Antibody drug conjugates and formulations thereof

Abstract

Provided are an anti-CD20 antibody drug conjugate, a preparation comprising the antibody drug conjugate, a composition comprising the antibody drug conjugate, and a medical use of the antibody drug conjugate.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biological medicine, in particular to an antibody drug conjugate, a preparation comprising the antibody drug conjugate, a composition comprising the antibody drug conjugate, and a medical use of the antibody drug conjugate. BACKGROUND

[0002] At present, the treatment of non-Hodgkin's lymphoma (NHL) includes traditional surgical treatment, chemotherapy, radiotherapy, bone marrow or hematopoietic stem cell transplantation, immunotherapy and targeted therapy. Traditional chemical drug treatment has certain curative effect on NHL patients, but it has great systemic toxic side effects, poor tolerance, and is prone to recurrence, which limits the use of patients. Targeted drugs, especially antibody drugs, are concerned due to their significant curative effect and small systemic toxic side effects during treatment.

[0003] In recent years, several CD20-targeted monoclonal antibody drugs have been developed (such as rituximab, ), and its anti-tumor activity has been verified in clinical trials.

[0004] However, with the in-depth clinical application, the limitations of rituximab treatment gradually emerged, that is, there are primary and acquired drug resistance in patients during treatment, which leads to the decrease or invalid of the therapeutic effect of rituximab. In addition, the effective rate of existing CD20-targeted monoclonal antibody monotherapy for tumor is not ideal, and it needs to be combined with chemotherapy drugs to improve the anti-tumor effect, which undoubtedly increases the risk of toxicity. Therefore, the existing CD20-targeted monoclonal antibody drugs represented by rituximab cannot meet the needs of the treatment of relapsed or refractory B-cell NHL (including DLBCL and FL, etc.) in clinical practice.

[0005] In view of the above problems existing in the existing CD20-targeted monoclonal antibody drugs, it is the main purpose of developing the CD20-targeted ADC drug ADC-1 to provide a treatment method with better curative effect, lower toxic side effect, and less drug resistance. SUMMARY

[0006] The inventors of the present application prepared an anti-CD20 antibody drug conjugate through a large number of experiments and creative labor, and confirmed that it has good biological activity and preparation stability, thereby completing the present application.

[0007] Therefore, in the first aspect of the present application, the present application provides an antibody drug conjugate, which has the structure shown in formula I,

[0008] Ab-(L-D) p

[0009] Formula I

[0010] wherein:

[0011] Ab is an anti-CD20 mAb, which is any antibody targeting CD20, such as rituximab or a biosimilar thereof;

[0012] D is a cytotoxic agent, which is Monomethyl auristatin E (MMAE);

[0013] L is a linker, which is 6-maleimidocaproyl-valine-citrulline-para-aminobenzoxycarbonyl (MC-vc-PAB), for connecting the anti-CD20 mAb and the cytotoxic agent;

[0014] p is 3.6-4.0.

[0015] In some embodiments, p is 3.7-3.9.

[0016] In some embodiments, p is 3.8.

[0017] In some embodiments, L-D in Formula I is vc-MMAE, which has the structure as shown in the following formula:

[0018]

[0019] In some embodiments, the antibody drug conjugate has the structure as shown in the following formula:

[0020]

[0021] wherein:

[0022] Ab is an anti-CD20 mAb, which is any antibody targeting CD20, such as rituximab or a biosimilar thereof,

[0023] p is 3.6-4.0.

[0024] In some embodiments, p is 3.7-3.9.

[0025] In some embodiments, p is 3.8.

[0026] In a second aspect of the present application, the present application provides an antibody drug conjugate formulation, which comprises:

[0027] an antibody drug conjugate at a concentration of 1-60 mg / mL (such as 1 mg / mL, 2 mg / mL, 3 mg / mL, 4 mg / mL, 5 mg / mL, 6 mg / mL, 7 mg / mL, 8 mg / mL, 9 mg / mL, 10 mg / mL, 13 mg / mL, 15 mg / mL, 17 mg / mL, 19 mg / mL, 20 mg / mL, 25 mg / mL, 30 mg / mL, 35 mg / mL, 40 mg / mL, 45 mg / mL, 50 mg / mL, 55 mg / mL, or 60 mg / mL);

[0028] 5-35 mM (such as 5 mM, 8 mM, 9 mM, 10 mM, 11 mM, 12 mM, 15 mM, 20 mM, 25 mM, 30 mM, or 35 mM) of histidine buffer, pH 5.0-6.2 (such as 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, or 6.2);

[0029] 2-10% (such as 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10%) of sucrose; and

[0030] 0.01-0.1% (such as 0.01%, 0.015%, 0.02%, 0.025%, 0.03%, 0.031%, 0.032%, 0.033%, 0.034%, 0.035%, 0.036%, 0.037%, 0.038%, 0.039%, 0.04%, 0.045%, 0.05%, 0.055%, 0.06%, 0.065%, 0.07%, 0.08%, 0.09%, or 0.1%) of Tween 80;

[0031] the antibody drug conjugate has a structure shown in Formula I I,

[0032] Ab-(L-D) q

[0033] Formula I I

[0034] wherein:

[0035] Ab is an anti-CD20 mAb, which is any antibody targeting CD20, such as rituximab or a biosimilar thereof;

[0036] D is a cytotoxic agent, which is Monomethyl auristatin E (MMAE);

[0037] L is a linker for connecting the anti-CD20 mAb and the cytotoxic agent, the linker being 6-maleimidocaproyl-valine-citrulline-p-aminobenzoxycarbonyl (MC-vc-PAB);

[0038] q is 3.3-4.3 (such as 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, or 4.3).

[0039] In some embodiments, q is 3.6-4.0.

[0040] In some embodiments, q is 3.7-3.9.

[0041] In some embodiments, q is 3.8.

[0042] In some embodiments, the concentration of the antibody drug conjugate is 1-20 mg / mL.

[0043] In some embodiments, the concentration of the antibody drug conjugate is 1-10 mg / mL.

[0044] In some embodiments, the concentration of the antibody drug conjugate is 3-7 mg / mL.

[0045] In some embodiments, the concentration of the antibody drug conjugate is 5 mg / mL.

[0046] In some embodiments, the concentration of the histidine buffer is 5-15 mM.

[0047] In some embodiments, the concentration of the histidine buffer is 8-12 mM.

[0048] In some embodiments, the concentration of the histidine buffer is 10 mM.

[0049] In some embodiments, the pH of the histidine buffer is 5.8-6.2.

[0050] In some embodiments, the pH of the histidine buffer is 5.6-6.0.

[0051] In some embodiments, the pH of the histidine buffer is 5.4-6.0.

[0052] In some embodiments, the pH of the histidine buffer is 5.4-6.2.

[0053] In some embodiments, the concentration of the sucrose is 3-9%.

[0054] In some embodiments, the concentration of the sucrose is 4-8%.

[0055] In some embodiments, the concentration of sucrose is 5-7%.

[0056] In some embodiments, the concentration of sucrose is 6%.

[0057] In some embodiments, the concentration of Tween 80 is 0.02-0.06%.

[0058] In some embodiments, the concentration of Tween 80 is 0.02-0.05%.

[0059] In some embodiments, the concentration of Tween 80 is 0.03-0.04%.

