 80. The pH is adjusted to 6.5 with 0.5mol / L hydrochloric acid, filtered through a 0.22μm membrane, and stored at 4℃.

[0169] 2. Reduction of TRS001 (molar ratio: mAb:TCEP≈1:2.8)

[0170] The TRS001 stock solution (55 mg / mL) prepared in Example 1 was thawed overnight (stabilization chamber) at 25°C. 1616 mL of buffer A was added to a reactor (pre-filled with 0.1 mol / L NaOH for over 24 hours and then thoroughly washed), followed by 1.8 mL of 100 mmol / L TCEP solution and mixing (100 rpm, 5 min). 182 mL of the TRS001 stock solution was added, and the mixture was stirred at 100 rpm. The reduction reaction was maintained at 25°C for 90 min.

[0171] 3. Combine (molar ratio: mAb:vcMMAE≈1:6)

[0172] Set the temperature control reaction bath to 4°C (addition with ice cooling). Maintain a stirring speed of 100-200 rpm. Add 10.5 mL of vcMMAE (50 mg / mL) (purchased from Levena Biopharma) to 189.5 mL of 50% ACN, then carefully add and mix very slowly through a sample addition tube (controlling the sample addition time to within 10 min), gently adding all materials dropwise to the reaction system from the previous step. After the vcMMAE solution has been completely added, maintain the reaction time for 1 h.

[0173] 4. Stop the reaction

[0174] Add 10 mL of buffer B to the reactor within 10 minutes to stop the coupling reaction. After stopping the coupling reaction, proceed to the next step of buffer replacement.

[0175] 5. Buffer replacement

[0176] Transfer the coupling solution to a TFF system (pre-soaked and washed with 0.1 mol / L NaOH). Replace buffer A with buffer C and then filter using a 0.22 μL Rapid-Flow apparatus. The residual amount of buffer A in the final solution should be less than 0.25%. The replaced TRS005 stock solution can be stored at 2–8 °C.

[0177] The final antibody-drug conjugate TRS005 had a DAR of approximately 3.2. The drug loading determination results for the antibody-drug conjugate are as follows: Figure 3 As shown.

[0178] Example 5: Consistency Experiment of Antibody-Drug Conjugate (TRS005) Preparation

[0179] Example 2 was repeated to obtain three batches of products. These were:

[0180] C_16205_20160106001

[0181] C_16205_20160106002

[0182] C_16205_20160106003

[0183] The DAR of the three batches was precisely controlled at 4.2 ± 0.3. The C4-RPLC and hydrophobic interaction chromatograms of the three batches are as follows: Figure 4 and Figure 5 As shown.

[0184] The results showed that the antibody-drug conjugates prepared by the method of the present invention exhibited good consistency among different batches.

[0185] Example 6: Flow Cytometry Detection Method for Antibody-Drug Conjugate (TRS005)

[0186] 1. Reagents and Cells

[0187] Sheath fluid: filtered deionized water;

[0188] DPBS (1x): Purchased from Gibco, catalog number: 14190-136;

[0189] Anti-human IgG antibody (FITC), purchased from abcam, catalog number: ab81051;

[0190] Raji cells were purchased from the Cell Bank of the Chinese Academy of Sciences, catalog number: TCHu44.

[0191] Ramos cells were purchased from Nanjing Kebai Biotechnology Co., Ltd.

[0192] 2. Equipment

[0193] BD Accuri C6 flow cytometer: purchased from BD Biosciences.

[0194] 3. Methods

[0195] Instrument startup: Place a tube of freshly drawn ultrapure water into the injection needle of the flow cytometer, turn on the instrument, and use Fast mode to rinse the instrument with water for 5 minutes. Prepare 8-peaks and 6-peaks quality control samples according to the instrument manual to calibrate the four channels of the flow cytometer.

[0196] Cell preparation: Take Raji or Ramos cells in logarithmic growth phase, centrifuge at 1000 rpm for 5 minutes, resuspend in DPBS, and adjust the viable cell density to 2 x 10⁶ cells / mL. 6 Cells / mL, aliquoted into 1.5mL centrifuge tubes, 1mL / tube.

[0197] Sample dilution: Dilute the sample to be tested (TRS005RS, TRS005 with a DAR of approximately 4.2) to 1000 μg / mL. Add 10 μL to each 1 mL of cell suspension and mix thoroughly by pipetting to achieve a final sample concentration of 10 μg / mL.

[0198] Antibody binding: Place the cell suspension containing the sample on ice and incubate for 30 minutes.

