CD20-TARGETED ANTIBODY COUPLING PHARMACEUTICAL PREPARATION

Abstract

A CD20-targeted antibody coupling pharmaceutical preparation, specifically a preparation comprising a CD20-targeted antibody coupling medication represented by formula I and an excipient. The antibody coupling pharmaceutical preparation has prominent antitumor effect.

Claims

1. An antibody-drug conjugate pharmaceutical preparation, wherein the preparation comprises: (a) antibody-drug conjugates with a structure as shown in formula I:
mAb-(L-D)n  I wherein, mAb represents a recombinant anti-CD20 monoclonal antibody, which is rituximab or a biosimilar thereof; D represents a small molecule toxin, which is one or more monomethyl auristatin; L is a linker connecting the antibody and the small molecule toxin; n is the average number of the small molecule toxins conjugated to the antibody; and n is an integer or non-integer of 4.2±1; and “—” is a bond; and (b) a carrier or excipient.

2. The antibody-drug conjugate pharmaceutical preparation of claim 1, wherein D is monomethyl auristatin-E (MMAE), monomethyl auristatin-D (MMAD), monomethyl auristatin-F (MMAF), or a combination thereof.

3. The antibody-drug conjugate pharmaceutical preparation of claim 1, wherein L is maleimidocaproyl-valine-citrulline-p-aminobenzyloxycarbonyl.

4. The antibody-drug conjugate pharmaceutical preparation of claim 1, wherein the structure of the antibody-drug conjugate is shown as the following formula: ##STR00005##

5. The antibody-drug conjugate pharmaceutical preparation of claim 1, wherein the preparation is an injection.

6. The antibody-drug conjugate pharmaceutical preparation of claim 1, wherein the preparation is a solution or a lyophilized agent.

7. The antibody-drug conjugate pharmaceutical preparation of claim 1, wherein the carrier or excipient is selected from the group consisting of a pH buffer, an osmotic pressure regulator, a lyophilized powder excipient, a protein protecting agent, a solubilizer, and water for injection.

8. The antibody-drug conjugate pharmaceutical preparation of claim 1, wherein the preparation method for the antibody-drug conjugate comprises the following steps: (1) performing a reduction reaction between the recombinant anti-CD20 monoclonal antibody and a reducing agent, thereby obtaining a reaction system comprising a reduced recombinant anti-CD20 monoclonal antibody; and the molar ratio of the monoclonal antibody to the reducing agent is 1:2.9 to 1:3.1; and (2) performing a coupling reaction between the reaction system of step (1) and a solution of a small molecule toxin in acetonitrile and water to form the antibody-drug conjugates according to claim 1; and the molar ratio of the monoclonal antibody in step (1) to the small molecule toxin is 1:7.0 to 1:8.0.

9. Use of the antibody-drug conjugate pharmaceutical preparation according to claim 1 for preparing anti-tumor drugs.

10. A non-therapeutic method for inhibiting tumor cells, wherein the method comprises the step of adding the antibody-drug conjugate pharmaceutical preparation according to claim 1 to a tumor cell-containing system.

Description

DESCRIPTION OF THE FIGURES

[0051] FIG. 1 is a hydrophobic interaction chromatogram to identify the drug-loading capacity of antibody-drug conjugate TRS005.

[0052] FIG. 2 shows the possible toxin binding sites and drug carrying numbers of the monoclonal antibody-drug conjugate formed.

DETAILED DESCRIPTION OF THE INVENTION

[0053] Through extensive and intensive researches, the inventors have unexpectedly discovered a CD20-targeted antibody-drug conjugate pharmaceutical preparation with high efficiency and optimized drug-loading capacity. The present invention has been completed on this basis.

[0054] Antibody

[0055] Antibodies suitable for the present invention are antibodies that target CD20, and are recombinant anti-CD20 monoclonal antibodies. The recombinant anti-CD20 monoclonal antibody may be rituximab, or it may be a biosimilar thereof.

[0056] Small Molecule Toxin

[0057] Small molecule toxins suitable for the present invention are compounds with high cytotoxicity. Specifically, the small molecule toxin is one or more monomethyl auristatin; more preferably, the small molecule toxin is monomethyl auristatin-E (MMAE), monomethyl auristatin-D (MMAD), monomethyl auristatin-F (MMAF), or a combination thereof.