[0060] In some embodiments, the concentration of Tween 80 is 0.035%.

[0061] In some embodiments, the formulation comprises:

[0062] the antibody drug conjugate at a concentration of 5 mg / mL;

[0063] 10 mM histidine buffer, pH 5.8;

[0064] 6% sucrose; and

[0065] 0.035% Tween 80.

[0066] It should be noted that "2-10% sucrose" refers to the mass / volume concentration (w / v %), which means that in 1000 mL of the antibody drug conjugate formulation, the mass of sucrose is 20-100 g. "0.01-0.1% Tween 80" refers to the mass / volume concentration (w / v %), the meaning of which can be understood similarly as described above for "2-10% sucrose".

[0067] In addition, the concentration of "histidine buffer" refers to the concentration of histidine and histidine hydrochloride, and the pH is obtained by adjusting the ratio of histidine and histidine hydrochloride.

[0068] In some embodiments, the antibody drug conjugate formulation is stable for 6 weeks at 25 ± 2°C / 60% ± 5% RH when the pH is 5.0-6.2 (e.g., 5.6-6.0).

[0069] In some embodiments, the antibody drug conjugate formulation is colorless and clear when stored at 25°C for 6 weeks when the pH is 5.0-6.2 (e.g., 5.6-6.0).

[0070] In some embodiments, the pH of the antibody drug conjugate formulation does not change when stored at 25°C for 6 weeks when the pH is 5.0-6.2 (e.g., 5.6-6.0).

[0071] In some embodiments, the antibody drug conjugate formulation has no change in concentration when stored at 25°C for 6 weeks at a pH of 5.0-6.2 (e.g., 5.6-6.0).

[0072] In some embodiments, the antibody drug conjugate formulation has no more than 2% (or no more than 1%) decrease in SEC monomer % content when stored at 25°C for 6 weeks at a pH of 5.0-6.2 (e.g., 5.6-6.0).

[0073] In some embodiments, the antibody drug conjugate formulation has no more than 2% (or no more than 1%) increase in SEC high polymer % content when stored at 25°C for 6 weeks at a pH of 5.0-6.2 (e.g., 5.6-6.0).

[0074] In some embodiments, the antibody drug conjugate formulation has no significant change in DAR value when stored at 25°C for 6 weeks at a pH of 5.0-6.2 (e.g., 5.6-6.0).

[0075] In some embodiments, the antibody drug conjugate formulation has no change in appearance when stored at 40°C for 4 weeks at a pH of 5.0-6.2 (e.g., 5.6-6.0).

[0076] In some embodiments, the antibody drug conjugate formulation has no change in pH when stored at 40°C for 4 weeks at a pH of 5.0-6.2 (e.g., 5.6-6.0).

[0077] In some embodiments, the antibody drug conjugate formulation has no change in concentration when stored at 40°C for 4 weeks at a pH of 5.0-6.2 (e.g., 5.6-6.0).

[0078] In some embodiments, the antibody drug conjugate formulation has no more than 5% (or no more than 4%) decrease in SEC monomer % content when stored at 40°C for 4 weeks at a pH of 5.0-6.2 (e.g., 5.6-6.0).

[0079] In some embodiments, the antibody drug conjugate formulation has no more than 5% (or no more than 4%) increase in SEC high polymer % content when stored at 40°C for 4 weeks at a pH of 5.0-6.2 (e.g., 5.6-6.0).

[0080] In some embodiments, the antibody drug conjugate formulation has no significant change in DAR value when stored at 40°C for 4 weeks at a pH of 5.0-6.2 (e.g., 5.6-6.0).

[0081] It is noted that the term "no significant change" or "no change" means no statistically significant change.

[0082] In addition, it is noted that the "antibody drug conjugate" above refers to a composition of ADC molecules with the same or different DAR values.

[0083] Specifically, the present application provides a composition comprising a plurality of anti-CD20 ADC molecules. In some cases, each ADC in the composition described herein comprises the same number of one or more drug molecules. In other cases, each ADC in the composition described herein comprises a different number of one or more drug molecules.

[0084] In the antibody drug conjugate described herein, each anti-CD20 antibody can be conjugated with 1, 2, 3, 4, 5, 6, 7, 8 or more drug molecules (preferably 2, 4, 6 or 8, more preferably 2 or 4).

[0085] The drug antibody ratio (DAR) refers to the number of molecules of anti-cancer drug conjugated to the anti-CD20 antibody. The number of molecules of anti-cancer drug contained in the ADC described herein is usually an integer, and when the number of molecules of anti-cancer drug contained in the ADC described herein (e.g., p in Formula I, or q in Formula II) is a fraction, the fraction refers to the average number of molecules of anti-cancer drug per anti-CD20 antibody in a composition comprising a plurality of ADC molecules.

[0086] In a third aspect of the present application, the present application provides a method for preparing the aforementioned antibody drug conjugate preparation, comprising:

[0087] reducing the anti-CD20 mAb and a reducing agent to obtain a reduced anti-CD20 mAb, the anti-CD20 mAb being any antibody targeting CD20, such as rituximab or a biosimilar thereof;

[0088] conjugating the reduced anti-CD20 mAb and vcMMAE;

[0089] quenching the conjugation reaction;

[0090] buffer exchanging the quenched conjugation reaction product to obtain the antibody drug conjugate preparation, the antibody drug conjugate preparation comprising:

[0091] the antibody drug conjugate is at a concentration of 1-60 mg / mL (such as 1 mg / mL, 2 mg / mL, 3 mg / mL, 4 mg / mL, 5 mg / mL, 6 mg / mL, 7 mg / mL, 8 mg / mL, 9 mg / mL, 10 mg / mL, 13 mg / mL, 15 mg / mL, 17 mg / mL, 19 mg / mL, 20 mg / mL, 25 mg / mL, 30 mg / mL, 35 mg / mL, 40 mg / mL, 45 mg / mL, 50 mg / mL, 55 mg / mL, or 60 mg / mL);

[0092] 5-35 mM (such as 5 mM, 8 mM, 9 mM, 10 mM, 11 mM, 12 mM, 15 mM, 20 mM, 25 mM, 30 mM, or 35 mM) histidine buffer, pH 5.0-6.2 (such as 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, or 6.2);

[0093] 2-10% (such as 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10%) sucrose; and

[0094] 0.01-0.1% (such as 0.01%, 0.015%, 0.02%, 0.025%, 0.03%, 0.031%, 0.032%, 0.033%, 0.034%, 0.035%, 0.036%, 0.037%, 0.038%, 0.039%, 0.04%, 0.045%, 0.05%, 0.055%, 0.06%, 0.065%, 0.07%, 0.08%, 0.09%, or 0.1%) Tween 80.

[0095] In some embodiments, the antibody drug conjugate is at a concentration of 1-20 mg / mL.

[0096] In some embodiments, the antibody drug conjugate is at a concentration of 1-10 mg / mL.

[0097] In some embodiments, the antibody drug conjugate is at a concentration of 3-7 mg / mL.

[0098] In some embodiments, the antibody drug conjugate is at a concentration of 5 mg / mL.

[0099] In some embodiments, the histidine buffer is at a concentration of 5-15 mM.

[0100] In some embodiments, the histidine buffer is at a concentration of 8-12 mM.