[0199] Endocytosis: After the ice bath, centrifuge the cell suspension at 1500 rpm for 5 minutes, discard the supernatant, resuspend in 1 mL of pre-chilled DPBS, and aliquot 200 μL / tube into four 1.5 mL centrifuge tubes. One tube serves as a negative control and is placed on ice, while the other three tubes serve as parallel experimental groups and are placed in a 37°C water bath. Incubate for 2 hours.

[0200] Staining: Dilute the secondary antibody anti-human IgG antibody (FITC-labeled) 100-fold with DPBS. After incubation, centrifuge the cell suspension at 1500 rpm for 5 minutes, discard the supernatant, resuspend in 250 μL of pre-chilled DPBS, add 50 μL of diluted secondary antibody solution to each suspension, mix well by pipetting, and place on ice for 30 minutes.

[0201] Instrument detection: After the ice bath, centrifuge the cell suspension at 1500 rpm for 5 minutes, discard the supernatant, resuspend in 200 μL of pre-cooled DPBS, and then perform instrument detection.

[0202] Cells from the negative control group, treated with ice bath, were gated, and the average fluorescence intensity at FL1 was recorded for each tube. Flow cytometry scatter plots and single-parameter histograms of the cells are shown below. Figure 6A , Figure 6B As shown.

[0203] The endocytosis rate of the sample is calculated using the following formula based on the average fluorescence intensity.

[0204] Sample internalization rate % = (Average fluorescence intensity of negative control group - Average fluorescence intensity of experimental group) / Average fluorescence intensity of negative control group × 100%

[0205] The endocytosis results of Ramos cells and Raji cells are as follows: Figure 6C , Figure 6D As shown. The results indicate that the antibody-drug conjugate TRS005 of the present invention has excellent internalization efficacy.

[0206] Example 7 Tumor Cell Inhibition Experiment

[0207] 1. Reagents

[0208] RPMI1640, purchased from Gibco, item number 11875-093;

[0209] FBS, purchased from CellMax, item number SA212.02;

[0210] Streptomycin / penicillin dual antibody, purchased from HyClone, lot number: SV30010;

[0211] DPBS (Dulbecco's phosphate buffer solution) was purchased from Gibco, catalog number 14190-136;

[0212] CCK-8 reagent, purchased from Biolite, catalog number 35004.

[0213] 2. Cells

[0214] Raji cells were purchased from the Cell Bank of the Chinese Academy of Sciences, catalog number: TCHu44.

[0215] 3. Methods

[0216] Cell Culture and Plating: Raji cells with a viability greater than 80% in the logarithmic growth phase and before passage 30 were selected for the experiment. Raji cells were removed from the culture flask and added to a 50 mL centrifuge tube, where they were mixed by pipetting. The cells were centrifuged at 1000 rpm (approximately 188 g) for 5-6 minutes. The supernatant was discarded, and the cells were resuspended in an appropriate volume of diluent. Cells were counted, and the cell density was adjusted to approximately 8 × 10⁶ cells / mL using pre-warmed complete culture medium (RPMI 1640 + 10% FBS + 1% penicillin and streptomycin).5 Cells / mL for cell seeding: Add 50 μL of cells to each target well and 100 μL of sterile water or DPBS to the edge wells. Incubate in a CO2 incubator at 37°C with 5% CO2.

[0217] Sample dilution: TRS005 standard (one batch of calibrated TRS005 product with a DAR of approximately 4.2), sample (one batch of uncalibrated TRS005 product with a DAR of approximately 4.2), and QC80% (TRS005 diluted to 80% concentration with a DAR of approximately 4.2) were diluted to 60, 60, and 48 μg / mL, respectively, and then serially diluted using 96-well plates, followed by 3-fold serial dilutions using complete culture medium.

[0218] Sample addition and incubation: Add 50 μL of diluted standards, samples, and QC to each well of the cell culture plate. Incubate the cell culture plate in a CO2 incubator for approximately 72 hours.

[0219] Color development: After incubation, add 10 μL of CCK-8 reagent to each well for color development and incubate in a carbon dioxide incubator for 3-4 hours.

[0220] Plate reading: Load the plate into the microplate reader and read the plate using 450nm as the reading wavelength and 650nm as the reference wavelength.

[0221] Data processing: The concentration value was plotted on the x-axis, and the difference between the absorbance at 450 nm and 650 nm was plotted on the y-axis. A four-parameter fitting model was used: Y = (AD) / (1 + (X / C)). B The data is analyzed using the formula A+D, where A represents the asymptote estimate on the curve; B represents the slope of the curve; D represents the asymptote estimate below the curve; and C is its EC50 value.