[0058] Wherein, the molecular structure of MMAE is shown below:

##STR00002##

[0059] Linker

[0060] A linker (L) suitable for the present invention is used to connecting the antibody and the small molecule toxin of the present invention. Specifically, the linker is maleimidocaproyl-valine-citrulline-p-aminobenzyloxycarbonyl, such as the one shown in the following formula:

##STR00003##

[0061] Antibody-Drug Conjugate

[0062] An antibody-drug conjugate is provided in the present invention, which comprises (a) a recombinant anti-CD20 monoclonal antibody and (b) a cytotoxic small molecule toxin connected together through a linker (L).

[0063] 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 connected through a linker.

[0064] Specifically, the antibody-drug conjugate has a structure as shown in formula I:

mAb-(L-D)n  I

[0065] wherein,

[0066] mAb represents an antibody of the present invention;

[0067] D represents a small molecule toxin of the present invention;

[0068] L is a linker connecting the antibody and the small molecule toxin;

[0069] n is the average number of the small molecule toxin conjugated to the antibody; and n is an integer or non-integer of 4.2±1; and “—” is a bond.

[0070] In another preferred embodiment, n is an integer or non-integer of 4.2±0.5.

[0071] In another preferred embodiment, n is an integer or non-integer of 4.2±0.3.

[0072] In another preferred embodiment, the structure of the antibody-drug conjugate is shown as the following formula:

##STR00004##

[0073] Preparation Method for the Antibody-Drug Conjugate

[0074] A conjugating method for antibody-drug conjugates is provided in the present invention, which conjugates a small molecule toxin to an antibody through a specific linker, and greatly improves the killing activity of the antibody to tumor cells without changing the affinity of the antibody.

[0075] First, the small molecule toxin is connected to the linker by conventional chemical synthesis methods, and a commercially available small molecule toxin connected with a linker may also be used.

[0076] Then, the free sulfhydryl groups formed by the reduction of four pairs of interchain disulfide bonds of a recombinantly expressed monoclonal antibody molecule is connected with the maleimide group in a linker to which a small molecule toxin has been connected.

[0077] The resulting monoclonal antibody-drug conjugates have a mixture of multiple drug-loading numbers in solution due to heterogeneity of reaction sites. The possible toxin binding sites and drug loading numbers of the monoclonal antibody-drug conjugates formed are shown in FIG. 2.

[0078] It can be known from the above that there are five forms of monoclonal antibody-drug conjugates formed theoretically that carry different numbers of drugs, which are 0, 2, 4, 6, and 8, respectively. In order to evaluate the number of drugs loaded, an internationally accepted evaluation method can be used, that is, counting the average molar ratio of the toxin drugs to antibodies (Drug Antibody Ratio, DAR).

[0079] The DAR value of the antibody-drug conjugates of the present invention is an integer or non-integer of 4.2±1; preferably, an integer or non-integer of 4.2±0.5.

[0080] Antibody-Drug Conjugate Pharmaceutical Preparation

[0081] An antibody-drug conjugate pharmaceutical preparation is also provided in the present invention, which comprises (a) an effective amount of antibody-drug conjugate of the present invention (as an active ingredient), and (b) at least one pharmaceutically acceptable carrier or excipient.

[0082] For preparation, the active ingredient is usually mixed with a carrier or an excipient, or diluted with a carrier or an excipient, or enclosed in a carrier which may be in the form of capsule or sachet. When the excipient acts as a diluent, solid, semi-solid or liquid materials can be used as the excipient. Therefore, the preparation may be a solution, a sterile injectable solution or the like.

[0083] Suitable excipients comprise lactose, glucose, sucrose, sorbitol, mannitol, starch, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, and the like. Preparations may also comprise wetting agent, emulsifier, preservative (such as methyl and propyl hydroxybenzoate) or the like.

[0084] The preparation can be formulated in single-dose or multi-dose form, both of which comprise calculated predetermined amount of the antibody-drug conjugate of the present invention to provide desired therapeutic effect, as well as suitable pharmaceutical excipients.