[0101] In some embodiments, the concentration of the histidine buffer is 10 mM.

[0102] In some embodiments, the pH of the histidine buffer is 5.8-6.2.

[0103] In some embodiments, the pH of the histidine buffer is 5.6-6.0.

[0104] In some embodiments, the pH of the histidine buffer is 5.4-6.0.

[0105] In some embodiments, the pH of the histidine buffer is 5.4-6.2.

[0106] In some embodiments, the concentration of the sucrose is 3-9%.

[0107] In some embodiments, the concentration of the sucrose is 4-8%.

[0108] In some embodiments, the concentration of the sucrose is 5-7%.

[0109] In some embodiments, the concentration of the sucrose is 6%.

[0110] In some embodiments, the concentration of the Tween 80 is 0.02-0.06%.

[0111] In some embodiments, the concentration of the Tween 80 is 0.02-0.05%.

[0112] In some embodiments, the concentration of the Tween 80 is 0.03-0.04%.

[0113] In some embodiments, the concentration of the Tween 80 is 0.035%.

[0114] In some embodiments, the reducing agent is DTT.

[0115] In some embodiments, the preparation method comprises:

[0116] 1) Take 10 mg of anti-CD20 mAb, use 15 mL of 30 KD ultrafiltration device to replace into the reducing buffer (25 mM sodium borate, pH 8.0, 25 mM NaCl, 5 mM EDTA) for three times; the final volume is about 1 mL, transfer to a new Eppendorf centrifuge tube (weighed), and weigh; detect the protein concentration, calculate the total amount of protein;

[0117] 2) To the antibody, add 2.0-3.0 times the molar number of DTT, incubate at room temperature for 2 hours, continuously mix; use 15 ml of 30KD ultrafiltration device to replace into conjugation buffer (50mM Tris, pH7.2, 150mM NaCl, 5mM EDTA), replace for three times; take the concentrated liquid, measure the protein concentration by A 280 (280nm wavelength absorbance) and weigh, calculate the total protein amount; take 10 μl sample, measure the free thiol number by Ellman's method;

[0118] And calculate the molar concentration of free thiol by the following formula (wherein, A 412 represents the absorbance at 412nm wavelength):

[0119]

[0120] b: cuvette light path length (usually 1cm)

[0121] Calculate the free thiol molar number according to the molar concentration of free thiol and the total protein solution volume;

[0122] 3) To the reduced antibody, add 1.0-1.5 times the molar number of vc-MMAE (dissolved in DMSO) of free thiol, mix, then incubate at room temperature for 2 hours, intermittently mix; to the reaction system, add 20 times the molar number of N-acetyl cysteine in the reaction liquid of the input vc-MMAE, mix, stand for 5 minutes;

[0123] 4) Use 15 ml of 30KD ultrafiltration device to replace into conjugate storage solution (10mM Histidine, 6% Sucrose, 0.035% PS80, pH5.8), replace for three times, to obtain the antibody drug conjugate preparation, store at 4°C.

[0124] In the fourth aspect of the present application, the present application provides a composition containing the aforementioned antibody drug conjugate, or the aforementioned antibody drug conjugate preparation, or the antibody drug conjugate preparation prepared by the aforementioned method.

[0125] In some embodiments, the composition further contains at least one pharmaceutically acceptable carrier, diluent or excipient.

[0126] In some embodiments, the composition further comprises a chemotherapeutic agent known to treat tumors, such as Adriamycin, cyclophosphamide, and taxanes [Taxol and Taxotere, Xeloda, Gemzar, Navelbine, tamoxifen, aromatase inhibitors (Arimidex, Femara, Arvi), 5-FU plus leucovorin, camptosar, oxaliplatin, cisplatin, carboplatin, estramustine, Novantrone, prednisone, Oncovin, doxorubicin, prednisolone, and the like, or combinations thereof.

[0127] In some embodiments, the composition further comprises an immunosuppressive agent selected from the group consisting of: (1) glucocorticoids, such as cortisone and prednisone; (2) microbial metabolites, such as cyclosporine and mycophenolic acid; (3) antimetabolites, such as azathioprine and 6-mercaptopurine; (4) polyclonal and monoclonal anti-lymphocyte antibodies, such as anti-lymphocyte globulin and OKT3; and (5) alkylating agents, such as cyclophosphamide. Specifically, the immunosuppressive agent is, for example, methylprednisolone, prednisone, azathioprine, mycophenolate mofetil, sirolimus, tacrolimus, rapamycin, mycophenolic acid, mizoribine, cyclophosphamide, fingolimod, and the like.

[0128] In a fifth aspect of the present application, the present application provides use of the aforementioned antibody drug conjugate, or the aforementioned antibody drug conjugate formulation, or the aforementioned antibody drug conjugate formulation prepared by the aforementioned method, or the aforementioned composition in the preparation of a medicament for preventing and / or treating a CD20-expressing cancer or an immune disease.

[0129] In some embodiments, the CD20-expressing cancer is a lymphoma or a leukemia.

[0130] In some embodiments, the lymphoma is a non-Hodgkin lymphoma, a B-cell non-Hodgkin lymphoma, a follicular non-Hodgkin lymphoma, a small lymphocytic lymphoma, or a diffuse large B-cell lymphoma.

[0131] In some embodiments, the leukemia is a chronic lymphocytic leukemia, a hairy cell leukemia, a B-cell prolymphocytic leukemia, or an acute lymphoblastic leukemia.

[0132] In some embodiments, the CD20-expressing immune disease is rheumatoid arthritis, granulomatosis with polyangiitis, Wegener's granulomatosis, microscopic polyangiitis, or multiple sclerosis.

[0133] In some embodiments, the rheumatoid arthritis is severe active rheumatoid arthritis that has failed treatment with at least one TNF antagonist.

[0134] Preferably, in some embodiments, the CD20-expressing cancer is a CD20-positive B-cell lymphoma.

[0135] In some embodiments, the CD20-positive B-cell lymphoma is a CD20-positive B-cell lymphoma that is resistant to an anti-CD20 mAb (e.g., rituximab).

[0136] In some embodiments, the lymphoma is a non-Hodgkin's lymphoma.

[0137] In some embodiments, the lymphoma is a non-Hodgkin's lymphoma that is resistant to an anti-CD20 mAb (e.g., rituximab).

[0138] In some embodiments, the non-Hodgkin's lymphoma is a diffuse large B-cell lymphoma.

[0139] In some embodiments, the non-Hodgkin's lymphoma is a diffuse large B-cell lymphoma that is resistant to an anti-CD20 mAb (e.g., rituximab).

[0140] In a sixth aspect of the present application, the present application provides a method for preventing and / or treating a CD20-expressing cancer or an immune disease, comprising administering to a subject in need thereof a prophylactically and / or therapeutically effective amount of the aforementioned antibody-drug conjugate, or the aforementioned antibody-drug conjugate formulation, or the antibody-drug conjugate formulation prepared by the aforementioned method, or the aforementioned composition.

[0141] In some embodiments, the CD20-expressing cancer is a lymphoma or a leukemia.

[0142] In some embodiments, the lymphoma is a non-Hodgkin's lymphoma, a B-cell non-Hodgkin's lymphoma, a follicular non-Hodgkin's lymphoma, a small lymphocytic lymphoma, or a diffuse large B-cell lymphoma.