[0222] The results are as follows Figure 7 As shown in the figure. The results indicate that the antibody-drug conjugate prepared in this invention exhibits stable cytotoxicity and excellent tumor cell inhibition.

[0223] Example 8: In vivo antitumor activity

[0224] 1. Experimental animals and their rearing environment

[0225] Species: BALB / c nude mouse

[0226] Animal sex: Male rat

[0227] Purchased at age: 4-5 weeks

[0228] Living environment: SPF grade

[0229] 2. Cells

[0230] Ramos cells were purchased from Nanjing Kebai Biotechnology Co., Ltd.

[0231] Daudi cells were purchased from the Cell Bank of the Chinese Academy of Sciences Type Culture Collection Committee.

[0232] PRMI1640 culture medium was purchased from Corning, lot number: 04216004.

[0233] The matrix adhesive was purchased from Corning, batch number: 60111319.

[0234] 3. Test Methods

[0235] Ramos cells and Daodi cells were cultured and expanded to 6.0 × 10⁶ cells per 10 ... 8 One, centrifuged to collect cells and adjust concentration and The matrix adhesives are mixed at a volume ratio of 1:1 and then 1×10 7 200 μl / mouse was injected subcutaneously into the right rib area of ​​BALB / c nude mice.

[0236] When the average tumor volume reaches 100-150mm 3 At that time, random grouping:

[0237] Negative control group: Vehicle;

[0238] Rituximab group: Rituximab (3 mg / kg);

[0239] Rituximab (6 mg / kg);

[0240] Rituximab and toxin combination group: Rituximab + MMAE (3mg / kg);

[0241] TRS005 DAR 4.2 (0.75 mg / kg) group: TRS005 (0.75 mg / kg);

[0242] TRS005 DAR 4.2 (1.5 mg / kg) group; TRS005 (1.5 mg / kg);

[0243] TRS005 DAR 4.2 (3mg / kg) group; TRS005 (3mg / kg).

[0244] Ten nude mice were used in each group. The drug was administered via tail vein. During the administration, the body weight and the long and short diameters of the tumors in the BALB / c nude mice were measured and recorded.

[0245] Tumor volume calculation formula: = major diameter × minor diameter × minor diameter / 2

[0246] 4. Statistical Analysis

[0247] Tumor volume and weight are expressed as mean ± standard deviation (mean ± SD). Statistical analysis was performed using one-way ANOVA, with p < 0.05 considered statistically significant and p < 0.01 considered highly significant.

[0248] 5. Results:

[0249] TRS005 showed a very significant therapeutic effect on Ramos and Daudi xenograft BALB / c nude mouse models (see...). Figure 8 , Figure 9 , Figure 10 , Figure 11 Its efficacy is 6-8 times that of Rituximab.

[0250] Figure 8 and Figure 9 This indicates a significant difference in efficacy between TRS005 and rituximab (MabThera). Specifically, the tumor inhibition rate in the TRS005 (3 mg / kg) group was 100%, and the tumor inhibition rate in the TRS005 (0.75 mg / kg) group was similar to, or even slightly better than, that in the rituximab (6 mg / kg) group.

[0251] Figure 10 and Figure 11 This indicates a significant difference in efficacy between TRS005 and rituximab (MabThera). Specifically, the tumor inhibition rate in the TRS005 (3 mg / kg) group was 100%, and the tumor inhibition rate in the TRS005 (0.75 mg / kg) group was similar to, or even slightly better than, that in the rituximab (6 mg / kg) group.

[0252] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. A method for preparing an antibody-drug conjugate targeting CD20 or a pharmaceutically acceptable salt thereof, characterized in that, The method includes the following steps: (1) The recombinant anti-CD20 monoclonal antibody and the reducing agent were subjected to a reduction reaction in PB reaction buffer at pH 7.6 at 25±1℃ for 90±10 minutes to obtain a reaction system containing the reduced recombinant anti-CD20 monoclonal antibody; wherein the recombinant anti-CD20 monoclonal antibody is rituximab or its biosimilar, the reducing agent is tris(2-carboxyethyl)phosphine, and the molar ratio of the recombinant anti-CD20 monoclonal antibody to the reducing agent is 1:2.9 to 1:3.1; (2) The solution of vcMMAE in acetonitrile and water is added dropwise to the reaction system of step (1) to allow the reduced recombinant anti-CD20 monoclonal antibody and vcMMAE to undergo a coupling reaction at 4±0.5℃ for 60±10 minutes; wherein the volume ratio of acetonitrile and water is 1:1, and the molar ratio of the recombinant anti-CD20 monoclonal antibody to vcMMAE is 1:7.0~1:8.0; (3) The coupling reaction is terminated by adding L-cysteine ​​termination buffer to the reaction system of step (2), wherein the L-cysteine ​​termination buffer is prepared by dissolving L-cysteine ​​in DTPA solution; and (4) After the coupling reaction in step (3) is terminated, the buffer is replaced and the coupling solution is transferred to the TFF system. The original PB reaction buffer is changed to the formulation buffer and then filtered using a 0.22-micron Rapid-Flow device.