[0085] The preparation can be administered by conventional routes including, but not limited to, intramuscular, intraperitoneal, intravenous, subcutaneous, intradermal, topical administration and the like.

[0086] When the preparation is used, a safe and effective amount of the antibody-drug conjugate is administered to a human wherein the safe and effective amount is preferably in the range of 0.001 to 3 milligrams per kilogram of body weight, more preferably in the range of 0.01 to 2 milligrams per kilogram of body weight. Of course, the route of administration, the patient's health status and other factors should be considered for the specific dose, which are within the scope of skills of skilled practitioners.

[0087] In addition, the antibody-drug conjugate pharmaceutical preparation of the present invention may further comprises other therapeutic drugs, including but not limited to cyclophosphamide, doxorubicin, vincristine, prednisone, PD1 antibodies, CTLA4 inhibitors, or a combination thereof

[0088] Use

[0089] A method for treating mammalian diseases using the antibody-drug conjugate pharmaceutical preparation of the present invention is also provided in the present invention. Preferably, the disease is a CD20 targeting disease such as a tumor, such as a lymphoma (e.g., B-cell non-Hodgkin's lymphoma) or leukemia (chronic lymphocytic leukemia).

[0090] The Beneficial Effects of the Present Invention Compared with Prior Arts

[0091] A preparation comprising an antibody-drug conjugate with optimized drug-loading capacity and a suitable carrier, diluent or excipient is provided in the present invention. The preparation has high drug activity, high stability, low toxicity, and excellent druggability.

[0092] The present invention will be further illustrated below with reference to the specific examples. It should be understood that these examples are only to illustrate the invention, but not to limit the scope of the invention. The experimental methods with no specific conditions described in the following examples are generally performed under the conventional conditions (e.g., the conditions described by US Sambrook et al., Molecular Cloning Laboratory Guide (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the manufacturer's instructions. Unless indicated otherwise, all percentage and parts are calculated by weight. Unless otherwise stated, the raw materials, reagents, cell strains or instruments used in the examples of the present invention are commercially available or conventional.

Example 1 Preparation of Antibody (TRS001)

[0093] Coding sequences are fully synthesized based on the amino acid and nucleotide sequences of light and heavy chains of rituximab. The suspension-adapted CHO-K1 (from ATCC, USA) were used as the host cells. Monoclonal antibody light chain (LC) and heavy chain (HC) dual expression plasmids, i.e. pcDNA3-RX-Neo and pcDNA3-RX-GS, were constructed using pcDNA3.0-based expression vector. Both expression plasmids carry genes for the light and heavy chains of the antibody (the light and heavy chains of TRS001 are completely identical to the light and heavy chain sequences of commercially available rituximab) and carry corresponding selection markers. The host cells were co-transfected with the two expression plasmids and stable transfected cells were selected with double selection markers. And then several candidate monoclonal cell lines were screened out step by step using semi-solid medium cloning method. Monoclonal cell lines used for production of antibody (TRS001) were then selected by shaking flask and reactor cultivation.

[0094] The monoclonal cell line was sequenced, and sequencing results showed that the sequences encoding the light and heavy chains of TRS001 were completely identical to those of commercially available rituximab. Moreover, the physical and chemical characteristics such as molecular weight of produced rituximab biosimilar are the same as those of rituximab.

[0095] The monoclonal cell line selected by the above steps was used as the final production cell line, and a recombinant anti-human CD20 monoclonal antibody TRS001 was prepared by referring to the production method provided by rituximab original medicine patent (CN93121424.6) for subsequent examples.

Example 2 Preparation of Antibody-Drug Conjugate (TRS005, Whose DAR is Approximately 4.2)

[0096] 1. Preparation of Solutions and Materials

[0097] Buffer A: 0.05 mol/L PB Reaction Buffer (pH7.6)

[0098] A 0.2 mol/L PB storage buffer (pH 7.6) was prepared using a solution of 0.2 mol/L NaH.sub.2PO.sub.4.H.sub.2O and 0.2 mol/L Na.sub.2HPO.sub.4.7H.sub.2O (using Millipore purified water), filtrated through a 0.22 μm membrane (Nalgene Rapid-Flow Unit) and stored at 4° C., and RT equilibrated and diluted to 0.05 mol/L as a reduction buffer.