[0143] In some embodiments, the leukemia is a chronic lymphocytic leukemia, a hairy cell leukemia, a B-cell prolymphocytic leukemia, or an acute lymphoblastic leukemia.

[0144] In some embodiments, the CD20-expressing immune disease is rheumatoid arthritis, granulomatosis with polyangiitis, Wegener's granulomatosis, microscopic polyangiitis, or multiple sclerosis.

[0145] In some embodiments, the rheumatoid arthritis is severe active rheumatoid arthritis that has failed treatment with at least one TNF antagonist.

[0146] Preferably, in some embodiments, the CD20-expressing cancer is a CD20-positive B-cell lymphoma.

[0147] In some embodiments, the CD20-positive B-cell lymphoma is a CD20-positive B-cell lymphoma that is resistant to an anti-CD20 mAb (e.g., rituximab).

[0148] In some embodiments, the lymphoma is a non-Hodgkin's lymphoma.

[0149] In some embodiments, the lymphoma is a non-Hodgkin's lymphoma that is resistant to an anti-CD20 mAb (e.g., rituximab).

[0150] In some embodiments, the non-Hodgkin's lymphoma is a diffuse large B-cell lymphoma.

[0151] In some embodiments, the non-Hodgkin's lymphoma is a diffuse large B-cell lymphoma that is resistant to an anti-CD20 mAb (e.g., rituximab).

[0152] In some embodiments, when the subject is an NHL PDX model, the subject in need thereof is administered a dosage of 0.3-10 mg / kg (e.g., 0.3 mg / kg, 1 mg / kg, 3 mg / kg, 10 mg / kg).

[0153] In some embodiments, when the subject is an NHL PDX model, the subject in need thereof is administered a dosage of 0.3-3 mg / kg, 1-3 mg / kg, 1-10 mg / kg, or 3-10 mg / kg.

[0154] In some embodiments, when the subject is a human, the subject in need thereof is administered a dosage of 0.1-5 mg / kg (e.g., 0.1 mg / kg, 0.3 mg / kg, 1 mg / kg, 3 mg / kg, or 5 mg / kg).

[0155] In some embodiments, a prophylactically and / or therapeutically effective amount of the aforementioned antibody drug conjugate, or the aforementioned antibody drug conjugate formulation, or the aforementioned method of making an antibody drug conjugate formulation, or the aforementioned composition is administered to a subject in need thereof once every 1, 2, 3, 4, 5, or 6 weeks.

[0156] In some embodiments, a prophylactically and / or therapeutically effective amount of the aforementioned antibody drug conjugate, or the aforementioned antibody drug conjugate formulation, or the aforementioned method of making an antibody drug conjugate formulation, or the aforementioned composition is administered to a subject in need thereof once every 18-24 days.

[0157] In some embodiments, the application further comprises administering to the subject in need thereof an additional chemotherapeutic drug or an immunosuppressive agent for treating the tumor.

[0158] In some embodiments, the chemotherapeutic drug is, for example, Adriamycin, cyclophosphamide and taxanes [Taxol and Taxotere], Xeloda, Gemzar, Navelbine, tamoxifen, aromatase inhibitors (Arimidex, Femara, Aranas), 5-FU plus leucovorin, camptosar, oxaliplatin, cisplatin, carboplatin, estramustine, Novantrone, prednisone, Oncovin, doxorubicin, prednisolone, etc., or a combination thereof.

[0159] In some embodiments, the immunosuppressive agent is selected from the group consisting of: (1) glucocorticoids such as cortisone and prednisone; (2) microbial metabolites such as cyclosporine and mycophenolic acid, etc.; (3) antimetabolites such as azathioprine and 6-mercaptopurine, etc.; (4) polyclonal and monoclonal anti-lymphocyte antibodies such as anti-lymphocyte globulin and OKT3, etc.; (5) alkylating agents such as cyclophosphamide. Specifically, the immunosuppressive agent is, for example, methylprednisolone, prednisone, azathioprine, mycophenolate mofetil, sirolimus, tacrolimus, rapamycin, mycophenolic acid, mizoribine, cyclophosphamide, fingolimod, etc.

[0160] In a seventh aspect of the present application, the present application provides the aforementioned antibody drug conjugate, or the aforementioned antibody drug conjugate formulation, or the aforementioned antibody drug conjugate formulation prepared by the aforementioned method, or the aforementioned composition for use in preventing and / or treating a CD20-expressing cancer or an immune disease.

[0161] In some embodiments, the CD20-expressing cancer is a lymphoma or a leukemia.

[0162] In some embodiments, the lymphoma is a non-Hodgkin lymphoma, a B-cell non-Hodgkin lymphoma, a follicular non-Hodgkin lymphoma, a small lymphocytic lymphoma, or a diffuse large B-cell lymphoma.

[0163] In some embodiments, the leukemia is a chronic lymphocytic leukemia, a hairy cell leukemia, a B-cell prolymphocytic leukemia, or an acute lymphoblastic leukemia.

[0164] In some embodiments, the CD20-expressing immune disease is rheumatoid arthritis, granulomatosis with polyangiitis, Wegener's granulomatosis, microscopic polyangiitis, or multiple sclerosis.

[0165] In some embodiments, the rheumatoid arthritis is severe active rheumatoid arthritis that has failed treatment with at least one TNF antagonist.

[0166] Preferably, in some embodiments, the CD20-expressing cancer is a CD20-positive B-cell lymphoma.

[0167] In some embodiments, the CD20-positive B-cell lymphoma is a CD20-positive B-cell lymphoma that is resistant to an anti-CD20 mAb (e.g., rituximab).

[0168] In some embodiments, the lymphoma is a non-Hodgkin lymphoma.

[0169] In some embodiments, the lymphoma is a non-Hodgkin lymphoma that is resistant to an anti-CD20 mAb (e.g., rituximab).

[0170] In some embodiments, the non-Hodgkin lymphoma is a diffuse large B-cell lymphoma.

[0171] In some embodiments, the non-Hodgkin lymphoma is a diffuse large B-cell lymphoma that is resistant to an anti-CD20 mAb (e.g., rituximab).

[0172] In some embodiments, the subject is a NHL PDX model and the dose administered is 0.3-10 mg / kg (e.g., 0.3 mg / kg, 1 mg / kg, 3 mg / kg, 10 mg / kg).

[0173] In some embodiments, the subject is a NHL PDX model and the dose administered is 0.3-3 mg / kg, 1-3 mg / kg, 1-10 mg / kg, or 3-10 mg / kg.

[0174] In some embodiments, the subject is a human and the dose administered is 0.1-5 mg / kg (e.g., 0.1 mg / kg, 0.3 mg / kg, 1 mg / kg, 3 mg / kg, or 5 mg / kg).

[0175] Beneficial effects:

[0176] 1. ADC-1 is an ADC conjugated by vcMMAE from Rituximab biosimilar MAB801, killing CD20-positive non-Hodgkin lymphoma (NHL) cells by binding to the CD20 receptor on the surface of tumor cells, endocytosis, and releasing MMAE to control tumor growth and cause tumor regression.