2. The method according to claim 1, characterized in that, In step (1), the molar ratio of the recombinant anti-CD20 monoclonal antibody to the reducing agent is 1:2.95 to 1:3.

05.

3. The method according to claim 1, characterized in that, In step (1), the molar ratio of the recombinant anti-CD20 monoclonal antibody to the reducing agent is 1:2.99 to 1:3.

01.

4. The method according to claim 1, characterized in that, In step (1), the molar ratio of the recombinant anti-CD20 monoclonal antibody to the reducing agent is 1:

3.

5. The method according to any one of claims 1 to 4, characterized in that, In step (2), the molar ratio of the recombinant anti-CD20 monoclonal antibody to vcMMAE is 1:7.2 to 1:7.

7.

6. The method according to any one of claims 1 to 4, characterized in that, In step (2), the molar ratio of the recombinant anti-CD20 monoclonal antibody to vcMMAE is 1:7.4 to 1:7.

6.

7. The method according to any one of claims 1 to 4, characterized in that, In step (2), the molar ratio of the recombinant anti-CD20 monoclonal antibody to vcMMAE is 1:7.

5.

8. The method according to any one of claims 1 to 4, characterized in that, The preparation buffer is an HT preparation buffer, and 1L of the HT preparation buffer contains 3.18g of L-histidine, 70g of trehalose dihydrate and 0.2mL of Tween 80. The pH is adjusted to 6.5 using 0.5mol / L hydrochloric acid and filtered through a 0.22μm membrane.

9. The method according to any one of claims 1 to 4, characterized in that, In step (1), the recombinant anti-CD20 monoclonal antibody and the reducing agent are subjected to a reduction reaction at 25°C for 90 minutes in PB reaction buffer at pH 7.6, and the molar ratio of the recombinant anti-CD20 monoclonal antibody to the reducing agent is 1:3; in step (2), the reduced recombinant anti-CD20 monoclonal antibody and vcMMAE are subjected to a coupling reaction at 4°C for 60 minutes, and the molar ratio of the recombinant anti-CD20 monoclonal antibody to vcMMAE is 1:7.

5.

10. The method according to claim 8, characterized in that, In step (1), the recombinant anti-CD20 monoclonal antibody and the reducing agent are subjected to a reduction reaction at 25°C for 90 minutes in PB reaction buffer at pH 7.6, and the molar ratio of the recombinant anti-CD20 monoclonal antibody to the reducing agent is 1:3; in step (2), the reduced recombinant anti-CD20 monoclonal antibody and vcMMAE are subjected to a coupling reaction at 4°C for 60 minutes, and the molar ratio of the recombinant anti-CD20 monoclonal antibody to vcMMAE is 1:7.

5.

11. The antibody-drug conjugate targeting CD20 or a pharmaceutically acceptable salt thereof obtained by the method according to any one of claims 1 to 10, wherein the structural formula of the antibody-drug conjugate targeting CD20 is as follows: The mAb is rituximab or its biosimilar; the average number of MMAEs conjugated to the recombinant anti-CD20 monoclonal antibody n is 4.2 ± 0.5, and the proportion of naked antibodies without MMAE conjugation is less than 3%.

12. The antibody-drug conjugate targeting CD20 according to claim 11, or a pharmaceutically acceptable salt thereof, characterized in that, n is 4.2 ± 0.

3.

13. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises the antibody-drug conjugate targeting CD20 as described in claim 11 or 12, or a pharmaceutically acceptable salt thereof, as well as histidine hydrochloride, trehalose, and Tween 80.

14. Use of the antibody-drug conjugate targeting CD20 according to claim 11 or 12, or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition according to claim 13, in the preparation of a medicament targeting CD20-positive tumors.

15. The use according to claim 14, characterized in that, The tumor is a lymphoma.

16. The use according to claim 14, characterized in that, The tumor is a B-cell lymphoma.

17. The use according to claim 14, characterized in that, The tumor is a B-cell non-Hodgkin lymphoma.

18. The use according to claim 14, characterized in that, The tumor is chronic lymphocytic leukemia.

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