[0099] Reducing Agent: 100 mmol/L TCEP Solution

[0100] 1.43 g of TECP (tris (2-carboxyethyl) phosphine) was dissolved in 50 mL of pure water to form a 100 mmol/L solution, which was equally divided to 1 ml/bottle and stored at −80° C.

[0101] Buffer B: 10 mg/mL L-Cysteine Termination Buffer

[0102] 1 g of L-cysteine was dissolved in 100 ml of a 0.1 mM DTPA (diethylenetriaminepentaacetic acid) solution, and the obtained solution was filtered through a 0.22 μm membrane and stored at −80° C.

[0103] 50% ACN Solution

[0104] A 50% acetonitrile solution was prepared by mixing the same volume of ACN and ultrapure water, and filtered through a 0.22 μm membrane and stored at 4 T.

[0105] Buffer C: HT Preparation Buffer

[0106] 1 L HT preparation buffer contains 3.18 g of L-histidine, 70 g of trehalose dihydrate, and 0.2 mL of Tween 80. The pH was adjusted to 6.5 using 0.5 mol/L hydrochloric acid, and the obtained buffer was filtered through a 0.22 μm membrane and stored at 4° C.

[0107] 2. Reduction of TRS001 (Molar Ratio: mAb:TCEP≈1:3)

[0108] The TRS001 stock solution (55 mg/mL) prepared in Example 1 was thawed at 25° C. overnight (stabilization chamber). 1616 mL of buffer A was added in a reactor (the reactor was pre-filled with 0.1 mol/L NaOH for more than 24 hours and cleaned), then 2 mL of 100 mmol/L TCEP solution was added and mixed (100 rpm, 5 minutes). 182 mL of TRS001 stock solution was added and stirred at 100 rpm, and the reduction reaction continued at 25° C. for 90 min.

[0109] 3. Binding (Molar Ratio: mAb:vcMMAE≈1:7.5)

[0110] The temperature-controlled reaction bath was set to 4° C. (added with ice for cooling). The stirring speed was kept at 100-200 rpm. 13.2 mL of vcMMAE (50 mg/mL) (purchased from Levena Biopharma company) was added to 186.8 mL of 50% ACN, and then the solution was carefully added through the sample addition tube and mixed very slowly (the sample addition time was controlled within 10 min). All the materials were gently dropped into the reaction system obtained from the above steps. The reaction time was maintained for 1 h after the vcMMAE solution was completely fed.

[0111] 4. Termination of the Reaction

[0112] 10 mL of buffer B was added into the reactor within 10 minutes to stop the conjugating reaction. After the conjugating reaction was stopped, the next step of buffer replacement was performed.

[0113] 5. Buffer Replacement

[0114] The conjugating solution was transferred to a TFF system (pre-soaked with 0.1 mol/L NaOH and cleaned). Buffer A was changed to buffer C and then filtrated with a 0.22 microliter Rapid-Flow device. The residual rate of buffer A in the final solution should be less than 0.25%. The TRS005 stock solution after buffer replacement can be stored at 2-8° C.

[0115] Finally, the antibody-drug conjugate TRS005 with a DAR of about 4.2 was obtained. The result of identification of drug-loading capacity of the antibody-drug conjugate was shown in FIG. 1.

Example 3 Lyophilized Preparation of Recombinant Anti-CD20 Monoclonal Antibody-Drug Conjugate

[0116] Preparation Comprises:

[0117] Rituximab, histidine hydrochloride, alginic acid, mannitol, polysorbate 80, and water for injection.

[0118] The obtained preparations were tested by stability experiments and the results indicate that they fully meet the requirements of pharmaceutical preparations.

[0119] All the documents cited herein are incorporated into the invention as reference, as if each of them is individually incorporated. Further, it would be appreciated that, in light of the above described teaching of the invention, the skilled in the art could make various changes or modifications to the invention, and these equivalents would still be in the scope of the invention defined by the appended claims of the application.