[0177] 2、ADC-1 in a variety of CD20-expressing NHL cell lines, as well as a variety of CD20-expressing human NHL PDX tumor models, all showed significant inhibitory effects on tumor cell growth, especially for Drug-resistant NHL PDX models also showed significant tumor growth inhibition;

[0178] 3、ADC-1 preparation process, the whole reaction process only in a container to complete the order, without intermediate purification steps, better than other preparation processes;

[0179] 4、ADC-1 preparation process, the whole process only needs one ultrafiltration purification step, without chromatography process, better than other preparation processes;

[0180] 5、ADC-1 has good formulation stability. BRIEF DESCRIPTION OF DRAWINGS

[0181] Figure 1 HIC-HPLC spectrum of ADC-1 of the embodiment of the application, wherein Minutes represents minutes;

[0182] Figure 2 ADC-1 of the embodiment of the application and Representative cell killing curve in Daudi cell lines, wherein Inhibition represents inhibition rate, and Concentration represents concentration;

[0183] Figure 3 ADC-1 of the embodiment of the application and Representative cell killing curve in Jeko-1 cell lines, wherein Inhibition represents inhibition rate, and Concentration represents concentration;

[0184] Figure 4 ADC-1 of the embodiment of the application and Representative cell killing curve in Raji cell lines, wherein Inhibition represents inhibition rate, and Concentration represents concentration;

[0185] Figure 5 ADC-1 of the embodiment of the application and Representative cell killing curve in Ramos cell lines, wherein Inhibition represents inhibition rate, and Concentration represents concentration;

[0186] Figure 6 ADC-1 of the embodiment of the application and Schematic diagram of the effect on tumor volume of CD20-positive human lymphoma (NHL) PDX model LYM#004;

[0187] Figure 7 ADC-1 and ADC-1 in this embodiment of the invention Schematic diagram of the effect on tumor volume of CD20-positive human lymphoma (NHL) PDX model LYM#013;

[0188] Figure 8 ADC-1 and ADC-1 in this embodiment of the invention Schematic diagram of the effect on tumor volume of CD20-positive human lymphoma (NHL) PDX model LYM#016;

[0189] Figure 9 This diagram illustrates the effect of pH on the SEC stability of the active substance ADC-1 (10mM histidine buffer system). The left figure shows the trend of SEC monomer content of ADC-1 in different pH systems with 10mM histidine, while the right figure shows the trend of SEC polymer content of ADC-1 in different pH systems with 25°C for 6 weeks and 40°C for 4 weeks. Detailed Implementation

[0190] The embodiments of the present invention will be described in detail below with reference to examples. However, those skilled in the art will understand that the following examples are for illustrative purposes only and should not be considered as limiting the scope of the invention. Unless otherwise specified in the examples, conventional conditions or conditions recommended by the manufacturer are followed. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0191] In this invention, unless otherwise stated, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the terms and laboratory procedures related to protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, and immunology used herein are all widely used terms and routine procedures in their respective fields. To better understand this invention, definitions and explanations of relevant terms are provided below.

[0192] Unless otherwise stated, in this invention, any concentration range, percentage range, proportion range or numerical range shall be understood to include any integer value within the range, and, where appropriate, fractional values ​​within the range.

[0193] In the present application, the term "antibody" refers to an immunoglobulin molecule that is composed of two identical pairs of polypeptide chains, each pair having one "light" (L) chain and one "heavy" (H) chain. The light chain can be classified as kappa and lambda. The heavy chain can be classified as mu, delta, gamma, alpha, or epsilon, and the antibody can be classified as IgM, IgD, IgG, IgA, and IgE, respectively, depending on the class of the heavy chain. Within the light and heavy chains, the variable and constant regions are joined by a "J" region of about 12 or more amino acids. The heavy chain further comprises a "D" region of about 3 or more amino acids. Each heavy chain is composed of a heavy chain variable region (V H ) and a heavy chain constant region (C H ). The heavy chain constant region is composed of three domains, C H 1, C H 2, and C H 3. Each light chain is composed of a light chain variable region (V L ) and a light chain constant region (C L ). The light chain constant region is composed of one domain, C L . The constant regions of antibodies can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and components of the complement system, Clq. V H and V L regions can also be subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FRs). Each V H and V L is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of each heavy / light chain pair (V H and V L ) form the antibody binding site. The assignment of amino acids to each region or domain follows the definition set out in Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)), or Chothia & Lesk (1987) J. Mol. Biol. 196:901-917; Chothia et al. (1989) Nature 342:878-883.

[0194] The antibody suitable for the present application is an antibody targeting CD20, and the anti-CD20 mAb is any antibody targeting CD20, such as rituximab or its biosimilars. Among them, the biosimilars refer to the antibody products with the same sequence as rituximab, the same physicochemical properties and biological activity, and the same clinical safety and effectiveness as rituximab.

[0195] In the present application, the structure of MMAE is:

[0196]

[0197] In the present application, the drug:antibody ratio (DAR) or drug loading is represented by p or q, i.e. Formula I: Ab-(L-D) p or Formula II: Ab-(L-D) q The average number of drug moieties (i.e. cytotoxic agents) per antibody in the molecule of Formula I or II can be an integer or a fraction. ADCs of Formula I include a collection of antibodies conjugated to a range (3.6-4.0) of drug moieties, and ADCs of Formula II include a collection of antibodies conjugated to a range (3.3-4.3) of drug moieties. The average number of drug moieties per antibody in an ADC preparation from a conjugation reaction can be verified by routine means such as mass spectrometry, ELISA assays, HIC and HPLC. The quantitative distribution of ADCs in terms of p or q can also be determined. In some cases, it can be desirable to isolate, purify and verify homogenous ADCs having a certain value of p or q from ADCs having other drug loadings by means such as reverse phase HPLC or electrophoresis.

[0198] In certain embodiments, less than the theoretical maximum number of drug moieties are conjugated to the antibody in the conjugation reaction. Generally, the antibody does not contain many free and reactive cysteine thiol groups that can attach drug moieties; in fact, most of the cysteine thiol groups in the antibody are present as disulfide bridges. In certain embodiments, the antibody can be reduced under partial or complete reducing conditions with a reducing agent such as dithiothreitol (DTT) or tricarbonylethylphosphine (TCEP) to generate reactive cysteine thiol groups.

[0199] The antibody drug conjugate of the present application can be used in combination with known chemotherapeutic agents or immunosuppressive agents for treating tumors, such as Adriamycin, cyclophosphamide, and taxanes [Taxol and Taxotere], Xeloda, Gemzar, Navelbine, tamoxifen, aromatase inhibitors (Arimidex, Femara, Aranas), 5-FU plus leucovorin, camptosar, oxaliplatin, cisplatin, carboplatin, estramustine, novantrone, prednisone, oncovin, doxorubicin, prednisolone, etc., or combinations thereof; and immunosuppressive agents selected from the group consisting of (1) glucocorticoids such as cortisone and prednisone; (2) microbial metabolites such as cyclosporin and mycophenolic acid; (3) antimetabolites such as azathioprine and 6-mercaptopurine; (4) polyclonal and monoclonal anti-lymphocyte antibodies such as anti-lymphocyte globulin and OKT3; and (5) alkylating agents such as cyclophosphamide. In particular, the immunosuppressive agent is, for example, methylprednisolone, prednisone, azathioprine, mycophenolate mofetil, sirolimus, tacrolimus, rapamycin, everolimus, cyclosporin, mycophenolic acid, mizoribine, cyclophosphamide, fingolimod, etc.

[0200] In the present application, "treatment" refers to clinical intervention in an attempt to alter the natural course of the individual or cell being treated, and can be performed either for prophylaxis or during the course of clinical pathology. Desirable effects of treatment include preventing occurrence or reoccurrence of disease, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, preventing metastasis, decreasing the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis. In some embodiments, antibodies or antibody drug conjugates of the application are used to delay the onset of disease or disorder, or to slow the progression of disease or disorder. The above parameters for assessing the success of treatment and improvement in disease can be readily measured by those skilled in the art. For cancer treatment, efficacy can be measured, for example, by assessing the time to disease progression (TTP) and / or determining the response rate (RR).

[0201] In the present application, "subject" refers to a vertebrate. In certain embodiments, the vertebrate refers to a mammal. Mammals include, but are not limited to, domestic animals such as cows, swine, and horses; pets such as dogs, cats, and horses; primates; mice, and rats. In certain embodiments, the mammal refers to a human.

[0202] In the present application, an "effective amount" means the amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result. A "therapeutically effective amount" of a substance / molecule of the present application can vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the substance / molecule to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of the substance / molecule are outweighed by the therapeutically beneficial effects. A "prophylactically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result. Generally, but not necessarily, since a prophylactic dose is used in subjects prior to or at the early stages of disease, the prophylactically effective amount will be less than the therapeutically effective amount. In the case of cancer, a therapeutically effective amount of a drug can reduce the number of cancer cells; reduce the tumor size; inhibit (i.e., slow to some extent, preferably stop) cancer cell infiltration into peripheral organs; inhibit (i.e., slow to some extent, preferably stop) tumor metastasis; inhibit, to some extent, tumor growth; and / or relieve to some extent one or more of the symptoms associated with the cancer.

[0203] For the prevention or treatment of disease, the appropriate dosage of an antibody drug conjugate of the present application (when used alone or in combination with one or more other additional therapeutic agents such as chemotherapeutic agents) will vary depending on the type of disease to be treated, the type of antibody drug conjugate, the severity and course of the disease, whether the antibody drug conjugate is for preventive or therapeutic use, previous therapy, the patient's clinical history and response to the antibody drug conjugate, and the judgment of the treating physician. Suitable dosages are readily determined by those skilled in the art using dosages conventionally used for the given therapeutic agent. It is also contemplated that the therapeutic agent can be administered in dosages that are less than those normally used for monotherapy with the therapeutic agent alone. For example, dosages of the antibody drug conjugate can be in the range of about 0.1 mg / kg to about 5 mg / kg. Thus, a patient can be administered one or more doses of about 0.1 mg / kg, 0.3 mg / kg, 1.0 mg / kg, 3.0 mg / kg, or 5 mg / kg (or any combination thereof) of the antibody drug conjugate.

[0204] A "pharmaceutically acceptable carrier" when used in the present application includes a pharmaceutically acceptable carrier, excipient, or stabilizer, which is nontoxic to the cells or mammal being exposed thereto at the dosages and concentrations employed. Typically, a physiologically acceptable carrier is an aqueous pH buffered solution. Examples of physiologically acceptable carriers include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid; low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, sucrose, trehalose, or dextrins; chelating agents, such as EDTA; sugar alcohols, such as mannitol or sorbitol; salt-forming counterions, such as sodium; and / or nonionic surfactants, such as TWEEN TMPolyethylene glycol (PEG) and PLURONICS TM .

[0205] In this invention, the 20 common amino acids and their abbreviations follow their usual usage. See Immunology-ASynthesis (2nd edition, ESGolub and DRGren, Eds., Sinauer Associates, Sunderland, Mass. (1991)), which is incorporated herein by reference.

[0206] The present invention will be further explained and described below with reference to specific embodiments.

[0207] Example 1: Construction of a cell line for the production of antibody MAB801 (the naked antibody portion for antibody-drug conjugate ADC-1)

[0208] Analysis of the commercially available drug Rituximab (MabThera) by LC / MS / MS The amino acid sequences of the light and heavy chains of rituximab were determined. Based on the sequence information of the patent (Anderson-1998, patent number: US5736137A), the complete gene sequences of the light and heavy chains were further confirmed. The light and heavy chain gene DNA of MAB801 were obtained through gene synthesis. Then, using a dual plasmid vector expression system (from Invitrogen), light chain (LC) and heavy chain (HC) gene expression plasmids were constructed. The two plasmids were co-transfected into mammalian host cells CHO DG44 (from Invitrogen). After two-step pressure screening, a cell population integrating both light and heavy chain gene sequences was obtained. The antibody expression capacity of the cell population was assessed to be satisfactory. Single-cell clones were screened using semi-solid culture medium. The obtained single-cell clones were screened through batch feeding experiments to select the cell line with the highest antibody expression level. Based on this cell line, a production cell bank of MAB801 was prepared.

[0209] Gene sequencing of the monoclonal cell line showed that the sequences encoding the light and heavy chains of MAB801 were completely identical to those of commercially available rituximab.

[0210] Example 2: Preparation of antibody MAB801 (for the naked antibody portion of antibody-drug conjugate ADC-1)

[0211] The preparation of MAB801 adopts the flow cell culture process widely used in the field of antibodies at present. The process, starting from the cell seed recovery of the working cell bank, includes the main steps of shake flask inoculation, shake flask cell expansion, reactor step-by-step cell expansion culture, and then inoculation into a production reactor for flow culture. Then, after the purification process including fermentation broth clarification treatment (depth filtration), Protein A affinity chromatography, low-pH virus inactivation and depth filtration, PB incubation, anion exchange chromatography, cation exchange chromatography, virus removal filtration, and tangential flow ultrafiltration diafiltration, etc., the product is obtained by filtration and dispensing.

[0212] The test results show that the molecular weight and other physicochemical indexes of the produced rituximab biosimilar are highly similar to those of rituximab.

[0213] Example 3 ADC-1—Preparation method of a CD20 antibody conjugate

[0214] 1) Take 10 mg of MAB801 antibody, and replace into a reducing buffer (25 mM sodium borate, pH 8.0, 25 mM NaCl, 5 mM EDTA) using a 15 mL 30 KD ultrafiltration device for three times; the final volume is about 1 mL, which is transferred to a new Eppendorf centrifuge tube (weighed), and weighed; detect the protein concentration, and calculate the total amount of protein.

[0215] 2) Add 2.5 times the molar number of DTT to the antibody, incubate at room temperature for 2 hours, and continuously mix; replace into a conjugation buffer (50 mM Tris, pH 7.2, 150 mM NaCl, 5 mM EDTA) using a 15 ml 30 KD ultrafiltration device for three times. Take the concentrated liquid, and detect the free thiol number by the Ellman's method; 280 Detect the protein concentration, and weigh, to calculate the total amount of protein; take 10 μl of the sample, and detect the free thiol number by the Ellman's method;

[0216] And calculate the molar concentration of the free thiol according to the following formula:

[0217]

[0218] b: The light path length of the cuvette (usually 1 cm)

[0219] According to the molar concentration of the free thiol and the total protein solution volume, the number of moles of the free thiol is calculated.

[0220] 3) Add 1.1 times the number of moles of vc-MMAE (dissolved in DMSO) to the reduced antibody, mix, and react at room temperature for 2 hours, with intermittent mixing. Add 20 times the number of moles of N-acetylcysteine to the reaction system, mix, and stand for 5 minutes.

[0221] 4) Use a 15ml 30KD ultrafiltration device to replace the conjugate storage solution (10mM histidine, 6% sucrose, 0.035% PS80, pH 5.8) three times to obtain the antibody-drug conjugate ADC-1, which is stored at 4℃. The concentration of the active ingredient - antibody-drug conjugate - is 5mg / mL.

[0222] Example 4: Determination of drug / antibody ratio in ADC-1

[0223] The prepared antibody-drug conjugate ADC-1 was analyzed by HIC-HPLC (Ouyang-2013) to determine the drug-antibody ratio (DAR). See [link to HIC-HPLC analysis]. Figure 1 The average drug loading rate (DAR) was calculated to be 3.8 based on the peak area of ​​the spectrum.

[0224] Example 5: In vitro cytotoxic activity of ADC-1 against CD20-expressing NHL cells

[0225] To investigate the cytotoxic effect of ADC-1 on CD20-expressing NHL cells, four CD20-expressing NHL cell lines—Daudi, Jeko-1, Raji, and Ramos—were selected. Cell proliferation inhibition assays were used. The assay reagents were used to determine ADC-1 and a commercially available reference drug. The results of the study on the killing effect on the above four cell lines are shown in Table 2 and... Figure 2 , Figure 3 , Figure 4 and Figure 5 As shown.

[0226] Table 2: ADC-1 and Killing effects in four CD20-expressing NHL cell lines

[0227]

[0228] Note: CD20 expression levels are referenced from Barth-2015 and Law-2004. IC 50 The value is the IC calculated from two 96-hole boards. 50 The average value; ND indicates that the highest inhibition percentage is less than 50%, therefore IC 50 The value cannot be calculated.

[0229] Experimental results showed that ADC-1 had significant cytotoxic effects on four CD20-expressing NHL cell lines, and the effects were significantly better than those of commercially available reference drugs.

[0230] Example 6 Tumor inhibition effect of ADC-1 in NHL PDX models

[0231] Human tumor tissue xenograft models (PDX models) are tumor models established by using human primary tumor tissues in immunodeficient mice, which retain the heterogeneity, molecular diversity and histological characteristics of primary tumors to the greatest extent, and have high predictive value for the clinical therapeutic effect of drugs. In recent years, they have been increasingly applied in cancer research (Hidalgo-2014). In order to effectively evaluate the efficacy of ADC-1 in future clinical indications, experiments were carried out in 3 CD20-expressing human lymphoma (NHL) PDX models. In all experiments, commercially available CD20-targeted mAb drugs were used as reference drugs to compare the tumor inhibition activity. Among the 3 NHL PDX models, 2 were resistant to the reference drugs.

[0232] The information of the 3 PDX models used in the study is shown in Table 3.

[0233] Table 3: PDX model information used in in vivo pharmacodynamic experiments

[0234]

[0235] 1. Efficacy study of ADC-1 in CD20-positive NHL PDX model LYM#004

[0236] LYM#004 is a drug-resistant DLBCL PDX model, and the experimental results are shown in Figure 6 Table 4. After administration of ADC-1 at doses of 1, 3 and 10 mg / kg, the T / C (%) on Day 18 was 45.90% (P>0.05), 4.46% (P<0.001) and 2.05% (P<0.001), respectively, and the TGI% was 54.10%, 95.54% and 97.95%, respectively. After administration of ADC-1 at a dose of 3 mg / kg, all tumor parts were regressed on Day 18; after administration of ADC-1 at a dose of 10 mg / kg, 5 / 8 of the tumors were partially regressed and 3 / 8 of the tumors were completely regressed on Day 18. After administration at doses of 3 and 10 mg / kg, the T / C (%) on Day 18 was 50.76% (P>0.05) and 51.50% (P>0.05), respectively, and the TGI% was 49.24% and 48.50%, respectively.

[0237] The experimental results showed that ADC-1 (3 and 10 mg / kg) had significant tumor growth inhibition activity, and ADC-1 (1 mg / kg) and the reference drugs ADC-1 (0.3, 1 and 3 mg / kg) and the reference drug Rituximab (3 mg / kg) all had no significant anti-tumor activity. The tumor-bearing mice had good tolerance to ADC-1 and Rituximab.

[0238] 2. Efficacy study of ADC-1 in CD20-positive NHL PDX model LYM#013

[0239] LYM#013 is a DLBCL PDX model, and the experimental results are shown in Table 2. After administration of ADC-1 at doses of 0.3, 1 and 3 mg / kg, the T / C (%) on Day 28 was 31.90% (P<0.05), 0.00% (P<0.001) and 0.00% (P<0.001), respectively, the TGI% was 68.10%, 100.00% and 100.00%, respectively, 2 / 8, 0 / 8 and 0 / 8 of the tumors were partially regressed, and 0 / 8, 8 / 8 and 8 / 8 of the tumors were completely regressed, respectively. Figure 7 After administration at a dose of 3 mg / kg, the T / C (%) on Day 28 was 79.86% (P>0.05), and the TGI% was 20.14%. The experimental results show that ADC-1 (0.3, 1 and 3 mg / kg) has a significant effect on inhibiting tumor growth, and has a certain dose dependence, while the reference drug Rituximab (3 mg / kg) has no significant anti-tumor activity. The tumor-bearing mice have good tolerance to ADC-1 and Rituximab.

[0240] 3. Efficacy study of ADC-1 in CD20-positive NHL PDX model LYM#016

[0241] LYM#016 is a DLBCL PDX model, and the experimental results are shown in Table 3.

[0242] After administration of ADC-1 at doses of 0.3, 1 and 3 mg / kg, the T / C (%) on Day 14 was 37.76% (P>0.05), 4.27% (P<0.01) and 1.91% (P<0.01), respectively, the TGI% was 62.24%, 95.73% and 98.09%, respectively. After administration of ADC-1 at a dose of 1 mg / kg, all the tumors were partially regressed on Day 14; after administration of ADC-1 at a dose of 3 mg / kg, 6 / 8 of the tumors were partially regressed, and 2 / 8 of the tumors were completely regressed on Day 14. Figure 8 After administration at a dose of 3 mg / kg, the T / C (%) on Day 14 was 30.98% (P>0.05), and the TGI% was 69.02%.

[0243] ​​​​The results showed that ADC-1 (1 and 3 mg / kg) had significant inhibitory effects on tumor growth, while ADC-1 (0.3 mg / kg) and the reference drug (3 mg / kg) had no significant anti-tumor activity. The tumor-bearing mice had good tolerance to ADC-1 and .

[0244] The results of the in vivo efficacy test of ADC-1 in three human-derived lymphoma (NHL) PDX models positive for CD20 are shown in Table 4.

[0245] Table 4: In vivo efficacy results of ADC-1 (i.v., q4d x 4) in CD20-positive human lymphoma PDX models

[0246]

[0247]

[0248]

[0249] Note: “ / ” means not applicable. The formula for calculating the tumor volume (TV) is: TV = l x w 2 / 2, where l and w represent the length and width of tumor measurement, respectively. The relative tumor volume (RTV) was calculated according to the measurement results, RTV = V f / V0, where V0 is the tumor volume measured at the time of administration (i.e., Day 0), and V f is the tumor volume measured on the last day. The relative tumor proliferation rate T / C (%) = (RTV of the administration group / RTV of the vehicle group) x 100%. The tumor growth inhibition rate TGI% = (average tumor volume of the vehicle group - average tumor volume of the administration group) / average tumor volume of the vehicle group x 100%. “++++” means T / C (%) ≥ 0 and ≤ 10%, “+++” means T / C (%) > 10% and ≤ 20%, “++” means T / C (%) > 20% and ≤ 40%, and “+” means T / C (%) > 40%. The anti-tumor activity of ADC-1 and The strength of the anti-tumor activity is determined according to the T / C (%) value of the highest dose in the model.

[0250] In summary, the in vivo efficacy test results show that ADC-1 has significant tumor growth inhibition effects in three PDX models of human lymphoma (NHL) (all models are DLBCL), and the tumor-bearing mice show good tolerance to ADC-1. It is worth noting that two of the three DLBCL PDX models are resistant to chemotherapy drugs.

[0251] Effect of pH on the stability of ADC-1 formulation

[0252] Based on the formulation research and previous experience of the inventors of the present application, the formulation prescription was finally determined as 5 mg / mL antibody drug conjugate (naked antibody drug conjugate without buffer and other components), 10 mM histidine buffer (pH 5.8), 6% sucrose, and 0.035% Tween 80 (PS80).

[0253] To investigate the effect of different pH of ADC-1 in 10 mM histidine solution on its quality, it was changed to prescription F1-F4 (pH: 5.8-7.0) to be investigated, and was placed at 25±2℃ / 60%±5%RH and 40±2℃ / 75%±5%RH for 6 weeks and 4 weeks, respectively. At the set time points, samples were taken for analysis, and the detection items included appearance, pH, protein concentration, SEC, iCIEF, and HIC. If the sample appeared obvious opalescence, it would not be subjected to subsequent related analysis. The specific experimental design is shown in Table 5.

[0254] Table 5: Experimental design for the effect of pH (5.8-7.0) on the stability of ADC-1 (10 mM histidine buffer)

[0255]

[0256] Note: W represents week, i.e. 2W is 2 weeks; and represents detection.

[0257] The experimental results showed that when ADC-1 was in 10 mM histidine buffer, its stability decreased with the increase of pH.

[0258] After being placed at 25℃ for 6 weeks, all samples were colorless and clear solutions, and the pH and concentration did not change. The iCIEF purity decreased, the acidic peak content increased, and the change rate accelerated with the increase of pH. The SEC purity showed a downward trend, the high polymer (HMW) content increased, and the change rate accelerated with the increase of pH. The DAR value did not show obvious change.

[0259] After being placed at 40℃ for 4 weeks, the sample stability showed the same trend, but the change range intensified. When the pH was 6.7 and 7.0, the appearance of the sample changed after one week at 40℃, and the opalescence degree intensified with the increase of pH. The UV detection result deviated from the actual value due to the reading distortion caused by the existence of particulate matter (opalescence). The SEC purity decreased, the high polymer and low molecular weight fragment (LMW) contents increased, and the change rate accelerated with the increase of pH. The iCIEF purity decreased, the acidic peak content increased, and the basic peak content decreased, and the change rate accelerated with the increase of pH.

[0260] A trend plot of SEC purity of the sample as a function of pH is shown in Figure 9 .

[0261] While the specific embodiments of the application have been described in detail, those skilled in the art will appreciate that various modifications and alternatives to those details could be developed in light of the overall teachings of the disclosure. The foremost scope of the application is given by the appended claims and any equivalents thereof.

Claims

1. An antibody drug conjugate formulation comprising: an antibody drug conjugate at a concentration of 1-60 mg / mL; 5-35 mM histidine buffer, pH 5.8-6.2; 2-10% (w / v) sucrose; and 0.01-0.1% (w / v) polysorbate 80; said antibody drug conjugate having a structure according to Formula I, Ab-(L-D) q Formula I wherein: Ab is an anti-CD20 mAb, said anti-CD20 mAb being rituximab; D is a cytotoxic agent, said cytotoxic agent being monomethyl auristatin E (MMAE); L is a linker, for linking said anti-CD20 mAb and said cytotoxic agent, said linker being 6-maleimidocaproyl-valine-citrulline-p-aminobenzoxycarbonyl (MC-vc-PAB); q is 3.3-4.

3.

2. The antibody drug conjugate formulation of claim 1, wherein, said formulation comprising: said antibody drug conjugate at a concentration of 1-10 mg / mL; 5-15 mM histidine buffer, pH 5.8-6.2; 3-9% sucrose; and 0.02-0.06% polysorbate 80.

3. The antibody drug conjugate formulation of claim 1, wherein, said formulation comprising: said antibody drug conjugate at a concentration of 3-7 mg / mL; 8-12 mM histidine buffer, pH 5.8-6.2; 5-7% sucrose; and 0.03-0.04% polysorbate 80.

4. The antibody drug conjugate formulation of claim 1, wherein, said formulation comprising: said antibody drug conjugate at a concentration of 5 mg / mL; 10 mM histidine buffer, pH 5.8; 6% sucrose; and 0.035% polysorbate 80.

5. The antibody drug conjugate formulation of any one of claims 1-4, wherein, q is 3.6-4.

0.

6. The antibody drug conjugate formulation of any one of claims 1-4, wherein, q is 3.7-3.

9.

7. The antibody drug conjugate formulation of any one of claims 1-4, wherein, q is 3.

8.

8. A composition comprising the antibody drug conjugate formulation of any one of claims 1-7.

9. The composition of claim 8, wherein, said composition further comprising at least one pharmaceutically acceptable carrier, diluent, or excipient.

10. Use of the antibody drug conjugate formulation of any one of claims 1-7 or the composition of any one of claims 8-9 for the manufacture of a medicament for the prevention and / or treatment of a CD20-expressing cancer or an immune disorder, said cancer being non-Hodgkin's lymphoma or chronic lymphocytic leukemia, said immune disorder being rheumatoid arthritis.

11. The use of claim 10, wherein, said non-Hodgkin's lymphoma being small lymphocytic lymphoma or diffuse large B-cell lymphoma.

12. The use of claim 10, wherein, said non-Hodgkin's lymphoma being B-cell non-Hodgkin's lymphoma or follicular non-Hodgkin's lymphoma.

13. The use of claim 10, wherein, said rheumatoid arthritis being severe active rheumatoid arthritis that has failed treatment with at least one TNF antagonist.

14. The use of claim 10, wherein, said non-Hodgkin's lymphoma being anti-CD20 mAb-resistant non-Hodgkin's lymphoma.

15. The use of claim 10, wherein, said non-Hodgkin's lymphoma being rituximab-resistant non-Hodgkin's lymphoma.

16. The use of claim 10, wherein, said non-Hodgkin's lymphoma being anti-CD20 mAb-resistant diffuse large B-cell lymphoma.

17. The use of claim 10, wherein, said non-Hodgkin's lymphoma being rituximab-resistant diffuse large B-cell lymphoma.

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